CHARACTERIZATION OF
WCWRGNANS FURMED BY
A CELL~FREE SYSTEM
FROM W MNOR

Emma for the W of Ph. 6.
MICHEGAR STATE UM‘JERSW
GENRE MEMO. 3R...
E 3 7,5

This is to certify that the

thesis entitled

CHARACTERIZATION OF APIOGALACTURONANS FORMED BY A
CELL-FREE SYSTEM FROM LEMNA MINOR

 

presented by

Leonard Mas caro , Jr.

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in Biochemistry

 

 

PM KW

Major professor

Date February 11, 1975

 

0-7 639

 

?
I

   
     
   

    

‘5"

? snuggle. av

‘nnns 2 sons 3
l 800', BlNDERY INC.

LlBRARY BINDFRS
meavom. mm;

‘W

’_

3,.

1
\ ABSTRACT

CHARACTERIZATION OF APIOGALACTURONANS
FORMED BY A CELL-FREE SYSTEM
FROM LEMNA MINOR

BY

Leonard Mascaro, Jr.

The products formed from UDP-D-apiose and UDP-
D-galacturonic acid in a cell-free system from £2223
miner have been characterized. Characterization experi-
ments have established that the products are apiogalac-
turonans.

UDP-D-[U-14C1Apiose (containing some UDP-D-
[U-14C1xylose) and UDP-D-[U-14C]ga1acturonic acid were
incubated with a particulate enzyme preparation from
E, miggg. At the end of the incubation the reaction
mixtures were extracted with methanol and water. The
radioactive material synthesized from UDP-D-[U-14C1-
apiose and UDP-D-[U-14clgalacturonic acid and remaining

in the insoluble residue is referred to as D-[U-14

l

C]apiose
product and D-[U- 4C]galacturonic acid product, respec-
tively. Based on radioactivity measurements extraction

of the products with ammonium oxalate solubilized

87% of the D-[U-14C1apiose product and 91% of the

Leonard Mascaro, Jr.

D-[U-14

C]galacturonic acid product. The radioactive
materials that were solubilized by ammonium oxalate
treatment are referred to as D-[U-14C1apiose solubilized

product and D-[U-14

C]galacturonic acid solubilized pro-
duct. Fungal pectinase could hydrolyze both solubilized
products. Hydrolysis of the D-[U-14C]apiose solubilized
product at pH 1 showed that 75% of the radioactivity was
present in D-[U-14C1apiose and 25% in D—[U-14C1xylose
(25%). The D-[U-14C]galacturonic acid solubilized pro-
duct was found to contain its radioactivity in D-[U-14CJ-
galacturonic acid and a small amount (less than 6%) in
D-[u-14c1xylose.

D-[U-14C1Apiose solubilized product released

-14c]apiose and [U-l4C1apiobiose when hydrolyzed at

D- [U
pH 4. A similar release of apiobiose has been reported
.for authentic apiogalacturonans. When [U-14C1apiobiose

1

was isolated from the D-[U- 4C]apiose product, reduced

by NaBH4 treatment, and hydrolyzed, the radioactivity

l4C]apiose and D-[U-14C1apiitol.

was found equally in D-[U-
This indicates that both moieties of the apiobiosyl side
chains were synthesized in XiEEQ’

Five fractions were obtained when D-[U-14C1-
galacturonic acid was chromatographed on a DEAE-Sephadex

column. The fraction which eluted with 0.25 M NaCl

(fraction D) contained 40% of the recovered

Leonard Mascaro, Jr.

radioactivity. When similarly chromatographed on the
DEAE-Sephadex column, the D-[U-14C1apiose solubilized
product was also recovered in 5 fractions that eluted

in the same positions of the gradient as seen for the
D-[U-14C]galacturonic acid product. The fractions were
labelled, in order of elution, A through E. The fractions

l4c]-

eluted from the column in order of increasing D-[U-
apiose content. Fraction D which eluted with 0.25 M
NaCl contained 21% of the recovered radioactivity.
Analysis of this fraction showed that 88% of its radio-
activity was contained in D-[U-14C]apiose and the
remainder in D-[U-14C1xylose. Hydrolysis of fraction D
at pH 4 released 63% of the D-[U-14CJapiose, the
majority as [U-14C]apiobiose.

D-[U-l4

C]Apiose solubilized product was also
synthesized in the presence of exogenous UDP-D-galac-
turonic acid and was chromatographed on the DEAE-Sephadex
column. The chromatogram showed that there was increased
synthesis of the more acidic products. Fraction D which
eluted with 0.25 M NaCl contained 53% of the radioactivity
recovered from the column. The amount of radioactivity

1

in Fraction D contained in D-[U- 4C]apiose was 95% with

the rest contained in D-[U-14C]xylose. Hydrolysis at

pH 4 released 81% of the D-[U-l4

C]apiose in Fraction D.
Gel chromatography of D-[U-14C]apiose product

and D-[U-14CIgalacturonic acid product showed that both

Leonard Mascaro, Jr.

contained molecules of different sizes. Both products
eluted from a Bio-gel P-300 column over its entire
fractionation range. Chromatography on a Bio—gel P-lOO
column showed that the D-[U-14CIga1acturonic acid
solubilized product contained molecules of smaller size
than did the D-[U-14C1apiose solubilized product.
Dialysis in water or chromatography on DEAE-Sephadex
caused a decrease in the amount of high molecular weight
material in the solubilized products as determined by
gel-chromatography.

Large, medium, and small molecular weight com-
ponents of the D-[U-14C1apiose solubilized product were
isolated by chromatography on Bio-gel P-300 and were
found not to vary significantly in D-[U-14C1apiose and
D-[U-14clxylose content. Addition of UDP-D-galacturonic
acid to the reaction mixture used to synthesize D-[U-14CJ-
apiose solubilized product resulted in an increase in the
size of the small molecular weight components. As
D-[U-14C]galacturonic acid product was synthesized for
increasing lengths of time the size of the product was
also increased. On the other hand, as the length of

l4C]apiose product

incubation used to synthesize D-[U-
was increased the percent of radioactivity found in the
large molecular weight component decreased.

These results show that the particulate enzyme

preparation from E. minor synthesizes a product from

Leonard Mascaro, Jr.

UDP-D-apiose and UDP-D-galacturonic acid which has a
structure similar to the apiogalacturonans of the cell
wall of E, E3325. The data obtained from the gel
chromatography experiments are consistent with a
mechanism of synthesis where D-apiose side chains are

added after formation of the polygalacturonic backbone.

CHARACTERIZATION OF APIOGALACTURONANS
FORMED BY A CELL-FREE SYSTEM

FROM LEMNA MINOR

 

BY

4 DWC‘
Leonard Mascaro, Jr.

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Biochemistry

1975

To Kathryn

and my parents

ii

ACKNOWLEDGMENTS

I would like to thank Dr. Paul K. Kindel for
his guidance and advice during the course of this research
project. He has served both as a colleague and a friend.
I would also like to thank Dr. Y. T. Pan and
Edwin Leinbach for their comradeship and help. Mr.
Leinbach prepared the UDPlU-14C1GalUA used in this
work. The technical assistance of Mrs. Beth Laine and
Mrs. Linda Bissett is also appreciated.
I thank the members of my guidance committee,
Drs. Loran Bieber, William Wells, Derek Lamport, Deborah
Delmer, and Clarence Suelter for the time that they have
spent reviewing my work. Drs. John Speck and A. Kivilaan
have also given me much advice on polysaccharide chemistry.
I also especially thank my wife, Kathryn, for
the loving encouragement and sound scientific advice that

she has given me.

iii

TABLE

LIST OF TABLES . . . .
LIST OF FIGURES. . . .
LIST OF ABBREVIATIONS. .
INTRODUCTION. . . . .
LITERATURE REVIEW . . .

The Plant Cell Wall. .

Components of the Cell

Morphology of Cell Wall Synthesis

OF

CONTENTS

Wall
Structure of the Cell Wall.

Role of the Golgi Apparatus

Orientation of Cellulose Microfibrils

Biosynthesis of Cell Wall Polysacchrides.

Synthesis of Glucans.

Synthesis of Matrix Polysaccharides.
Possible Role of Lipid Intermediate.
Solubilization of Polysaccharide Synthesizing

System. . . . .
D-APiOSe o o o o 0

Chemistry . . . .
Occurrence in Nature.

Identification of Apiogalacturonans in L.

minor . . . . .
UDPAp1 . . . . .

In Vitro Synthesis of Apiogalacturonans

iv

Page
viii
ix

xii

27
28

28
28

29
31
33

EXPERIMENTAL PROCEDURES . . . . . . . . .

Materials . . . . .
General Methods . . .
Culture of L. Minor. .
Paper Chromatography . . . .
DEAE-Sephadex Column Chromatography . .
Isolation of Particulate Enzyme Preparation.
Biosynthesis of Radioactive Product and Solu-
bilized Product . . . . . . . . .
Partial Acid Hydrolysis with Fuming HCl . .
Hydrolysis at pH 1 . . . . . . . . .
Hydrolysis at pH 4 . . . . . . .
Hydrolysis of Solubilized Product with Fungal
Pectinase . . . . . . . . . . . .

RESULTS 0 O O O I O O O O O O O O O
Biosynthesis of Product and Solubilized Product

Biosynthesis of D-[U-14C]Apiose Product and
Solubilized Product . . . . . .

Biosynthesis of D-[U-14C1Galacturonic Acid,
D-[U-14C]Glucuronic Acid, and D-[U-14C]-
Xylose Products and Solubilized Products .

Addition of UDPGalUA, UDPGlcUA, and UDPXyl to
the Reaction Mixture Used to Synthesize
D-[U-14C]Apiose Product and Solubilized
Product . . . . . . . . . . . .

Partial Acid Hydrolysis of D-[U-14C1Apiose
Product . . . . . . . . . . . .
Acid Hydrolysis at pH 1 . . . . . . . .

Hydrolysis of D-[U-14C1Apiose Solubilized
Product . . . .
Hydrolysis of D-[U-l‘iCJGalacturonic1 Acid, D-
[U-1C]Glucuronic Acid, and D-[U-14CI-
Xylose Solubilized Products. . . . . .
Hydrolysis of Authentic Apiogalacturonan from
Whole Plants. . . . .
Hydrolysis of D-[U-15C1Apiose Solubilized
Product Synthesized in the Presence of
Nonradioactive Sugar Nucleotides . . . .

Hydrolysis of D-[U-14C]Apiose Solubilized Product

at pH 4 O O O O O O O O O O O O O

Page
36
36
37
38
39
39
4O
41
44
44
45
45
47

47

47

50

51

51
53

53

58

62

64

66

Gel Chromatography of D-[U-1

Hydrolysis of D-[U-14C]Apiose Solubilized Pro-
duct Synthesized in the Absence and Pre-
sence of Nonradioactive Sugar Nucleotides .

Release of D-[U-14C1Apiose and [U-14C]Apio-
biose as a Function of Hydrolysis Time . .

Hydrolysis of [U-14C]Apiobiitol . . . . .

Sodium Chloride Fractionation of D-[U-14C]-
Apiose Solubilized Product. . . . . . .

DEAE-Sephadex Column Chromatography . . . .

Chromatography of D-[U-14C]Apiose Solubilized

Product . . . .
Chromatography of D-[U-14C1Galacturonic Acid
Solubilized Product . . .

Chromatography of D-[U-14C]Apiose Solubilized
Product Synthesized in the Presence of
UDPGalUA . . . . .

Chromato raphy of D-[U-14C]Glucuronic Acid and
D-[U-1C]Xylose Solubilized Products. . .

Chromatography of D-[U-14 C]Apiose Solubilized
Products Synthesized in the Presence of
UDPGlCUA and UDPXyl . . . . .

Characterization of Fractions Obtained from
the 1DEAE-Sephadex Chromatography of D-
[U-14C]Apiose Solubilized Product Syn-
thesized in the Absence and Presence
of Nonradioactive Sugar Nucleotides . . .

Rechromatography of Fractions Obtained from
DEAE-Sephadex Column Chromatography of
D-[U-14C]Apiose Solubilized Product . . .

Product and D-[U-14C]Galacturonic Acid
Solubilized Product . . . . .

"Degradation" of D- U-14C]Apiose Solubilized

Product and D-[U- 4C]Ga1acturonic Acid
Solubilized Product . .

Effect of UDPGalUA on the Size of D-[U-14C]-
Apiose Solubilized Product. . . . .

Effect of Incubation Time on the Size of D-

[U-14C1Apiose Solubilized Product and D-
[U-1 4C]Ga1acturonic Acid Solubilized
Product . . . . . . . . .

Pectinase Hydrolysis of Solubilized Products .

Hydrolysis of D-[U-14C]Apiose Solubilized
Product . . . .

Hydrolysis of D-[U-14CIGalacturonic Acid
Solubilized Product . . . . . . . .

vi

4C]Apiose Solubilized

Page

66
70
70

73
76

76

79

82

88

91

92

98

103

109

123

123
131

131

136

Page

DISCUSSION 0 O O O O O O O O O O O O O 140
Characterization of Solubilized Products . . . 140
Identification of D-[U-14C1Apiose Solubilized

Product as an Apiogalacturonan . . . . . . 145
Possible Mechanisms of Synthesis . . . . . 157
Degradation of D-[U-14C]Apiose and D-[U-14C1-

Galacturonic Acid Solubilized Products . . . 161
Summary . . . . . . . . . . . . . . 164

LIST OF REFERENCES . . . . . . . . . . . 166

vii

LIST OF TABLES
Table Page

1. Biosynthesis of D-[U-14C]A iose, D-[U-14C]-
Galacturonic Acid, D-[U- 4C]G1ucuronic
Acid, and D-[U-14C]Xylose Products and
Solubilized Products . . . . . . . . 48

2. Synthesis of D-[U-l4CJApiose Product and
Solubilized Product in the Presence and
Absence of Nonradioactive Sugar Nucleo-
tides . . . . . . . . . . . . . 52

3. Acid Hydrolysis of D-[U-14C]Apiose Solu-
bilized Product with Various Concentrations
of Trifluoroacetic Acid . . . . . . . 56

4. Hydrolysis at pH 1 of D-[U-14CJApiose Solu-
bilized Product Synthesized in the Presence
of Nonradioactive Sugar Nucleotides . . . 65

5. Hydrolysis at pH 4 of D-[U-14C]Apiose Solu-
bilized Product Synthesized in the Absence
and Presence of Nonradioactive Sugar
Nucleotides . . . . . . . . . . . 69

6. Distribution of Radioactivity in Fractions
Obtained After DEAE- Sephadex Chroma-
tography of D-[U-14C]Apiose Soluble
Product Synthesized in the Presence of
Varying Quantities of UDPGalUA. . . . . 86

7. Release of D-[U-14C]Apiose and [U-14C1Apio-
biose from Fractions Obtained from DEAE-
Sephadex Column Chromatography of D-
[U-14C]Apiose Solubilized Product Synthe-
sized in the Absence of Nonradioactive
Sugar Nucleotide and in the Presence of
UDPGalUA . . . . . . . . . . . . 94

8. Rechromatography of Fractions Obtained from

DEAE-Sephadex Column Chromatography of
D-[U-14C]Apiose Solubilized Product . . . 99

viii

Figure
1.
2.

10.

LIST OF FIGURES

Diagrammatic representation of the cell wall

Radiochromatogram scan of the products
obtained from the partial acid hydrolysis

Radiochromatogram scan of the products
obtained after hydrolysis at pH 1 of
D-[U-14C]apiose solubilized product . .

Radiochromatogram scan of the products
obtained from the hydrolysis at pH 4 of
D-[U-14C]apiose solubilized product syn-
thesized in the presence of UDPGalUA . .

Time-course of the hydrolysis at pH 4 of D-
[U-14C]apiose solubilized product synthe-
sized in the presence of UDPGalUA . . .

Radiochromatogram scan of the products
obtained from the acid hydrolysis of
[U-I4C1api0biit01 o o o o o o o o

DEAE-Siphadex column chromatogram of D-
[U-1 C]apiose solubilized product . . .

DEAE-Sephadex column chromatogram of D-
[U-14C]galacturonic acid solubilized
product. . . . . . . . . . . .

DEAE-Sephadex column chromatogram of D-
[U-14C]apiose solubilized product synthe-
sized in the presence of UDPGalUA . . .

DEAE-Sephadex column chromatograms of D-
[U-14C]glucuronic acid solubilized
product (a) and D-[U-14C]xylose solu-
bilized product (b). . . . . . . .

ix

Page

55

6O

68

’72

75

78

81

84

90

Figure Page

11. Bio-Gel P- 300 column chromatograms of D-
[U-14C]apiose solubilized product and D-
[U- 4C]galacturonic acid solubilized
product . . . . . . . . . . . . . 105

12. Bio-Gel P- 300 column chromatograms of D—
[U-14C]apiose solubilized product (a) and
the rechromatography of selected fractions
(b) O O O O O O O O O 0 O O O O 107

13. Bio-Gel P- 100 column chromatograms of D-
[U—14C]apiose solubilized product (a) and
D-[U-14C]ga1acturonic acid solubilized
Product (b). . . . . . . . . . . . 111

14. Bio-Gel P-300 column chromatograms of non-
dialyzed, water dial zed, and sodium phos-
phate dialyzed D-[U- 4C]apiose solubilized
product . . . . . . . . . . . . . 114

15. Bio-Gel P-3OO column chromatograms of D-
[U-14C]apiose solubilized product (a) and
the rechromatography of a selected fraction
after dialysis in water (b) . . . . . . 118

16. Bio-Gel P-300 column chromatography of D-
[U-14C]apiose solubilized product after
dialysis in sodium phosphate buffer and
after elution from a DEAF-Sephadex column. . 122

17. Bio-Gel P-300 column chromatograms of D-
[U-14C]apiose solubilized product synthe-
sized in the presence and absence of
UDPGalUA. . . . . . . . . . . . . 125

18. Bio-Gel P- 100 column chromatograms of D-
[U-14C]ga1acturonic acid solubilized pro-
duct synthesized in 0.5, 2, and 15 min. . . 127

19. Bio-Gel P- -300 column chromatograms of D-
[U-14C]apiose solubilized product synthe-
sized in 0.5 min and 15 min . . . . . . 130

20. Bio-Gel P- -100 column chromatograms of D-
[U-14C]apiose solubilized product that was
untreated and was treated with pectinase . . 133

Figure Page

21. Bio-Gel P-30 column chromatogram of D-
[U-14C]apiose solubilized product that
was treated with pectinase . . . . . . 135

22. Bio-Gel P-lOO column chromatograms of D-
[U-14C]ga1acturonic acid solubilized
product treated and untreated with
fungal pectinase . . . . . . . . . 138

xi

BSA

GDPGlc

GDPMan

UDPAp1

UDPAra

UDPGalUA

UDPGlc

UDPGlcUA

UDPXyl

UTP

LIST OF ABBREVIATIONS

bovine serum albumin

guanosine 5'-(a-D-glucopyranosy1 pyrophos-
phate)

guanosine 5'-(a-D-mannopyranosy1 pyrophos-
phate)

uridine 5'-(a-D—apio-D-furanosyl pyrophosphate)

uridine 5'-(a-L-arabinopyranosy1 pyrophos-
phate)

uridine 5'-(a-D-galactopyranosyluronic acid
pyrophosphate)

uridine 5'—(a-D-g1ucopyranosy1 pyrophosphate)

uridine 5'-(a-D-glucopyranosyluronic acid
pyrophosphate)

uridine 5'-(a-D-xylopyranosy1 pyrophosphate)
uridine triphOSphate
total volume

void volume

xii

INTRODUCTION

Cell-free preparations from a number of plants
have been prepared which contain glycosyl transferase
activities that are believed to be involved in cell wall
synthesis. Recently, a particulate enzyme preparation

was isolated from Lemna minor (duckweed) that incorporated

 

radioactivite sugars from UDP[U-14C]Api and UDP[U-14C]-
GalUA into products which were insoluble in methanol and
water.

The cell wall of E. minor contains large quanti-
ties of apiogalacturonans. If the products synthesized
in_zi££2 with the cell-free system from L, minor have a
structure similar to authentic apioqalacturonans then
this will confirm that the particulate enzyme prepar-
ation contains an enzymatic system capable of synthesiz-
ing an actual cell wall polysaccharide.

The goal of this research was to characterize

14C]Api and UDP[U-14C]-

the products obtained when UDP[U—
GalUA were incubated with particulate enzyme preparation
from E. minor and to determine whether the products

synthesized in vitro are the same as authentic

apiogalacturonan. A second goal was to gain insight
into the mechanism of cell wall synthesis by studying
the structure of the products synthesized under dif-
ferent reaction conditions. Some of these data have

been presented previously (1).

LITERATURE REVIEW

The Plant Cell Wall

 

Components of the Cell Wall

Plant cells contain a cell wall exterior to the
plasmalemma. The cell wall is important for the
structure and growth of the cell. This wall is composed
of lignin, carbohydrate, and protein.

Extraction of the cell walls of higher plants
with such divalent metal chelating agents as ammonium
oxalate, sodium hexametaphosphate, or EDTA results in
the release of a polysaccharide fraction rich in D-galac-
turonic acid from the cell wall (2). This material has
been termed pectin and is a complex mixture of acidic and
neutral polysaccharides. Such neutral sugars as D-galac-
tose, L-arabinose, D-xylose, L-rhamnose, and L-fucose
have been found in pectin (2). D-Galacturonic acid is
present in the cell wall as an a-l,4-ga1acturonan with
small amounts of L-rhamnose contained in the backbone
(2, 3). The galacturonic acid resudues are often found
esterified at carbon atom 6 with methanol (3). Some
of the galacturonans as isolated in pectin are free of

neutral sugars while other galacturonans contain large

amounts of neutral sugars bound as side chains to the
uronic acid backbone. A neutral polysaccharide found in
pectin is a B-l,4-D-galactan with side chains of
L-arabinose. This arabinogalactan can occur in pectin
as a pure polysaccharide or as neutral blocks connected
to galacturnans (3, 4). Other neutral polysacchrides
isolated from pectin are D-galactan and L-arabinan (2).

Those polysacchrides in the cell wall which are
neither pectins nor the 8—1,4-glucan, cellulose, are
known as hemicelluloses (5). These polysacchrides are
not structurally related to cellulose. Hemicellulose
polysaccharides are generally solubilized from the cell
wall by alkali extraction and the following polysacchride
structures are representative of them: B-l,4-xy1ans with
side chains of L-arabinose and 4-0-methyl glucuronic acid,
B-l,4-mannans which occur alone or with side chains of
D-glucose or D-galactose, and B-l,3-galactans with
L-arabinose side chains (5).

Several different proteins are contained in
plant cell walls. These proteins are involved in the
metabolism of the wall and as structural components.

The cell walls of Zea mays and Avena sativa both contain

 

proteins capable of autolytically degrading the wall
(6, 7). A hydroxyproline-rich protein was found in the
primary cell wall of sycamore and bean cells grown in

suspension culture (8). This protein was given the

name "extensin" by Lamport and was postulated to be a
structural component in the wall which acts to orientate
the matrix polysacchrides and control cell elongation

(9). During cell elongation there is need for an increase
in the plasticity of the cell wall and extensin may
function by allowing the cellulose microfibrils to slide
over one another (9). The hydroxyproline and serine
residues of extensin are attached O-glycosidically to
L-arabinose and D-galactose, respectively, and therefore
extensin seems to be linked to arabinogalactan (10, ll,

12).

Structure of the Cell Wall

 

The cell wall, as seen with the electron micro-
scope, has an ordered structure. Figure 1 is a diagram-
matic representation of the cell wall of a mature plant
cell (13). The cell wall is divided into two major
layers; the primary wall which is formed during cell
growth and the secondary wall which is formed during
cell differentiation (14). The period of cell differen-
tiation with resultant formation of the secondary wall
is referred to as secondary thickening. The primary wall
consists of a loose, random network of cellulose micro-
fibrils embedded in a matrix of pectins and hemicelluloses
(14). The secondary wall is divided into three layers,
each of which has a highly organized microfibril

structure that is orientated differently in each of

.Amoav Ham3 Hams on» no cowumuemmmummu OHuMEEmnm6flo .H musmwm

 

.3; tween.

.93. 1381.25 raucouom

'U'UUUUU"
.3

8.3. 2521.9: Emucooom .2. ---..-

3) .96. 3:51.25 beecoumm

 

the layers (14). Analysis of birch, ash, sycamore, and
pine cell walls showed that the secondary wall contained
cellulose, hemicellulose, and lignin, but not pectin (15).

The diameter of individual cellulose microfibrils
is variable. Electron microsc0py of negatively stained
cellulose has shown that the individual microfibrils are
made up of smaller, highly crystalline fibers having an
average diameter of 35 A (16). The arrangement of micro-
fibrils in the polysaccharide matrix gives the cell wall
the same increase in strength as seen in filament-wound
reinforced plastic (17). The size of cellulose molecules
isolated from primary and secondary walls differ.
Primary wall cellulose in cotton balls has a nonuniform
degree of polymerization of between 2,000 and 6,000
while secondary wall cellulose has a uniform degree of
polymerization of 14,000 (M.W.=2.3 x 106) (18). Max-
Figini has stated that there must be two different
mechanisms of cellulose synthesis to account for the
formation of these two cellulose species and that the
uniform size of secondary wall cellulose suggests a
template mechanism of synthesis (18).

Albersheim and co-workers have used chemical
and enzymatic methods to degrade the extracellular and
cell wall polysacchrides of suspension cultured sycamore
cells (19, 20, 21). Characterization of the fragments

obtained from cell wall degradation allowed these

investigators to postulate that the primary cell wall
contained 4 major components (21). The first component
consisted of elementary fibrils of cellulose; hydrogen
bonded together to form microfibrils. The second com-
ponent was a 8-1, 4-glucan with side chains of xylose and
fucosyl-galactose. The third component was a pectin con-
sisting of a backbone of rhamnogalacturonan with side
chains of arabinan and 4-linked galactan. The fourth
component was a hydroxyproline-rich protein with
arabinosyl tetrasacchride and 3, 6-1inked galactan
attached, respectively, to the hydroxyproline and

serine residues of the protein. Albersheim and co-
workers have also postulated the following arrangement
for these four components in the primary wall (21).
Xyloglucan is hydrogen bonded to the cellulose fibrils.
This is a very tight bond because of the large number of
available hydrogen-bonding sites and there is enough
xyloglucan available in the wall to encapsulate all the
cellulose fibrils in a monolayer of xyloglucan (20).

The reducing ends of the xyloglucans are covalently bound
to the galactan side chains of the pectin. The 3, 6-linked
arabinogalactan side chains of the protein are in turn
covalently bound to the pectin backbone. This tenative
model of the primary wall indicates that all the
components of the wall are tightly bound to form a

single macromolecule. As Kleegstra gt 31. have

suggested, this model leads to interesting speculation‘
about the mechanism of cell wall extension although other
structural models for the primary wall could be derived
from their data (21). Xyloglucans in cell suspension
cultures of red kidney beans have been shown to have a
nearly identical structure to the sycamore cell xylo-

glucan (22).

Morphology of Cell Wall Synthesis

 

Role of the Golgi Apparatus

 

The involvement of the golgi apparatus in plant
cell wall synthesis can be deduced from electron micro-
graphic studies of cell plate formation during cell
division. The cell plate is formed by the fusion of
small vesicles (23). These vesicles are derived from
the golgi apparatus and their movement to the area of
plate formation seems to be directed by microtubules
(24, 25). Membranes from the coalescing vesicles con-
stitute the plasmalemma of the new cell surface (26).

Further confirmation for the role of the golgo
apparatus was obtained by autoradiographic studies.
When intact root cells from wheat were soaked in a
solution of [3H]glucose for 5 min by Northcote and
Pickett-Heaps the radioactivity in the cells was found
to be localized in the cisternae of the golgi apparatus
(27). Further treatment of the root cells for a short

period with nonradioactive glucose caused the

10

radioactivity originally present in the golgi apparatus
to be transferred to the golgi associated vesicles in

the cytoplasm (27). Longer periods with nonradioactive
glucose resulted in the localization of the radioactivity
in the cell wall and slime layer (27). Similar auto-
radiographic evidence for the involvement of the golgi
apparatus in cell wall synthesis was also obtained with
sycamore seedlings (28).

Homogenization and fractionation of cells pre-
viously incubated with radioactive glucose have also shown
that the resulting radioactive polysacchrides were
localized in the golgi apparatus (29, 30, 27). The
radioactive polysacchrides were identified as pectins and
hemicelluloses (29, 30, 27). The absence of radioactivity
in cellulose suggests that the golgi apparatus is not the
site of synthesis for this glucan. I have previously
cited data from the analysis of woody tissue (15) which
stated that the secondary wall formed during cell dif-
ferentiation did not contain pectin. However, in a
fractionation experiment with differentiating maize cells
soaked in radioactive glucose, radioactive pectin was
found in the golgi apparatus (29). This indicates that
some pectin is synthesized during synthesis of the
secondary cell wall. Golgi apparatus were also the
site of pectin synthesis in the elongation of Lilium

longiflorum pollen tubes (31). Although the previously

 

11

discussed data all support the theory that the golgi
apparatus is the site for synthesis of the plant cell

wall matrix polysacchrides there have been other
organelles proposed. For instance, Villemez‘gg'gl. have
used centrifugal fractionation to isolate the particles
reaponsible for in vitro synthesis of a variety of poly-
sacchrides in onion (32). Their data led them to state
that the organelle reSponsible for polysacchride synthesis
was of very large size and was most probably the plasma
membrane.

The location of the polysaccharide synthesizing
system in the golgi apparatus leads to a speculative
theory about the control mechanism for cell wall synthesis
at different stages of cell growth and differentiation.
Northcote has postulated that since many of the enzymes
responsible for the interconversion of sugars are membrane
bound they may be contained in the golgi apparatus (33).
Therefore, the golgi apparatus could control the type of
polysacchride synthesized by controlling the synthesis
of the appropriate precursors (33). Experimental evidence
for this theory is not well documented although the
appearance of UDP-glucose-4-epimerase activity in the

green alga Acetabularia mediterranea is correlated with

 

the development of the galactose-containing cap of this

organism (34).

12

Orientation of Cellulose
MicrofibriIs

 

The organelle responsible for cellulose synthesis
and the mechanism for orientation of the microfibrils in
the wall are not clearly understood. Microtubules are
thought to have a role in cell wall and microfibril syn-
thesis. In secondary thickening microtubules with the
same orientation as the neighboring cellulose microfibrils
are found adjacent to the cell wall (35, 36). This
observation suggests that microtubules control the orien-
tation of the microfibrils either by being the site of
cellulose synthesis or by directing the matrix poly-
sacchrides into the wall and thus directing the orien-
tation of the developing microfibrils (36, 33).

Another organelle thought to be involved in the
synthesis of cellulose microfibrils is a particle found
attached to the plasmalemma. The 60 to 150 2 particle
is clearly seen sculptured to the plasmalemma of freeze-
etched yeast cells (37). Electron micrographs obtained
by Moor and Muhlethaler indicated that the particles
formed hexagonal arrangements on the plasmalemma and
were penetrated by short fibrils, believed to be cellu-
lose, which disappeared into the inner layer of the cell
wall (38). In some cases the particles formed rows that
have the same orientation as the adjacent microfibrils in
the wall (39). These particles are presently thought to

be the actual site of cellulose synthesis (33).

13

Although previously described data indicated
that the golgi apparatus is not involved in cellulose
synthesis, there is an important exception. The marine

alga Pleurochrysis sherffelii has a cell wall composed

 

of scales containing cellulose. An electron micrographic
study of this alga show that the scale is completely
synthesized in the organism's single golgi apparatus and
then transported through the cytoplasm and deposited
outside the cell by golgi derived vesicles (40). There-
fore, in this organism cellulose is synthesized inside
the golgi apparatus.

Biosynthesis of Cell Wall
Polysacchrides

 

£2.21EE2 sugar nucleotides are the substrates
for the enzymes synthesizing plant cell wall polysac-
chrides. The energy required to form a glycosidic bond
requires that the monosacchrides be activated prior to
incorporation into polysacchride (41). Nucleoside
diphosphate sugars are excellent donors of sugars because
of their high negative free energy of hydrolysis (AG°).
UDPGlc, for instance, has a AG° of hydrolysis equal to

-7.600 kilocalories (41).

Synthesis of Glucans

 

The polysaccharide whose in vitro biosynthesis

has been studied in the greatest detail is the B-l,4-

14

glucan, cellulose. Research in this area is often
contradictory and is complicated by the fact that under
certain conditions a B-l,3-glucan is also synthesized
by particulate enzyme preparations.

In 1958 Feingold EE.E£° first reported that

particulate enzyme preparations from Phaseolus aureus

 

(mung bean) could incorporate radioactivity from UDP—

14ClGlc into a polysaccharide identified by partial

[U-
acid hydrolysis as B-l,3-glucan (42). The B-l,3-glucan
in plants is given the common name callose, but it is not
a normal constituent of the plant cell wall (43). How-
ever, the synthesis of callose is known to be an impor-
tant response to cell wounding and callose is a major
component of sieve plates and pollen tubes (43).

Although the synthesis of B-l,4-glucan from UDPGlc was
not observed by Feingold £5 31. in g. aureus other
researchers using approximately the same techniques have
reported the simultaneous production of B-l,3- and
B-l,4-glucan with particulate enzyme preparations from

g. aureus and Lupinus 21223 (42, 44, 45). After publi-
cation of these conflicting experiments Hassid and co-
workers undertook a careful study of the g. aureus and

L. 21225 systems and announced that only B—l,3-glucan

was synthesized from UDPGlc (46). To heighten the
uncertainty of the identity of the UDPGlc product from

E. aureus it should be noted that one of the original

15

reports identifying B-l,4-glucan as a product of UDPGlc
came from Hassid's laboratory (44). A partial explanation
for the discrepancy in the identity of the product syn-
thesized from UDPGlc with a. aureus particulate enzyme
preparations was proposed by Clark and Villemez. They
reported once again that this enzyme preparation could
synthesize a mixture of B-l,3- and B-l,4-glucans from
UDPGlc but that the relative amounts of the two glucans
produced was dependent on the temperature used to germi-
nate the a. aureus seedlings (47). Although they could
not explain why other researchers were unable to find
evidence of B-l,4-glucan synthesis they felt that this
may be the result of subtle differences in methods of
enzyme preparation (47).

In contrast to the g. aureus system, the ability
of particulate enzyme preparations from.A!gna sativa
(oat) seedlings to synthesize B-l,3- and B-l,4-glucans
from UDPGlc is well documented (48, 49). The concen-
tration of UDPGlc in the reaction mixture determined
whether production of B-l,3- or B-l,4-glucan predomi-
nated (49). The particulate enzyme preparation had

5 5 M for the

UDPGlc Km's of 1.1 x 10’ M and 6 x 10"
synthesis of B-l,4- and B-l,3-g1ucan, respectively
(49). Particulate enzyme homogenates from wheat seedlings

and Lilium multiflorum endosperm also exhibit a UDPGlc

16

concentration dependent synthesis of B-l,3- and B-l,4-
glucans (50, 51).

Separate enzyme systems seem to be responsible
for the synthesis of B-l,3- and B-l,4-glucans. Tsai
and Hassid have "solubilized" the two activities by
digitonin extraction and separated them from one another
by absorption on hydroxylapatite gel and elution with
phosphate buffer (51). Studies seeking to identify the
organelles responsible for B-l,3- and B-l,4-glucan
synthesis have provided additional evidence for the
existence of separate enzyme systems. Ray gt_al. have
used a combination of velocity and isopycnic density
gradient centrifugation to isolate the enzymatic
particle in pea seedling homogenates which are
responsible for the synthesis of B-l,4-glucans from
UDPGlc and GDPGlc (52). Electron micrographs showed
that the isolated fraction with glucan synthetase
activity contained condensed golgi dictyosomes and free
dictyosomal membranes (52). An elegant study by Morré
and co-workers showed that two organelles were responsible
for glucan synthesis in onion stem: plasma membrane and
golgi apparatus (53). Both of these organelles were
able to synthesize B-l,3- and B-l,4-glucans but at
1.5 uM UDPGlc concentration the majority of the B-l,4-
glucan synthesis occurred in the golgi dictyosome

fraction and at 1 mM UDPGlc concentration the majority

17

of the B-l,3-glucan synthesis occurred in the plasma
membrane fraction (53).

The discoveries by Morré and Ray that golgi
membranes could synthesize B-l,4-glucan from UDPGlc
seems to conflict with the previously discussed data on
the absence of in 3133 cellulose synthesis in golgi
apparatus (29, 30, 27). Morré has stated that B-l,4-
glucan synthesis in his golgi preparations may be part
of a synthesis system for glyc0proteins or hemicellulose
polysaccharides (53). It is also possible that the golgi
apparatus contained inactive cellulose synthesizing
enzymes prior to their transport to the plasmalemma and
the isolation of the golgi caused activation of these
enzymes (53).

Plant particulate enzyme preparations can also
use GDPGlc to synthesize glucans. A particulate enzyme
preparation from P. aureus could incorporate D-glucose
from GDPGlc into a polysaccharide component (54, 55).
The polysaccharide was identified as cellulose since it
released a series of B-l,4-glucan oligosaccharides
after partial acid hydrolysis (55). The absolute identity
of this material as cellulose, however, is unclear
because the incorporation of D-glucose from GDPGlc in
a. aureus was affected by the addition of GDPMan to the
reaction mixture (56, 57). D-Mannose and D-glucose were

both incorporated into an alkali insoluble material and

18

enzymatic degradation and acetolysis have shown that the
material was a B-l,4-glucomannan (56). Addition of
GDPMan to a reaction mixture containing GDP[U-14C]Glc

and a particulate enzyme preparation from E. aureus

did not affect the rate of D-[U-14C]glucose incorporation
but did increase the total amount of incorporation (56,
58). The increase in total D-glucose incorporation as
the result of the presence of GDPMan is believed to be
caused by the synthesis of additional D-mannose-containing
D-glucose acceptor. Addition of GDPGlc to a reaction
mixture containing GDP[U-14C]Man and the particulate
enzyme preparation caused an increase in the rate of
incorporation of D-[U-14C1mannose and a decrease in the
total amount of D-[U-14C]mannose incorporated (58).

It should be noted that in order to measure the initial
rate of D-glucose and D-mannose incorporation the reaction
mixture was extracted with lipid solvents in order to
remove glycoproteins that were also synthesized (58).
Synthesis of B-l,4-glucans with GDPGlc has also been
reported with particulate enzyme preparations from

£3 §123§_and pea seedlings (51, 52). The L, glbgg
preparation also contained UDPGlc-Z-epimerase activity
and D-mannose was incorporated from the resulting GDPMan
(52). In the case of a. aureus Villemez has stated that
GDPGlc is the substrate for the synthesis of a gluco-

mannan rather than cellulose (58).

19

The role of UDPGlc and GDPGlc in cellulose syn-
thesis was also studied in cotton. Barber and Hassid
reported that a particulate enzyme preparation prepared
from 4- to 8-day-old cotton balls could synthesize cellu—
lose from GDPGlc and the extent of D-glucose incorporation
was stimulated by the presence of GDPMan (59). Enzyme
prepared from older balls was inactive (59). As pre-
viously mentioned it has been proposed that cellulose in
the primary and secondary walls is synthesized by dif-
ferent mechanisms (18). Marx-Figini, in interpreting
Barber's and Hassid's experiment, felt that the 8-1,
4-glucan synthesized from GDPGlc in cotton represented
synthesis of primary wall cellulose (60). He stated that
Barber and Hassid were probably unable to synthesize
secondary cell wall cellulose in their enzyme preparation
because the isolation of the enzyme would have destroyed
the template mechanism necessary for synthesis of homo-
logous secondary wall cellulose (18, 60). Later work
by Delmer and Beasley with intact isolated cotton fibers
showed that at the stage of growth when primary wall is
produced the fibers incorporated D-glucose from GDPGlc
into alkali insoluble glucan (61). As the fibers aged
formation of the primary wall ceased and secondary wall
synthesis began (61). During this same time period the
fibers lost the ability to synthesize glucan from GDPGlc

but gained the ability to synthesize glucolipids from

20

UDPGlc (61). Delmer and Beasley explained these results
by suggesting that in cotton GDPGlc is used for synthesis
of cellulose only in the primary wall (61). This theory
leads to interesting speculation about the control of
cellulose synthesis, but must be tempered by the knowledge
that the identity of the B-l,4-glucans synthesized from
UDPGlc and GDPGlc as cellulose in both the P. aureus and
cotton systems is not positive. In fact, x-ray diffraction
studies of glucans synthesized ig_!i££g with a particulate
enzyme preparation from g. aureus suggested that only low
molecular weight oligosaccharides were synthesized from
UDPGlc and GDPGlc (62).

The bacterium Acetobacterium xylinum can synthe-

 

size fibrils of B-l,4-glucan which are identical in
structure to the cellulose fibrils found in plants (63).
Fibrils are formed extracellularly and since they are
easily isolated, cellulose synthesis in A. xylinum has
been extensively studied (63, 64). Intact cells will

14C]glucose into

incorporate radioactivity from D-[U-
cellulose (65). Cell-free homogenates obtained from

A, xylinum.were able to synthesize cellulose fibrils
when supplied with D-glucose and ATP. Later work with
cell-free homogenate prepared by sonication showed that

UDPGlc was a precursor of cellulose of A. xylinum

synthesis (66).

21

Synthesis of Matrix Poly-
saccharides

A variety of researchers have investigated the
ability of particulate enzyme preparations from different
plants to incorporate monosaccharides from sugar nucleo-
tides into suspected cell wall matrix polysaccharides.
Although many different sugars have been investigated,
little success has occurred beyond announcements that
particular enzyme activities have been discovered. The
inability of researchers to go beyond this point and to
investigate the mechanism of cell wall polysaccharide
synthesis attests to the complexity of this system.

Incubation of a particulate enzyme preparation

14ClGlcUA resulted in

from immature corn cobs with UDP[U-
the incorporation of radioactivity into a water-insoluble
fraction (67). Extraction of the insoluble material with
ammonium oxalate and alkali showed that the radioactivity
was incorporated into both pectin and hemicellulose poly-
saccharides (67). Hydrolysis of the fractions followed

by paper chromatography of the hydrolysates showed that
the radioactivity was contained in D-xylose, L-arabinose,
glucurono-galactose, glucurono-xylose, D-galacturonic
acid, and D-glucuronic acid with the majority of the
radioactivity contained in D-xylose (67). The particulate

enzyme preparations must have contained UDPGlcUA carboxy-

1yase (EC, 4.1.1.35), UDPGlCUA-4-epimerase (EC, 5.1.3.6),

22

and UDPAra-4-epimerase (EC, 4.1.3.5) activities to account
for the presence of these radioactive sugars in the poly-
saccharides. The D-galactose residue in glucurono-
galactose was not radioactive (67). Therefore, D-galactose
must have been incorporated into the acceptor molecule
prior to the preparation of the particulate enzyme.

As described previously the D-glucuronic acid
residues in hemicellulose polysaccharides are often
methylated (5). Methylation occurs by the enzymatic
transfer of methyl groups from S-adenosyl-L-methionine to
the glucuronic residues of previously synthesized hemi-
cellulose (68).

Incorporation of D-xylose and L-arabinose from
their respective uridine sugar nucleotides into hemi-
cellulose was demonstrated with particulate enzyme prepar-
ations from immature corn cobs and £23 gays seedlings
(69, 70). The product was soluble in alkali and
identification of the oligosaccharides obtained after
partial acid hydrolysis showed that the product consisted
of a D-xylan backbone with L-arabinose side chains (69,
70). Another matrix polysaccharide synthesized with
g. aureus seedling particulate enzyme was D-galactan.

The substrate was UDPgalactose and the resulting water
soluble polysaccharide had a molecular weight greater

than 4,600 (70).

23

Particulate enzyme preparations from g. aureus
and tomatoe could synthesize polygalacturonic acid when
supplied with UDPGalUA (72, 73). Since UDP-methylgalac-
turonic acid will not function in this synthesis, it is
assumed that methylation of D-galacturonic acid occurs
after synthesis of the polysaccharide (72). The enzymatic
introduction of methyl ester groups into pectin was
demonstrated in P, aureus with S-adenosyl-L-methionine
as the substrate (74). Methylation occurred with endo-
genous pectin but not with exogenous pectin that was
present in the reaction mixture (75). This suggests that
the location of the esterification system is within a
membrane-bound organelle. Studies by Kauss g£_gl, have
shown that pectin methyl esterase when added to the par-
ticulate enzyme preparation would not degrade methyl-
esterified pectin synthesized in vitro unless the lipid
membrane of the particulate enzyme was disrupted by
treatment with detergent or phospholipase A (73).

The structure of radioactive polygalacturonic
acid synthesized from UDP[14CIGalUA was investigated by
enzymatic degradation with exopolygalacturonate transeli-
minase (76). This enzyme degrades polygalacturonic acid
from the reducing end (77). When the product synthesized
in 11352 was treated with this enzyme, radioactivity was
released solely as unsaturated digalacturonic acid (76).

The amount of unsaturated digalacturonic acid released

24

was proportional to length of treatment (76). Since syn-
thesis of polysaccharides by glycosylation occurs from the
nonreducing end, the results of the transeliminase treat-
ment indicate that the polygalacturonic acid was labelled
throughout the chain and incorporation of galacturonic
acid did not occur by the addition of a few residues to
the ends of preformed chains.

gossible Role of Lipid
Intermediate

 

The role of polyisoprenol sugar intermediates
in the synthesis of components of the bacterial cell wall
is well established (78). The possibility that similar
lipid intermediates may function in plant cell wall syn-
thesis has been investigated by several research groups.

Cell-free preparations from A, xylinum incor-
porated radioactivity from UDP[14CJGlc into lipid
material (79). Radioactive sugar was released from
this material by treatment in 0.01 N H2804 at 100°C or
in 0.1 N LiOH at room temperature (79). The radioactive
products released from the glycolipid were identified
as glucose, cellobiose, and possibly other higher
intermediates (79). This suggests that in this
organism a polyprenoidpyrophosphate may be an inter-
mediate in cellulose synthesis.

Jung and Tanner have shown that the synthesis of

yeast "mannan" from GDPMan proceeds through the high

25

energy lipid intermediate, dolichol monophosphate
mannoside (80). The identity of the lipid as dolichol
was verified by mass spectroscopy (80). Yeast "mannan“
proved to be a heterogeneous glycoprotein and the manno-
lipid acted only to transfer a mannosyl residue to the
protein (81). The rest of the mannose residues were
added directly to the mannan from GDPMan without the
intervention of dolichol monophosphate (81).

Several researchers have demonstrated the
existence of dolichol isoprenoid compounds in higher
plants. Both Kauss and Villemez have isolated a mannosyl
lipid from a reaction mixture containing particulate
enzyme preparation from A. aureus and GDP[U-14C]Man (82,
83). The lipid was tentatively identified as an iso-
prenoid by a preliminary mass spectral analysis and the
fact that $2.!izg studies showed that the lipid was syn-
thesized from 5-[3Hl-DL-mevalonic acid (82, 84). The
mannosyl lipid had a high transfer potential as evidenced
by the fact that the sugar was released by acid hydrolysis
at pH 2 (84). In addition, the incorporation of mannose
into lipid could be reversed by addition of GDP but not
GMP to the reaction mixture (84). Villemez, when studying
the synthesis of the mannosyl lipid from GDPMan, isolated
a low molecular weight membrane-bound protein from
E, aureus which contained D-mannose oligosaccharides

(84). A similar glyc0protein was also isolated when

26

the particulate homogenate was incubated with GDPIU-14C1-

Glc (84). He postulated that this glyc0protein may also
be an intermediate in cell wall polysaccharide synthesis
and could obviate the need for a primer polysaccharide
(34).

Storm and Hassid were unable to demonstrate the
transfer of D-mannose from endogenous mannosyl lipid to
polysaccharide in A. aureus particulate enzyme prepar-
ations (85). This experiment indicated that the mannosyl
lipid is not an intermediate in cell wall polysaccharide
synthesis. Particulate enzyme preparations from g. aureus
did transfer D-mannose from GDPMan to the exogenous lipids
phytol phosphate, phytanol phOSphate, dolichol monophos-
phate, and betulaprenol phosphate (86, 87). Since
addition of these exogenous lipids allow for the syn-
thesis of larger quantities of glycolipids, it is hoped
that this will help in elucidating the possible role of
glycosyl isoprenoids in plant cell wall synthesis.

Particulate enzyme preparations from cotton
fibers have also shown evidence of containing lipid
intermediates. Radioactive D-glucose and D-mannose were
incorporated into an acid lipid fraction when UDPGlc and
GDPMan, respectively, were incubated with particulate
enzyme (88). The synthesis of the glycosyl lipids was
reversed by the addition of UDP and GDP to the reaction

mixture (88). Isolated glycosyl lipids were labile to

27

acid hydrolysis at pH 2 (88). The free lipid chromato-
graphed with an Rf similar to ficoprenyl phosphate and
the natural acceptor in the particulate enzyme preparation
could be replaced by ficoprenyl phosphate (88).
Solubilizatigngf Polysaccharide
SyntheSiEing System

The inability of researchers to solubilize the
plant cell wall polysaccharide synthesizing systems has
hampered research efforts in understanding the mechanism
of this complex process. The partial "solubilization"
of the UDPGlc and GDPGlc dependent glucan synthesizing
activities in g. aureus, A. sativa, and A. ElRE§.bY
extraction with digitonin has been reported (89, 57,
90). However, the procedure does not result in complete
solubilization and there are still particles contained in
the "solubilized" supernatant (89). Digitonin, therefore,
probably did not cause a true release of particulate
enzyme from membrane complexes. Heller and Villemez have
reported the solubilization of the glucomannan synthe-
sizing system in E. aureus by the use of Triton X-100
extraction (91). The solubilized enzyme activity could
transfer sugar moieties from GDPGlc and GDPMan to
suspected polysaccharides, but could not use UDPGlc,
UDPGal, UDPXyl, UDPArab, or UDPGlcUA (91). Solubili-
zation with Triton x-100 resulted in a 3.5 increase in

the specific activity of the GDPMan activity and the

28

activity remained in solution after centrifugation at
300,000 x g for 40 min (91). The Trition solubilized

activity was not purified further.

D-Apiose

Chemistry

In 1901 Vongerichten discovered that apiin, a
flavonoid glycoside found in parsley, contained a pre-
viously unknown pentose sugar which he named apiose (92).
Vongerichten later reported that apiose has a branched
chain structure (118). The structure of the naturally
occurring apiose was finally elucidated as 3-C-hydroxy-
methyl-aldehydo-D-glycero-tetrose (94). The Fisher (I)
and Haworth (II) structures of D-apiose are shown.
Structure II is one of 4 possible cyclic isomers of
D-apiose. A detailed description of the chemistry of

D-apiose is found elsewhere (95).

Occurrence in Nature

 

The first example of the occurrence of D-apiose
in compounds other than a flavonoid glycoside was
reported by Bell EE.21° These results suggested that
D-apiose was a constituent of the polysaccharide

fraction of the marine alga Posidonia australis (96).

 

Besides A. australis the following plants have also
been shown to possess polysaccharides containing

D-apiose: Zostera marina (97, 98, 99), Lemna gibba

29

(100), A. nana (101), A. pacifica and Phyllospadix (98),

 

and A. 91225 (101).

All reports on the occurrence of D-apiose in
nature have been limited to members of the plant kingdom.
However, D-apiose is a common constituent of plants.
Duff has surveyed 175 plants and found that D-apiose was
contained in the acid hydrolysates of at least 60% of
the plants tested (101). According to Duff, one of the
plants with the highest content of D-apiose was A. AAAQE
(duckweed) (101). D-apiose was localized in the cell
wall of A. AAAQA (101).

Identification of Apiogalac-
turonans in L. minor

Beck (102) and Hart and Kindel (103) extracted
cell wall material from A. 31225 with ammonium oxalate
and isolated a family of polysaccharides containing
D-apiose and D-galacturonic acid. Both groups were able
to further fractionate the polysaccharides after ammonium
oxalate extraction. Beck isolated two fractions con-
taining D-apiose and D-galacturonic acid, one of which
also contained D-xylose and D-galactose (103). Hart and
Kindel used a combination of NaCl fractionation and
DEAE-Sephadex chromatography to fractionate their
material into a series of polysaccharides containing
D-apiose and D-galacturonic acid (102). The content

of D-apiose in these polysaccharides varied from 7.9

30

to 38.1% by weight (102). The fractions were reported

to contain only D-apiose and D-galacturonic acid although
the techniques used by these workers were specific only
for these two sugars (102). Hart and Kindel did report,
however, that in analyzing the D-apiose content of the
polysaccharides one and sometimes two faint spots not
corresponding to D-apiose were found when the acid
hydrolysate of the polysaccharide fractions were chroma-
tographed on paper (102). The identities of these spots
were not investigated.

The following information on the isolation and
partial characterization of the polysaccharides containing
D-apiose isolated from A. 21225 was obtained from Hart
and Kindel (102). When they extracted cell wall prepar-
ations from A. AAAQE_with 0.5% ammonium oxalate, 14% of
the wall material was solubilized. This solubilized
material contained 20% of the D-apiose originally present
in the cell wall. After fractionation of the solubilized
material, all the polysaccharides isolated were of the
same general type: D-galacturonans with side chains of
D-apiobiose. The sensitivity of the apiogalacturonans
to pectinase degradation was found to be dependent on the
D-apiose content of the polysaccharides. Those apiogalac-
turonans of low D—apiose content and insoluble in
l M NaCl were degraded by pectinase, while those of

high D-apiose content and soluble in l M NaCl were not

31

degraded. Resistant apiogalacturonans could be degraded
by pectinase if the D-apiobiose side chains were first
removed from the polysaccharide by mild acid hydrolysis.
Formaldehyde release after periodate oxidation of apio-
galacturonans showed that about 50% of the D-apiose
molecules were substituted at either the 3 or 3' position.
Mild acid hydrolysis of the ammonium oxalate
extracted material resulted in the release of a disac-
charide from the polysaccharide which was identified as
apiobiose (III) (104). Purified apiogalacturonan
fraction IIa (as defined by Hart and Kindel [102]) was
used to study the release of apiose from apiogalacturonan.
Hydrolysis of fraction IIa at pH 4 for 3 hr at 100°C
resulted in near total release of the D-apiose in the
polysaccharide as apiobiose (104). The rate of D-apiose
release from polysaccharide was pH dependent. The rate
declined steadily from pH 3.5 to almost zero at pH 6.5
(104). These results led Hart and Kindel to propose that
the structure of apiogalacturonans (IV) in A. AAAQE con-
sisted of a backbone of a-l, 4-linked polygalacturonic

acid with side chains of apiobiose (102, 104).

UDPAEi

D-apiose is synthesized enzymatically in plants

by the conversion of UDPGlcUA to UDPApi and CO (105,

2

106). The enzyme responsible for this reaction has been

32

 

 

H O
\ //
H-C-OH
HOH C-C-OH
2 l
CHZOH
OH OH
(I) (II)
D—apiose D—apiosylfuranose
(III)
apiobiose
Api Api Api
l I l
Api Api Api
l I
-Ga1UA-GalUA-GalUA-GalUA-GalUA—Ga1UA-GalUA-
(IV)
apiogalacturonan

List of structures. The dotted lines indicate undetermined
stereochemistry.

33

partially purified from A. EABQE and has been given the
common names UDPGlcUA cyclase and D-apiose synthetase
(93, 107, 108). UDPApi was found to be an extremely
labile compound as demonstrated by its half-life of
97.2 min when stored at pH 8.0 and 25°C (106). However,
under proper conditions UDPApi can be stored for long
periods of time. When stored at pH 6.4 and -20°C 94%
of the UDPApi was still intact after 120 days (108).
In Vitro Synthesis of Apio-
galacturonans

32.21332 synthesis of apiogalacturonans in
A. Eiflgi should, for a number of reasons, be an excellent

system for studying cell wall polysaccharide synthesis.

1. Apiogalacturonans are presumably of physiological
significance because of the large quantity con-

tained in the cell wall of A. minor.

2. Because of the large quantities of apiogalac-
turonan in A. minor cell walls the apiogalac-
turonan synthesizing system should be easy to

detect.

3. We can compare the structure of authentic apio-
galacturonans with AA vitro synthesized D-apiose

containing compounds.

4. Procedures for characterizing apiogalacturonans

are already developed.

34

5. Apiogalacturonans can be solubilized by rela-

tively mild treatment.

From previous discussion of the mechanism of
cell wall polysaccharides synthesis one would expect that
sugar nucleotides containing D-apiose and D-galacturonic
acid would be the precursors of apiogalacturonan. Kindel
has reported that radioactivity from UDP[14CJApi was
incorporated into material insoluble in water and
methanol when the sugar nucleotide was incubated with a
particulate enzyme preparation from A. EEBQE (109).
This material was solubilized when extracted with 0.5%
ammonium oxalate (109). Although UDP[14C]Xyl was also
present in the reaction mixture, Kindel did not observe
D-[U-14CIXyl incorporated into the methanol and water
insoluble material (109). Hydrolysis of the material

l4C]apiobiose side

at pH 4 resulted in the release of [U-
chains thus suggesting that D-[U-14C]apiose was incor-
porated into apiogalacturonans or a compound of similar
structure (107).

The incorporation of D-apiose with the particulate
enzyme preparation from A. AAAQE has been further investi-
gated by Pan and Kindel (110 and unpublished results).
They found that the enzyme responsible for the transfer
of D-apiose from UDPApi has a Km of 4.9 uM and maximal

activity at pH 5.7. They have also found that the

particulate enzyme preparation from A. minor could

35

l 1

transfer D-[U- 4Cl-xylose from UDP[U- 4C]Xyl to a methanol
and water insoluble material. This activity has a Km of
12.7 uM and maximal activity at pH 5.7. Leinbach and
Kindel have found that particulate enzyme preparation

14C]galacturonic

from A. minor can also incorporate D-[U-
acid from UDP[U-14C1GalUA into a methanol and water
insoluble material (119). This activity exhibited a

Km of 8 uM and maximal activity at pH 6.2.

EXPERIMENTAL PROCEDURES

Materials

 

DEAE-Sephadex A-25 (particle size 40-120 u,
capacity: 3.5 1 0.5 mequivalents/g) was purchased from
Pharmacia Fine Chemicals. Bio-Gel P-30 (100-200 mesh),
Bio-Gel P-100 (50-150 mesh), and Bio-Gel P-300 (50-100
mesh) were obtained from Bio-Rad Laboratories. UDPXyl
and UTP were purchased from Schwarz/Mann and P-L Bio-
chemicals, respectively. UDPGlcUA, UDPGalUA, and fungal
pectinase were purchased from Sigma Chemical Co. The
pectinase was partially purified by the manufacturer who
stated it still contained several other enzymes. UDP-
[U-14C]GlcUA (190 mCi/mmole), UDP[U-14C]Xyl (156 mCi/

14C]galactose-l-P (214 mCi/mmole) were

obtained from New England Nuclear Corp. UDP[U-14C1GalUA

mmole), and D-[U-

was synthesized from D-[U-14C]galactose-l-P and UTP by

the method of Feingold SE 21' (59). D-Apiose, apiobiose,

14C]22° sodium chloride soluble apiogalac-

1

D-apiitol, [
turonan fraction, [ 4C]60° sodium chloride soluble apio-
galacturonan fraction and 22° sodium chloride insoluble

apiogalacturonan fraction were prepared by Hart and

36

37

Kindel (102, 104). The specific activity of the [14C]
apiogalacturonan fractions ranged from 14,000 to 16,000
dpm per mg.

UDP[U-14C1Api was enzymatically synthesized from
UDP[U-14C]GlcUA (108, Pan and Kindel, unpublished experi-
ments). From the data of 11 individual preparations of
UDP[U-14C1Api, the percent of the total radioactivity in

1

the final solution present in UDP[U- 4C]Api, UDP[U-14C1Xy1,

and UDP[U-14C]GlcUA ranged from 61 to 72, 24 to 40, and

1

0 to 8%, respectively. UDP[U- 4C]Xyl was simultaneously

14C]Api because of the presence

synthesized with UDP[U-
of UDPGlcUA carboxy-lyase I activity in the UDPGlcUA
cyclase preparations. The mixture of the three radio-
active sugar nucleotides will be referred to as "UDP-
[U-14C]Api." In the present paper, values of radio-
activity and sugar nucleotide content reported for
"UDP[U-14C]Api" are the sum of the total amounts con-
tained in UDP[U-14C]Api, UDP[U-14CJXy1, and UDP[U-14C]-

1

GlcUA. UDP[U- 4C]Api solution was stored frozen at -90°C.

General Methods

 

Radioactivity was detected on paper chromatograms
with a Packard radiochromatogram scanner, model 7201
(Packard Instrument Co. Inc.). All other radioactivity
measurements were made with a Packard Tri-Carb liquid-
scintillation counter, model 3310, with one of the

following scintillation solutions: (A) Bray's solution

38

(111); (B) 5.5 g 2,5-diphenyloxazole and 0.1 g 1,4-bis
[2-(4-methyl-5-phenyloxazolyl)]-benzene in 667 ml of
reagent grade toluene and 333 ml of Triton X-100;

(C) 4.0 g 2,5-bis-2-(5-tert-butylbenzoxazolyl)-thiophene
in 1 liter of reagent grade toluene. Aqueous solutions
of radioactive material (1.0 to 1.5 ml) were counted in
10 m1 of scintillation solution A or B. Radioactive
compounds on paper were counted directly by complete
immersion of the paper in scintillation solution C. The
counting effeciencies with scintillation solutions A, B,
and C were 77, 81, and 63%, respectively.

Solutions were concentrated under reduced pressure
by rotary evaporation at temperatures below 30°C. Gel
chromatography was performed with columns prepared as
recommended by the manufacturer; elution was performed
at room temperature with descending flow. Dialysis was
performed with tubing that had been freshly prepared by
heating 30 min at 100°C. Means and mean deviation were
reported for experimental values based on more than one

determination.

Culture of L. Minor

 

A. minor was grown as described by Kindel and
Watson (108). The plants used in the preparation of the
particulate enzyme preparation were grown on 4 liters of

inorganic medium in small plastic pans (29 x 33 cm).

39

After the fronds had multiplied to the point of covering
the surface of the medium (18 to 22 days) A. minor were

removed as needed.

Paper Chromatography

 

Descending paper chromatography was used and was
performed with washed Whatmann No. 3MM paper. Washing
was done with 0.1 M citric acid followed by water.
Chromatography was performed at 22°C and the following

solvents were used: (A) ethyl acetate - H20 - acetic

acid - formic acid (18:4:3:l, by vol), (B) n-propanol -

ethyl acetate - H O (85:10:5, by vol), (C) isopropanol -

2

H 0 (9:1, v/v), (D) H O saturated butanol, (E) ethyl

2 2

acetate - acetic acid - pyridine - H O (5:1:5:3, by vol).

2
Nonradioactive sugars were detected on chromatograms by
spraying with aniline hydrogen pthalate (112) or by

using the AgNO dip method (113).

3

DEAE-Sephadex Column Chromatography

 

Columns of defined DEAE-Sephadex A-25 were
treated with 3 bed volumes of 0.05 M phosphate buffer,
pH 7.7, before the sample was applied. Samples were
dialyzed in 0.05 M sodium phosphate buffer, pH 7.7, for
2 hr with one buffer change midway through the dialysis,
before being applied to the column. The rate of sample
application was equal to the rate used to operate the

column. After the sample was applied the column was

40

treated with additional buffer followed by a step gradient
of 0.1 to 0.3 M NaCl in 0.05 M sodium phOSphate buffer,

pH 7.7. Except for Figure 10 the salt gradient described
on the figures indicates the first column fraction that
each step of the gradient was applied. The column was
operated at 4°C.

Isolation of Particulate Enzyme
Preparation

 

 

A. miggg_was collected on 4 layers of cheese-
cloth, washed thoroughly with distilled water, freed of
excess water with absorbent paper, and weighed. The
remaining steps were performed at 4°C. The fronds were
ground to a smooth consistency (for l to 2 min) with a
mortar and pestle in 0.05 M sodium phosphate buffer,
pH 7.3, containing 1% BSA (wt/vol) and 1 mM MgC12.
Two ml of buffer were used for every 1 g wet weight of
plant material. The resulting homogenate was filtered
through 4 layers of cheesecloth and the filtrate was
centrifuged for 10 min at 480 g. The precipitate was
discarded and the supernatant solution was centrifuged
for 8 min at 34,800 g. The supernatant solution was
discarded and the precipitate was gently resuspended in
0.1 M sodium phosphate buffer, pH 6.0, containing 1%
BSA (wt/vol) and 10% sucrose (wt/vol) with a Ten Broeck

glass homogenizer. For every 10g wet weight of A. minor

used, 0.5 ml of the buffer was used for resuspension.

 

41

This was called the particulate enzyme preparation. The

final pH of the particulate enzyme preparation was 6.1.

Biosynthesis of Radioactive Product
and SqubIliz d Product

 

 

Radioactive product which was insoluble in

l4C]Api,"

methanol and water was formed when "UDP[U-
UDP[U-14C]GalUA, UDP[U-14C]GlcUA, or UDP[U-14C]Xy1 was
incubated with particulate enzyme preparation. A
reaction mixture was prepared which contained 50 ul of
particulate enzyme preparation and either 0.17 or 0.33
nmoles "UDP[U-14C]Api" (60,000 to 120,000 dpm), 0.05 to
0.10 nmole of UDP[U-14C]GalUA (25,000 to 36,000 dpm),

0.1 to 0.3 nmole of UDP[U-14C]G1CUA(55,000 to 124,000
dpm) or 0.08 nmole of UDP[U-14C1Xyl (91,200 dpm). Eleven
different preparations of "UDP[14C]Api" were used in the
experiments reported in the present paper. The propor-
tions of the three sugar nucleotides present in the

l4C]Api" were stated earlier in this

solutions of "UDP[U-
section. In specified experiments UDPGalUA, UDPXyl, or
UDPGlcUA was added to the reaction mixtures containing
"UDP[U-14C1Api." The volume of the reaction mixtures con-
taining "UDP[U-14C1Api" or UDP[U-14C]GalUA were 70 ul.
The volume of the other reaction mixtures will be stated
in the appropriate places in the Results.

The reaction mixtures were incubated for 15 min

at 25°C. At the end of the incubation period 1 m1 of

75% aqueous methanol (v/v) containing 1% KCl (wt/vol) was

42

added to the mixture and the precipitate was collected
by centrifugation at room temperature. The supernatant
solution was discarded and the precipitate was then
extracted at room temperature as follows: twice with

1 m1 portions of 75% aqueous methanol containing 1%

KCl, twice with 1 ml portions of methanol, and once with
a 1 m1 portion of water. The radioactive material
retained in the residue remaining after these
extractions is referred to as the product in this
dissertation. In the experiments reported here,

D'[U-MC]apiose product, D-[U-l4

4

C]galacturonic acid
product, D-[U-l C]glucuronic acid product, and D-[U-14C]-
xylose product all refer to the products synthesized from

14C]G1cUA, and

"UDP[U-14C]Api," UDP[U-14C]GalUA, UDP[U-
UDP[U-14C]Xy1, respectively. When UDPGalUA, UDPGlcUA,
or UDPXyl were added to the reaction mixture used to syn-

l4C]apiose product it will be stated

thesize D-[U-
explicitly that the D-[U-14C]apiose product was syn-
thesized in the presence of a specific nonradioactive
sugar nucleotide.

Radioactive material was solubilized from the
product by suspending the product in freshly prepared
1% ammonium oxalate, pH 6.5, and incubating for 15 min
at 50°C. After incubation the suspension was centri-

fuged and the supernatant solution was removed. This

procedure was repeated 3 additional times. In the first

43

extraction 0.3 ml of ammonium oxalate solution was used
while in each of the subsequent 3 extractions 0.1 m1 of
ammonium oxalate solution was used. The four extracts
were combined. The radioactive material contained in
the combined extracts is referred to as solubilized
product in this dissertation. The same terminology used
in the preceding paragraph is used to describe which
radioactive sugar nucleotides were used to synthesize
the solubilized product and whether nonradioactive sugar
nucleotides were present in the reaction mixture, A.g.,
D-[U-14C]apiose solubilized product synthesized in the
presence of UDPGalUA. The amount of radioactivity in the
residue remaining after the extractions with ammonium
oxalate were completed was determined by adding 3 drops
of 2% NaOH to the residue, heating the suspension briefly
in a boiling water bath, applying the suspension to a

2 cm x 6 cm piece of chromatography paper, and then
assaying the paper for radioactivity by using scintil-
lation solution C. In almost all experiments reported
the solubilized products from several reaction mixtures
were combined and then used in the various experiments.
Whenever the solution containing solubilized product

was concentrated it was first dialyzed either in 0.05

M sodium phosphate buffer, pH 6.8 or 7.7, or in water.

44

Partial Acid Hydrolysis with
Fuming HCl

 

 

Radioactive product was suspended in concentrated
HCl to which an equal volume of fuming HCl (concentrated
HCl saturated with HCl gas at 4°C) was added (42). The
mixture was incubated at 27°C for 60 min. After incu-
bation the mixture was diluted with 10 volumes of water
and the HCl was removed by concentrating the sample to
dryness at 40°C. The dilution and concentration was
repeated 2 additional times. The resulting hydrolysis

products were identified by paper chromatography.

Hydrolysis at pH 1

 

The radioactive sample in solution was placed in
a 1 dram screw-cap vial. A quantity of 2 M trifluoro-
acetic acid equal to one-tenth the volume of the sample
was then added to the vial. The pH of the resulting
solution was tested with pH paper. If the pH was above
1.5 then additional 2 M trifluoroacetic acid was added
until the pH was between 1 and 1.5. In this dissertation
these conditions will be referred to as hydrolysis at
pH 1. The solution was heated in an autoclave for
30 min at 121°C and 15 lb pressure, and it was then
spotted on chromatography paper. The paper was developed
in the appropriate solvent, scanned, and the radioactive
areas were cut out and counted in scintillation solution

C. Recovery of radioactive material on paper

45

chromatograms after hydrolysis at pH 1 was routinely

between 85 and 90%.

Aydrolysis at pH 4

 

The radioactive sample in solution and an equal
volume of 0.5 M sodium acetate buffer, pH 4.0, were
placed in a l dram screw-cap vial. The vial was heated
for 3.5 hr at 100°C and the resulting hydrolysate was
spotted directly on chromatography paper. The paper
was developed with solvent A, scanned, and the radio-
active areas were cut out and counted in scintillation
solution C. The high concentration of acetate buffer
used in this procedure was necessary because of the
inherent buffering capacity of some of the samples.
Recovery of radioactive materials on paper chromatograms
after hydrolysis at pH 4 was routinely between 85 and
95%.

Hydrolysis of Solubilized Product
WIth’FungaI’Pectinase

 

 

Radioactive solubilized product was dialyzed in
water at 4°C as described in the individual experiments
in order to remove ammonium oxalate. Portions of the
solution of dialyzed solubilized product were mixed with
a quantity of 0.5 M sodium acetate buffer, pH 4.5, equal
to one-tenth the volume of the portion. The concentration

of pectinase in the 0.5 M acetate buffer was 20 mg/ml.

46

The solution was incubated at 37°C for the times stated

in the individual experiments.

RESULTS

Biosynthesis of Product and
Solubilized’Product

 

 

Aiosynthesis of D-[U-14C]Apiose
Product and SolubiliZed Product

 

 

When the procedure for the biosynthesis of radio-
active product described in the Experimental Procedures
was used with "UDP[U-14C]Api," 35% of the total radio-
activity in the assay was incorporated into D-[U-14C]-
apiose product (Table l).

14C]apiose

Much of the radioactivity in D-[U-
product was solubilized by treatment with ammonium oxalate.
The temperature needed to solubilize the maximum amount

14

of D-[U- C]apiose product was determined. Identically

prepared samples of D-[U--14

C]apiose product, each con-
taining 13,000 dpm, were extracted with 1% ammonium
oxalate at 22°C, 30°C, 40°C, and 50°C. This was done by
extracting each sample of D-[U-14C]apiose product for

15 min with 0.3 m1 of 1% ammonium oxalate followed by

3 additional 15 min extractions each with 0.1 ml of 1%
ammonium oxalate at the appropriate temperature. The

remainder of the procedure was the same as that described

in the Experimental Procedures except that the temperature

47

48

.uospoum poNAHHQsHOm on mpcommmuuouo

.ooEuomumm mucmeflummxo mo mosses
may ucmmmummu mammnpcmuom ca mosam> one .H: om mums mmusuxfis cowuoomu ecu mo moEsHo>
map .wamuwauogmoo com «DUHUHUVHIDHmQD Eoum poscoum mo mammzucmm men mom .mouscmooum

Hmucoeflum xm on» :H confluence mm meHUVHIDHmoD cam adooawfioealoamoo edaaooHOVHIDHmoD
s.Hm¢_ovHIDHmoDe Eoum couwmosucmm mos uosooum omnwaflodaom coo uosooumm

 

 

Ade ma Lav He mefloeHnDLEQD
Ame m h we Ame m h mm dooao_oeauaamoo
are H s Ho Lee G h as «aaooeoeauoemoo
loo m s no ice m h mm seda_oeansaeoo=
Awe Awe .musuxaz soauoomm on» as com:
nucmEpmmHB muoaoxo ESHcOEEd mm uosooum oucw owumuom uofluouaosz Momsme IDH

oouwawosaom uoscoum mo ucsoem (HoocH mua>wuomoflomm ea

 

endoscope omnsahnsaom one nuooooud mnoasx_oeauoguo poo .osoa deco
unsusaofiosanoguo .oaoa oaeousuonaoonoeaueauo .mnosdmnoeauaauo mo mendnudanoem .H mamas

49

was varied as indicated. Based on radioactivity measure-

l4C]apiose product

ments the following amounts of D-[U-
were solubilized by ammonium oxalate treatment: 40% at
22°C, 50% at 30°C, 75% at 40°C, and 87% at 50°C. These
results show that 1% ammonium oxalate readily solubilizes

14C]apiose product and that the extent of solubili-

D-[U-
zation is dependent on the temperature at which the
extraction is performed.

Water will also solubilize D-[U-14C]apiose product
but not to the same extent as 1% ammonium oxalate. The
procedure described in the preceding paragraph was used
to extract the D-[U-14C]apiose product except that the
1% ammonium oxalate was replaced by‘water. The amount
of D-[U-14C]apiose product solubilized was 10, 11, and
38% at extraction temperatures of 22°C, 30°C, and 50°C,
respectively.

Completeness of solubilization at 50°C was
measured by extracting D-[U-14C]apiose product (25,000
dpm) with 0.3 ml of 1% ammonium oxalate followed by 5'
additional extractions with 0.1 ml of 1% ammonium
oxalate. Except for the 2 additional ammonium oxalate
extractions the procedure is identical to the isolation
of solubilized product described in the Experimental
Procedures. The amount of radioactivity solubilized by
each extraction was determined with scintillation

solution B. The following amounts of radioactivity,

50

in order of extraction, were recovered in each extract:
18,184, 2,063, 1,704, 1,605, 759, and 421 dpm. These
results indicate that 4 extractions at 50°C with 1%
ammonium oxalate are adequate to isolate D-[U-14C1apiose
solubilized product.

Biosynthesis of D-[U-14C]Galacturonic

Acid, D-[U-14C]Glucuronic Acid, and

5:10-14C]Xylose Products and
Sb.ubilIzed Products

1

 

 

 

 

 

[U- 4C]Ga1UA, UDP[U-14C]GlcUA, and UDP[U-14C]Xy1
were used to synthesize the respective products and
solubilized products (Table l). Radioactivity was
incorporated into product from all the radioactive sugar

14C]G1cUA was not as efficient

nucleotides although UDP[U-
a donor as the other sugar nucleotides. The amount of
radioactivity in the three solubilized products varied

14C]-

(Table 1). Based on radioactivity measurements D-[U-
galacturonic acid product was solubilized slightly better
than D-[U-14C]apiose product but both D-glucuronic acid
and D-xylose products had significantly less of their
radioactivity solubilized by ammonium oxalate treatment.
The completeness of extraction of these three solubilized
products was investigated and was found to be similar to
the D-[U-14C]apiose solubilized product. When the 4
solubilized products described in Table l were dialyzed

at 4°C for 2 hr the recovery of radioactivity in non-

diffusible material was greater than 75%.

51

Addition of UDPGalUA, UDPGlcUA, and
UDPXyl to the Reaction Mixture usEH
£9 Synthesize D-[U—14C1Apiose
Product and Solubilized PrBHuct

 

 

 

 

The effect of adding UDPGalUA, UDPGlcUA, and
UDPXyl to the reaction mixture used to synthesize
D—[U-14C]apiose product and solubilized product was
determined (Table 2). These data demonstrate that of
the three nonradioactive sugar nucleotides used, only
UDPGalUA caused a significant increase in the incorpor-

14C]apiose product

ation of radioactivity into both D-[U-
and solubilized product. Addition of UDPXyl to the
reaction mixture caused a large decrease in the formation
of D-[U-14C]apiose solubilized product as only 32% of

the D-[U-14C]apiose product synthesized in the presence
of UDPXyl was solubilized by ammonium oxalate treatment.
Addition of UDPGlcUA resulted in no significant change

in the incorporation of radioactivity into D-[U-14C]-
apiose product, although, the amount of product solu-
bilized by ammonium oxalate treatment decreased to 81%.
The results in Table 2 also show that almost all of each
solubilized product was nondialyzable.

Partial Acid Hydrolysis of
- U- C]Apiose Product

 

 

D-[U-14C]Apiose product was subjected to partial
acid hydrolysis. The hydrolysate was chromatographed
on paper with Solvents A and B and the radioactive

compounds on the chromatograms were located with a

52

.mmsao>

some momasoamo on com: mums one UoEMOMHmQ uses one» mucoeflummxm Hosofl>fioofi mo Henson

0:» mumcmammc monocucmumm ca mucosa: was .cowusHOm wmfiaovalagmas may cw ucommum
muw>fiuomowcmn Houou can no memos can so omuoasoaoo mums m05am> Hoscfl>flocHo

.mmsao> mmocu mcHEESm one muoaoxo Edwcoesm spas mc0fluomuuxo may

Hmumo mcflcflmeou osoflmmu waosaomcfl we» cw can uosooum cmuaawosaom who cw mufi>fluooowcmu

mo pesoso moo mswudmoofi mo ooswsumumo mos uoopoum comm cw muw>fiuom0Hcon mo ucSOEm

one .nn H scoop mosses summon a so“: no N now ooe do .o.c me .udmmsn decompose season

2 mo.o so commaowo mos uoscoum coNflawosHOm zoom .ooooo mos Huxmos owns a: me can

cocoa mums macaomao can <Damwmos cons an on mp3 muusust coauomou on» NO mEsHo> Hmcwm

one .ompoowocfi mm amxmos no .dooawmoo edoamumas mo mmHOEc v Hmnuwm cmswmucoo mmusuxfle

cofiuommu may no mouse .momcmno mcflsoHHOH map How umooxo monotonoum Houcmeummxm on»
CH cmowuommo mm counmoum mums posooum couwaaosaOm one uosooum mmOHQ¢H0vHIDHIom

 

maneumamwosoz
mos omen uosoonm
touwawosHow
wooedafloeau9_uo

uoscoum

ooNAHsnsHom
”WOHQ¢HUVHIDHUQ

uosooum
whofla«_oeauoluo

H
(V

o
\O
(V

m.mm Amv h.N

H

Adv m.ma ANV o.v H m.v~ Amv m.o

H
W)

o
o
"3

Ace m.o

H
0‘

o
‘0
W1

w.mm Amy m.o

H

m.HH ANV m.o

H

Amy w.m

H
F-

0
‘fi
F)

Amy o.N

H
[x

O
F!
V'

m.mm Amv m.N

H

m.mm ANV 5.0

H

Ame o.H

 

Awe “we Lee Lee
Huxmo: «oedema: anaconda msoz

 

cowuooum

hcowuooum comm cw ucomoum huw>fluooowcom mo pesoe<

ommowuomaosz Homsm o>auomowoousoz mo unsound one
mocmmmum on» so uosooum omuwawosHom one posooum omowmdfiovalbala mo mwmonusmm .N Manda

53

radiochromatogram scanner (Figure 2). Three radioactive
compounds were located, one of which was at the origin and
is presumed to be unhydrolyzed product while the other

two compounds had Rf values identical to D-xylose and
D-apiose in the two solvents. This establishes that

14C]apiose

D-[U-14C]xylose was incorporated into D-[U-
product from UDP[U-14C]Xy1 contained in the UDP[U-14C]-

Api solution.

Acid Hydrolysis at pH 1

Aydrolysis of D-[U-l4
Solfibilized Product

 

C]Apiose

 

 

Partial acid hydrolysis cannot be used to quanti-

14C]apiose and D-[U-14C]-

tate the relative amounts of D-[U-
xylose contained in D-[U-14C]apiose product because of
incomplete hydrolysis. Treatment of cell wall poly-
saccharides with l M trifluoroacetic acid for 1 hr and
121°C has been reported to result in complete hydrolysis
(7). When tested in D-[U-14C]apiose solubilized product,
1 M trifluoroacetic acid hydrolysis did result in complete
hydrolysis as defined by the total release of radio-
activity as monosaccharides. However, after paper
chromatography only 71% of the radioactivity hydrolyzed
was recovered on the chromatogram (Table 3). In order

to optimize the recovery of radioactivity after hydrolysis

with trifluroracetic acid samples of D-[U-14C]apiose

solubilized product were hydrolyzed with either 1.0 M,

54

.< ucm>aom as
women so poemmumoumeouao mm3 mummmaouomn may one mmusomooum Houcmeflummxm
map as confluommp mo mamaaouomn owoo Hofluuom Op UmuomflQSm mp3 uosooum

mmowmmmu IDHIQ one .H: mom mo wEsHo> Hmcwm o no: scans musuxHE cowuommu

ea
may on compo mums maumz mo mmHOEn N can coaumummmum memucm muoasofluumm

mo an oom umcu pmmoxm monsomooum Houcmefluomxm who cw confluommp mm omummmud

mm: uosooum omOHmmHU IDHIQ .uosooum mmOHmmHU IDHIQ mo mflmmaouomn meow

ea ea
Howuumm on» Eonw Umcflouoo monotone on» no snow Eoumoumsonnooaoom .m musmflm

55

N ousmflm

325m 5

 

 

.9 Q

mmo_n_<‘

mmo._>x

U (5110
~ asNOdsz-m 30103130

 

 

56

.< ucm>Hom nuHs coEHONHmm mp3 annoumouoeouno modem .mouoooooum HmucmEHumdxm

may cH H mm on mHmmaoucmc oHom
.mHSmmmum mocsom mH one oHNH
mHMH> one .2H0>Huoommmu .2 Ho.o
map chuoo ou mHMH> HosoH>HocH
can .2 o. N m0 H2 N. m .2 o. N mo
CH omUMHm cons mums 28mm ooov

now cmoHHommc mm mos ousomooum one mo umUCHmEmH one
no so H condos can m>MHoouso so cH cmomam cosh mums
new .mo.o .H.o .o.H mo mCOHumuucmoooo Hmch omuHmmo
ou compo mums oHom OHHmomouosHmHHu 2 N.o mo H: m.oa
H: m. 0H .2 m. NH mo H: NH coo mHmH> moolsmuom Educ H
.H: OONV mummmHMHc mo mmHmEmm .cHE om cam om Hmumm

0N2 mo woodman nqu u: N now 0N2 cH use so oonHoHo one monotonoum HoucoEHuwmxm
on» cH omoHuommU mo pwummmum mos posooum UGNHHHQSHom omOHQNHUVHIDHIQm

 

 

 

N.mm m.m o.mN N.mm Ho.o
n.0h e.mN m.m o.mm mo.o
m.Nn m.VN m.N N.mm H.o
m.on m.>N o.N v.Hn o.H
S; S; :3 3305

N ossomfioo H ccsomEoo GHOHHO Awe mHmmHouohm

Emumouoeouno on» sH com: oHoe

venom so 29H>Huom0Hpmm UHHmomouosHmHHa

»UH>HuomoHoom ooum>ooom mo uGSOE< mo mum>oomm Hmuoe mo coHuoHucoosoo

 

ncoo ndoHuo> equ uosoouo omNHHHndHom mnoHdano

ocHo< oHumomouosHMHuB mo mcoHueuucmo
eduaeuo mo mHmNHouon oHo< .m mamas

57

0.1 M, 0.05 M, or 0.1 M trifluoroacetic acid for 1 hr at
121°C. Two radioactive compounds were found when the
hydrolysates were analyzed by paper chromatography

(Table 3). The best recovery of radioactivity was ob-
tained with a trifluoroacetic acid concentration of 0.1 M.
For all four acid concentrations the percent of recovered
radioactivity found in Compound 2 was nearly identical.
The amount of recovered radioactivity found in Compound 1
was also nearly identical for acid concentrations of l,
0.1, and 0.05 M. However, Compound 1 was not released
when the D-[U-14C1apiose solubilized product was hydrolyzed
with 0.01 M trifluoroacetic acid. In another experiment
hydrolysis times of 30 and 60 min with 0.1 M trifluoro-
acetic acid resulted in identical recoveries of radio-
activity on the chromatograms and identical quantities

of recovered radioactivity in Compounds 1 and 2. There-
fore, I decided that optimum results would be obtained

if solubilized products were hydrolyzed at pH 1 for 30
min at 121°C.

Samples of D-[U-14C]apiose solubilized product
were hydrolyzed at pH 1 as described in Experimental
Procedures and the resulting hydrolysates were chroma-
tographed in Solvents A - D. All four chromatograms
showed that 98% of the recovered radioactivity was con-
tained in two monosaccharides which had R values of

f

D-apiose and D-xylose. Compounds 1 and 2 discussed

58

in the preceding paragraph, corresponded to D-xylose and
D-apiose, respectively. Figure 3 shows the result of
chromatographing the hydrolysate in Solvent A. These

results established that the particulate enzyme preparation

1

from A. minor can incorporate D-[U- 4C]apiose and D-[U-14C]-

xylose from their respective sugar nucleotides into

D-[U-14C]apiose solubilized product and hence into

4

D-[U-14C]apiose product. D-[U-1 C]Glucuronic acid was

not detected in the D-[U-14C]apiose solubilized product.

1

The determination of the amounts of the D-[U- 4C]xylose

1

and D-[U-14C]apiose contained in the D-[U- 4C]apiose

product will be discussed later.

H drolysis of D—[U-14C]Galacturonic
Ac1 , D— U-14C]Glucuronic Acid, and
D-lU-14C xylose Squbilized
PrOducts

 

 

 

 

Samples of D-[U-14C]galacturonic acid, D-[U-14C]-
glucuronic acid, and D-[U-14C]xylose solubilized products
described in Table l were dialyzed for 2 hr in water at
4°C and hydrolyzed in 1 M trifluoroacetic acid for 30 min
at 121°C and 15 pounds pressure in an autoclave. All
procedures for the hydrolysis except for the l M acid
concentration were identical to that described in the
Experimental Procedures for hydrolysis at pH 1. Each
sample contained 5,000 to 7,000 dpm. Paper chromato-

graphy was used to identify the radioactive hydrolysis

59

.mwm moB mEmumouoEouno was Co huH>Huom0HUmn

mo mum>oomm .< qu>Hom CH momma Co pocmoumoumsouso mp3 mummmHouoan ecu

oCo monotonoum HmquEHummxm 02» CH omQHuommp mm H mm on UmumHouomn moz

Leap

pCm

mos UCC
oonaaaosaon

mHmmHouown

ooo.oc ooooond ooneaaoseon onoedoloesuoluo oonmaoao was .cHs oo

om Houmm Hopes mo mmmCCCo CHHB Dov Hm H2 N How nouns CH UmNmHmHU
mousomooum HmquEHummxm map CH UmoHuommo mo coummmum mmz poscoum

dmoHda2oe (CLIQ .uosooud o0NHHHndH0m mmoHdomo uaLuo o0 H mm up

H VH
umumo poCHouoo muodooum map mo Coon EmumoudEOHCUOHpmm .m mHCmHm

60

m ousmHm

525m 5

 

 

 

\‘1

 

mmo_n_<

O

mmo._>x

o .3. 2225028

u (61.10

 

 

3SNOd S38 80133130

61

products. Recovery of starting radioactivity on the
chromatograms ranged from 75 to 80%.

The hydrolysates obtained from D-[U-14C]galac-
turonic acid solubilized product were chromatographed in
solvents A and E. Four radioactive compounds were
detected on the chromatograms. One compound contained
60% of the recovered radioactivity and had the same Rf
as D-galacturonic acid. These 2 solvents resolve
D-galacturonic acid and D-glucuronic acid. A second
compound with an Rf of D-xylose in Solvent A contained
less than 6% of the recovered radioactivity. The
remainder of the recovered radioactivity was contained
in two compounds which were not identified but presumably
are oligosaccharides containing uronic acid as they did
not move far from the origin.

The hydrolysates obtained from D-[U-14C]glucuronic
acid solubilized product were chromatographed in Solvents
A, C, and E. Four radioactive compounds were detected
on the chromatograms. Interestingly, one compound con-
tained 64% of the recovered radioactivity and had the
same Rf as D-xylose. Of further interest was that the
second major radioactive compound, which contained 23%
of the recovered radioactivity, had the same Rf as
D-galacturonic acid rather than D-glucuronic acid. The
two unidentified radioactive compounds with the smallest

Rf values are again presumed to be oligosaccharides.

62

Chromatography in Solvent C of the hydrolysate
obtained from D-[U-14C]xylose solubilized product showed
that the recovered radioactivity was contained in a
single compound which had the same Rf as D-xylose.
Solvent C is capable of resolving D—xylose and
L-arabinose.

From these results we conclude that the particu-
late enzyme preparation from A. AAAQE apparently con-
tained UDPGlcUA carboxy-lyase and UDPGlcUA 4-epimerase
activities that convert UDP[U-14C1GlcUA to UDP[U-14C1Xy1
and UDP[U-14CJGalUA. The particulate enzyme preparation
either does not contain UDPAra 4-epimerase activity to
convert UDP[U-14CJXyl to UDP[U-14C]Ara or it cannot

1

incorporate L-[U- 4C]arabinose into solubilized product.

gydrolysis of Authentic
ApiogalaCturonan from
Whole Plants

 

Since previously described experiments showed
that the particulate enzyme preparation from A. EAAQA
incorporated D-[U-14C1xylose into product we decided to
hydrolyze authentic apiogalacturonans (isolated from
whole plants) with trifluoroacetic acid in order to
determine whether they contained D-xylose also. Two
apiogalacturonan fractions, isolated by Hart and Kindel

l4C]22°C sodium chloride soluble fraction

(104) [
(7,000 dpm) and [14c160°c sodium chloride soluble

fraction (9,400 dpm), were hydrolyzed at pH 1 as

63

described in the Experimental Procedures. The hydroly-
sates were analyzed by paper chromatography in Solvent A.
Recovery of radioactivity on each chromatogram was 85%.
Two radioactive compounds were detected in the hydrolysate
of the [14C]22°C sodium chloride soluble apiogalacturonan
fraction; one at the origin and the other with a Rf of
D-apiose. The compound with a Rf of D-apiose contained
40% of the recovered radioactivity. Four radioactive
compounds were detected on the chromatogram of the
hydrolysate of the [14C160°C sodium chloride soluble
apiogalacturonan fraction. One compound contained 26%

of the recovered radioactivity and was located at the
origin. Another compound contained 5% of the recovered
radioactivity and had the same Rf as D-galacturonic acid.
The two remaining compounds had the same Rf values as
D-xylose and D-apiose and contained 27 and 41%, respec-
tively, of the recovered radioactivity. The compound
corresponding to D-xylose was eluted from the chromatogram
and when re-chromatographed on paper in Solvent B it had
the same Rf as D-xylose. Compounds with the Rf values

of D-apiose and D-xylose were also identified when the
hydrolysate from the [14C]60°C sodium chloride soluble
apiogalacturonan fraction was chromatographed in

Solvents C and D. These results indicate that D-xylose
was contained in the apiogalacturonan fraction isolated

from intact plants at 60°C.

64

Hydrolysis of D-[U-14C]Apiose Solubilized
Prodibt Synthesizedmin the Presence
deronradioactive Sugar Nudleotides

 

D-[U-14C]Apiose solubilized products synthesized
in the absence and presence of UDPGalUA, UDPGlcUA, and
UDPXyl were hydrolyzed at pH 1 and the hydrolysates were

1 1

analyzed for D-[U- 4C]apiose and D-[U- 4C]xylose content

(Table 4). These data show that the relative amounts of

D-[U-14C]apiose and D-[U-14

C]xylose incorporated into
D-[U-14C]apiose solubilized product were affected by the
presence of nonradioactive sugar nucleotides in the
reaction mixture. In addition to this data the data in
Table 6 show that the percent of radioactivity contained
in D-[U-14C]apiose increased with increasing amounts of
UDPGalUA present in the reaction mixture. A decrease in
the percent of D-[U-14C1xylose incorporated in the
presence of UDPXyl is not surprising because of the
resultant dilution of UDP[U-14C1Xyl. Addition of
UDPGlcUA to the reaction mixture would also cause the
formation of UDPXyl in the reaction mixture because of

the presence of UDPGlcUA carboxy-lyase activity in the

particulate enzyme preparation.

65

.ooECOHHom mos uCoEHHomxo nooo

moEHu mo uonECC onu uComonoH mHmonuCoHom CH moCHo> one .omOmeHUvHIDHIQ CH ooCHmu

(Coo mos omonoHUVHIDHIo CH oouo>ooou DOC wuH>Huoo0Hoom .C uCobHom nuHs mnmoumou

IoEouno Momma an touMHoCo osos monommHouomn one .mousooooum HouCoEHuomxm one CH

oonHuonoo no H me so omnmaouoms mums N manna sH omnHuonmo memoa can .asoaodoa

.CDHmquD mo ooComon on» CH poNHmonqum muosooum ooNHHHnCHom omOHmoHovHIDHIQ
ooumfloHo one can suppose ooNHHHnoHon mmoHdnnoeHuoluo oonsaoHo when

 

 

 

H.m N.mm “He medoo
H.OH 0.0 n o.Hm INC «aoHomoa
m.H v.0 n m.mm Ame CoHoomo:
m m.N H N.mb Avv oCOC
“we
omOmeHOvHIDHIo omonmmovHIDHIo CH ousuxH2 COHuooom 09
omoHQCHUVHIDHID oouo>ooom huH>Huoo0Hoom popom oUHuooHosz Hmmsm

 

omooHuooHOCz Homsm o>Huoo0HooHCoz mo ooComon onu
CH ooNHmonqum uospoum ooNHHHnCHom omOHCCHovHIDHIQ mo H mm um mHmmHouomm .e mHmCB

66

Hydrolysis of D-[U-14C]Apiose Solubilized
Product at pH‘4

 

 

Hydrolysis of D-[U-14C]Apiose Solubilized
Aroduct Synthesized in the Absence and
Presence of Nonradioactive Sugar
Nucleotides

 

D-[U-14C]Apiose solubilized product was hydrolyzed
at pH 4 and the hydrolysate was chromatographed on paper
(Figure 4). Three peaks were obtained. The largest peak
was due to material at the origin of the chromatogram
which we assumed is unhydrolyzed D-[U-14C]apiose solu-
bilized product. The other two peaks were due to

14C]apiose and [U-14C]apiobiose since they had the

D-[U-
same Rf values as the authentic compounds on the chroma-
togram. No D-[U-14C1xylose was detected on the chroma-
togram. The procedure would detect as little as 6% of

l4C]apiose

the D-[U-14C1xylose present in the D-[U-
solubilized product.

D-[U-14C]Apiose solubilized products synthesized
in the absence and presence of UDPGalUA, UDPGlcUA, and
UDPXyl were hydrolyzed at pH 4 and the amounts of

14C]apiobiose released from

D-[U-14C]apiose and [U-
each were determined (Table 5). These hydrolysis exper—
iments demonstrate that the presence of nonradioactive
sugar nucleotides during synthesis resulted in the
formation of a solubilized product which released an

1

increased amount of D-[U- 4C]apiose and [U-14C]apiobiose

when hydrolyzed at pH 4.

67

.wme mos Eoumouoeouno on» Co >DH>Huoo0Hoou mo >uo>ooom
.Hn m mos oEHu mHmmHouomn on» Donn umooxo monsooooum HouCoEHHomxm onu
cs oooHuouoo on e no on consaonoss was lame ooo.oe .Ha come .aoaooooo co

ooComoum onu CH oouHmonqum .uosooum ooNHHHnCHom omOHmonu IDHIQ ooumeHc

N

vH

one .un H Houmm nouns mo omCono o nuHB Uov so an N COM 0 m CH CoumHoHo

mos posooum CoNHHHnCHom omonoHU (Dana one .H: me mo oECHo> HCCHm

«H
m can oCo CDHCUCQD mo moHOEC v ooCHmuCoo moHCuxHE COHuoooH one .mousooo

(one HouCoEHuomxm onu CH ConHuomoo mo mouomoum mo3 .CDHoomoD mo ooComoum

one on oouanosusmn .uosoone ooueaflnsaon onoedanoe (alto .aoanodoo no

H

ooComon onu CH CoNHmonuCem uosooum ooNHHHnCHOm omonoHU IDHIQ mo v mm um

VH
mHthouown onu Eouw moCHouno muospoum onu mo Coon EououoEounUOHoom .v oHCmHm

68

v oquHm

mozgm E

 

 

O

mmoE<

.0

mmo._>x

mmoao :2

u (5110

 

 

 

3SNOd 538 80133130

69

.ooEHOMHom

mm3 uCoEHuomxo nooo moEHu mo HonECC on» uComonou mHmonuCouom CH mosHm> one
.wOOH ou Hoswo mH omonoHU HIDHIQ CH euH>Huoo0HooH onu oHnou mHnu CH oHOMouone
.omOHnOHmoHUVHIDH Ho omOHmowU HIDHIQ mo CommoHoH mos noHn3 muosnoum ooNHHHnCHOm
on» CH noCHouCoo omonoHU HID no mo uCoouom on» uComonou mosHo> one .hnmoumou
IoEouno women »n UonHoCo Hos monommHouoen one .mousooooum HooCoEHuomxm on» CH
omnHuonmo on e mm on omanonoHs mums N manna cH oonHuonwo Hexaoe pen .aooaodoo
.CDHoUmoD mo oOComon onu CH ooNHmononem monotone ooNHHHnCHOm omonoHo IDHIQ
coanmHo on» oCo uosooum ooNHHHnCHOm omoHQCHUvHIDHIo CoueHMHo oneo eH

 

 

o.NH m.c~ .HV Hexdos
m.mN v.mH .HV «DOHUmQD
m.o H v.mN m.N H m.0N ANV «DHoomQD
e.o n o.mH e.o n o.HH Ame oeoz
Awe Ame oHCuxH2 COHuooom oe
omoHnOHQCHUVHIDH omOHmnnovHIDHIQ nooon ooHuooHoCz Momsm

 

mmooHuooHosz Homsm o>Hu0C0HooHCoz mo ooComon oCo ooComnC on»
CH ooNHmonuCem uosooum ooNHHHnCHom omonCHUeHIDHIo mo e no no mHmeHouomm .m mqmne

70

Release of D-[U-14C]Apiose and
lU-14C1Apiobiose as a Function
deydrolysid'Time

 

 

Samples of D-[U-14C]apiose solubilized product
synthesized in the presence of UDPGalUA were subjected
to hydrolysis at pH 4 for varying periods of time
(Figure 5). These results showed that release of
radioactivity from the D-[U-14C]apiose solubilized
product was essentially complete after a 3 hr hydrolysis
period although 50% of the D-[U-14C]apiose originally
contained in the sample had not been released. We also

14C]apiose

investigated the hydrolysis at pH 4 of D—[U-
solubilized product and found that its hydrolysis at

pH 4 was also complete after 3 hr.

Hydrolysis of [U-14C1Apiobiitol

 

I determined whether both moieties of the [U-14C]-

apiobiose side chains of D—[U-14C1apiose solubilized

product were synthesized AH vitro. [U-14C]Apiobiose was

l4C]apiose solubilized product syn-

isolated from D—[U-
thesized in the presence of 4 nmoles of UDPGalUA by
hydrolysis at pH 4 and was purified by paper chroma-

l4C]apiobiose

tography in Solvent A. A solution of [U-
(6,500 dpm) was titrated to pH 10 with 0.5 M NaOH and
was then made 80 mM in NaBH4. The solution was allowed
to stand 16 hr at 22°C after which it was acidified to
pH 3 with acetic acid. Addition of methanol and

repeated concentration of the solution under reduced

71

.ooCHEuouoo mos omOHnOHmm

(HUvHIDH CCo omOHmmHU IDHIQ mo oomooHoH pCo CHmHHo onp um uosoonm UoNHHHnCHom

VH

CH mCHCHCEoH omOHmoHUVHIDHIQ mo uCoouom ono e mm on oEHn mHmeHouown nooo mom

.2 uCo>Hom CH momma Co Conmoumouoeouno onos monommHouomn on» oCm monsooooum HouCoE
IHuomxm onu CH nonHuomoo mm H me no CoumHouoen ouoB monEom .CDHoumoD mo oOComoum

on» CH oouHmonuC>m uosconm omOHmmnoe (9.19 on» mo uCouCoo omononoe (Data on»

H H
oCHEuouoo on uoouo CH .eHo>Huoommou .wmm CCo .mm .em .Nm .me .mm .mm oCoB un OH

oCo .m .mN.m .v .m .N .H mo moEHo mHmeouoen How meoumoumeouno onu Co muH>HuoooHoou
mo 2Ho>ooou one .Coms ouos Mn 0H oCo .m .mN.m .v .m .N .H mo moEHo mHmeHouomn ponu
umooxo monsooooum HouCoEHuomxm on» CH nonHuomoo on v mm no ooNeHouomn ouos “ECU ooo.OH

.H: OONV .CDHoUmQD mo oOComoum on» CH coNHmonunmm .uosooum ooNHHHnCHom omonoHU (CHIC

vH
coueHoHo onu mo monEom .v oHCmHm mo UComoH onu oCo monsooooum HouCoEHuomxm onu

CH conHHomoo mo ooNeHoHn CCC pouomoum mos .CDHowmQD mo ooComoum onu CH ooNHmoanmm

.oosooum UoNHHHndHom omonmHUv IDHIQ .CDHoumoD mo ooComon onu CH ooNHmonunem uoscoum

H
ooNHHHnCHom omOHmoHUVHIDHIo mo v mm on mHmmHoucen onu mo omuCOOIoEHe .m oquHm

72

m oHCmHm

585 us: 28:23;
a e m

 

_ _ _
mmoE<

 

Ilfl. H.IIIIIIII

III? \
m . llllll‘“
lllllll ' | I. I II |I.I.I|

mmoao _n_<

 

(.1
r... O

1
2.2%

 

\ :30...

 

 

R

6%) 3SOIdV

8H

73

pressure removed the borate as methyl borate. The
reduced disaccharide was purified by paper chromatography
in Solvent A and hydrolyzed in 0.04 M trifluoroacetic

14

acid for 30 min at 121°C. Conversion of [U- C]apiobiose

to [U-14C]apiobiitol after NaBH4 treatment was greater
than 90%. The hydrolysate was analyzed by paper chroma-
tography in Solvent E (Figure 6). Two radioactive com-
pounds were obtained which had the same Rf values as
D-Apiitol and D-apiose. The amount of recovered radio-

activity in D-[U-14C]apiitol and D--[U--14

C]apiose was
50.2 and 49.8%, respectively. The amount of radioactivity
from [U-14C]apiobiitol that was recovered in D-[U-14C1-

l4C]apiose was 92.6%. This experiment

apiitol and D-[U-
established that synthesis of both moieties of the
[U-14C]apiobiose side chains occurred AH yAggg.

Sodium Chloride Fractionation of D-[U-14C]-

Apiose solubilized Produdt

Hart and Kindel had shown that after 0.5%
ammonium oxalate extraction authentic apiogalacturonans
were fractionated into two components when treated with
l M NaCl (102). Apiogalacturonans of high D-apiose con-
tent were soluble in 1 M NaCl while apiogalacturonans
of low D-apiose content were insoluble in 1 M NaCl (102).

I have investigated the solubility of D-[U—14

C]apiose
solubilized product in 1 M NaCl. D-[U-14C]Apiose

solubilized product was dialyzed in water at 4° for

74

.muHCmom CH

nonHuomoo mo Conmoumouoeouno UCC CoueHouomn mos UCo omoHnOHmoHUeHIDH
Eoum ooummoum mos HouHHQOHQCHUVHIDH .HouHHnOHmonoeHIDH mo mHmmHouomn

oHoo onu Eoum poCHouno muosooum onu mo Coom EoumouoaounUOHUom .m ousmHm

75

m ousmHm

5sz E

 

 

.O

mmo E< .5: _n_<

U (6110

 

 

3SNOd 538 80103130

76

2 hr with changes of dialysis water after 30 and 60 min.
The dialysate was diluted 1 to 10 with water. One mg of
authentic 60° sodium chloride insoluble apiogalacturonan
fraction was dissolved in 500 pl of the diluted dialysate.
The solution was warmed to 22°C and stirred while 500 pl
of 2 M NaCl was added dropwise. A precipitate formed
during the addition of the NaCl. The suspension was
centrifuged and the supernatant was removed. The pre-
cipitate was washed once each with 500 pl portions of

2 M and l M NaCl and then was dissolved in 1 ml of water.
The radioactivity in the NaCl soluble and insoluble
fractions was assayed with scintillation solution B.

The NaCl soluble fraction contained 1958 dpm and the
NaCl insoluble fraction contained 71 dpm. The results
indicate that D-[U-14C1apiose solubilized product has

a high D-apiose content.

DEAE-Sephadex Column
Chromatography

 

QHromatography of D-[U-14C1Apiose
Solubilized Product

 

Column chromatography with DEAE-Sephadex separ-

ated the 0-[0-14

C]apiose solubilized product into 5
fractions (Figure 7). Recovery of solubilized products
from the column was usually between 60 and 90% and
in the experiment depicted in Figure 7, 81% of the

14

D-[U- C]apiose solubilized product was recovered.

One radioactive fraction was eluted with 0.1 M NaCl,

77

.m one .o .0 .m .C mCoHuooum Snow on ooCHnEoo Con» ouos mCOHpooum CEsHoo
nouCOHpCH one .m COHuCHom COHuoHHHoCHom nuHs euH>Huoo0Hoou How ooeommm

ouos CCC mCoHuooum CECHoo on» Comm oo>OEoH ouoz muosdHHn .oquHm ono

CH nouooHCCH Common ouonmmonm CH Hooz mo pCoHooum moon onu nuHs Conn pCo
UouooHHoo ouos m on m mCOHuoon CECHoo oHHnB Common ouonmmonm nqu Couoouu

mos CECHoo one .CECHoo onn Co oooon mos name ooo.ovv onEom HE m.H

on» me couooHHoo ouos N UCC H mCOHuooum CECHOU .oouooHHoo onos mCOHuoouw

HE o.H oCo un\HE 0N no: open son CECHoo one .mousooooum HouCoEHuomxm

ono CH ponHuomoo mo A80 m x ECHU HoCuouCH so m.ov CECHoo xoconmomlmnmo C CO
Conmoumouoeonno CCC Couomoum mos uosooum ooNHHHnCHom omoHQCHUvHIDHIQ .uosooum

noNHHHnCHom omOHmoHUVHIDHIo mo Eonmouofiouno CECHoo xooonmomumdmo .e ousmHm

78

e ousmHm

$2222 20 .55.“.

 

 

 

 

 

 

 

s s
. locotno
/ o\ {loyal \
I yo _ ’0
o x
O
l / l
0
<1
... L
ml m . Q q . o ..¥Wm¢ 1!, 4x a It
. _oezzm.o__oezs_m~.e_ _ce22~.e_ . ooze; .
P

 

 

S

A1|A|10V0|0V8

S

(N0|10V8:| 83d WdG)

79

two with 0.2 M NaCl, one with 0.25 M NaCl, and a small
fraction with 0.3 M NaCl. The indicated column fractions
were combined to give 5 large fractions which were
labelled, in order of their elution, A through E, with
fraction A being eluted first and fraction E last. The
percentage of recovered radioactivity contained in
fractions A, B, C, D, and E was 15, 25, 32, 21, and 2%,

respectively.

Chromatography of D-[U-14C]Galacturonic

Acid SOIubilized Product

 

 

When D-[U-14C]galacturonic acid solubilized pro-
duct was chromatographed on a DEAR-Sephadex column 2
major fractions and 3 minor fractions were recovered

(Figure 8). Recovery of D-[U--l4

C]galacturonic acid
solubilized product from the column in this experiment
was 76%. Radioactive material eluted from the column in
the same positions in the elution gradient as was seen
for the chromatography of D-[U-14C]apiose solubilized
product depicted in Figure 7 although the amount of
radioactivity in the fractions was different. In

Figure 8 the percentage of recovered radioactivity

found in fractions A, B, C, D, and E was 3, 7, 41, 40,

and 6%, respectively.

80

.m CCC .o .0 .m .C mCOHuomHm Snow on ooCHnEoo ouos mCoHuoon

CECHoo ooHCOHoCH one .HE N mo oECHo> m CH CECHoo on» Co Cooon

mos “amp ooo.mHV onEom one .5 oHCmHm mo UComoH on» CH oonHuomoo

no “so m x ECHU HoCuouCH Eo m.ov CECHoo xoomnmomtmnmo C CO Conmoum
Iouosouno mos oCo monsooooum HouCoEHuomxm on» CH ponHHomoo mm oouomoum

mos nosooum nouHHHnCHOm oHoo OHCOHCHUCHCOHU IDHIQ .uosooum ooNHHHnCHOm

CH
oHoo UHCousuooHomHUVHIDHIQ mo Eoumouofiouno CEdHoo xonmnmomlmnma .m oHCmHm

81

m oHCmHm

$9222 20 :05:
cc ON

 

 

 

 

.11 1 "1T 1
m. o o e a

P L
d

 

. . .
Was. 2 No .er 2 he .an 2 Ne _ 8.2 2 2

 

§

(Nouovui 83d Wdfl) AllA|10V0l0V8

S

82

ghromatographyof D-[U-14C1Apiose
Solubilized Productis ntheSized
ih the Presence of UDEGalUA

 

Comparison of the two elution profiles shown in
Figures 7 and 8 indicated that a larger percentage of

radioactivity present in D-[U---14

C]galacturonic acid
solubilized product had was recovered in fractions C,

D, and E than was recovered in the same fractions when
D-[U-14C1apiose solubilized product was chromatographed
on DEAE-Sephadex. If D-[U-14C1apiose and D-galacturonic
acid are incorporated from their respective sugar nucleo-
tides into the same polysaccharides then the acidity of
the polysaccharide should increase because of the

increased ratio of D-galacturonic acid to D-[U-14

C]apiose.
D-[U-14C1Apiose solubilized product synthesized in the
presence of UDPGalUA would therefore, when chromatographed
on the DEAR-Sephadex column, have a higher percentage of
radioactivity recovered in the more acidic fractions,
which elute with the higher salt concentrations, than
would D-[U-14C1apiose solubilized product synthesized

in the absence of UDPGalUA. When D-[U-14C1apiose
solubilized product, synthesized in the presence of
UDPGalUA, was subjected to DEAE-Sephadex column chroma-
tography 5 fractions were obtained (Figure 9). In the
experiment reported recovery of radioactivity from the

column was 78%. Radioactive material eluted from the

column in the same positions in the elution gradient as

83

.m can ~o .0 .m .d mcowuomum EHOw

ou omcHnEoo mumB mcowuomum cadaoo topmowocfl one .HE m.H mo mEsHo> m as
cEdHoo may :0 cmomam mm3 “Eat ooo.mmv onEmm one .5 musmflm mo ccmmma

mcu cw omnwuomoc mm “Eu m x Ewan Hmcumucw Eu m.ov CEsHoo xmomnmmmnmdma m

:0 omcmmumoumsouso mmz cam mousomUOHm Hmucmsfiummxm map can N manna CH
ombwuommo mm omummmum mmB .doamomos mo mocmmmum 0:» ca Umuwmmsucmm .uosooud
consawnsaom omofim¢HUvHIDHIQ .moamomoo mo mocmmmum on» Ca omnammnucmm uosooum

tonwaflnsaom mmOflmmHUv IDHIQ mo Emnmoumeouno GEdHoo xmomcmmmlm<mo .m musmwm

H

84

m musmwm

mum—2:2 2o :05:
cu

 

— P

1 d

m

r _

 

. 82 2% .

 

 

 

» p L
d 1 1d

a o m
as. s as. omz 2 N...

_

p 5
W J-

<
8oz E fie

P
d

 

L
c

E

§

(NOIJDVEH 83d WdG) All/\llOVOIGVH

 

85

was seen in Figures 7 and 8. In Figure 9 the percentage
of recovered radioactivity contained in fractions A, B,

C, D, and E was 6, 9, 22, 53, and 7%, respectively.

These data show that the D-[U-14C1apiose solubilized
product synthesized in the presence of UDPGalUA contained
an increased amount of radioactive material that eluted

as fractions D and E with a concurrent decreased amount of
radioactive material that eluted as fractions A, B, and C

compared to the results obtained when the D-[U-l4

C]apiose
solubilized product was synthesized in the absence of
UDPGalUA. The increase in the amount of material which
eluted in fractions D and B when UDPGalUA was present
during synthesis confirms the theory that both D-[U-14C1-
apiose and D-galacturonic acid were incorporated into the
same polysaccharide.

The optimum concentration of UDPGalUA needed to
cause maximum formation of fractions D and E and minimum
formation of fractions A through C was determined
(Table 6). These results showed that the maximum shift
of radioactivity into fractions D and E occurred when
4.0 nmoles of UDPGalUA was present. The presence of
16.0 nmoles of UDPGalUA yielded results that were basi-
cally the same as those obtained when 4.0 nmoles of

UDPGalUA was present. However, D-[U-14

C]apiose solu-
ibilized product synthesized in the presence of 0.5 nmoles

(Bf UDPGalUA showed an elution pattern intermediate

86

.H mm as mwmhaouohc an omCHEHmuwo mums mooam> ommna .mm0axxHUvHIDHIQ cw ucmmmum was
pontoum touwaflnsaom mmOHQMHUeHIDHIQ on» GM muw>fiuomoflomu may mo HoocfimEoH mnan

.wnmmumoumfiouno cadaoo Hmumm Umum>oomu hufi>wuomoflcmu
mo uCSOEm Hmuou on» Eouw omumasoamo mum: m canons» d mc0fluomum ca cmum>oomu >uw>wuom
Iowomn mo unsoam may now connedmu mmoam> one .5 musoflm cw owumoflocfi mm mcofluowuw
cEsHoo mamm on» mcflcHnEoo wn omchuno mum3 m cmsounu d mcofluomum .aam>wuommmmu
.ucmmmum «Damomoa msocmooxm mo mmHOEc o.mH can .o.v .m.o nufi3 omnwmmsucmm uosooum
omuflHAQSHOm nuw3 mucmeummxm may MOM mmm can .nn .Hm can ucmmwum «madame: msocmmoxm
uoonuwz Umuwmmnucmm uosooum omuwafinsHom mmOAQMHUVHIDHIQ suw3 mucmeummxm 03u on» How
wam can mm mm3 mcEsHou may :0 omomHm >uw>wuomowtmu on» mo hum>oowm .mHm>wuommmmu .Edo
ooo.vv can .ooo.mm .ooo.hv omcflmucoo «panama: mo meOEc o.mH can .o.v .m.o mo mocmmmum
map cw oouwmmcucmm mmHQEmm on» can Eat ooo.ov omcwmucoo comm «Samoan: mo mucmmnm may
cw omnwmmcucmm uosooum omuflawnsaom mm0ammnou nDHIQ mo mmamemm N one .ummmsn mo HE
m.H cw comm wumz mcEsHoo map so cocoam uosooum monwaancaom mmoamm_u HIDHIQ mo mwamsmm
one .5 musmwm mo ocmmma may cw ponwuommo mm A80 m x Emwt Hmcum Ga 50 w.ov cesaoo
xoomnmmmlm¢mo m co omnmmuooumsouco mum: muooooum omnwafinsHom one .>Hm>fiuoommmu .H:
mm tam .vh .mh mumz mousuxHE cowuowwu may no moEdHo> HMCfim on» can monouxwe Goduommu
mumwumoummm on» ou omocm mm3 «Damomoo mo mmaoec o.mH no .o.v .m.o was» umooxm monsoon
Ioum Hmucmefluomxm on» Ca confluomco mm who: mmusuxwe cowuommu one .mmuscmooum Hmucma
Iflummxm may cw confluommp mm omumdmum mm3 uosooum ownwaflnsaom mmoflmdgovalbulom

 

 

 

m em mm m m oa o.oH

e mm mm m 0 mm o.v

m mm mm m «a om m.o

N am mm mm ma om mac:

v ow mm Nm Hm me ago:

Amy “we Ame Awe “we Awe ammaoecv

m o o m 4 mmohm<

IHUVHIDHIQ ma ucmmmum uosooum ousuxwz coauommm

cowuomum scum ca omum>oomm UmnwafinsHom mmon<~UvHIDHIa on popvd dnamumno
>ua>wuomowomm mo uGsOE¢ cw >uw>wuowoaomm mo uc505¢

 

m<bamumoo mo mmwnwucmso mcwhum>
mo mocmmmum 0:» ca powwmmnucwm uostoum mansaom mmme4~Uvaloulo mo msmuumouME
lounu ampmnmomlmdmo Hound omcwmuno mcofiuomum cw wuw>wu080flomm mo GOMusnwuumwo .w mum<a

87

between that synthesized in the absence of UDPGalUA and
that synthesized in the presence of 4.0 nmoles of
UDPGalUA. The results from the chromatography of two

1

samples of D-[U- 4C]apiose solubilized product synthe-

sized in the absence of UDPGalUA demonstrate that the

14C]apiose solubilized

ion-exchange chromatography of D-[U-
product was very reproducible (Table 6).

I have determined that the increased recovery of
radioactivity in fractions D and E, seen when UDPGalUA
was added to the reaction mixture, did not occur if
UDP[U-14C1Api and UDPGalUA were incubated separately
with the particulate enzyme preparation. The experiment
was performed by incubating 4 nmoles UDPGalUA with 50 pl
of particulate enzyme preparation in a reaction volume
of 54 pl for 15 min at 25°C. A control incubation was
prepared by incubating 4 pl of H20 with 50 p1 of par-
ticulate enzyme preparation for 15 min at 25°C. After
incubation the two reaction mixtures were extracted as
described for the preparation of product and solubilized
product in the Experimental Procedures. The solubilized
extracts (0.5 ml) from the incubations with UDPGalUA
and water were each combined with 0.5 ml samples of
‘D-[U-14C1apiose solubilized product (30,000 dpm) and
were then dialyzed in phosphate buffer and chromato-
{graphed on columns of DEAE-Sephadex as described in the

legend of Figure 7. Recovery of radioactivity from the

88

columns was 84% for the control experiment and 76% for
the UDPGalUA experiment. A comparison of the elution
profiles obtained from the two chromatographies showed
that they were identical to each other and to Figure 7.
Therefore, addition of solubilized material synthesized
from UDPGalUA alone did not affect the migration of
D-[U-14C1apiose product through the DEAR-Sephadex

column.

Chromato ra h of D-[U-14C1Glucuronic
Acid and D-iU-13CIXonse Solu-

bilized Products

 

 

D-[U-14C]Glucuronic acid and D-[U-14C1xylose
solubilized products were chromatographed on DEAE-

14C]-

Sephadex columns (Figure 10). The recovery of D-[U-
glucuronic acid and D—[U-14C1xylose solubilized product
from the ion-exchange columns in the experiments
depicted in Figure 10 were 65 and 45%, respectively.
Since the volume of NaCl solution in each of the steps
in the gradient described in Figure 10 is different
than in the gradients used in the previously discussed
ion-exchange experiments, it is not possible to compare
the percentage of radioactivity contained in Fractions

14

1k, B, C, D, and E for the D-[U- C]glucuronic acid and

l1-[U-14C1xylose solubilized products. However, the
elution profile for the D- [0-14C]glucuronic acid
8<Dlubilized product is similar to that seen in Figure 8

14

fCDr the D-[U- C]galacturonic acid solubilized product.

89

Figure 10. DEAR-Sephadex column chromatograms of
D-[U-14C]glucuronic acid solubilized product (a) and
D-[U-14C1xylose solubilized product (b). D-[U-14C1-
glucuronic acid and D-[U-14C1xylose solubilized products
were prepared from reaction volumes of 55 and 70 pl,
respectively, as described in the Experimental Pro-
cedures. The solubilized products were chromatographed
on DEAE-Sephadex columns (0.8 cm internal diam x 10 cm)
as described in the Experimental Procedures. The columns
were developed identically. The column flow rate was

25 ml/hr and 2 ml fractions were collected. D-[U-14C1-

14C]xylose solubilized products

glucuronic acid and D-[U-
containing 24,000 dpm and 16,000 dpm, respectively, in

a 4 ml volume were placed on the column as column
fractions 1 and 2 were collected. The columns were
treated with phosphate buffer while column fractions

3 to 8 were collected and then in order with 16 ml each
of 0.1 M, 0.2 M, and 0.25 M NaCl in phosphate buffer

and 20 ml of 0.3 M NaCl in phosphate buffer. The
fractions where the various NaCl solutions eluted were
determined by conductivity measurements in an identical
experiment except that no solubilized product was present
and are indicated on the figure. Aliquots were removed

from the column fractions and were assayed for radio-

activity with scintillation solution B.

1500

1000

600

RADIOACTIVITY (DPM PER FRACTION)

90

 

 

 

l 1
0.1M 0.2M 0.25M 0.3M

rNaCl ' NaCl rNaCI 'NaCI‘

 

 

r J

20 40
FRACTION NUMBER

Figure 10

 

 

91

This is unexpected since a previous hydrolysis experi-
ment disclosed that the majority of the radioactivity
in the D-[U-14C]glucuronic acid product was contained

in D-[U-14C1xylose. The elution profile for the

14

D-[U- C]xylose solubilized product is similar to

l4C]apiose solubil-

that seen in Figure 7 for the D-[U-
ized product. This shows that D-[U-14C1xylose was
incorporated into an acidic polysaccharide.
Chromatography of D-[U-14C1Apiose

o 1 ize roductsSgntfiesized

in the Presence of UDP cUA

and UDnyl

 

 

 

 

 

The elution of D-[U-l4

C]apiose solubilized pro-
ducts synthesized in the presence of UDPGlcUA and

UDPXyl through the DEAF-Sephadex column was investi-
gated. These experiments were performed to show whether
the addition of UDPGlcUA and UDPXyl to the reaction
mixture would cause the same changes in ion-exchange
properties that were seen when UDPGalUA was present
during synthesis. The dialyzed D-[U-14C1apiose solu-
bilized products synthesized in the presence of UDPGlcUA
and UDPXyl described in Table 2 were used in this
experiment. Three ml samples of the two solubilized
products, each containing 69,000 dpm, were chromato-
graphed on columns of DEAR-Sephadex (0.8 cm internal

diam x 10 cm) as described in the legend of Figure 7

except that 2 ml fractions were collected at a rate

92

of 25 ml/hr. The same fractions as described in
Figure 7 were combined to form fractions A through E.

Recovery of D-[U-14

C]apiose solubilized products syn-
thesized in the presence of UDPGlcUA and UDPXyl from
the DEAF-Sephadex column in these experiments was 80
and 75%, respectively. For D-[U-14C]apiose solubilized
product synthesized in the presence of UDPGlcUA the
percentage of recovered radioactivity contained in
fractions A, B, C, D, and E was 12, 10, 19, 46, and
10%. For D-[U-14CIapiose solubilized product syn-
thesized in the presence of UDPXyl the percentage of
recovered radioactivity contained in fractions A, B,
C, D, and E was 14, 15, 34, 29, and 3%. Comparison

of these values with the ones in Table 6 shows that
addition of UDPGlcUA to the reaction mixture caused
nearly the same change in distribution of radioactivity
in the fractions that addition of UDPGlcUA caused.
Addition of UDPXyl caused a slight increase in the
amount of radioactivity recovered in fraction D at the
expense of fraction C.

Characterization of Fractions Obtained from
the—fifiifi:§ephadex—Chromatography of’

5=Tfi=T¢EiApiase Solubilized’froduct

Synthesized in the Absence and Pre-

sence ofNonradioactIVeSugar
Nucleotides

 

 

 

 

The results described in the preceding sections

4

established that D-[U-l C]apiose solubilized product

93

synthesized in the presence and absence of nonradioactive
sugar nucleotides could be separated into 5 fractions

by DEAF-Sephadex column chromatography. The fractions
were characterized on the basis of their content of

14C]apiose and D-[U-14C]xylose and on their ability

1 1

D-[U-

to release D-[U- 4C]apiose and [U- 4C]apiobiose after
pH 4 hydrolysis.
Table 7 shows the results of the characteri-
zation of the individual fractions obtained from DEAE-
Sephadex chromatography of D-[U-14CJapiose solubilized
product synthesized in the absence of nonradioactive

14C]apiose solubilized pro-

sugar nucleotide and D-[U-
duct synthesized in the presence of UDPGalUA. These
data show that the fractions differed in their

D-[U-14C]apiose and D-[U-l4

C]xylose content as well
as in their ability to release D-[U-14C]apiose and
[U-14C]apiobiose when hydrolyzed at pH 4. The results

l4C]apiose

also indicate that regardless of whether D-[U-
solubilized product was synthesized in the presence or
absence of UDPGalUA, the fractions were eluted from
the column in the order of their increasing D-[U-14C]-
apiose content. I examined fractions A through D in
order and found that each fraction contained a greater

14C]apiose than

percentage of its radioactivity in D-[U-
did the preceding fraction, as determined by hydrolysis
at pH 1. Examination of the fractions by hydrolysis

at.pH 4 in the same order also showed that each

94

mo moamaom osu endopmouoeouco pom .H: mm mo3 monoust cowuooon on» no oEoHo>

Hocwm ocu poo «Deacon: mo moaoec v oocwoucoo «Deoomoa mo oocomoum on» cw uooooum

oouHHHndaom oNHmoSHGMm on com: monopxwe coHHoooH one .oowzu ooEHOMHom mo3 HcoEHHomxo

zoom .e ousmwm mo ocomoa onu Ugo monotonoum HoucoEHuomxm onu ca oonwuomoo mo .60 ma x

Soap Hocuouce Eo m.ov ceoaoo xooocmomlmdmo o co cosmoumouosonno can monotonoum Houcoe
Ifluomxm on» cw oonwuoooo mo touomonm mo3 uosooum touwaensaom omowmdflovHIDHIoo

 

 

 

 

 

 

 

 

 

¢.H ~.o H v.ev n.o H ~.mm m.o H N.mm o
m.o m.m H v.am m.m H w.mm ~.H H 5.0m U
.l l- I meow Um
I I I o we to
doaoomoo osocommxm mo oocomoum onu cw oouwmonucmm uosooum noneaensaom
m.~ m.a H m.mv v.a H o.mH ~.~ H m.em o
H.~ H.@ H H.m~ m.m H H.~H m.~ H e.mm o
m.o e.a H v.m m.~ H v.~H N.o H m.~e m
v.o m.H H m.m v.0 H a.m m.~ H m.mo m
moommoonsz Howsmo>euommwoouc02I
msocomoxm mo ooconnd on» cw ooNHoonucmm Hosooum couwawasaom
Awe “we .wv
omowmogovanHIo oomownowmd onowmd nooono cesaou
I I_o use I_o Ioeuo I_o Iaeno 2H accumum xmcmrmmmum<mo
omownoemdaova DH «a mo oaoweva euw>wmooowoom mo HcsoE< Eoum coauooum

e no no mammaouoem

 

odoamomoo mo oocoooum

on» cw poo oowuooaosz Hmmsm o>HuoooHomucoz mo oncomnm on» cw oonwmocucem

uosooum wouHHHnsHom mmon<_ovanoeun «0 mammumoumsouno asaaou xoumrammumema
Eoum oocHouno ocowuooum Eouu ooownowm¢HU¢HIoH poo omoamdnovHIoan mo omooaom .e anode

9S

.oouHHEo mmz v mm Ho mwmhaouoem .ooEuomuom mo3 H mm Ho mammaouoen oamcwm do

.omownowmonov IDH Ho omowmofiovalsgla mo oomooaou oo3 nown3 mcoeuooum
on» nH oonwmunoo oooemofiovanHIQ mo ucoouod onu ucomoumou mosao> one .mouoooo
loam HounoEHuomxm onu cw oonwuoooo no v no no ooumaouomn ouoz mcowuuoum oneo

.omoHMxHUvHIDHIo cw ucoooum mo3
euw>wuoooHoon on» no Hooceoeou one .ooowmoHUvHID_IQ cw ucoooum mo3 nown3 Eoumouoaouno
on» some oopo>ooou eue>euooowoou mo Hcoouom on» Hcomoumou mosao> one .oousooo

Ioum HoucoEHuomxm onu cw toneuoooo no H no no ooueaouoen ouoz ocoeuoouu onen

.nucoswuomxo ouooeaooo

onu How oouuooou ouo nowuoe>oo cooE poo mcooz .4 uco>Hom ca oonmoumouofiouno ouos
mouooeaonoem .ooueaouoen too .Uoom Ho oououunoonoo .un we now Uov no nouns ca ooueaowo
opoz ocoHuooum one .aoaoomoo mo oonomoum on» cw ponwmonuceo Hooooum ooueawnsHOm
omowmoHUvHIoHIo nHH3 mucoEHuomxo 03» onu How web one mm Ugo oowuooaoon woman o>euoooHoou
Icon mo oocomno onu cw powwoonuchm uooooum oouwawnsaomgo HIDHIQ nuH3 ounoEHuomxo 039
on» MOM wmv ono Hm mo3 :EsHoo onu scum muw>wuooowoou mo wuo>ooom .e ousmwm cw ooan
IEoo ouo3 umnu ocowuooum cesaoo wouonasc oeoo on» mcwcwnsoo an cocaouno onoz a nmsounu
4 ocowuooum .oouooaaoo ouo3 ncoeuoouu HE o.m coo Hn\HE om oo3 ouou 30am :EsHoo one
.Emo ooo.ooa can ooo.mva ponwoucoo poo Hommsn mo HE v.m cw nooo ouo3 floaoomoo mo oocoo
Ioum on» n« nouwmonucho uusoouo oouwawnsaom ooowmoHUvaIDHIQ mo ooamson onu 0cm emu
ooo.o~m one ooo.mma nonwoucoo coo Hommsn no as m onu ca nooo ouo3 oowuooaosn woman
o>Huoo0eooHnoc mo oocoono on» cw powwoonuneo uosooum touwaandaoo ooOAQMHUvHIDHIa

voucwucou .b qudfi

96

fraction was capable of releasing a greater percentage of
its D-[U-14C]apiose as [U-14C1apiobiose and D-[U-14C1apiose
than the preceding fraction. I also found, when the
fractions were subjected to hydrolysis at pH 4 in the

same order, that they released an increasing percentage

of their D-[U-14C]apiose as [U-14C]apiobiose rather than

as D-[U-14C]apiose as shown by the increase in the
[U-14C1apiobiose to D-[U-14C]apiose ratio (Table 7).

Once again, there were differences between iden-
tical fractions obtained from D-[U-14C]apiose solubilized
products synthesized in the absence and presence of
UDPGalUA (Table 7). The fractions obtained after chro-

14

matography from D-[U- C]apiose solubilized product syn-

thesized in the presence of UDPGalUA contained a greater

1

percentage of their radioactivity in D-[U- 4C]apiose

than did the corresponding fractions obtained from solu-
bilized product synthesized in the absence of UDPGalUA
(Table 7). After hydrolysis of fractions C and D at

4C]apiose and [U-14C]apio-

14

pH 4 a higher yield of D-[U--1
biose was obtained from D-[U- C]apiose solubilized
product synthesized in the presence of UDPGalUA than from
D-[U-14C]apiose solubilized product synthesized in the
absence of UDPGalUA. This is seen in fraction D, for

l4C]apiose solu-

example, where fraction D from D-[U-
bilized product synthesized in the absence of UDPGalUA

contained 88% of its radioactivity in D-[U-14C]apiose

97

of which 62% of the D-[U-14C]apiose was released after

hydrolysis at pH 4 as D-[U-l4clapiose and [U-14CJapio-

1

biose. Fraction D obtained from D-[U- 4C]apiose solu-

bilized product synthesized in the presence of UDPGalUA
contained 95% of its radioactivity in D-[U—14C]apiose
of which 81% of the D-[U-14C]apiose was released after

hydrolysis at pH 4. However, the ratio of [U-14

1

C]-
apiobiose to D-[U- 4C]apiose was lower in fractions
obtained from solubilized product synthesized in the pre-
sence of UDPGalUA than in the fractions obtained from

D__[U_14

C]apiose solubilized product synthesized in the
absence of UDPGalUA (Table 7).

Fraction D obtained from the DEAR-Sephadex
chromatography of D-[U-14C]apiose solubilized product
synthesized in the presence of UDPGlcUA was also char-
acterized. The fraction D material was obtained from

the DEAF-Sephadex chromatography of D-[U-14

C]apiose
solubilized product synthesized in the presence of
UDPGlcUA which was described in the previous section.
Fraction D was dialyzed 12 hr in H20 at 4°C and concen-
trated to 1.1 ml. Two samples, each containing 4800 dpm
in 500 pl of solution, were hydrolyzed; one at pH 1

and the other at pH 4 as described in the Experimental
Procedures and the hydrolysates were chromatographed

on paper in Solvent A. Hydrolysis at pH 1 showed that

95% of the recovered radioactivity was contained in

D-[U-14C1apiose. The remainder of the radioactivity

98

was contained in D-[U-14C]xylose. Hydrolysis at pH 4

resulted in the release of 25.2% of the radioactivity

in D-[U-l 1

_14

4C]apiose as D-[U- 4C]apiose and 58.8% as

C]apiobiose. The ratio of [U-14C1apiobiose to
14

[U

D-[U- C]apiose was 2.3. These results showed that

1

fraction D from D-[U- 4C]apiose solubilized product

synthesized in the presence of UDPGlcUA and fraction D

14

from D-[U- C]apiose solubilized product synthesized in

14

the presence of UDPGalUA were similar in D-[U- C]apiose

and D-[U-14C]xylose content and in their ability to

14 14

release D-[U- C]apiose and [U- C]apiobiose after

hydrolysis at pH 4.

Rechromatography of Fractions Obtained
from fifiKE-SepfiadexColumn Chroma-

togra h of D-lU-I4CJApiose
SoIEBiIizea Product

The homogeneity of the fractions collected from

 

 

 

the DEAF-Sephadex column was investigated by chroma-
tographing D-[U-14C]apiose solubilized product on a
column of DEAF-Sephadex, isolating fractions A, B, C,
and D, rechromatographing the fractions on a second
column of DEAF-Sephadex, and determining the D-[U-14C1-
apiose content of the different fractions before and
after rechromatography (Table 8). Fraction B rechroma-
tographed as a single component in its original position
in the gradient as evidenced by the single peak that

was obtained. Fractions A, C, and D each yielded two

99

on» no muoseHH¢ .ooom pm He H.H 0» umumuuamocoo ecu us me you 0.4 pm Hmums

nH oonHoHo ono3 mnowuooum “now one .nEnHoo on» scum oouo>ooou mo: Honu mue>wuoo

Iowoou on» no .>Ho>euoommou .wma ono .mm .ma .mm oonHoHnoo o ono .0 .m .n mnowuooum

.e onnmflm nH oouoowonfl mm o nmnounu n mnoHHomum snow on ooanEoo ono3 mnoeuoonm nEnHoo

oEom one .mve mo3 nEnHoo onu Eoum mue>wuooowoou mo >no>ooon one .oouooeaoo ouo3

mnowuoonm HE m ono Hn\HE om mo3 oHoH 30am nEnHoo one .Hommnn mo HE v nH mo3 nEsHoo

map on totem rent ooo.oomo «Agata use .5 musmHm cH emnHuomme mm 1Ho> He m.e .50 me x

Soap Honnoune Eo m.ov nEnHoo xooonmomlmnmo o no oonmonmouofionno ono monsooooum Hounoa
Iwnomxm on» nH oonwuomoo mo oonomoum oo3 uonooum oonwawnnaOm oooHQnHUvaIanloo

 

 

m.Hm 5.0N m

e.mm m.vm o v.mm o

m.mm m.vm o

m.mh v.mm um m.hm U

o.mm m.mm m m.mm m

H.mm N.Mb m

on H.mn a «.mm a

A V m m Mme A v

w n on ouofiounu w
anmmumoumsouno uncomm Hound cannon nmnmmumoumeouno assnoo
n onooom munonomeou Howoz xooonmomo umuwm A xooonmom
Houmn unounoo nH omnHmHnoo emmnmmomuwmum Hound unounoo swwnmmowmwwm
ooowmdnu IDHIQ euw>wuooowoom m . m oooflmmgo Isula m .u m

we ooHo>ooom mo unnoen ea

 

mauscoum wouHHHnsHom mmonanovHIsHIn Ho anamumou
IoEounU nEnHou xooonmomlmdmo Baum oonwouno mnowuooum mo mnmonmouofiounoom .m mqmne

100

.nnomonm mm3 >nn>nnoo0noon nnononumnmnn omnooon ooumaouoen non mos Honuonozo

.omoHexHUvHIDHIQ nn nnomoum mo3 mnw>nnoo

Ionoou onn mo noonwoeou one .omonmoHOvHIDHIQ nn nnomoum mo3 nonnz EoumonoEonno

uomom onn Eonm oouo>ooou >nn>nnoo0noon mo nnoonom onn nnoooumou monao> one .monnooo
Ionm HonnoEnnomxm onn nn oonnuomoo no H me no poneaonoen ouo3 mnownooum onen

.« nno>Hom nn mnmonmonoEouno Momma an Gonna

Iuonoo mo: monomenouoen onn nn omoaexnovaloalo ono omonmoHUvHIDHIQ mo nnnoEo one

.H me no nonmaonoen ono .Uoom no oononnnoonoo .oov no Honoz nH Mn on How nonmaowo
onoz mnonnooum oonnneoo onn ono oonnnEoo onos nanaoo xooonmomIMdmo onoooo onn Eonm
mnownooum nEnHoo onowumonmmd .»Ho>wnooomon .o ono .0 .m .4 mnonnooum How wme ono
.ooa .mm .mm mo3 nEnHoo onn Eonm enw>wnooonoou mo eno>ooom .oEnHo> HE A no nEnHoo
onn moHo>oo on too: mo3 .oEnHo> HE m.e mo nanaoo onn no poms ono onn on onnm nH
HonOHnHomonm .nnowooumImono Huoz d .nao> HE H .50 m x sown Honnonnw Eo m.ov Gannon
xooonmomImnmo onooom o nmnonnn .aaononmmoo .oonmmnmonoeounoou mos nonnooum oonounnoo
Inoo nooo mo noonnoeou one .H an no oonaaonoen onos AH: ommv ononnoonu oononnnoonoo

oonnnnnou .m anode

101

peaks on rechromatography. The 2 components of fraction
A chromatographed in the positions expected of fractions
A and B. The 2 components of fraction C chromatographed
in the position expected of fractions B and C combined
and in the position expected of fraction D. The 2 com-
ponents of fraction D chromatographed in the positions
expected of fractions D and E. The fourth column of data
in Table 8 shows what percentage of the radioactivity
recovered after rechromatography was contained in each
of the fractions. For the rechromatography of fractions
A, C, and D greater than 85% of the radioactivity
recovered from the column was present in the 2 fractions
reported in each Table 8. The rest of the recovered
radioactivity was spread over the remainder of the column.
For the rechromatography of fraction B, 30% of the radio-
activity recovered did not elute with the single fraction
reported in Table 8. Most of the radioactivity not con-
tained in the reported fraction was present in the
positions expected of fractions C and D as a trailing
shoulder of the single large fraction.

The amount of radioactivity that was present in
each of the fractions after rechromatography was not
invariant. An identical rechromatography experiment was

performed, except that analyses for D-[U--l4

14

C]apiose and
D-[U- C]xylose were not made. In this experiment the

fractions rechromatographed in the same positions in

102

the elution gradient as in the experiment described in
Table 8. The percentages of radioactivity which were
recovered from the column in the individual fractions
after rechromatography were as follows: for fraction A,
52.5% of the recovered radioactivity was found in the
position of fraction A and 37.9% in the position of
fraction B, for fraction B, 81.5% of the recovered
radioactivity was found in the position of fraction B,
for fraction C, 58.2% of the recovered radioactivity was
found in the combined positions of fractions B and C and
32.8% in the position of fraction D, and for fraction D,
72.5% of the recovered radioactivity was found in the
position of fraction D and 13.9% in the position of
fraction E.

l4C]apiose content of

A comparison of the D-[U-
fractions before and after rechromatography was made
(Figure 8). In the case of fractions A, B, and D there
were no changes in the D-[U-14C]apiose content even
though rechromatography had resulted in fractions A and
D each being eluted as two separate fractions. However,
the two components that were recovered from rechroma-

tography of fraction C had different D-[U-14C1apiose

content from each other and from the original fraction C.

103

Gel Chromatography of D-[g:E4C]Apiose Solubilized
Product and'D- U-quTGalacturonic
Acid Solubilized Product

14

 

Column chromatography of D-[U- C]apiose and

l4C]galacturonic acid solubilized products on Bio-

D-[U-
Gel P-300 showed that both solubilized products con-
sisted of molecules of different sizes (Figure 11).
Dextrans with a weight average molecular weight in the
range 5,000 to 125,000 are fractionated by this gel
(114). The chromatoqrams in Figure 11 show that

1 14C]galacturonic acid solu-

D-[U- 4C]apiose and D-[U-
bilized products both eluted from the column over the
entire fractionation range. Based on radioactivity
measurements the recoveries of D-[U-14C]apiose and
D-[U-14C]galacturonic acid solubilized products from

the column were 79 and 71%, respectively. Whether the
peak of radioactive material that was eluted in the void
volume of the column is also of variable size has not
been investigated. A peak of radioactive material

always eluted at V when D-[U-14CJga1acturonic acid

t
solubilized product was chromatographed on the Bio-Gel
P-300 column. However, the peak of radioactive material
depicted in Figure 11 which eluted at Vt when D-[U-14CJ-
apiose solubilized product was chromatographed on Bio-
<3el P-300 was not always seen (Figure 12a). The

1

(appearance of this peak with D-[U- 4C]apiose solubilized

product was random between experiments ; the conditions

104

.noHnoon nooo nH oonooHHoo moB noan mnH>HnoooHoon oono>ooon Honon

mo nnoonom onn nnomonmou Awe enH>HnomoHoon mo monHo> .m noHnnHom nOHnoH
IHHnnHom nnHz enH>HnoooHoon now ooeommo onoz ono mnOHnoonm nEnHoo onn Eonw
oo>oEon ono3 mnonoHHd .nn\HE m mo onon onm o no oonooHHoo onoz mnOHnooum
HE ono .nEnHoo onn no ooOMHm ouoz .HE m.o mo oEnHo> o nH .wHo>Hnoonon
.Emo ooo.mH ono ooo.H~ manHonnoo .mnonooum ooNHHHnnHOm oHoo UHnonnnooHom

IHUVHIDHIQ ono omonnnov IDHIQ .m.m no .uowmnn onmnomonm EnHoOm z no.0

H
nnH3 ooQOHo>oo ono oononnHHHnoo moz nOHnB n50 ov x EoHo HonnonnH Eo Hv

nEnHoo oomIm HooIon o no oonmonmonoeonno ono monsoooonm HmnnoEHnomxm onn nH

ooanomoo mo oonomonm onoz mnonoonm ooNHHHnnHom oHoo oHnonnnOMHomHo IDHIQ onm

VH
IDHIQ ono nonoonm

omonnnUv IDHIQ .nonoonm ooNHHHnnHOm oHoo UHnonnnooHomHU

H
ooNHHHnnHom omoHQMHUv

«H

HIDHIQ mo mamnmonoEonno nEnHoo oomlm HooIOHm .HH ouanm

 

 

Q (.0 O ’ . . ..
Z3 ,3
< .v'
o f"
2 j
C) o
o: 4.,
D o
I— ,o
O —"‘” ‘
:5 .3
'.
Ca
in.
.3
0:”
3% .
o g
D— I
< .‘0"
Q:
0'.‘
- coo-IIIII' O.
O —’ O O a. . . .....

 

 

(%) All/\IlOVO IGVH

 

10

FRACTION NUMBER

Figure 11

106

.nOHnooum nooo nH oonooHHoo mo3 noan mnH>HnoooHoon nono>ooon Honon

mo nnoonom onn nnomonmou Amy enH>Hnoo0Hoon mo monHo> .HH ouanm mo onomoH onn
nH ooanomoo mo nEnHoo oomlm HooIOHm onn no mHonouomom oonmonmonofionnoou nonn
moz nOHnoonw oononnnoonoo nooo mo noonHoEon one .monnoooonm HonnoEHnomxm onn
nH ooanomoo mo H mm no woNeHOnoen ono oo>OEoH onoz nOHnoon nooo mo AH: oomv
mnOHnnom .oomm no HE e.o on oononnnoonoo mo3 noHnB mo nooo .HHH ono .HH

.H noHnoonm EHOM on ooanEOo onoB now nH mnOHnooum nESHoo tonooHonH one .wmn
mo3 nEnHoo onn Eonm enH>Hnoo0Hoou mo >no>ooon one .m nOHnnHom nOHnoHHHnnHom
nnHz mnH>Hnoo0HooH MOM ooeommo onoz ono mnOHnoonm nEnHoo onn Eonw oo>oeon onoz
mnonoHHn .HH onanm mo onomoH onn nH oonHHomoo mo nEnHoo oomIm HooIon o no
oonmonmonoeouno ono ooem no HE m.o on oononnnoonoo mos nEmo ooo.eMHV onomeHoHo
onn mo onEom HE m.m n .nn H nonmo omnono nommnn o nnHz 00v no nn m now

.m.m no .nommnn ononmmonm anHUOm z mo.o nmnHomo oouwHoHo mo: monsoooonm HonnoE
IHuomxm onn nH ooanomoo mo oonomonm AHE v nH .Emo ooo.mmHv nonooum ooNHHHnnHom

omOHmnmov IDHIQ .nnv mnOHnoonm oonooHom mo unmoumonofionnoon onn ono nov nonconm

H
ooNHHHnnHom omOHmoHUVHIDHIQ mo mEonmonoEonno nanHoo oomlm HooIOHm .NH onanm

 

 

 

 

 

 

 

(96) All/\ILOVOIGVU

107

RADIOACTIVITY (94.)

 

 

 

(96) MIA IlOVO IO V8

I--- H
‘o ...... 0‘ III
0 In
.—I
l l
O,
0".
0".
o’.‘..
0"..
0".
O... I
R v’ R
1.. J
fig
0‘..
00‘... i
/ ..."o
r ’x
a" .‘I
a Q. a».
\ \ \K.
n\
I
2 2
.Q
J l
m
c: U\ ed :3
N H
I .——0

FRACTION NUMBER

Figure 12

108

for its appearance were not investigated. Comparison
of the two chromatograms in Figure 11 showed that the
D-[U-14C]apiose solubilized product contained more
molecules of large size which eluted in the void volume
than did the D-[U-14C]galacturonic acid solubilized
product.

The homogeneity of the fractions obtained from
the chromatography of D-[U-14C]apiose solubilized product
on the Bio-Gel P-300 column was investigated (Figure 12).

D-[U-l4

C]Apiose solubilized product was chromatographed
on the Bio-Gel P-300 column and the fractions indicated
in Figure 12a representing large, medium, and small

14C]apiose solu-

molecular weight components of the D-[U-
bilized product were labelled fractions I, II, and III,
respectively, and were rechromatographed on the Bio-Gel
P-300 column. The results showed that fractions I, II,
and III had the same elution volumes on rechromatography
as they had in the initial chromatography on Bio-Gel
P-300 although each distributed itself over a larger
molecular weight range on rechromatography (Figure 12b).
Samples of D-[U-14C]apiose solubilized product prior to
rechromatography and fractions I, II, and III were hydro-
lyzed at pH 1 and chromatographed on paper with Solvent
A. Analysis of the chromatograms showed that the

14

D-[U- C]apiose solubilized product before chromatography

1

contained 79.5% of its radioactivity in D-[U- 4C]apiose.

109

Fractions I, II, and III contained 82.4, 76.8, and 80.4%,

1

respectively, of their radioactivity in D-[U- 4C]apiose.

The remainder of the radioactivity was contained in D-

[U-14C]xylose. The similar D-[U-14

14

C]apiose content of
the three fractions and of D-[U- C]apiose solubilized
product showed that D-[U-14C]apiose and D-[U-14C]xylose
were not concentrated in molecules of a particular size.
The chromatograms obtained from the chromatography

of D-[U-14C]apiose and D-[U--l4

C]galacturonic acid solu-
bilized products on a Bio-Gel P-100 column were dissimilar
(Figure 13). D-[U-14C]Apiose solubilized product eluted
from the column predominantly at the V0 (Figure 13a).
There was no elution of radioactive material at Vt' On
the other hand, D-[U-14C]galacturonic acid solubilized
product eluted from the column over the entire fraction-
ation range of the Bio-Gel P-100 column. These results
indicate that the smallest molecules of D-[U-14CJgalac-
turonic acid solubilized product are smaller than the

1

smallest molecules of D-[U- 4C]apiose solubilized product.

"Degradation" of D-[p—14CJApiose Solubilized
product andD-[U-I‘EJGalacturonic AcId’
Sclubilized Product

 

14

If D-[U- C]apiose solubilized product was

dialyzed in water prior to column chromatography on
Bio-Gel P-300 there was a significant change in elution
profile when compared with the chromatogram obtained

1

from nondialyzed D-[U- 4C]apiose solubilized product

110

Figure 13. Bio-Gel P-lOO column chromatograms of

14C]apiose solubilized product (a) and D-[U-14C]-

1

D-[U-

galacturonic acid solubilized product (b). D-[U- 4C]-

Apiose and D-[U--14

C]galacturonic acid solubilized pro-
ducts were prepared as described in the Experimental
Procedures and chromatographed on a Bio-Gel P-lOO
column (1 cm internal diam x 40 cm) which was equil-
ibrated and developed with 0.05 M sodium phosphate

1 14C]galac-

buffer, pH 6.8. D-[U- 4C]Apiose and D-[U-
turonic acid solubilized products, containing 24,000
and 12,000 dpm, respectively, in a volume of 0.6

ml were placed on the column. One ml fractions were
collected at a flow rate of 20 ml/hr. Aliquots were
removed from the column fractions and were assayed
for radioactivity with scintillation solution B.
Based on radioactivity measurements the recoveries

14C]apiose and D-[U-l4

of D-[U- C]galacturonic acid
solubilized products were 95 and 79%, respectively.
Values of radioactivity (%) represent the percent

of total recovered radioactivity which was collected

in each fraction.

RADIOACTIVITY (%)

15

10

10

111

 

 

 

 

 

 

 

 

 

 

 

 

FRACTION NUMBER

Figure 13

112

(Figure 14). Dialysis in water resulted in a decrease
in the amount of radioactive material which eluted at
V0 and a concurrent increase in the amount of radio—
active material which eluted at Vt‘ However, dialysis
of D-[U-14C]apiose solubilized product in sodium phos-
phate buffer prior to chromatography resulted in no
change in elution from the Bio-Gel P-300 column
(Figure 14). This process which resulted in a decrease
in the amount of large molecular weight components of
the solubilized product as demonstrated by gel-chroma-
tography was named "degradation" by us.

I have attempted to reverse the "degradation"

14

of D-[U- C]apiose solubilized product caused by dialysis

in water by performing a second dialysis in sodium phos-

phate buffer. D-[U-l4

C]Apiose solubilized product was
prepared and divided into 2 samples. One sample, con-
taining 27,000 dpm in a volume of 0.5 ml, was chroma-
tographed on the Bio-Gel P-300 column. The other
sample containing 60,000 dpm was dialyzed in water

for 16 hr at 4°C with changes in water after 1 and

4.5 hr. The recovery of radioactive material after
dialysis in water was 65%. A sample of the water
dialysate, containing 17,000 dpm in a volume of 0.5 ml,
was chromatographed on the Bio-Gel P-300 column. The

remainder of the water dialysate was then dialyzed in

0.1 M sodium phosphate buffer, pH 6.8, at 4°C for 24 hr

113

.noHnoonm nooo nH oonooHHoo moB noHn3 enH>Hnoo0Hoon

oono>ooon Honon mo nnoonom onn nnomonmon Awe mnH>HnoooHoou mo monHo>
.eHo>Hnoommon .monEom ooNeHoHo ononmmonm ono .ooNeHoHo nono3 .oommHoHo
Inon onn now Mme ono .mm .OOH onoz nEnHoo onn Eonm moHHo>ooon onn mnnoE
IonnmooE enH>HnoooHoon no oomom .HH onanm mo onomoH onn nH ooanomoo mo
nEo mm x EoHo HonnonnH Eu HV nEnHoo oomlm HooIon o no oonmonmonoEonno

nonn onoB monEom oonnn one .mHo>Hnoomoon .wmm ono we mos ononmmonm ono
nonoz nH mHmmHoHo nonmo HoHnonoE o>Hnoo0Hoon mo moHno>ooom .e.n no .nomwnn
ononmmonm EnHWOm z mo.o nH onEom nonno onn Uno mono? nH onEom ono “nn H
nonmo nommnn mo omnono ono nnHz Dov no Mn m.m now UoNeHoHo ono3 monEom onn
mo oBe .HH: oom nH .Emo ooo.vmv monEom m onnH oooH>Ho mo? ono monsoooonm
HonnoEHnomxm onn nH ooanomoo mo oouomonm mo: nonoonm UoNHHHnnHom omonn

Ino IDHIQ .nonoonm ooNHHHnnHOm omOHmonUvHIDHIQ ooweHoHo ononmmonm EnHUoo ono

vH
.ooumHoHo nonoz .ooneHoHonon mo mEonmonoEonno nEnHoo oomnm HooIOHm .vH onsmHm

114

 

 

 

 

 

 

c:
LIJ
N /
33
<( ‘/”
— z-
a
CD‘\\*5>/‘/, .
o IN a’
-- > —-1-- / ,v’
0'.
9
‘ I
k A.
.‘
_ a ’
LLJ I
DJ
>_
._.|
<E i;
a .°
q
C)
o.
I.- .H -I-..
> m, o
Lu
DJ
>—
_l
<(
9
2
CD
2
l J
00 <1

(%) All/\llOVOIGVU

20

FRACTION NUMBER

 

Figure 14

115

with changes of buffer after 2 and 5 hr. Recovery of
radioactive material after dialysis in phosphate

buffer was 69%. A sample of the phosphate dialysate,
containing 13,000 dpm in a volume of 0.5 ml, was chroma-
tographed on the Bio-Gel P-300 column. In all cases
chromatography on the Bio-Gel P-300 column was performed
as described in the legend of Figure 11. The recoveries
of radioactive material from the columns for the non-
dialyzed sample, water dialyzed material, and water
dialyzed then phosphate dialyzed material 88, 88, and
92%, respectively. Comparison of the three Bio-Gel
P-300 chromatograms showed that dialysis in sodium phos-
phate buffer was unable to reverse the "degradation"
caused by dialysis in water.

14C]_

Dialysis in water also "degraded" D-[U-
galacturonic acid solubilized product. Although a
chromatogram from this experiment is not presented

D-[U-14

C]galacturonic acid solubilized product was
dialyzed for 3 hr in water at 4°C and chromatographed
on a Bio-Gel P-lOO column as described in Figure 13
except that 2 ml fractions were collected. A sample
of nondialyzed D-[U-14C]galacturonic acid solubilized
product was similarly chromatographed on the column.
Recovery of radioactivity after dialysis in water

was 78% and recoveries of radioactivity from the

Bio-Gel P-100 column after chromatography of non-

dialyzed solubilized product and solubilized product

116

dialyzed in water were 100 and 84%, respectively. The
water dialysate had a decreased amount of radioactive
material that eluted in the column V0 and an increased
amount of radioactive material eluted at Vt when com-
pared with the sample that was not dialyzed. D-[U-14C]-
Galacturonic acid solubilized product was also dialyzed
in 0.05 M sodium phosphate buffer (pH 6.8) for 3 hr at
4°C and was chromatographed on the Bio-Gel P-lOO column
as described above. The recovery of radioactivity after
dialysis was 98% and recovery after column chromatography
was 74%. "Degradation" did not occur during dialysis

in phosphate buffer as there was essentially no dif-

1

ference in the elution profiles of D-[U- 4C]galacturonic

acid solubilized product dialyzed in sodium phosphate

14

buffer and of nondialyzed D-[U- C]galacturonic acid

solubilized product.

When D-[U--14

C]apiose solubilized product was
chromatographed on a Bio-Gel P-300 column and then
dialyzed in water, it was not "degraded" (Figure 15).
After water dialysis fraction I still eluted at V0,
thus indicating that under these conditions "degra-
dation" did not occur. Although the data are not
presented here, degradation was also not obtained
when the V0 fraction obtained from the Bio-Gel P—lOO

14

column chromatography of D-[U- C]galacturonic acid

117

Figure 15. Bio-Gel P-300 column chromatograms of D-
[U-14C]apiose solubilized product (a) and the rechroma-
tography of a selected fraction after dialysis in

l4C]Apiose solubilized product

water (b). D-[U-
(200,000 dpm) was dialyzed in 0.05 M sodium phosphate
buffer, pH 6.8 concentrated, and chromatographed on a
Bio-Gel P-300 column as described in the legends of
Figures 11 and 12. The concentrated sample (160,000
dpm) was placed on the column in a volume of 0.5 ml.
Recovery of radioactivity after column chromatography
was 89%. The indicated column fractions in (a) were
combined to form fraction I. A sample of fraction I
(20,000 dpm) was dialyzed in water for 6 hr at 4°C with
changes of water after 2 and 4 hr. Recovery of radio-
active material after dialysis was 89%. The dialysate
was concentrated to 0.5 ml and was rechromatographed on

the Bio-Gel P-300 column as described in Figure 11.

Recovery of radioactivity from the column was 66%.

118

 

 

 

 

_ - T

 

m om m

95.55: n: 2%: E>E<oa§

 

FRACTION NUMBER
Figure 15

119

solubilized product was dialyzed in water at 4°C and
rechromatographed on the same column.

14C]Apiose solubilized product was also

D-[U-
"degraded" when chromatographed on DEAF-Sephadex even
though the material was never dialyzed in water.

D-[U-l4

C]Apiose solubilized product was prepared as
described in the Experimental Procedures and was
dialyzed in 0.05 M sodium phosphate buffer, pH 7.7,
containing 0.002% Hibitane (l, l'-hexamethyl lenebis
[5-(p-chlorophenyl)biguanide]) for 2 hr at 4°C. This
material is referred to as the P04 dialysate. A sample
of dialysate was chromatographed on the Bio-Gel P-3OO
column. A second sample of the P04 dialysate (23,000
dpm) in a volume of 2.5 ml was placed on a DEAE-Sephadex
column (0.8 cm internal diam x 5 cm) prepared as
described in the Experimental Procedures. The DEAE-
Sephadex column was washed with 7 ml of 0.05 M sodium
phosphate buffer, pH 7.7, containing 0.002% Hibitane.
The column was then developed with 10 ml of the above
phosphate buffer containing 0.3 M NaCl containing 0.002%
Hibitane as 1 ml fractions were collected at a flow rate
of 20 ml/hr. Aliquots of each column fraction were
assayed for radioactivity with scintillation solution B.
Total recovery of radioactive material from the column
was 68%. Column fractions 3 to 8, containing 53% of

the radioactivity originally placed on the column, were

120

combined and a 4 m1 sample was concentrated to 1.2 ml
at 22°C. This material is referred to as the DEAE-
Sephadex fraction. The DEAF-Sephadex fraction was also
chromatographed on the Bio-Gel P-300 column (Figure 16).
Comparison of the two chromatograms showed that "degra-
dation" had occurred. In this particular experiment
the Bio-Gel P-300 column was equilibrated and developed
with l M NaCl in order to minimize association between

molecules of D-[U-14

C]apiose solubilized product and
because of the high salt concentration of the DEAE-
Sephadex fraction after concentration. Hibitane was
added to the solutions to preclude the growth of
bacteria capable of metabolizing D-[U-14C]apiose solu-
bilized product.

In the experiment depicted in Figure 16 a sample
of D-[U-14C1apiose solubilized product (10,000 dpm)
was dialyzed at 4°C for 2 hr in water followed by 2 hr
in sodium phosphate buffer, pH 7.7. Solid NaCl was
dissolved in the dialysate to give a NaCl concentration
of l M and the dialysate was then chromatographed on the
Bio-Gel P-300 column as described in the legend of
Figure 16. Although the column chromatogram obtained
from the water dialysate is not shown in Figure 16, it
was nearly identical to the one obtained from the DEAE-
Sephadex fraction. These results show that the amount

of degradation caused by water dialysis and DEAE-Sephadex

chromatography was identical.

121

Figure 16. Bio-Gel P-300 column chromatography of

14

D—[U- C]apiose solubilized product after dialysis in

sodium phosphate buffer and after elution from a DEAE-

l4C]Apiose solubilized product

Sephadex column. D-[U-
was dialyzed in sodium phosphate buffer (PO4 dialysate
fraction) and eluted from a DEAF-Sephadex column (DEAE-
Sephadex fraction) as described in the text. Samples
of P04 dialysate fraction (7,000 dpm) and DEAF-Sephadex
fraction (3,000 dpm) in a volume of 0.5 ml were chroma-
tographed on a Bio-Gel P-3OO column (1 cm internal diam
x 21 cm) that was equilibrated and developed with l M
NaCl containing 0.002% hibitane. Prior to gel chroma-
tography, solid NaCl was dissolved in the P04 dialysate
fraction to give a NaCl concentration of l M. One ml
fractions were collected at a rate of 3 ml/hr. Based
on radioactivity measurements the recovery of the P04
dialyzed fraction and the DEAR-Sephadex fraction from
the column was 85 and 87%, respectively. Values of
radioactivity (%) represent the percent of total

recovered radioactivity which was collected in each

fraction.

RADI OACTI VITY (%)

10

122

 

 

1
Vt

PO DI ALYSATE *

I
OD

.6"
i o‘
g. o/ o\

 

 

:2/ °
Ix \

L

I
.
I
I
I
D
I
I
I
I

4
/ DEAE-SEPHADEX
“cg/FRACTION ..
.o' ‘-.

 

10
FRACTION NUMBER

Figure 16

 

123

Effect of UDPGalUA on the Size of D-[U-14C]-
Apiose Solubilized Product

 

 

l4C]Apiose solubilized products synthesized

D- [U-
in the absence and presence of UDPGalUA were chromato-
graphed on the same Bio-Gel P-300 column (Figure 17).
The recovery of radioactive material from the column
for D-[U-14C]apiose solubilized product synthesized in
the absence and presence of UDPGalUA was 72.3 and
89.6%, respectively. Comparison of the two elution
profiles showed there was little difference in the
amount of radioactive material which eluted in the
void volume of the column. However, there was a
shift in the position of the broad peak which repre-
sents the elution of the smaller molecules. These
results show that the presence of UDPGalUA during syn-
thesis caused a relative increase in the size of the
smaller radioactive molecules compared to those syn-
thesized in the absence of UDPGalUA.

Effect of Incubation Time on the Size of D-[U-14CJ-

Apiose Solubilized Product andD-TU-i35]Galac-
turonic Adid Solubilized Product

 

 

 

The length of the incubation period used in syn-

thesis of D-[U-14

C]galacturonic acid solubilized pro-
duct affected its size as determined by chromatography
with Bio-Gel P-lOO (Figure 18). For incubation times

of 0.5, 2, and 15 min, 9.1, 32.1, and 37.9%, respectively,

of the starting radioactivity was incorporated into

124

.noHnoonm HonoH>HonH nooo nH oonooHHoo mos noHnB mnH>Hnoo0Hoon oono>ooon
Honon mo nnoonom onn nnomonoon Awe mnH>Hnoo0Hoon mo monHo> .HH onnon

mo onomoH onn nH ooanomoo mo oonnHo ono nEnHoo oomIm HooIon onn on

He m.o no oeono> o cH coHHdoo cums «oncoooo redo ooo.mmv cooron coo redo
ooo.mmv nnH3 ooNHmonnnhm mnonooum ooNHHHnnHom omonnHUvHIDHIo .H: mm mo
oEnHo> HonHm o nH noHoomoD mo moHOEn v oonHonnoo «DHoomoD mo oonomonm onn nH

nonoonm ooNHHHnnHom omOHmoHU IDHIQ oNHmonnnem on noon onnanE nOHnooon one

vH
.monnoooonm HonnoEHnooxm onn nH ooanomoo mo oonomono moz nonoonm ooNHHHnnHom

omonnnu IDHIQ .noHoomoD mo oonomno Uno oonomonm onn nH ooNHmonnnem nonoonm

VH

ooNHHHnnHOm omOHmoHUo IDHIQ mo mEonmonoEouno nEnHoo oomIm HoonHm .eH onanm

H

125

 

 

 

‘

 

one-ounce... o ..._

 

 

10

l I To

Q N

(%) AllAllOVOIGVH

FRACTION NUMBER

Figure 17

126

.noHnoonm HonoH>HnnH nooo nH nonooHHoo mo3 noHnB

mnH>HnoooHoon nono>ooon Honon mo nnoonoo onn nnomonmon va enH>Hnoo

IoHoon no monHo> .mHo>Hnooomon .nHE mH ono .m .m.o no mnOHnonnonH HOW

soc oom.~n coo .ooo.on .oo~.m mo; noncond coonnnoonom mo oononomH mnH>Hooo
IoHoon no nnnoso one .mH onanm no onoooH onn nH noanomoo mo nEnHoo
OOHIm HooIon onn no nonoonmonoeonno onoz HE m.o no oEnHo> o nH mnonoono

UoNHHHnnHom oHoo OHnonnnooHomHU IDHIQ onn .nOHnoHomH nonmn .monnooo

«H
Ionm HonnoEHnomxm onn nH noanomoo mo whommo omonn Bonn oouomonm moz

nonoonm ooNHHHnnHom .nHE mH now nonno onn nno .nHE m MOM ono .nHE m.o
MOM ono “oomm no nononnonH onoz monnanE nOHnooon oonnn HHn .monnnooonm
HonnoEHnomxm onn nH noanomoo mo nouooono ouo3 nonoonm ooNHHHnnHOm oHoo

UHnonnnooHomHU IDHIQ no mHmonnnem onn now monnanE nOHnooon HoOHnnooH

VH
oonne .nHE mH ono .N .m.o nH ooNHmonnnem nonoonm UoNHHHnnHOm oHoo

UHnonnnooHomHUv IDHIQ no mEonoonoEOnno nEnHOo OOHIm HoUIon .mH onanm

H

127

 

 

12..
6

(%) AllAllOVOIGVH

 

I r
_. ' g?
o
, 9'
CA..
‘73... 0,0!
I; I!
6 r
Qf‘ o
I" J
.5" ’o’
I.— ——) D .—
>"" J a}, R
'4. If
‘ o'
\ as!
u駧é
E E \‘7”
55 0"
F— E:‘ cu ‘{;;:Z _. 83

 

10

FRACTION NUMBER

Figure 18

 

128

1

D-[U- 4C]galacturonic acid product and 96, 94, and 89%,

l4C]galacturonic acid product

respectively, of the D-[U—
was solubilized by ammonium oxalate extraction.

Recovery of radioactivity from the column was 84, 76,
and 83% for material synthesized with incubation times
of 0.5, 2, and 15 min, respectively. Comparison of the
elution profiles showed that as the reaction mixtures
were incubated for longer periods of time the percent of
radioactive material which eluted in the V0 increased.
These results indicated that the size of the molecules
of D-[U-14C]galacturonic acid solubilized product
increased with increasing incubation time.

The size of the D-[U-14C]apiose solubilized
product synthesized in the presence of UDPGalUA, as
determined by chromatography with Bio-Gel P-300, was
also affected by the length of the incubation period
used in synthesis (Figure 19). For the 0.5 and 15 min
incubations 6.4 and 44.1%, respectively, of the starting
radioactivity was incorporated into D-[U-14C]apiose
product of which 91.4 and 90.2%, respectively, was
solubilized by ammonium oxalate extraction. Recovery

l4C]apiose

of radioactivity from the column for D-[U-
solubilized product synthesized with incubation times
of 0.5 and 15 min was 83 and 77%, respectively. Com-

parison of the two elution profiles showed that

D-[U-14C]apiose solubilized product synthesized in

 

129

.nOHnOoMM Hon©H>HonH nooo nH nonooHHOO mo3 nOHnB enH>Hnoo

IOHooM noMo>OOoM Honon MO nnoOMom onn nnomoMmoM nwv mnH>HnooOHooM MO
monHo> .HH oManm MO onomoH onn nH nonHMOmoo mo AEO on x EoHo HonMonnH
Eo HV nEnHOO oomlm HoUIOHm o no nonmoMmOnoEOMno oMo3 .HE m.o MO oEnHO>

o nH .mnonUOMm ooNHHHnnHOm omOHmoHU IDHIQ one .noHnonnOnH nHE mH onn

oH
EOMM Eon ooo.mm nno nOHnonnOnH nHE m.o onn EOMM Eon oov.m moa nonoOMm

noNHHHnnHOm omOHoonU IDHIQ mo nonoHOmH MnH>HnoocHooM MO nnnOEo one

«H
.moMnooOOMm HonnoEHMomxm onn nH nonHMomoo mo meommo omonn EOMM ooMom

IoMm mo? nonoOMm ooNHHHnnHom .nHE mH MOM Monno onn nno nHE m.o MOM ono
.oomm no nononnonH oMoB whommo nnom .H: mm MO oEnHO> HonHM o non ono
anoomoD MO moHOEn v nonHonnOO oMnanE mommo noom .moMnUoOOMm HonnoE
IHMomxm onn nH nonHMomoo mo ooMoooMm oHoB nDHoomoD MO oonomoMm onn nH ooNHm

Ionnnem nonoOMo noNHHHnnHOm omOHooHUw IDHIQ MO mHmonnnmm onn MOM moMnanE

H
nOHnoooM HoOHnnonH Oze .nHE mH ono nHE m.o nH UoNHmonnnem nonoOMm noNHHHn

InHOm omOHmonu ID_IQ MO mEoMmonoEOMnO nEnHOO oomIm HooIOHm .mH oManm

VH

130

 

mH oManm

$9222 20 :05:

cm
_

2:23

 

(%) All/\llOVOIGVH

131

0.5 min contained a greater percentage of radioactivity
in molecules which eluted in the column VO than did
D-[U-14C]apiose solubilized product which was synthe-
sized in 15 min. These data suggest that acceptor
molecules of larger size were preferentially used in
the synthesis of the polysaccharide.

Pectinase Hydrolysis of Solubilized
figroducts

 

 

Hydrolysis of D-[U-14C]Apiose
SolubilizedEProduct

 

 

Untreated D-[U-14C]apiose solubilized product
and D-[U-14C1apiose solubilized product treated with
pectinase were chromatographed on a Bio-Gel P-lOO
column (Figure 20). Most of the untreated D-[U-14C]-
apiose solubilized product eluted from the column in
the V0. The treated material, on the other hand,
eluted over the entire fractionation range of the
column indicating that enzyme catalyzed hydrolysis of

14

the D-[U- C]apiose solubilized product had occurred.

The amount of D-[U-14C]apiose and D-[U-14C]-
xylose contained in the fragments resulting from
pectinase hydrolysis was investigated. D-[U-14C]Apiose
solubilized product was treated with pectinase and then
chromatographed on a column of Bio-Gel P-30 (Figure 21).
The fractions indicated in Figure 21 and labelled as

1, 2, and 3 were each concentrated to approximately

200 p1 at 30°C. The three samples were hydrolyzed at

132

.nonoOMm ooNHHHnnHOm

omOHmonU IDHIQ nonooMn omoanoom MOM wow ono nonoOMm ooNHHHnnHOm omOHmo

VH
IHUVHIDHIQ UonooMnnn MOM mew oMo3 nEnHOU OOHIm onn EOMM moHMo>Ooom .m nOHnoHOm
nOHnoHHHnnHom mnHmn en nowommo mo3 enH>HnooOHoom .nonooHHoo oMoB mnOHnooMM

HE mm.o ono Mn\HE m.H mos nEnHOO onn MO onoM 3OHM one .anHM nonooMmon

IoEOMnO moz onEom nonooMnnn onn “MoMMnn ononomonm EnHoom 2 Ho.o nnH3 nonoMnH
IHHnoo noon non nOHnB AEO OH x EoHn HonMonnH EO e.ov nEnHOO OOHIm HooIon o no
nonmoMmOnoEOMno oMo3 monEom onn nOHnonnOnH MonMn .erm no Mn mm MOM nononnonH
oMoB monEom o3n one .m.v mm .MoMMnn ononooo EnHoOm 2 m.o Mo H1 om ooooo moz
noonooMnnnv nOHnMom Monno onn oe .moMnnoOOMm HonnoEHMomxm onn nH nonHMomoo mo
omoanoom nnH3 oonooMn no: nonoOMm ooNHHHnnHOm MO nOHnMom ono .monnn onoMomom
nH oooon oMo3 .Emn ooo.m manHonnOo nooo .nonoOMd ooNHHHnnHOm omOHdo

Inc Ioeao .ooNMHoHc Mo coHHoHoo orn Mo 1H1 oomo mcoHoMoo Modem 039 .nr H

oH
ono m.o MonMo MonoB MO momnono nnHz Mn N MOM Dov no Monoz nH ooumHoHo mo3 ono

moMnooOOMm HonnoEHMomxm onn nH nonHMOmoo mo ooMomoMm mos nonnOMm ooNHHHnnHom

omOHmnnov IDHIQ .omoanoom nnHB nonooMn mo3 ono oonooMnnn mo: nonn nonnOMo

H
ooNHHHnnHOm omOHmoHUVHIDHIQ Mo mEoMoOnoEOMnO nEnHOO OOHIm HoUIOHm .om oManm

133

om oManm

mumsSz 20:05:
2

 

 

9:55. mm<z :omE

\

82m 5.2:
mm <2 .53

 

 

§

§

(NOIlGVUd 83d WdCl) All/\IlOVOIGVU

134

.m ono .m .H mnoHnooMM EMOM on ooanEOO nonn oMoB mnOHnooMM nEnHOO

nonoOHnnH one .wvm mo? nEnHOO onn EOMM HoHMonoE o>HnooOHooM MO >Mo>ooom

.m nOHnnHom nOHnoHHHnnHom nH enH>HnooOHooM MOM oomommo oMo3 mnOHnooMM nEnHOO
onn MO mnonoHHn .oonooHHOO oMo3 mnOHnooMM HE o.H ono Mn\HE om mos onoM

30HM nEnHoo one .m.m no .MoMMnn onoEMOM EanoEEo z mo.o nnHB oomoHo>oo

ono nonoMnHHHnoo mo3 nonn AEO me x EoHo HonMonnH EO m.ov nESHOO omIm

HooIOHm o no nonmoMmonoEOMno mo3 AH ommv nonoOMm ooNHHHnnHOm omOHmonUVHIDHIQ
onn nnoEnooMn oooanoom MonMo .moMnnoOOMm HonnoEHMomxm onn nH nonHMomoo

mo meow m MOM omoanOom nnHz nonooMn nonn mo3 AH: ome nH .Emo ooo.ovv
onomeoHo one .Mn v MonMo omnono Mono3 onO nnHz Mn vH MOM Mono3 nH noueHoHo

mos nonoOMm noNHHHnnHOm omOHmoHU

Ioeno one .coHnoHOm Hdnnoo usedoo Mo

vH H
H: om ono nOHnoMomoMm onenno onoHDOHnMom MO H: OOH nonHonnOO oMnanE nOHnoooM

onn nonn nmooxo moMnooOOMm HonnoEHMomxm onn nH nonHMomoo no noMomoMm moB

nonoOMm ooNHHHnnHOm omOHmnmov IDHIQ .omoanoom nnHB oonooMn mo3 nonn nonoOMm

H
ooNHHHnnHOm omOHmoHUVHIDHIQ MO EoMmonoEOMnO nEnHOO omlm HooIOHm .Hm oManm

135

 

 

 

 

 

 

_. (J?
—"——”'O”’
o
m O/
> I] \3
_ H I
o
3
~- /
N j
f/‘L—o’c’
C:—— ~__ __1
:, '_”’JL 0 “—4
c
c
c
l l

 

 

E ‘ é

(Nouovzu 83d woo) AllAllDVOIGVH

10

FRACTION NUMBER

Figure 21

 

136

pH 1 as described in the Experimental Procedures and the
hydrolysates were analyzed by paper chromatography with

Solvent A. All of the radioactivity in the samples was

present in D-[U-14C]apiose and D-[U-14C1xylose, however,

the relative amounts of the two sugars varied. The

14C]xylose in

amount of radioactivity present as D-[U-
fractions 1, 2, and 3 was 13, 8, and 64%, respectively.

In a second, similar experiment the amount of radio-
14

,- Eli—“UL?“ r.— 'J‘ L" '-

activity present as D-[U- C]xylose in fractions 1, 2,

and 3 was 15, 10, and 28%, respectively. These results

 

show that the small fragments of D-[U-14C]apiose solu-
bilized product isolated after pectinase treatment were
enriched in D-[U-14C]xylose.

Hydrolysis of D-[U-14C]Galacturonic
Acid SOlubilizedflPrOHuct

 

 

In order to investigate the pectinase suscepti-
bility of D-[U-14C]galacturonic acid solubilized product,
a sample of this material was chromatographed on a Bio-
Gel P-100 column and the radioactive material which
eluted at V0 was isolated and concentrated. This
material was divided into two portions; one of which
was treated with pectinase while the other was untreated.
The two samples were then chromatographed again on the
Bio-Gel P-lOO column (Figure 22). Recovery of radio-
active sample from the column was 87% for treated

material and 54% for untreated material. Most of the

137

.nOHnooMM nooo nH nonooHHOO mo3 nOHnB mnH>Hnoo

IoHooM ooMo>OooM HonOn MO nnoOMom onn nnomoMooM nwv mnH>HnooOHooM MO monHo>
.oonooHHOo oMo3 mnOHnooMM HE H ono mH oManm MO onomoH onn nH nonHMomoo mo OOHIm
HooIOHm no oonmoMmonoEOMno oMo3 monEom onn nOHnonoonH MonM< .Mn mH MOM erm

no nononnOnH oMoB monnn nnom .m.o no .MoMMnn ononooo EnHoOm z m.o MO H1 QMH
ooooo mos noonooMnnnv nOHnMom Monno onn oe .nH on ooooo HE\mE m MO noHnoMnnoO
InOo o no omoanoom manHonnOo .m.v no .MoMMnn ononooo EnHoOm 2 m.o MO H: ooH
non noonooan nOHnMom ono .naoo oomHv mnOHnMom H: oov Honoo m onnH nooH>Ho

ono oomm no HE m.o on nonoMnnoonoo mo3 HoHMonoE onn mHmeHoHo MonM< .ooo no

Mn N MOM 0mm nH noueHoHo nno ooanEOO oMo3 .O> onn on mnHonommoMMOo .HE om On «H
MO moEnHo> nOHnnHo nH nonnHo nOHnB HoHMonoE one .oonooHHOO oMo3 mnoHnooMM

HE m nonn nmooxo mH oManm Mo onomoH onn nH oonHMOmoo mo OOHIm HooIOHm MO

nEnHOO o no nonmoMmonoEOMno mo3 AHE m.o nH .Emo ooe.mv nonoOMm ooNHHHnnHOm one
.moMnooOOMm HonnoEHMomxm onn nH nonHMomon mo ooMomoMm mo3 nonoOMm ooNHHHnnHOm oHoo

OHnOMnnooHoUHU IDHIQ .omoanoom HomnoM nnHB oonooMnnn ono nonooMn nonnOMm ooNHHHn

«H
InHOm UHoo OHnOMnnooHomHUVHIDHIQ MO mEoMmOnoEOMnO nEnHOO OOHIm HooIOHm .Nm oManm

138

 

 

 

 

 

LIJQ
23
25
:o:
|._
82
0.:
J l
O l-fi

r-I

(%) AIIAIIOVOICIVII

 

10

FRACTION NUMBER

Figure 22

 

139

untreated material eluted in the V0 of the column while
the pectinase treated material eluted at the V of the

t
column. These results indicate that D-[U-14C]galacturonic
acid solubilized product was also capable of being

degraded by pectinase.

 

II—

DISCUSSION

Characterization of Solubilized Products

 

Procedures have been developed for the charac-
terization of radioactive polysaccharides synthesized
with a particulate enzyme preparation. Radioactive

14C]Api," UDP-

products were synthesized from "UDP[U-
[U-14C]GaluA, UDP[U-14C]GlcUA, and UDP[U-14C1Xyl in
large enough yields that the products could be par-
tially characterized (Table 1). However, not all of
the radioactivity in the reaction mixtures was incor-
porated into product. I have not attempted to isolate
or characterize the radioactive compounds in the
reaction mixtures that were not incorporated into pro-
duct. The inability of the particulate enzyme prepar-
ation to incorporate all of the radioactive sugar

into product under these reaction conditions is pre-
sumably caused by rapid inactivation of enzymatic
activity, metabolism of the sugar nucleotides by other
enzymatic pathways, or an insufficient quantity of
acceptor molecules for the transferase reactions or
all or a combination of these. Glycosyl transferase

activities in the particulate enzyme preparation from

140

141

E! minor are indeed unstable. Studies of the enzymes

14C]apiose and

responsible for the synthesis of D-[U-
D-[U-14C]galacturonic acid products by Pan, Leinbach
and Kindel have shown that the particulate enzyme
preparation lost 50% of both activities when stored

for 6 min at 25°C (unpublished results). Despite the

incomplete incorporation of radioactive sugars into

3- unn- Y-n‘

product the yields were sufficient to partially char-
acterize the products.

I have not characterized the insoluble radio-

 

active material remaining after ammonium oxalate

extraction of the products. In the case of D-[U-l4

C]-
glucuronic acid and D-[U-14C]xylose products the
insoluble radioactive material may be hemicelluloses
since D-glucuronic acid and D-xylose are important
constituents of hemicellulose polysaccharides. It is
also possible that the nonsolubilized materials have
structures identical to the solubilized products but
that ammonium oxalate extraction did not solubilize
them for unknown reasons.

l4C]xylose and D-[U-14CJ-

14

The presence of D-[U-
galacturonic acid in the D-[U- C]glucuronic acid indi-
cated that the particulate enzyme preparation contained
UDPGlcUA carboxy-lyase and UDPGlcUA 4-epimerase activi-
ties. The absence of D-[U-14C]apiose in the D-[U-14C]-

glucuronic acid solubilized product does not preclude

142

the existence of UDPGlcUA cyclase activity in the par-
ticulate enzyme preparation, however. The pH optima
for UDPGlcUA cyclase activity is 7.8 in potasium phos-
phate buffer and the incorporation assays were performed
at pH 6.1 (115).

The absence of incorporation of D-[U-14C]glu- F“
curonic acid into solubilized product may explain why

14C]glucuronic

the amount of radioactivity in the D-[U-
acid product was less than for the other 3 products

(Table 1) since before radioactivity could be incor-

 

porated into solubilized product D-[U-14C]GlcUA in the

1

reaction mixture had to be converted to UDP[U- 4C]Xyl

l4C]GalUA.

and UDP U-
I have not investigated whether the UDPGlcUA
4-epimerase and carboxy-lyase are membrane bound or
in organelles or both. E° minor seems to be an excel-
lent system for investigating Northcote's theory that
the synthesis of polysaccharides is controlled in the
golgi apparatus by control of the synthesis of the dif-
ferent sugar nucleotides (33). Plant cell wall syn-
thesis has the potential of being an important tool in
studying the development of cell surfaces because of
the change in the types of polysaccharides synthesized

when primary cell wall synthesis is replaced by

secondary cell wall synthesis.

143

The identity of the radioactive sugars contained
in the insoluble material remaining after ammonium
oxalate extraction was not determined. Other radio-

active sugars such as D-[U--l

4C]glucuronic acid and
L-[U-14C]arabinose may have been contained in this
material.

DEAE-Sephadex chromatography of the 4 solu-
bilized products showed that they were all negatively
charged since they were initially bound to the column
(Figures 7, 8, and 10). This result was expected for

the D-[U-l4 14

C]galacturonic acid and D-[U- C]glucuronic
acid solubilized products since they both contained
uronic acid sugars. However, since the D-[U-14C]apiose
and D-[U-14C]xylose solubilized products were also
bound by the DEAE-Sephadex column this suggests that
they also contained uronic acid residues.

Presumably the solubilized products were frac-
tionated on the DEAR-Sephadex column according to their
ratio of acidic to neutral sugars. The higher this
ratio the higher the concentration of NaCl necessary
to disassociate the solubilized product from the ion-
exchange column. The column chromatograms indicate

l4C]xylose solubilized

that the D-[U-14C]apiose and D-[U-
products contain less of the highly acidic material
than does the solubilized products synthesized from

the uronic acids (Figures 7, 8, and 10). Hart and

144

Kindel have reported that the polygalacturonic acid
backbone of apiogalacturonan, obtained by mild acid
hydrolysis of apiogalacturonan, was recovered after
DEAF-Sephadex chromatography in a fraction which eluted
from the column in 0.3 M NaCl (102). When the D—[U-14C1-
galacturonic acid solubilized product was chromatographed
on the DEAE-Sephadex column most of the radioactive
material was recovered in fractions which eluted with
NaCl concentrations of 0.25 M or less (Figure 8).

This result suggests that the D-[U-14C]galacturonic

 

acid solubilized product contains some neutral sugars.
The fractions obtained from the DEAE-Sephadex
chromatography of D—[U-14C]apiose solubilized product
were not homogeneous as determined by rechromatography
on DEAE-Sephadex (Table 8). The lack of homogeneity
may have been caused by "degradation" as shown in
Figure 16. Rechromatography did not, however, change
the D-[U-14C]apiose content of the fractions recovered
from fractions A, B, and D. The significance of the

14C]apiose content of the fractions

change in D-[U-
recovered from the rechromatography of fraction C is
unknown.

The polysaccharide nature of the solubilized
products is evidenced by their high molecular weight

as shown by their impermeability to dialysis membranes

and the results obtained by gel chromatography of the

145

14

D-[U-14C]apiose and D-[U- C]galacturonic acid solu-

bilized products. From the elution of the D-[U-14CI-

14C]galacturonic acid solubilized

apiose and D-[U-
products through a column of Bio-Gel P-300 the molecules
had a disperse molecular weight range from at least

1

125,000 to 5,000 (Figure 11). The D-[U- 4C]galacturonic

acid solubilized product contained lower molecular

14C]_

weight molecules than were contained in the D-[U-
apiose solubilized product (Figure 13). These in yitrg
synthesized pectic materials are especially suited for
studying molecular size differences by gel chromatography
because they are solubilized by relatively gentle tech-
niques. However, the ammonium oxalate extraction pro-
cedure may result in some breakdown (116). Villemez has
described a procedure for the gel chromatography of the
acetate derivatives of in yitrg synthesized [14C]g1uco-
mannans (117). He resolved the [14C]glucomannan into

two fractions; one with a minimum molecular weight of
200,000 and the other with a minimum molecular weight

of 60,000.

Identification of D-[U-14CJApiose Solubilized
Prddhct as an Apiogalacturonan

 

Although direct evidence for the identification
of the D-[U-14C]apiose product as an apiogalacturonan
was not obtained, there is an abundance of indirect

evidence which confirms this conclusion.

146

D-[U-14C]Apiose solubilized product has many of the same
properties as authentic apiogalacturonan isolated from
intact plants.

Like authentic apiogalacturonan, the D-[U-14C]-
apiose product could be extracted by ammonium oxalate

treatment (Table l). I have shown that the solubili-

 

r=
zation is dependent on the presence of ammonium oxalate i
which is an indication of the pectic nature of the §
14 .
D-[U- C]apiose product.
The release of [U-14C]apiobiose from D-[U-14C]-
s

apiose solubilized product after hydrolysis at pH 4
(Figure 4) also shows that the solubilized product has
a structure similar to apiogalacturonan (104). Hart
and Kindel postulated that the cleavage of the glyco-
sidic bond between the apiobiose side chains and the
polygalacturonic acid backbone is the result of the
transannular participation of the free carboxylic group
of the D-galacturonic acid residue (104). Similar

l4C]apiobiose from the solubilized pro-

release of [U-
ducts suggests a similar structure of a polygalacturonic
acid backbone with apiobiose side chains.

1 l4C]galacturonic

The D-[U- 4C]apiose and D-[U-
acid solubilized products probably contain a backbone
of a-l,4-galacturonan since they were both hydrolyzed
by treatment with pectinase (Figures 20 and 22). It is

not surprising that the D-[U-14C]apiose solubilized

147

product was not hydrolyzed completely since side chains
of D-apiose are known to confer pectinase resistance on

the polygalacturonic acid backbone of apiogalacturonans

l

(102, 120). The small fragments of D-[U- 4C]apiose

solubilized product resulting from treatment with pec-
tinase were found to be enriched in D-[U-14C]xylose F
content (Figure 21). This result may be explained by

1

assuming that the D-[U- 4C]xylose residues are bound as

7.’ in... ".4 -

side chains to the polygalacturonic acid backbone but

do not confer resistance to pectinase hydrolysis.

 

I‘ll» ‘1 ‘urli

Therefore treatment with pectinase will result in the
cleavage of the backbone near the point of D-[U-14C1-
xylose attachment thus releasing small fragments of
polysaccharide containing D-[U-14C]xylose. A similar
release of small oligosaccharides containing D-xylose
and D-galacturonic acid was reported when a pectic poly-

saccharide isolated from Zosteraceae and containing D-

 

galacturonic acid, D-xylose, D-apiose, and other neutral
sugars was hydrolyzed with pectinase (120).
As was the case with authentic apiogalacturonans

l4C]apiose solubilized product

from intact plants, D-[U-
was fractionated by chromatography on a DEAE-Sephadex
column (Figure 7).

Additional evidence for the identification of

1

D-[U- 4C]apiose solubilized product as an apiogalacturonan

is seen by the effect of adding UDPGalUA to the reaction

148

l4C]apiose product.

mixture used to synthesize D-[U-
Addition of UDPGalUA to the "UDP-[U-14C]Api" reaction
mixture resulted in an increase in the incorporation of
radioactive sugars into the product (Table 2), an

l4C]apiose to D-[U-14CJ-

increase in the ratio of D-[U-
xylose (Table 4) an increase in susceptibility to
hydrolysis at pH 4 (Table 5), a change in chromatography
properties when chromatographed on DEAE-Sephadex
(Figure 9), and an increase in the size of the molecules
in the small molecular weight fraction (Figure 17).
These changes indicate that addition of UDPGalUA to
the reaction mixture resulted in the incorporation of
D-[U-14C]apiose and D-galacturonic acid into the same
molecules of polysaccharide.

Additional galacturonans would be synthesized
when UDPGalUA is present in the "UDP-[U-14C]Api"
reaction mixture. The increase in incorporation of

14C]apiose product synthesized

radioactivity into D-[U-
in the presence of UDPGalUA (Table 2) could be explained
by the presence of additional galacturonan acceptors

for the incorporation of radioactive side chains.

l4C]apiose

The increase in the ratio of D-[U-
to D-[U-14C]xylose seen in the D-[U-14C]apiose solu-
bilized product could have resulted from the dilution
of UDP-[U-14C]Xyl by UDPXyl synthesized in the reaction

mixture from UDPGalUA. This conversion of UDPGalUA to

 

In.-

149

UDPXyl is possible because of the presence of UDPGlcUA
4-epimerase and UDPGlcUA carboxy-lyase activities in

the particulate enzyme preparation. However, the amount
of conversion of UDPGalUA to UDPXyl cannot be large since
the D-[U-14C]apiose product synthesized in the presence
of UDPGalUA did not exhibit the lower amount of solubili- I.
zation with ammonium oxalate treatment that was seen
when D-[U-14C]apiose product was synthesized in the

presence of UDPXyl. Also, there was little D-[U-14C]-
l

 

xylose found in D-[U- 4C]galacturonic acid solubilized
product. Therefore, the decrease in content of
D-[U-14C]xylose with addition of UDPGalUA to the
reaction mixture may be caused instead by UDPGalUA
effecting the control mechanisms for D-apiosyl and
D-xylosyl transferase activities. Pan and Kindel
(unpublished results) have found that addition of
UDPGalUA to the reaction mixture used to synthesize
D-[U-14C]apiose product resulted in an increased rate

1

of D-[U- 4C]apiose incorporation and a decreased rate

of D-[U-14C]xylose incorporation.

The increase in the release of D-[U-14

C]apiose
and [U-14C]apiobiose after hydrolysis at pH 4 from
D-[U-14C]apiose solubilized product synthesized in the
presence of UDPGalUA (Table 5) may be the result of

increased incorporation of D-galacturonic acid into

the D-[U-14C]apiose containing polysaccharide thus

150

resulting in a more acidic polysaccharide which can
better participate in the hydrolysis.

It has been reasoned that if D-apiose and D-
galacturonic acid are incorporated into the same poly-

1

saccharide then D-[U- 4C]apiose solubilized product syn-

thesized in the presence of UDPGalUA should have a
l

r-
higher ratio of D-galacturonic acid to D-[U- 4C]apiose

then would D-[U-14C]apiose solubilized product synthesized

‘ “.St'1'\.~‘.)l‘",o'c’ e!

in the absence of UDPGalUA. The polysaccharide with

the higher ratio would be more acidic than the other.

 

'—

As determined by DEAR-Sephadex chromatography,
D-[U-l4clapiose solubilized product synthesized in

the presence of UDPGalUA was more acidic than the
product synthesized without UDPGalUA (Figures 7 and 9).
The more acidic polysaccharides elute from the DEAE-
Sephadex column in fractions D and E. Twenty-three per-
cent of the recovered radioactivity was contained in

14C]apiose solu-

these 2 fractions combined when D-[U-
bilized product synthesized in the absence of UDPGalUA
was chromatOgraphed. Sixty percent of the recovered
radioactivity was contained in fractions D and E combined
when D-[U-14C1apiose solubilized product synthesized in
the presence of UDPGalUA was chromatographed. The
increase in the amount of highly acidic polysaccharide

fraction was dependent on the amount of UDPGalUA added

to the reaction mixture (Table 6).

151

When I first saw the change that addition of
UDPGalUA to the reaction mixture caused in the chroma-
tography of D-[U-14C]apiose solubilized product on the
DEAF-Sephadex column, I thought that a galacturonan may
have been synthesized from UDPGalUA which then complexed
with the D-[U-14C]apiose solubilized product. The change
in the elution profile would have then been caused by

l4C]apiose product

the galacturonan dragging the D-[U-
with it rather than the D-galacturonic acid being

incorporated into the D-[U-14C]apiose solubilized pro-
duct. This theory is incorrect, however, since mixing
galacturonan synthesized from UDPGalUA with D-[U-14C]-
apiose solubilized product did not change the elution

profile of D-[U-l4

C]apiose solubilized product on the
DEAR-Sephadex column.
Characterization and comparison of the DEAE-

l4C]apiose solu-

Sephadex fractions obtained from D—[U-
bilized product synthesized in the absence and presence
of UDPGalUA showed that UDPGalUA affected the D-[U-14C]-

l4C]xylose ratio and susceptibility to

apiose/D-[U-
hydrolysis at pH 4 of corresponding fractions (Table 7).
These results show that the higher the D-[U-14C]xylose
content of a fraction the lower the acidity was as
determined by elution order from the DEAE-Sephadex
column. Fraction D eluted from the DEAF-Sephadex

column in a similar position in the gradient as the

152

22° sodium chloride soluble apiogalacturonan IIa fraction
characterized by Hart and Kindel (102). When hydrolyzed
at pH 4 this authentic apiogalacturonan totally released
its D-apiose (104). In the case of fraction D obtained

from D-[U-14

C]apiose solubilized product synthesized in
the presence of UDPGalUA 81% of the incorporated D-[U-14CJ-
apiose was released after hydrolysis at pH 4 (Table 7).
This shows that fraction D has similar hydrolysis
properties at pH 4 to the authentic apiogalacturonan
fraction.

Although Hart and Kindel did not find D-xylose
in the apiogalacturonans that they isolated from the
cell wall of E. minor (102), I was able to demonstrate

l4C]xylose

by hydrolysis at pH 1 the existance of D-[U-
in the [14C]60° sodium chloride soluble apiogalacturonan
fraction isolated by them. D-Xylose seems to be a normal
constituent of the apiogalacturonan fractions isolated
at higher temperatures as evidenced by these results
and those of Beck (103) albeit D-xylose is not contained
in the fractions isolated by ammonium oxalate extraction
at 22°C.

Beck was unable to establish whether D-apiose
and D-xylose were contained in his 60°C extract as
separate apiogalacturonan and xylogalacturonan poly-

saccharides or as an apioxylogalacturonan (103). He

did suggest that both pentoses were contained in the

153

same polysaccharide because the D-xylose and D-apiose
containing polysaccharides were not separated from one
another by DEAE-Cellulose chromatography (103). The
characterization experiments performed with D-[U-14C]-
apiose solubilized product also did not determine

whether D-[U-l

4C]xylose and D-[U-14C]apiose were F
incorporated in vitro into the same polysaccharide.

However, I was unable to completely separate incor-
l

 

porated D-[U-14C]apiose from D-[U- 4C]xylose by DEAE-

Sephadex chromatography (Tables 7 and 8) and this also

 

suggests that both pentoses were incorporated into the
same molecules. A possible experimental approach to
answer this question would be to prepare an affinity
column for D-apiose using an antibody specific for this
sugar. If the D-[U-14C]apiose and D-[U-14C1xylose

incorporated in the D-[U-l4

C]apiose solubilized product
were contained in different polysaccharide molecules
than the D-apiose affinity column would bind the
D-[U-14C1apiose containing material but not the
D-[U-14C]xylose containing material.

Evidence that D-[U-14C]xylose was incorporated
into the apiogalacturonan molecules, however, is seen
from the effect that the presence of UDPXyl in the

reaction mixtures used to synthesize D-[U-14

C]apiose
product has on the properties of the resultant product.

The changes seen when UDPGlcUA was added are also

154

probably due in part by incorporation of D-xylose
because of the presence of UDPGlcUA carboxy-lyase
activity in the particulate enzyme preparation. There
is less conversion of UDPGlcUA to UDPGalUA in the
reaction mixture than there is conversion to UDPXyl as

shown by the larger quantity of D-[U--14

1

C]xylose as

compared to D-[U- 4C]galacturonic acid contained in the

D-[U-l4

C]glucuronic acid solubilized product.

Addition of UDPGlcUA and UDPXyl to the reaction
mixtures used to synthesize D-[U-14C]apiose product
resulted in a decreased solubilization of radioactive
material by ammonium oxalate extraction (Table 2).
Addition of these nonradioactive sugar nucleotides may
have promoted the synthesis of a D-apiose containing

polysaccharide which was not a pectin. Approximately

80% of the D-apiose in the cell wall of E. minor was

 

not extracted by ammonium oxalate and the structure
of this D-apiose-containing material is unknown (102).

The large amount of D-[U-l4

C]apiose product synthesized
in the presence of UDPXyl which was not solubilized by
ammonium oxalate treatment may indicate that the cell
wall of E. miggg contains an apioxylan. However,

the ammonium oxalate solubilized and nonsolubilized
materials may also have the same structure except that

the nonsolubilized material has more D-xylose in the

side chains. The D-xylose side chains for unknown

155

reasons may cause the polysaccharide to be less sus-
ceptible to solubilization by ammonium oxalate.
Addition of UDPXyl to the reaction mixture would
increase the number of D-xylose side chains in the
product and thus decrease the amount of material solu-
bilized. This last possibility agrees with the obser-
vation that D-xylose containing polysaccharides were
extracted from the cell wall of E. miggg only at
higher temperatures (103).

When hydrolyzed at pH 4 D-[U-14C]apiose solu-
bilized product, synthesized in the presence of

l4C]apiose

UDPXyl, released a larger amount of D-[U-
as D-[U-14C]apiose than did D-[U-14C]apiose solubilized
product synthesized in the absence of nonradioactive
sugar nucleotide (Table 5). There was no increase in

the amount of [U-1

4C]apiobiose released. This suggests
that D-xylose was incorporated into the polysaccharide

and this affected the hydrolysis at pH 4 in a presently
unknown manner.

The experimental results described above all
show that an apiogalacturonan, containing some D-xylose
side chains, can be synthesized with the particulate
enzyme preparation from E. miggg. However, the results

14C]apiose solu-

obtained from hydrolyzing the D—[U-
bilized product at pH 4 are not exactly the same as

was obtained by Hart and Kindel (104) with authentic

156

apiogalacturonans. When they hydrolyzed an apiogalac-
turonan fraction, which had been purified by DEAE-
Sephadex chromatography, at pH 4 all of D-apiose was
released from the polysaccharide and recovered as
apiobiose (104). On the other hand, I was unable to
obtain total release of D-[U-14C]apiose from any of
the fractions of D-[U-14C]apiose solubilized product
by hydrolysis at pH 4 (Tables 5 and 7). I also found

1

that the ratio of [U-14C]apiobiose to D-[U- 4C]apiose

14C]apiose solu-

released from the fractions of D-[U-
bilized product was lower than was reported by Hart
and Kindel (104). However, hydrolysis of the [14C]-
22°C and 60°C sodium chloride soluble apiogalacturonan
fractions of Hart and Kindel at pH 4 also resulted in

14C]apiose from the polysac-

incomplete release of D-[
charides (Hart and Kindel, unpublished results). In
the case of the [14C]60°C sodium chloride soluble

l4C]apiose

apiogalacturonan fraction only 41% of the D-[
was released from the polysaccharide after a 3 hr
hydrolysis period and the ratio of [14C]apiobiose to

D_[14

C]apiose was 2.7 (Hart and Kindel, unpublished
results). This may indicate that the completeness

of hydrolysis at pH 4 may depend on small differences
in structure or preparation of apiogalacturonan. Some

of the D-[U-14C]apiose may be contained in the solu-

bilized product as monosaccharide side chains.

157

Possible Mechanisms of Synthesis

 

I do not know whether apiogalacturonans were
synthesized d3 2232 by the particulate enzyme prepar-
ation from E. miggg. However, the amount of radio-
activity contained in the largest molecules of D-[U-l4C]-
galacturonic acid increased as the reaction mixture was Fe
incubated for longer periods of time (Figure 18). One

1

interpretation of this is that more than one D-[U- 4C]-

galacturonic acid residue was incorporated into each

1

molecule of D-[U- 4C]galacturonic acid solubilized

 

Jill-Wu”! "Kl _" , Z." O-

product. In order to determine whether dg’ggyg syn-
thesis has occurred additional experiments will have to
be performed to determine whether the reducing ends
and interior residues of the D-[U-14C]galacturonic
acid solubilized product were synthesized £2.2i2523
There is a second interpretation for the results
shown in Figure 18. It is still possible to incorporate
a single D-[U-14C]galacturonic acid residue into pre-
formed chains and obtain an increase in the relative
amount of radioactivity into the large molecular weight
fraction with increasing synthesis times if the rate
of incorporation into large chains is faster than into
small chains.
There are at least 2 possible mechanisms for
synthesis of apiogalacturonans. The first mechanism is

similar to one seen in bacterial systems where an

158

oligosaccharide-repeating unit containing D-apiose

and D-galacturonic acid is first synthesized as part
of a lipid intermediate and then transferred to the
growing polysaccharide (78). This mechanism allows

for a highly uniform structure. A second mechanism

of synthesis is that the polygalacturonic acid backbone

be synthesized first after which the pentose sugar side

chains are incorporated. This mechanism is similar
to the one found for the methylation of pectins (74,
75).

 

H-

Based on the gel chromatography experiments
some proposals about the mechanism of apiogalacturonan
synthesis can be made. Since D-[U-14C]apiose is incor-
porated in the absence of exogenous UDPGalUA and the
D-[U-14C]galacturonic acid solubilized product is
smaller than the D-[U-14C]apiose solubilized product
(Table 13), it is doubtful that synthesis occurs by
means of an apiogalacturonasyl lipid intermediate.
Therefore synthesis most likely occurs by the formation
of the polygalacturonic acid backbone followed by
incorporation of the D-apiose side chains. Such a
synthesis mechanism can be used to explain why the
percent of D-[U-14C]apiose solubilized product of high
molecular weight decreased when the incubation times
were increased from 0.5 to 15 min (Figure 19) even

14

though in the case of D-[U- C]galacturonic acid

159

solubilized product the percent of high molecular weight
material increased as the length of incubation increased
(Fraction 18). At the beginning of the £2 vitro incor-

l

poration of D-[U- 4C]apiose the largest molecules of

polygalacturonic acid are probably used preferentially

as acceptor molecules for D--[U--l

4C]apiose transfer. Fe
As the incorporation reaction proceeds then the smaller
molecules of polygalacturonic acid would act as

acceptors and thus increase the fraction of lower

 

molecular weight D-[U-14C]apiose solubilized product.

winning". . 1.92...

An alternate explanation for the results shown
in Figure 19 is that there is a degradative enzyme in
the particulate enzyme preparation which hydrolyzes the
backbone of the galacturonan acceptor. Therefore,
initially the D-[U-14C]apiose side chains were incor-
porated into large molecular weight polysaccharides, but
as the synthesis reaction proceeded, the apiogalacturonans
and the galacturonan acceptors would be cleaved into
smaller pieces by the degradative enzyme. This theory

l4C]galacturonan

is unlikely, however, since the D-[U—
did not show evidence of being cleared during synthesis
(Figure 18).

As discussed previously the D-[U-14C]galac-
turonic acid solubilized product probably contains

neutral sugar side chains. Some of these neutral

sugar side chains may be the result of the conversion

160

of UDP[U-14C]GalUA to UDP[U-14C]Xyl. A second possi-
bility is that the particulate enzyme preparation con-
tains acceptor molecules for D-galacturonic acid
transfer which contain neutral sugars. This suggests
that apiogalacturonan synthesis occurs by alternating

periods of backbone elongation with periods of incor-

poration of the side chains. I do not know if the par-

-_. ‘fii

ticulate enzyme preparation contains nonradioactive

sugar nucleotides when isolated. If it did this would
4

 

also explain why the D-[U--l C]galacturonic acid solu-

‘7

bilized product contains neutral sugars.

Since both residues in [U-14C]apiobiose were
incorporated 12.21EE2 this suggests that incorporation
of the two D-[U-14C]apiose residues of the disaccharide
into the apiogalacturonan occurred simultaneously.
Otherwise some of the [U-14C]apiobiose molecules would
have contained an 12.2122 synthesized D-apiose moiety
at the reducing end of the disaccharide. A reaction
mechanism involving incorporation of intact apiobiose
residues through a lipid intermediate could account for
these results. Pan and Kindel were unable to isolate
a D-[U-14C1apiose containing lipid intermediate from
this system, however (unpublished results). A second
mechanism can be imagined where the polygalacturonic
acid acceptor was bound to an enzyme complex containing

2 apiosyl transferase enzymes. The first transferase

161

would transfer D-apiose to a D-galacturonic acid residue
in the backbone. The second transferase would imme-
diately transfer another D-apiose to the first apiosyl
residue.

Degradation of D-[U-14C]Apiose and

D-TU-15C]Galacturonic Acid
Solubilized Products

1

 

 

 

The mechanism by which D-[U- 4C]apiose and
D-[U-14C]galacturonic acid solubilized products were
"degraded" by dialysis in water or by chromatography
on DEAE-Sephadex is unknown (Figures 14 and 16). Any
possible mechanism must be able to account for the
absence of "degradation" when the solubilized products
were dialyzed in sodium phosphate buffer (Figure 14).
The mechanism would also have to explain why dialysis
in water does not "degrade" fractions of solubilized
products that have been chromatographed on a column of
Bio-Gel equilibrated with sodium phosphate buffer.

The "degradative" process may indicate that the
solubilized product consists of aggregates of non-
covalently bound polysaccharide molecules which are
disassociated by dialysis in water or by DEAE-Sephadex
chromatography. However, this hypothesis seems
questionable since the "degradation" was not reversed

by dialysis in sodium phosphate buffer. Also this

hypothesis does not explain why solubilized product

162

recovered after gel chromatography was not degraded
by water dialysis (Figure 15).

If the solubilized products are in solution as
single molecules then "degradation" may result from
the enzymatic cleavage of glycosidic bonds. However,
the inactivation of such enzymes by sodium phosphate
buffer is hard to understand. Another possible expla-
nation for the "degradation" mechanism is that during
dialysis in water or chromatography on DEAR-Sephadex
the larger molecules of solubilized product were
selectively lost. The decrease in the amount of
radioactive material contained in the V0 of the gel
chromatography column after dialysis or DEAR-Sephadex
chromatography would be caused by this selective loss
of large molecular weight material. The amount of
radioactive material not recovered after dialysis or
DEAE-Sephadex chromatography is large enough to support
this mechanism although it too is highly speculative.

An understanding of this "degradation" process
may be important in the study of cell wall structure.
This is especially true in the light of recent models
of cell wall structure based on extensive cross linking
of polysaccharides and proteins (21). 1

14

I do not know whether the D-[U- C]apiose and

1

D-[U- 4C]galacturonic acid solubilized products used

in the gel-chromatography experiments were degraded

163

during the solubilization procedure with ammonium
oxalate. Such degradation of pectic polysaccharides

by ammonium oxalate treatment has been reported (116).
Initial experiments by Leinbach and Kindel suggest that

l4C]galacturonic acid product with

extraction of D-[U-
sodium hexametaphosphate rather than ammonium oxalate I
resulted in a greater percentage of the radioactivity I

being isolated in the V0 when chromatographed on a Bio-

Gel P-300 column (Leinbach and Kindel, unpublished

 

results). Apiogalacturonans are not methyl esterifred
(102) so that degradation of the solubilized products
by B-elimination is not probable.

It is possible that as polysaccharides were syn-
thesized in the particulate enzyme preparation they
were also degraded enzymatically. This possibility
was not investigated and finding proper control experi-
ments for such an investigation is not practical. The
obvious experiment of isolating solubilized product,
determining its molecular weight distribution by
chromatographing a portion on a gel chromatography
column, incubating the rest of the solubilized product
with particulate enzyme preparation, and then rechroma-
tographing on the gel chromatography column to see if
there is any change in the molecular weight distribution
is not valid. This is true because the polysaccharide

may be synthesized within a membrane-bound organelle (7S).

164

Addition of isolated solubilized product to the par-
ticulate enzyme preparation will not result in the
polysaccharide being in the same environment as it

was synthesized in since it will not be able to pene-
trate the lipid membrane. This is important because
the organelle may protect the newly synthesized poly-
saccharide from degradative enzymes in the cytoplasm

or in other organelles. Addition of isolated solu-
bilized product to the particulate enzyme preparation
may result in the exposure of the product to degradative
enzymes that it would normally be protected from before

extraction from the reaction mixture or vice versa.

 

Summary

I have shown that the particulate enzyme prepar-
ation from E. miggg is capable of synthesizing D-apiose
and D-galacturonic acid containing polysaccharides with
structures similar to the apiogalacturonan components
of the cell wall. The iEIXiEEE synthesized apiogalac-
turonans seem to contain side chains of D-xylose also.
Studies with E. EEBQE on the cell-free incorporation

of D-[U-14C]apiose and D-[U-l4

C]galacturonic acid, from
their respective sugar nucleotides, into polysaccharides
can be done with the knowledge that an actual component
of the plant cell wall is synthesized. In addition I

have shown that the synthesis of cell wall polysaccharides

in the particulate enzyme preparation from E. minor is

165

an integrated system where the products formed are
influenced by the Species and relative amounts of sugar
nucleotides present in the reaction mixture.

The results of several experiments have allowed
for the proposal of a tentative mechanism of apiogalac-
turonan synthesis where the backbone of polygalacturonic
acid is synthesized first from UDPGalUA after which the

D-apiose containing side chains are added.

LIST OF REFERENCES

“.1330.” ‘- '-

 

'!

10.
11.

12.

LIST OF REFERENCES

Mascaro, L., and Kindel, P. K. (1974) Fed. Proc. 33,
1445.

Aspinqll, G. O. (1970) in The Carbohydrates (Pigman,
W., and Horton, D., eds.), Vol. IIB, pp. 515-536,
Academic Press, New York.

 

Stoddart, R. W., Barrett, A. J.p and Northcote, D. H.
(1967) Biochem. J. 102, 194-204.

 

Stoddart, R. W., and Northcote, D. H. (1967) Biochem.
g. 105, 45-59.

Whistler, R. L., and Richards, E. L. (1970) in The
Carboh drates (Pigman, W., and Horton, D., eds.),
V01. IIA, pp. 447-469, Academic Press, New York.

Katz, M., and Ordin, L. (1967) Biochim. Bigphys. Acta
141, 126-134.

 

Kivelaan, A., Bandurski, R. S., and Schulze, A.
(1971) Plant Physiol. fig, 389-393.

 

Lamport, D. T. A., and Northcote, D. H. (1960)
Nature 188, 665-666.

 

Lamport, D. T. A. (1965) in Advances in Botanical
Research (Preston, R. D., ed.), Vol. 2, pp. 151-
218, Academic Press, New York.

 

Lamport, D. T. A. (1969) Biochemistryg, 1155-1163.

 

Lamport, D. T. A. (1970) in Ann. Rev. Plant Physiol.
(Machlis, L., ed.), Vol. 21, pp. 235-270) Annual
Reviews Inc., Palo Alto.

 

Lamport, D. T. A. (1973) in Biogenesis of Plant Cell
Wall Polysaccharides (Loewus, F.,FEH.), pp. 149-
164, Academic Press, New York.

 

 

166

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

167

Timell, T. E. (1965) in Cellular Ultrastructure of
Woody Plants (Cote, W. A., Jr., ed.), pp. 127-
156, Syracuse University Press, Syracuse.

 

 

Shafizadeh, F., and McGinnis, G. D. (1971) in Adv.
Carbohydrate Chem. and Biochem. (Tipson, R. 8.,
ed.), V61. 267'pp. 297-349, Academic Press, New
York.

 

Thornber, J. P., and Northcote, D. H. (1961)

 

Muhlethaler, K. (1965) in Cellular Ultrastructure of
Woody Plants (Cote, W. A., Jr., edf), pp. 191-
198, Syracuse University Press, Syracuse.

 

 

 

Mark, R. (1965) in Cellular Ultrastructure of Woody T
Plants (Cote, W. A., Jr., ed.), pp. 493-533, 5
Syracuse University Press, Syracuse.

 

Marx-Figini, M., and Schulz, G. V. (1966) Biochim.
Biophys. Acta 112, 81-101. '"

 

Talmadge, K. W., Keegstra, K., Bauer, W. D., and
Albersheim, P. (1973) Plant Physiol. 51, 158-173.

 

Bauer, W. D., Talmadge, K. W., Keegstra, K., and
Albersheim, P. (1973) Plant Physiol. EE, 174-187.

 

Keegstra, K., Talmadge, K. W., Bauer, W. D., and
Albersheim, P. (1973) Plant Physiol. EE, 188-196.

 

Wilder, B. M., and Albersheim, P. (1973) Plant
Physiol. 2E, 889-893.

Pickett-Heaps, J. D., and Northcote, D. H. (1966)
Jo cello SC]... -1_' 109-120.

 

Whaley, W. G., and Mollenhauer, H. H. (1963)

Whaley, W. G., Dauwalder, M., and Kiphart, J. E.
(1966) J. Ultrastructure Res. EE, 169-180.

 

Frey-Wyssling, A., Lopez-Saez, J. F., and Mfihlethaler,
K. (1964) J. Ultrastructure Res. E2. 422-432.

 

Northcote, D. H., and Pickett-Heaps, J. D. (1966)
Biochem J. 2g, 159-167. '

Wooding, F. B. P. (1968) J. Cell Sci. 3, 71-80.

 

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

168
Bowles, D. J., and Northcote, D. H. (1972) Biochem.
g. 130, 1133-1145.

Harris, P. J., and Northcote, D. H. (1971) Biochim.
Biophys. Acta 237, 56-64.

 

Dashek, W. V., and Rosen, W. G. (1965) Plant
Physiol. 19 Sup. XXXIX.

Villemez, C. L., McNab, J. M., and Albersheim, P.
(1968) Nature 218, 878-880.

 

Northcote, D. H. (1968) in Plant Cell Organelles
(Pridham, J. B., ed.), pp. 179-197, Academic
Press, New York.

 

 

Zetsche, K. (1966) Biochim. Biophys. Acta 124,
332-338.

 

Ledbetter, M. C., and Porter, M. D. (1963) J. Cell.
Biol. E2, 239-250.

Hepler, P. K., and Nowcomb, E. H. (1964) J. Cell.
Biol. g9, 529-533.

Streiblova, E. (1968) J. Bacteriol. 32, 700-707.

Moor, H., and Mfihlethaler, K. (1963) J. Cell Biol.
£1, 609-628.

Robinson, D. G., and Preston, R. D. (1972) Planta

Brown, R. M., Jr., Franke, W. W., Kleinig, H. F.,
and Sitte, P. (1970) J. Cell. Biol. 32. 246-271.

 

Hassid, W. Z. (1969) Science 165, 137-144.

 

Feingold, D. S., Neufeld, E. F., and Hassid, W. Z.
(1958) J. Biol. Chem. 233, 783-788.

 

Currier, H. B. (1957) Amer. J. Bot. 11, 478-488.

 

Villemez, C. L., Franz, G., and Hassid, W. Z.
(1967) Plant Physiol. 13, 1219-1223.

 

Brummond, D. 0., and Gibbons, A. P. (1964) Biochem.
Eiopgys. Res. Commun. El, 156-159.

 

Flowers, H. M., Batra, K. K., Kemp, J., and Hassid,
W. Z. (1968) Plant Physiol. 33, 1703-1709.

 

 

47.

48.

49.

SO.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

169
Clark, A. F., and Villemez, C. L. (1972) Plant
Physiol. £2, 371-374.

Ordin, L., and Hall, M. A. (1968) Plant Physiol. 11,
473-476.

 

Tsai, C. M., and Hassid, W. Z. (1973) Plant Physiol.
EE, 998-1001.

 

Péaud-Lenvel, C., and Axelos, M. (1970) FEBS Letters

 

g, 224-228.

Smith, M. M., and Stone, B. A. (1973) Biochim.
Biophys. Acta 313, 72-94.

 

Ray, P. M., Shininger, T. L., and Ray, M. M. (1969)
Biochemistry_§i, 605-612.

 

Van Der Woude, W. J., Lembi, C. A., and Morré, D.
J. (1974) plant Physiol. 25, 333-340.

 

Elbein, A. D., Barber, G. A., and Hassid, W. z.

 

Barber, G. A., Elbein, A. D., and Hassid, W. Z.

 

Elbein, A. D. (1969) J. Biol. Chem. 244, 1608-1616.

 

Flowers, H. M., Batra, K. K., Kemp, J., and Hassid,
W. Z. (1969) J. Biol. Chem. 244, 4969-4974.

 

Villemez, C. L. (1971) Biochem. J. 121, 151-157.

 

Barber, G. A., and Hassid, W. Z. (1965) Nature 207,
295-296.

 

Marx-Figini, M. (1966) Nature 210, 754-755.

Delmer, D. P., Beasley, C. A., and Ordin, L. (1974)
Plant Physiol. E3, 149-153.

 

Robinson, D. G., and Preston, R. D. (1972) Biochim.
BiOphys. Acta 273, 336-345.

 

Muhlethaler, K. (1949) Biochim. Biophys. Acta 3,

 

Colvin, J. R. (1959) Nature 183, 1135-1136.

 

I»: a

0‘

‘ -1. fin'hr Fun.

‘1'
run..-

 

65.

66.
67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

170

Minor, F. W., Greathouse, G. A., Shirk, H. G.,
Schwartz, A. M., and Harris, M. (1954) J. Am.
Chem. Soc. 19' 1658-1661.

 

Glaser, L. (1958) J. Biol. Chem. 232, 627-636.

 

Kauss, H. (1967) Biochim. Biophys. Acta 148, 572-574..

 

Kauss, H., and Hassid, W. Z. (1967) J. Biol. Chem.

 

Pridham, J. B., and Hassid, W. Z. (1966) Biochem. J.
100, 21P.

 

Bailey, R. W., and Hassid, W. Z. (1966) Proc. Natl.
Acad. Sci. U.S. E2, 1586-1593.

 

 

McNab, J. M., Villemez, C. L., and Albersheim, P.
(1968) Biochem. J. 106, 355-360.

 

Villemez, C. L., Swanson, A. L., and Hassid, W. Z.
(1966) Arch. Biochem. Biophys. 116, 446-452.

 

Liu, T. Y., Elbein, A. D., and Su, J. C. (1966)
Biochem. Biophys. Res. Commun. E, 650-657.

 

Kauss, H., and Hassid, W. Z. (1967) J. Biol. Chem.

 

Kauss, H., Swanson, A. L., Arnold, R., and Odzuck,
W. (1969) Biochim. Bigpgys. Acta 192, 55-61.

 

Villemez, C. L., Liu, T. Y., and Hassid, W. Z.
(1965) Proc. Natl. Acad. Sci. U.S. 21, 1626-1632.

 

Macmillan, J. D., Phaff, H. J., and Vaughn, R. H.
(1964) Biochemistgy 2, 572-578.

 

Lennarz, W. J., and Scher, M. G. (1972) Biochim.
Biophys. Acta 265, 417-441.

 

Kjosbakken, J., and Colvin, J. R. (1973) in Eig-
genesis of Plant Cell Wall Polysaccharides
(Loewusi:F., edi), pp.*361-37l, Academic Press,
New York.

 

Jung, P., and Tanner, W. (1973) Eur. J. Biochem.
37, 1-6.

 

Babczinski, P., and Tanner, W. (1973) Biochem.
Biopgys. Res. Commun. E1. 1119-1124.

 

 

 

 

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

171

Kauss, H. (1969) FEBS Letters 2, 81-84.

 

Villemez, C. L., and Clark, A. F. (1969) Biochem.
BiOphys. Res. Commun. fig. 57-63.

 

Villemez, C. L. (1970) Biochem. Biophys. Res. Commun.
‘12, 636-641.

 

Storm, D. L., and Hassid, W. Z. (1972) Plant

Alam, S. S., Barr, R. M., Richards, J. B., and
Hemming, F. W. (1971) Biochem. J. 121, 19P.

 

Clark, A. F., and Villemez, C. L. (1973) FEBS
Letters 22, 84-86.

Forsee, W. T., and Elbein, A. D. (1973) J. Biol.
Chem. 248, 2858-2867.

 

Tsai, C. M., and Hassid, W. Z. (1971) Plant Physiol.
11, 740-744.

 

Liu, T. Y., and Hassid, W. Z. (1970) J. Biol. Chem.
245, 1922-1925.

 

Heller, J. S., and Villemez, C. L. (1972) Biochem. J.
128, 243-252.

 

Vongerichten, E. (1901) Ann. Chem. 218, 121-136.

 

Sandermann, A., and Grisebach, H. (1970) Biochim.
Biophys. Acta 208, 173-180.

 

Hudson, C. S. (1949) in Advances in Carbohydrate
Chem. (Pigman, W. W., and Wolfrom, M. L., eds.),
Vol. 4, pp. 57-74, Academic Press, New York.

 

Hart, D. A. (1969) Ph.D. Thesis, Michigan State
University, East Lansing, Michigan.

Bell, J. D., Isherwood, F. A., and Hardwick, N. E.
(1954) J. Chem. Soc., 3702-3706.

 

Bacon, J. S. D. (1963) Biochem. J. fig. 103P.

 

Ovoda, R. G., Vaskovsky, V. B., Ovodov, Y. S.
(1968) Carbohyd. Res. g, 328.

 

Williams, D. T., and Jones, J. K. N. (1964) Can. J.
Chem. 1E, 69-72.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

172

Beck, E., and Kandler, O. (1965) Z. Naturforsh
ZOB, 62-67.

Duff, R. B. (1965) Biochem. J. 21, 768-772.

 

Hart, D. A., and Kindel, P. K. (1970) Biochem. J.
116, 569-579.

 

Beck, E. (1967) Z. Pflanzenphysiol. Bd. El, 444-461.

 

Hart, D. A., and Kindel, P. K. (1970) Biochemistgy
2, 2190-2196.

Sandermann, H., Tisue, G. T., and Grisebach, H.
(1968) Biochim. Biophys. Acta 165, 550-552.

 

Kindel, P. K., and Watson, R. R. (1973) Biochem. J.
133, 227-241.

 

Gustine, D. L., and Kindel, P. K. (1969) J. Biol.
Chem. 244, 1382-1385.

 

Kindel, P. K., Gustine, D. L., and Watson, R. R.
(1971) Fed. Proc. 29, 1117.

 

Kindel, p. K. (1973) in Biogenesis of plant Cell
Wall Polysaccharides (Loewus, F., ed.), PP. 85-
94, Academic Press, New York.

Pan, Y. T. (1974) Fed. Proc. 32, 1445.

 

Bray, G. A. (1960) Anal. Biochem. E, 279-285.

 

Partridge, S. M. (1949) Nature, Lond., 164, 443.

Trevelyan, W. E., Procter, D. P., and Harrison,
J. W. (1950) Nature, Lond., 166, 444-445.

Anderson, D. M. W., and Stoddart, J. F. (1966)
Carbohydrate Res. 2. 104-114.

 

Gustine, D. L., Yuan, D. H., and Kindel, P. K.
(1975) Arch. Biochem. BiOphys., in press.

 

Anderson, D. M. W., Bews, A. M., Garbutt, S., and
King, N. J. (1961) J. Chem. Soc., 5230-5234.

 

Villemez, C. L. (1974) Arch. Biochem. Biophys. 165,
407-412.

 

 

 

173

118. Vongerichten, E., (1902) Ann. Chem., 321, 71-83.

 

119. Leinbach, E. L., and Kindel, P. K. (1974) Abstracts

 

of Papers, 168th National Meeting, Biol. 149,
American Chemical Society, Washington, D. C.

 

 

120. Ovodou, Y. 8., Ovodovo, R. G., Bondarenko, D. D.,
and Krasikovo, I. N. (1971) Carbohydrate Res.
Lg, 311-318.

 

 

 

 

rs! .M 4.5.153. $le any“, . ,