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This is to certify that the

dissertation entitled
Purification and Structural and Kinetic
Characterizations of a-Galactosidases

A and B from Human Liver
presented by

Francis E. Wilkinson

has been accepted towards fulfillment
of the requirements for

Ph.D. degree inBiochemistm

 

Date October 11, 1985

 

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

RETURNING MATERIALS:
1V1531_] Place in book drop to
remove this checkout from
w your record. FINES will
- be charged if book is
returned after the date
stamped below.

 

 

 

 

 

 

PURIFICATION AND STRUCTURAL AND KINETIC CHARACTERIZATIONS

OF a-GALACTOSIDASBS A AND 8 FROM HUMAN LIVER

by

Francis Eugene Wilkinson

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1985

 

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ABSTRACT

Purification and Structural and Kinetic Characterization

of a-Galactosidases A and B from Human Liver

Francis Eugene Wilkinson

a-Galactosidases A and B were purified to homogeneity
in higher yields than has been reported for either enzyme
from any other human tissue by a combination of a newly
developed procedure to separate the a-galactosidases and
previously published procedures. The d—galactosidase A
preparation, but not B preparation, hydrolyzed a natural
substrate, Gal<fl—4GalBl-461081*1'-Ceramide, which indicates
that the new method for separating the a-galactosidases is
successful (Previous workers separated most of the
a-galactosidase A from crgalactosidase B). The subunit
molecular weights are 47,800 and 46,800 daltons for the A
and B enzymes, respectively.

The amino acid compositions of both a-galactosidases
were determined and were found to be fairly similar. Both
a—galactosidases were carboxymethylated and then submitted
for Neterninal sequencing. Each preparation yielded a
unique N-terninal sequence, indicating that they are
honodilers (the native molecular weights for both proteins

had previously been shown to be about 100,000 daltons).

Nearly complete homology was found in the N-terminal region
of these two proteins. These N-terminal sequences
represent the first information obtained about the amino
acid sequence of any human lysosomal glycosidase.

Tryptic peptides of both ckgalactosidases were
generated and separated by reversed-phase high performance
liquid chromatography. Sufficient quantities of only
crgalactosidase B peptides were obtained for Neterminal
sequencing. Peptides accounting for twelve per cent of
(rgalactosidase B were sequenced. None of these
a—galactosidase B peptides was found to have a sequence
similar to the crgalactosidase A tryptic peptides
determined by others, suggesting that the homology between
crgalactosidases A and B is possibly restricted to the
N-terminal region.

Some kinetic parameters of both enzymes were
determined. The specific activities were determined to be
45.2 and 4.18 umoles of 4—methylumbelliferyl-a
-Q-galactoside (4-MU—d-Gal) hydrolyzed per minute per mg of
the A and B enzymes, respectively. a-Galactosidases A and B
hydrolyzed the 4—MU—a-Gal with Km values of 1.83 and 13.1
mM, respectively. The Vmax were determined to be 52.9 and
13.9 umol of 4-Mka—Ga1 hydrolyzed per minute per mg of
protein of the A and B enzymes, respectively. Both
a—galactosidases A and B were competitively inhibited by
ngalactose with respect to the hydrolysis of 4—MU—a—Gal;

Ki values of 16.7 and 27.9 mM, respectively, were

observed. N-Acetyl-Q;galactosamine was found to be a
competitive inhibitor of a-galactosidase B with a Ki of
1.65 mM, but it did not inhibit a—galactosidase A.

Conduritol C epoxide, a structural analogue of
wgfgalactose, inactivated argalactosidase A, and very
slowly B, in a time-dependent fashion. g—Galactose, a
competitive inhibitor of a-galactosidase A. blocked the
time-dependent inactivation of (rgalactosidase A by
conduritol C epoxide. These data strongly support the
conclusion that conduritol C epoxide is a suicide inhibitor
of a—galactosidase A. This is the first demonstration of a
suicide inhibitor of any lysosomal glycosidase. Conduritol
C epoxide appeared to be a competitive inhibitor of

crgalactosidase A with a Ki of 330 mM.

This thesis is dedicated to my parents, Mr. and Mrs.
Gilbert Wilkinson and to my brothers and sisters, whose
love I cherish and whose finanacial support is very

gratefully acknowledged.

I am also dedicating this work to those suffering from
Fabry's disease and the other lysosomal storage diseases

who have been praying for relief.

 

 

 

ACKNOWLEDGEMENTS

I would like to thank my major professor, Dr. Charles
Sweeley, for his support and intellectual stimulation. 1
would also like to thank Dr's. Richard Anderson, William
Deal, Walter Esselman, and William W. Wells for their input
as committee members. A special thanks goes to Dr. James L.
Fairley for his many suggestion during this project.

I would also like to thank the following post—docs,
graduate students, and others who worked with me in Dr.
Sweeley's lab over the past few years for their scientific
insights and friendship: Matt Anderson, Diane Bloomer, John
Burczak, Dorothy Byrne, Margaret Kabalin, Kimihiro Kanemitsu,
Jos Kint, Norm LeDonne, Mike Lockney, Richard Lynch, Lyla
Melkerson, Barb Myszkiewicz, Chris Marvel, Fumito Matsuura,
Joe Moskal, Mitsuru Nakamura, Rob Soltysiak, Mike Thome, Mr.
Wang, and Cliff Wong.

1 would like to thank others in the department who were
especially helpful: Doris Bauer, Betty Brazier. Steven
Brookes. Lydia Coleman, David DeWitt, Theresa Fillwock, Susan
Leavitt, Arlyne Garcia—Perez, and Dr. Clarence Suelter.

A special note of thanks goes to Dr. Shaun D. Black,
formerly at the University of Michigan but now at Ohio State
University, who gave me many helpful suggestions for the
sequencing work.

I would also like to thank all my friends both back home
and here in Michigan who have made these seven years Just
seem to fly by. And I should also thank all my
ex—girlfriends who married the proverbial someone else so I
could have enough time to do all this work.

 

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TABLE OF CONTENTS

page
ABSTRACT ...............................................
DEDICATION .............................................
ACKNOWLEDGEMENTS .......................................
LIST OF TABLES ........................................ vii
LIST OF FIGURES ....................................... viii
ABBREVIATIONS ......................................... ix
REVIEW OF THE LITERATURE .............................. 1
The Physiological Importance of Lysosomal Enzymes. 1
Biosynthesis of Lysosomal Enzymes ................. 6
Therapeutic Approaches ............................ 19
Organ Transplantation .......................... 19
Enzyme Replacement Therapy ..................... 21
a ~Galactosidases A and B: Association with Disease
States, Function, Biosynthesis, and Structures. 22
Fabry's Disease and its Association with
d—Galactosidase A Deficiency ................. 22
Purification of the a-Galactosidases ........... 26
Oligosaccharides on the d—Galactosidases ....... 27
Biosynthesis of (kGalactosidase A and B ........ 28

Biological Significance of d—Galactosidase B... 29

STATEMENT OF THE PROBLEM .............................. 35
MATERIALS AND METHODS ................................. 37
Materials ......................................... 37
Methods ........................................... 41
Synthesis of the Affinity Ligand ............... 41

Enzyme Assays .................................. 47

iv

 

Purification of<x~Galactosidases A and B from

Human Liver .................................. 49
General Methods ............................. 49
Affinity Chromatography ..................... 51

Gel Electrophoresis ............................ 53
Methods for Protein Determination .............. 54
Protein Alkylation ............................. 54
Carboxymethylation .......................... 54
Pyridethylation ............................. 54
Trypsin Digestion .............................. 55
HPLC Separation of Peptides .................... 55
Induction of Antibodies ........................ 57
Polyclonal .................................. 57
Monoclonal .................................. 57
Synthesis of Conduritol C Epoxide .............. 60
RESULTS ............................................... 69
Synthesis of the Affinity Ligand ................... 69

Purification of d—Galactosidases A and B from
Human Liver ....................................... 77

Chemical Characterizations of the Purified

a-Galactosidases A and B .......................... 87

Antibody Production ................................ 102

Polyclonal ...................................... 102

Monoclonal ...................................... 102

Synthesis of Conduritol C Epoxide .................. 110
1

Interpretation of fl-NMR Spectra of
Conduritol C and Conduritol C Epoxide .......... 123

Substrate and Kinetic Sudies of a—Galactosidases
A and B ........................................... 137

Hydrolysis of Gb03e30er ......................... 137

V

 

The Kinetics of wGalactosidase A ............... 142

The Kinetics of a—GalaCtosidase B ............... 142
Inhibition by Conduritol C Epoxide .............. 155
Tritiated Conduritol C Epoxide .................. 161
DISCUSSION ............................................ 169
The Purification Procedure ......................... 169
General Methods ................................. 169
Affinity Chromatography ......................... 170
Physical Characterization of the Purified
a—Galactosidases ................................ 174
Kinetic Characterizations .......................... 175
Inhibition with Conduritol C Epoxide ............... 177
BIBLIOGRAPHY .......................................... 181

vi

 

LIST OF TABLES

page

Active Site Sequences of Non~lysosomal

Glycosidases ................................... 5
Biosynthesis of Lysosomal Enzymes ................. 7
The Purification of arGalactosidases A and B from

Human Liver .................................... 78
Amino Acid Composition of Human Lysosomal Enzymes. 90
N—terminal Sequences of Human Lysosomal

Glycosidases ................................... 93
Peptides Sequences of Human a—Galactosidases

A and B ........................................ 109

Melting Points of Intermediates and Related Compounds
in the Synthesis of Conduritol C Epoxide ....... 113

vii

 

10.

11.

12.

13.

15.

16.

17.

18.

LIST OF FIGURES

The carbohydrate processing events ................
The major types of N—linked oligosaccharides ......
Glycoconjugates catabolized by a—galactosidase A..
Degradation of neutral glycoconjugates.- ...........
Glycoconjugates catabolized by a—galactosidase 8..
Synthesis of the affinity ligand ..................
Synthesis of conduritol C epoxide .................

13C—NMR spectrum of the Affinity Ligand .........

13C—NMR spectrum of a— and B—galactosylamine....

Coupled NMR spectra (1H—13C) of the affinity
ligand the galactosylamines ....................

Column chromatography of a—galactosidases A and B
on DE—52 .......................................

Column chromatography of a—galactosidase A on
Sephadex G—150 .................................

Dual affinity chromatographic separation of
a—galactosidases A and B .......................

Assessment of purity and molecular weight
determination of human liver d—galactosidases
A and B by SDS—gel electrophoresis .............

page
11

15

25

32

34

43

62

71

73

75

80

84

86

89

Primary separation of tryptic peptides of human liver

wgalactosidase A ..............................

95

Primary separation of tryptic peptides of human liver

(rgalactosidase B ..............................

Repurification of an a—galactosidase A peptide...

97

99

Primary separation of tryptic peptides of human liver

a—galactosidase B ..............................

101

The separation of two peptides of a—galactosidases B

for amino acid sequencing ......................

viii

104

 

 

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

The purification of a major peptide from a mixture of
three d—galactosidase B peptides for amino acid
sequencing ..................................... 106

The purification of a peptide for amino acid
sequencing from a complex mixture of
d—galactosidase B peptides ..................... 108

Infrared spectra of conduritol C tetraacetate ..... 112

Infrared spectra of conduritol B and conduritol C. 115

Mass spectra of conduritol B and conduritol C ..... 118

Infrared spectrum of conduritol C epoxide ......... 120

Mass spectra of conduritol B epoxide and

conduritol C epoxide ........................... 122
1H—NMR spectrum of conduritol C ................... 125
Decoupled 1H—NMR spectra of conduritol C .......... 127
1H—NMR spectrum of conduritol C epoxide ........... 132

Decoupled 1H—NMR spectra of conduritol C
epoxide ........................................ 134

Three—dimensional structures of conduritol C and

conduritol C epoxide ........................... 139
Hydrolysis of GbOse3Cer by a—galactosidases
A and B ........................................ 141

Lineweaver-Burk plot of the hydrolysis of
4—MU-a-Gal by crgalactosidase A ................ 144

The competitive inhibition of galactose of
4—MU-a-Gal hydrolysis by a—galactosidase A ..... 146

The effect of GalNAc on the hydrolysis of 4—MU—a—Gal
by wgalactosidase A ........................... 148

The competitive inhibition by conduritol C epoxide
of 4—MU- wGal hydrolysis by a—galactosidase A...150

Lineweaver—Burk plot of the hydrolysis of 4—MUai—Gal
by d-galactosidase B ........................... 152

Competitive inhibition by galactose of 4-MU—q—Gal
hydrolysis by a—galactosidase B ................ 154

Competitive inhibition by GalNAc of 4—MU—a—Gal
hydrolysis by (rgalactosidase B ................ 157

ix

40.

41.

42.

43.

Time—dependent inhibition of a—galactosidases A and B
by conduritol C epoxide ........................ 159

Protection of d—galactosidase A by galactose from
inhibition by conduritol C epoxide ............. 163

Purity determination of tritiated conduritol C
epoxide ....................................... 165

Inhibition of a—galactosidase A by tritiated
conduritol C epoxide .......................... 168

 

 

CBE

CCE

Con A

DIEM .
DE-52

DTT

FCS

wGal A
ckGal B
GalNAc
GbOse3Ceramide
gc-ms
GlcNAc

6M2

HFBA

HPLC

4—MU
4-MU-chal

o-Nka-GalNAc

PEG
PMSF
p—NP

p—NP— Ot-GalNAc

TFA

Abbreviations

conduritol B epoxide

conduritol C epoxide

Concanavalin A-Sepharose

Dulbelco's minimal essential medium
DEAE—cellulose

dithiothreitol

fetal calf serum

a—galactosidase A

a~galactosidase B
N—acetyl—Qfgalactosamine
Gala1-4Ga181-4qu31—1'-Ceramide

gas chromatography-mass spectroscopy
N—acetyl~gfglucosamine

The Tay-Sach's Ganglioside
heptafluorobutyric acid

high performance liquid chromatography
4—Methylumbelliferyl
4—Methylumbelliferylax—Q:Galactoside

o—Nitrophenyl—2-acetamido-2—deoxy-a
—Q:Galactoside

polyethyleneglycol
phenylmethylsulfonylfluoride
p—nitrophenol

p-Nitrophenyl~2—acetamido-2—deoxy-a
—Q;galactoside

trifluoroacetic acid

xi

'f
I m

"

1“

7|.

LITERATURE REVIEW

The Physiological Importance of Lysosomal Enzymes The
lysosome was recognized by deDuve as a distinct organelle
in 1955 (1,2), and he postulated that its complement of
hydrolytic enzymes can degrade any biomolecule (3).
Several of these enzymes were studied by the use of

synthetic substrates, while their ig_vivo functions were

 

often discovered through correlations with disease states.
An individual occasionally came to the attention of the
medical profession because of life—threatening conditions
which were due to a deficiency of one or more lysosomal
enzymes. (Most of the deficient enzymes are glycosidases,
but not all of them are even hydrolases: the terms
lysosomal enzyme and hydrolase will be used interchangeably
here). Such a deficiency commonly results in the
accumulation in the lysosome of the natural substrate(s) of
the enzyme. The accumulated material can disrupt normal
cellular function, which is why these deficiencies are
called lysosomal storage diseases. The lysosomes of some
diseases have a characteristic appearance depending upon
what material has accumulated. In several diseases, the
chemical structure of the accumulated material has been
determined and a correlation was made of an enzyme activity
found in normal tissue capable of degrading the material

with a deficiency of the enzyme in the tissues of the

afflicted individual (4). That a disease has not yet been
associated with the deficiency of a lysosomal enzyme may
reflect the severity of its absence so that the infant dies
either in.utero or shortly after birth. This is the case
in caprine B-mannosidosis (5,6).

The genetics of many of the lysosomal storage diseases
have been determined, and all studied to date have proven
to be recessive traits; two of these disorders, Fabry's and
Hunter's diseases, are sex—linked (7). None of these
diseases can be successfully managed. Studies are
continuing on biochemical characterizations of the
individual hydrolases as well as determinations of the
biosynthesis and normal function of each in order to
determine the molecular basis for the deficiency. The
results of these studies may be useful in effecting a cure
for these diseases.

A logical first step in the biochemical
characterization of a lysosomal hydrolase is the
purification of the enzyme so that a comparison can be made
between the normal and mutant enzymes. The following
lysosomal enzymes, among others, have been purified to
apparent homogeneity from human tissues:
N-acetylgalactosamine 6-sulfate sulfatase (8),
crN-acetylglucosaminidase (9), acid lipase (10),
arylsulfatases A (11) and B (12), aspartylglucosaminidase
(13), cathepsin D (14), crL—fucosidase (15),

a~galactosidases A (16) and B (17), galactocerebrosidase

 

‘/

I'V

(18), B—galactosidase A (19), glucosylceramidase (20),

2
B-glucuronidase (21), a—glucosidase (22), B-hexosaminidase
(23), and sphingomyelinase (24). Several of these enzymes
have been partially chemically characterized. Some of
these characterizations have included amino acid
composition (ll-15, 19, 21, and 24), partial amino acid or
cDNA sequence (25-27), carbohydrate composition (28,29), or
a general structure of the carbohydrate moieties attached
to the enzyme (30-33).

In general, these hydrolases degrade biomolecules that
are delivered to the lysosome by receptor—mediated
endocytosis (34) or by other mechanisms. The digestion
products are amino acids, simple sugars, lipids, and
nucleotides and are thought to be removed from the cell by
a process similar to a reversal of receptor—mediated
endocytosis.

These hydrolases generally have an acidic pH Optimum,
and ip_glgg_measurements have shown that lysosomes have an
internal pH of 4.7 to 4.8 (35). Some of the hydrolases
that degrade neutral glycosphingolipids and gangliosides
utilize a specific activator protein that appears to be
involved in both solubilizing the lipid and stabilizing the
glycosidase (36). The AB variant of 6M2 gangliosidosis
(37) and a variant of metachromatic leukodystrophy (38,39)
are due to deficiencies of the respective activator
proteins.

The kinetic parameters and substrate specificities of

4
many lysosomal hydrolases have been determined (4). The
determination of the mechanism of catalysis is especially
important since the inability of these enzymes to perform
properly has such drastic consequences. One of the methods
of determining the mechanism of catalysis has involved
suicide inhibitors. These have been used to isolate the
active site peptide of a few non<lysosomal glycosidases
(40-44): the results of these (Table 1) and other studies
on the mechanism of glycosidases have been reviewed (45).
Among the lysosomal glycosidases, van Diggelen gt_al; (46)
have reported an ig_gigg_inactivation of lysosomal
B-galactosidase by B-galactosyl p—nitrophenol triazene
which had previously been shown to be a suicide inhibitor
of §;_ggll_B-galactosidase (44). They (46) also showed
inhibition of the lysosomal B-glucosidase with the
B—glucosyl derivative. It has been suggested that
conduritol B epoxide is a suicide inhibitor of the
lysosomal enzyme B-glucocerebrosidase because injection of
conduritol B epoxide into mice significantly reduces the
levels of this enzyme in the brain, liver, and spleen (47)
and because it is a suicide inhibitor of two non-lysosomal
glycosidases (40,42). Conduritol B epoxide has been shown
to be a competitive inhibitor of calf brain B-glucosidase
(48). But to date no compound has been shown to be a
suicide inhibitor of any lysosomal glycosidase, nor has the
amino acid sequence around the active site of any lysosomal

glycosidase been determined.

Table 1. Active site sequences of non-lysosomal hydrolases.

1. Val—Met-Ser—Asngrp—Ala-Ala-His—His-Ala—Gly-Val—Ser—

2. Ile-Thr-Glx-Glx-Gly—Val-Phe—Gly—A§p;Ser—Glx—

3. lle-Agpret-Asn—Gln-Pro-Asn-Ser—Ser

4. Gln—Ala-Thr-Asn-Arg—Asn—Thr—Asp—Gly—Ser—Thr-Aspryr

5. Thr-Thr—Ala-Thr-Asp—lle—lle—Cys-Pro—M§£;Tyr-Ala-Arg

1. B—Glucosidase A from Aspeggillus wentii (40). The
active site label was conduritol B epoxide.

2. B-Glucosidase A from bitter almonds (41). The active
site label was 6-bromo-3,4,5~trihydroxy cyclonex-l-ene

oxide.

3. Rabbit intestinal sucrase'isomaltase complex (42).
The active site label was condurtiol B epoxide.

4. Hen egg lysosyme (43). The active site label was
2',3'-epoxypropyl~0—B-N-acetyl—Q—glucosaminyl*B—
(1-4)-N—acetyl—Qfglucosaminide.

5. E; coli B~galactosidase (44). The active site label
was B—ngalactopyranosylmethyl p—nitrophenyl triazene.

The underlined amino acid was derivatized by the
respective active site label.

6

Biggygghesis of Lysosomal Enzymes. The biosynthesis of the
lysosomal hydrolases is a complicated process that involves
at least two peptide cleavages, the action of
glycosyltransferases and glycosidases, and includes the
proper sorting of the enzymes within the endomembrane
system to either the lysosome or to secretory vesicles
(Table 2 lists the main steps of lysosomal enzyme
biosynthesis). Understanding the biosynthesis of these
enzymes is epecially important since a few of the lysosomal
storage disorders, l-cell disease and pseudo—Hurler

dystrophy (49,50) and a variant of GM gangliosidosis

2
(51-53), are due to an improper biosynthesis of the
respective lysosomal hydrolase. The early stage of the
biosynthesis of lysosomal enzymes is exactly the same as
that of secreted mammalian proteins, and is similar to the
synthesis of some secreted bacterial proteins (54). The
mRNA's coding for the pre-pro form of the lysosomal
hydrolases are translated on cytoplasmic ribosomes until a
leader sequence of about twenty, mostly hydrophobic amino
acids comprises the nascent chain (55). At this point a
complex of six proteins known as the signal recognition
particle, SRP, binds the leader sequence with high affinity
and interacts with the ribosome, thereby stopping further
translation (56). The complex than binds with a docking
protein (57) on the rough endoplasmic reticulum, the SRP is

released, the nascent peptide goes through a pore in the

membrane to the lumen of the endoplasmic reticulum, and

' fiyni)

Table 2. Biosynthesis of Lysosomal Enzymes.

1) Transcription of the gene coding for the lysosomal
enzyme and processing of the hnRNA to mRNA. These events
occur in the nucleus.

2) Translation begins on a cytoplasmic ribosome, and as
soon as the leader sequence has been translated, the Signal
Recognition Particle, SRP, binds the ribosome, and
translation ceases. The SRP-ribosome-mRNA-nascent protein
binds with a docking protein on the endoplasmic reticulum,
and translation resumes with the nascent protein being in
the lumen of the endoplasmic reticulum.

3) The leader sequence is proteolytically removed,and the
protein is glycosylated during translation. Both of these
events occur in the endoplasmic reticulum.

4) Carbohydrate processing events converting the

610 Man GlcNAc oligosaccharide to the high mannose,
complex, or hyLrid chains occur next. The enzymes
responsible for the initial reactions such as
a-glucosidases l and II are in the endoplasmic reticulum,
while those responsible for the last events such as the
glycosyltransferases involved in the formation of complex
chains are in the Golgi apparatus.

5) If the enzyme has subunits, the subunits presumably
associate in the Golgi apparatus before transport to
lysosome.

6) The lysosomal enzyme is then transported to the
lysosome where a C- or N-terminal peptide is removed from
the enzyme, and this cleavage is sometimes necessary for
the enzyme to be functional.

 

 

8
translation continues. An as yet unidentified protease
cleaves the leader sequence of hydrophobic amino acids from
the N—terminus shortly after translation resumes, yielding
the pro~form of the enzyme (55). The balance of the
nascent protein is then translated directly into the lumen
of the endoplasmic reticulum. Cell-free translation of
mRNA of secreted proteins in the absence of microsomes
results in the translation of the pre—pro form of the
lysosomal enzyme that cannot be further processed (55), the
implications of which will be discussed later.

Studying the carbohydrates and processing events of
the carbohydrate moieties on the lysosomal enzymes is of
special importance because 1) two lysosomal storage
diseases are due to a deficiency of a processing enzyme, 2)
the carbohydrates are involved in directing the lysosomal
enzymes to their proper cellular location, and 3) the
carbohydrates are involved in maintaining the lysosomal
enzymes in circulation. During translation the protein is
N-glycosylated on certain asparagine residues. Lennarz and
coworkers (58) found that a sequence of —Asn—X—Ser(Thr)- (X
probably cannot be Asp) is required for glycosylation
although not all such sequences are glycosylated.
Assessments of the three-dimensional structures of the
proteins around possible glycosylation sites showed that
those sites in B—turns or loops tend to be glycosylated,
while those in a-helices are not (59). Other factors may

be involved because bovine pancreatic ribonucleases A and B

8
translation continues. An as yet unidentified protease
cleaves the leader sequence of hydrophobic amino acids from
the N—terminus shortly after translation resumes, yielding
the pro-form of the enzyme (55). The balance of the
nascent protein is then translated directly into the lumen
of the endoplasmic reticulum. Cell-free translation of
mRNA of secreted proteins in the absence of microsomes
results in the translation of the pre—pro form of the
lysosomal enzyme that cannot be further processed (55), the
implications of which will be discussed later.

Studying the carbohydrates and processing events of
the carbohydrate moieties on the lysosomal enzymes is of
special importance because 1) two lysosomal storage
diseases are due to a deficiency of a processing enzyme, 2)
the carbohydrates are involved in directing the lysosomal
enzymes to their proper cellular location, and 3) the
carbohydrates are involved in maintaining the lysosomal
enzymes in circulation. During translation the protein is
N-glycosylated on certain asparagine residues. Lennarz and
coworkers (58) found that a sequence of —Asn—X—Ser(Thr)- (X
probably cannot be Asp) is required for glycosylation
although not all such sequences are glycosylated.
Assessments of the three—dimensional structures of the
proteins around possible glycosylation sites showed that
those sites in B-turns or loops tend to be glycosylated,
while those in a-helices are not (59). Other factors may

be involved because bovine pancreatic ribonucleases A and B

9
have the same amino acid sequence, but only the B form is
glycosylated.

An oligosaccharide is transferred §n_bloc from a

 

dolicholpyrophosphate lipid intermediate to the asparagine
by a membrane—bound enzyme. The transferred oligosaccharide
contains three glucosyl, nine mannosyl, and two
N—acetylglucosaminyl residues (60,61) (Figure 1 Structure
A). Subsequent glycoprotein processing, which includes the
removal and/or addition of sugars on the core
oligosaccharide, commences shortly after N—glycosylation.
The oligosaccharide processing generally results in various
kinds of high mannose chains, complex chains which usually
indicate the presence of sialic acid, or hybrid chains
which are a combination of high mannose and complex (Figure
2 shows representative examples of all three types of
chains). All three types of oligosaccharides have been
found on lysosomal hydrolases. The control mechanisms for
determining which types of oligosaccharides will be formed
have not yet been determined, but data support two possible
models. The first is that the control is inherent in the
conformation of the protein (62) such that some high
mannose chains become inaccessible to the processing
enzymes because of the folding of the nascent protein

(63). The second is that the complement of processing
enzymes in the respective cell type (64) varies among the
cell types so that the enzymes themselves regulate the

extent of processing. The main processing events and their

 

 

 

 

 

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regulation are presented briefly below.

The first step in the processing of N—linked
carbohydrates is the removal of the three glucosyl units
from the tetradecasaccharide by two unique enzymes,
a-glucosidases I and II. a—Glucosidase I removes the
terminal glucosyl residue and (kglucosidase II removes the
two remaining arglucosyl residues (Figure 1 Structure A to
B) (61,65). Both of these a—glucosidases have been
partially characterized and are localized in the rough
endoplasmic reticulum. Most N—linked oligosaccharides
studied to date are normally processed in this fashion, but
an exception is the human myeloma IgD:WAH in which there is
incomplete removal of the internal glucosyl residue (66).

High mannose chains result from the removal of few, if
any, a—mannosyl residues from the oligosaccharide by
a-mannosidase I (Figure 1 Structure B to C) (67).

The mannose-G-phosphate recognition system has been of
intense research interest for several years and has
recently been elucidated. In brief, primarily in
fibroblasts and a few other tissues, a GlcNAc-
phosphotransferase in the Golgi apparatus (68) transfers
GlcNAc-phosphate from UDP—GlcNAc to carbon six of one or
two of any of five mannosyl residues on the high mannose
chains (69) (Figure 1 Structure B the starred positions)
after the three glucosyl residues have been removed. This
GlcNAc~phosphotransferase is highly specific.

Non—lysosomal proteins having a high mannose chain as well

..
11
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17

as heat—denatured lysosomal enzymes are poor substrates for
the enzyme. It has been postulated that a structural
determinant in the protein portion of lysosomal hydrolases
is required for recognition by the GlcNAc-
phosphotransferase (70). This GlcNAc-phosphotransferase is
deficient in I-cell disease (49) and very low in
pseudo-Hurler dystrophy (50), two lysosomal storage
diseases in which functional lysosomal enzymes including
B—hexosaminidase and arylsulfatase A are secreted by
fibroblasts but are not retained by the cells (71). The
GlcNAc is removed by a Golgi apparatus—associated
a-N-acetyglucosaminylphosphodiesterase (68). The
mannose-B-phosphate then binds to a receptor protein of
Mr=215,000 (72,73) that directs the hydrolase to the
lysosome where it is thought that acid phosphatase removes
the phosphate from the mannose—6—phosphate (74). This
mannose-6-phosphate receptor also exists on the cell
surface and functions in directing extracellular
mannose-6-phosphate-containing lysosomal hydrolases to the
lysosomes via receptor—mediated endocytosis. It is
important to remember that this mannose—G-phosphate
recognition system functions mainly in fibroblasts because
l-cell patients have normal levels of lysosomal hydrolases
in other cell types (75,76). An additional mechanism of
transporting these enzymes to lysosomes must operate in
tissues other than fibroblasts. In this connection, a

second mannose-B—phosphate receptor of Mr=46,000 has

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18
recently been reported (77).

The complex chains are formed by a series of several
reactions. The first is the removal of the foura 1—2
linked mannosyl residues by a—mannosidase I (Figure 1
Structure B to C) (67), which is a membrane-bound enzyme
localized in the Golgi apparatus. The next step is the
addition of a GlcNAc residue by GlcNAc transferase I
(Figure 1 C to D), which is a necessary step for the
formation of both complex and hybrid carbohydrates (78).
Structure D shown in Figure 1 is necessary for the action
of (rmannosidase II, which removes two of the remaining
a-mannosyl residues (79) so that Structure D can be a
substrate for successive GlcNAc, galactosyl, sialyl, and
fucosyl transferases resulting in complex chains (80)
(Figure 2 Structure C). Structure D in Figure 1 can also
be the substrate for GlcNAc transferase III with Structure
F being the product of the reaction; this last structure
can be the substrate of the glycosyltransferases listed
above which results in a hybrid carbohydrate (Figure 28).

Once processing of the carbohydrate moieties, whether
high mannose or complex, has been completed, a high
molecular weight form of the enzyme can be secreted by the
cell, and a lower molecular weight protein is directed to
the lysosome. One or more peptides are generally removed
from the lysosomal hydrolase before its delivery to the
lysosome, and two lysosomal enzymes have been shown to be

formed by the removal of a C—terminal peptide (82).

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19

Many of the lysosomal enzymes are comprised of two or
more subunits, and the assembly of these subunits into
active enzymes is only now being addressed. Subunit
association in the one case studied to date occurs only
among the precursor subunits after most or all of the
carbohydrate processing reactions, but before the final
proteolytic cleavages and shipment of the enzyme to the
lysosome have occurred (51). It has recently been shown
(52,53) that a variant of Tay-Sach's disease is due to an
improper association of the cxand B chains of
B-hexosaminidase A so that the large molecular weight
precursor of the achain is not converted to the mature
form. The precursor of the mutant a chain appears to have
the same molecular weight and carbohydrate as the normal,
but the reason for the faulty assembly has not yet been

determined.

Therapeutic Approaches

Organ Transplantation. There is currently no effective
treatment for any of the lysosomal storage diseases in
contrast to the treatment of juvenile (Type I) diabetes
with insulin.. One of the current strategies is to reduce
the incidence of the respective diseases by performing
enzyme assays on amniotic cells derived from at-risk
patients and then performing a therapeutic abortion if the

patient lacks the respective lysosomal hydrolase. Research

rush-I .

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20

is continuing for a nore viable alternative.

Progress is especially being made in the area of organ
transplantation. A kidney transplanted into a Fabry's
patient (83) appears to metabolize the lipid that would
otherwise accululate there and cause kidney failure, which
is a lajor cause of death of Fabry's patients. However,
the transplanted kidney lay not secrete enough of the
deficient enzyme, (kgalactosidase A, to metabolize the
lipid accumulating in endothelial cells, the other major
site of accumulation, causing heart disease and death in
some Fabry's patients. Therefore the ideal organ for
transplantation therapy would be one that could be
transplanted with low risk to the patient and that is
either the only organ involved in the disease or one that
is able to secrete enzyne that can be endocytosed by the
organs where the substrate has accumulated.

Recently (84), an eight—nonth-old infant suffering
from Hurler's syndrome received a bone narrow transplant in
the hope that the bone narrow-derived leukocytes would
produce enough of the deficient enzyme, a—L—iduronidase, to
halt both the progression of the disease and reverse the
clinical synptons that had already occurred. The
non-neural tissues appeared nornal up to eleven months
after the bone narrow transplant, but the mental
development of the child was low perhaps due to his having
been kept in isolation for five months or to the inability

of the enzyne to cross the blood-brain barrier. A less

gm

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21

detailed report has appeared (85) about a bone marrow
transplant in an infant with metachromatic leukodystrophy.
This patient was progressing well up to ten months after
the bone marrow transplant. In addition, an adult cat
having arylsulfatase B deficiency received a bone marrow
transplant (86). The cat's physical and biochemical
symptoms were all markedly improved. including its skeletal
abnormalities. Collectively, these experiments indicate
that the plasma forms of these lysosomal hydrolases are
synthesized and secreted by bone marrow~derived cells and
that the plasma forms of these enzymes degrade the material
that would otherwise accumulate in the lysosome, possibly
by being endocytosed by the somatic cells where the
respective substrate would accumulate.

Enzyme ReplaCement Therapy. There are currently two
variations of enzyme replacement therapy. The first
involves the purification, or nearly total purification, of
the respective enzyme from human sources and injecting the
enzyme into the bloodstream. This procedure has both
positive and negative features. The positive side is that
since the enzyme is of human origin, it will not elicit an
antibody response. and the respective enzyme should
function ig_gigg_(87). The drawbacks to this procedure
include 1) the tissue source is frequently autopsy
specimens which are difficult to obtain, 2) there is only a
small quantity of these enzymes in human tissue, 3) the

purification procedures generally give low yields, 4) the

 

 

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22

infused enzyme may not reach the organ(s) where the
substrate has accumulated, 5) the infused enzymes can be
quickly removed from circulation by the Ashwell receptor
(88), and 6) the enzyme may not be able to cross the
blood—brain barrier. The second general strategy is the
infusion of genetically engineered enzyme. This would
require the isolation of the entire gene coding for the
lysosomal enzyme, which would have to be cloned into a
mammalian cell in order for the requisite carbohydrate
processing (55) necessary for uptake by the respective
cells and possibly for avoiding an immune response.
Another drawback is that neither the amino acid nor cDNA
sequence of any human lysosomal enzyme has yet been
completely determined. but 80% of the sequence of
a—L-fucosidase (25) and lesser amounts of three other human

lysosomal glycosidases have been determined (26—27).

a—Galactosidases A and 8: Association with Disease States,

Function, Biosynthesis, and Structures.

Fabry's Disease and its Association with a—Galactosidase A
Deficiency. Fabry's disease is a fatal, sex—linked
recessive error of glycosphingolipid metabolism. There is
a deficiency of an d—galactosidase so that lipids
accumulate in the tissues of Fabry's patients. This lipid
accumulation is responsible for a variety of ailments

including skin lesions, renal impairments, cardiovascular

 

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23

disease. ocular opacities, cataracts, retinal edema, and
dysfunction of the central nervous system and
gastrointestinal tract. The patient usually dies of kidney
failure in his fourth or fifth decade. Heterozygous
females are affected to a much smaller extent (89). The
accumulated lipid was identified by Sweeley and Klionsky
(90) as ceramide trihexoside, GbOseSCeramide, with
digalactosylceramide as a minor component. Wherrett and
Hakomori (95) found blood group B glycosphingolipids
accumulating in Fabry's patients whose blood type was B
(See Figure 3 for the structures of these and other
glycoconjugates likely to be metabolized by this enzyme).

Later Brady g£_al; (98) correlated the deficiency of
an enzyme, which they called ceramide trihexosidase, with
Fabry's disease. It was initially thought that this
ceramide trihexosidase was a B—galactosidase, since the
non—reducing terminus of ceramide trihexoside is
galactose. However, Kint (99) clearly demonstrated by the
use of a synthetic substrate that the deficient enzyme in
Fabry's disease is an wgalactosidase. Beutler and Kuhl
(100) and Kint g£_al; (101) found that there are two
different a—galactosidases, which they called the A and B
forms. It was reported that the A form is absent while the
B for- persists or even increases in Fabry's disease.

An activator protein, shown to be necessary for the in_
111g_hydrolysis of ceramide trihexoside. has been partially

purified from human liver (36). No case of Fabry's disease

 

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24

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25

 

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26

having a deficient activator protein has yet been reported.
Purification of the (rGalactosidases. Several attempts
were made to purify the two a—galactosidases to determine
if they have a precursor—product relationship. as had been
postulated (101), or are interconvertible (102), and to
determine the lesion responsible for Fabry's disease.

Major contributions towards the purification of these
enzymes were made by Beutler and Kuhl (103), who separated
the two wgalactosidases by DEAR-cellulose chromatography,
which is still used by some of the major investigators in
the field (16,104—106). Hayes and Beutler (107)
purportedly purified from placenta the A enzyme which has a
subunit of Mr=67,500, and a contaminant of Mr=47'000

that appeared as a diffuse band on an SDS-polyacrylamide
gel; this contaminant was probably a—galactosidase A. The
first successful purification of either a—galactosidase was
by Kusiak g£.g;; (17), who obtained the B form from human
placenta. This protein had a subunit Mr=47,700 and was
reported to be a homodimer. Their preparation of the A
form contained many contaminants as revealed by SDS—gel
electrophoresis and had a subunit Mr=57.700 which is
significantly higher than the Mr=49,800 that has since

been determined (16). Dean and Sweeley purified the B form
(104) and almost completely purified the A form (105) from
human liver. They also compared the kinetic parameters and
substrate specificities for both enzymes (108). The first

successful purification of a—galactosidase A was by Bishop

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27

and Desnick (16). Their purification scheme was a
tremendous improvement over others in terms of yield,
purity and the number of steps required; their success was
primarily due to the use of an affinity ligand developed by
Harpaz and Flowers (109) for the purification of coffee
bean a-galactosidase.

Oligosaccharides on the (rGalactosidases. Digestions of
the crgalactosidases with neuraminidase showed that the A
form has sialic acid while the B form does not (110,111).
It was inferred that the B form is a glycoprotein by its
binding to concanavalin A—Sepharose during purification
(17). The biological importance of the carbohydrate
moieties on a-galactosidase A was demonstrated by ig_g3;gL
experiments by Desnick §t_gi; (87). They infused partially
purified human splenic and plasma a—galactosidase A into
two brothers with Fabry's disease to determine: (1) what
effect these enzymes would have on the plasma level of
ceramide trihexoside, (2) if these enzymes would remain in
the plasma, and (3) if the gross symptoms would improve in
either patient. They found that, although the splenic form
has a higher specific activity than the plasma form, the
plasma form persisted much longer in circulation and

cleared much more of the circulating GbOse Cer than did

3
the splenic enzyme. It is thought that the Lg_vivo
efficacy of the plasma form relative to the splenic form

was due to the higher degree of sialylation of the plasma

form. The rapid clearance of the splenic enzyme is

 

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28

consistent with the clearance of desialylated glycoproteins
from serum by the Ashwell receptor (88). These experiments
also demonstrated the potential use of the plasma form of
wgalactosidase A for enzyme replacement therapy in Fabry's
patients.

Biosynthesis of a—Galactosidases A and B. Due to the
biological importance of the carbohydrate moieties of
wgalactosidase A and to determine the biosynthetic
processing events of lysosomal hydrolases, others in this
laboratory studied the carbohydrate processing events of
these two lysosomal glycosidases (30,31). The largest
glycosylated precursor of a—galactosidase A is Mr=58.000;
that of wgalactosidase B is Mr=65.000, while the

molecular weights of the fully processed enzymes are 49,000
and 48,000. respectively, which indicates that one or more
peptides are removed from the precursors of both enzymes.
Half of the carbohydrate chains of crgalactosidase A are
processed to the complex type having a tri— or
tetraantennay structure. while the other half are high
mannose chains with eight or nine mannoses. 0n the other
hand a—galactosidase B has only high mannose chains
containing 7 to 9 mannoses. A phosphorylated biosynthetic
intermediate of a—galactosidase B was found (30) in
fibroblasts but, not surprisingly, such an intermediate was
not found for <rga1actosidase A from Chang liver cells
(31). However, 50% of the a—galactosidase A purified from

human placenta (106) has one or more phosphorylated

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29

carbohydrate chains.
Biological Significance of a—Galactosidase B.
(rGalactosidase B has been studied primarily as an
adjunct to the studies of a—galactosidase A, and in a sense
has been seen as a contaminant that is difficult to
separate from a-galactosidase A. Until recently the
principal biological importance of (rgalactosidase B has
been its demonstration as an wN—acetylgalactosaminidase
(17,112,113). The porcine (114) and human (105) liver
enzymes have been shown to cleave the terminal
(rN—acetygalactosaminyl residue from the Forssman
glycolipid (See Figure 4 for the degradation of the
principal neutral glycosphinglipids by the lysosomal
glcosidases) and is presumably also involved in the
catabolism of blood group A antigens and
wN—acetylgalatosamine in O—linked glycoproteins (See
Figure 5 for the structures of these and other
glycoconjugates likely to be catabolized by
a—galactosidase 8). Julia Frei of this laboratory (119)
showed that the hepatic level of a—galactosidase B in a
patient who died of malignant histiocytosis was either 2 or
30% of normal, depending upon which artificial substrate
was used. The correlation between a-galactosidase B
deficiency and malignant histiocytosis has not been firmly
determined. Glucosylceramide is the only glycoconJugate
found thus far in higher concentration in malignant

histiocytosis relative to a normal control, but the patient

 

 

 

30

did not have any of the recognized forms of Gaucher's

disease.

 

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32

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33

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34

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A:

STATEMENT OF THE PROBLEM

Most of the lysosomal storage diseases have been associated
with a deficiency of one or another specific lysosomal
hydrolase. For most of these enzymes little is known about
the primary sequence, the lesion responsible for the
lysosomal storage disease or the molecular mechanism of
catalysis. Furthermore, our understanding of the
biosynthesis of these enzymes is incomplete. This project
was undertaken to characterize chemically the human
a—galactosidases and to determine their kinetic
properties. The specific goals of this project were as
follows:
a) To purify a—galactosidases A and B from human liver
and to determine enough of the primary sequence of each
enzyme so that cDNA probes based upon the amino acid
sequences could be synthesized and used by others to
isolate and sequence the genes of both a—galactosidases.
b) To induce monospecific antibodies to ~galactosidase
A so that others could study the biosynthesis of
wgalactosidase A.
c) To determine the kinetic parameters of both
a-galactosidases and to synthesize a suicide inhibitor
of one or both enzymes so that information pertinent to

the mechanism of catalysis could be obtained.

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36
These studies should lead to a better understanding of the
structures and mechanisms of catalysis of both
ckgalactosidases. and provide a basis for future
investigators to determine the entire structures of both
enzymes. Conceivably this could lead to an effective

treatment of Fabry's disease.

2'.‘ II 1

MATERIALS

Enzyme Assays

ethylenediamine
N—acetyl—Qfgalactosamine
4-MU—a—QfGalactoside
p—NP—a—GalNAc

Lactosylceramide
GbOSe3Cer

Sodium taurocholate

Preparation of the Affinity

6—aminohexanoic acid
ammonia, anhydrous
benzylchloroformate
CNBr

grgalactose

hydrogen gas

isobutyl chloroformate
Iatrobeads

methanol, anhydrous

Eastman Kodak, Co.
Rochester, NY

Sigma Chemical Co.
St. Louis, MO

Sigma Chemical Co.
St. Louis, MO

Sigma Chemical Co.
St. Louis, MO

Purified by John Burczak

of this laboratory
from bovine liver

Calbiochem
San Diego, CA

Resin

Sigma Chemical Co.
St. Louis, MO

Matheson

East Rutherford, NJ

Aldrich Chemical Co.,

Milwaukee, WI

Sigma Chemical Co.
St. Louis, MO

Sigma Chemical Co.
St. Louis, MO

Airco, inc.
Montvale, NJ

Eastman Kodak, Co.
Rochester, NY

latron Laboratories,

Tokyo, Japan

Inc.

inc.

reagent grade methanol
refluxed over magnesium
turnings and iodine then

redistilled

37

~—_——,

 

Pd on activated charcoal

Sepharose 4—B

Enzyme Purification

Ampholytes (pH 3 to 5)

concanavalin A—Sepharose

DE—52
(DEAE cellulose)

human liver

Hypatite C
(Hydroxylapatite)
a—methylmannoside

PMSF

Sephadex 0—150

Protein Derivitization and Peptide Generation andSeparation

38

Aldrich Chemical, Co., Inc.

Milwaukee, WI

Sigma Chemical Co.
St. Louis, MO

Bio—Rad Laboratories
Richmond, CA

Sigma Chemical Co.
St. Louis, MO

Whatman, Laboratory
Products Inc.
Clifton, NJ

autopsy specimens obtained
through the courtesy of Dr
Kevin Cavanaugh at lngham
County Hospital and Dr.

Harold Bowman at St. Lawrence

Hospital

Clarkson Chemical Co., Inc.

Williamsport, PA

Sigma Chemical Co.
St. Louis, MO

Sigma Chemical Co.
St. Louis, MO

Pharmacia Fine Chemicals,
Piscataway, NJ

Inc.

 

ifiondapak Phenyl column

dithiothreitol

heptafluorobutyric acid

HPLC solvent filters,

FHUP 04700 for organics,
and HATF 04700 for water

Waters Associates
Milford, MA

Boehringer Mannheim
Biochemicals
Indianapolis, IN

Aldrich Chemical Company,
Milwaukee, WI

Millipore Corp.
Bedford, MA

Inc.

 

39

HPLC solvents, water, Burdick and Jackson
acetonitrile, and Laboratories, Inc.
isopropanol Muskegon, MI
1
iodoacetic acid Sigma Chemical Co. g

St. Louis, MO

Sephadex G—50 Pharmacia Fine Chemicals, Inc.
Piscataway, NJ

Synchropak RP—8 column SynChrom,lnc.
250 X 4.1 mm Linden, IN
TPCK—trypsin A generous gift from

Dr. John Wilson of this
department

4—vinyl pyridine Aldrich Chemical Co., Inc.
Milwaukee, WI

Antibody Induction-Polyclonal and Monoclonal

 

albino Swiss mice, females Spartan Animal Services
Michigan State University

aminopterin Sigma Chemical Co.
St. Louis, MO

DMEM KC Biological, Inc.
Lenexa, KS

Fetal Calf Serum KC Biological, Inc.
Lenexa, KS

Freund's Complete Adjuvant Gibco Laboratories
Grand Island, NY

Freund's Incomplete Gibco Laboratories
Adjuvant Grand, Island, NY
glutamine KC Biological, Inc.

Lenexa, KS

horse serum Flow Laboratories, Inc.
McClean, VA

hypoxanthine Sigma Chemical Co.
St. Louis, MO

NCTC 109 MA Bioproducts
Bethesda, MD

POIyethylene Glycol 1000 Baker Chemical Co.
Phillipsburg, NJ

penicillin

Rabbit anti—mouse IgG

SP2/0-Ag14 mouse myeloma
cells

Staphylococcus aureus
cells, heat killed

thymidine

40

KC Biological, Inc.
Lenexa, KS

Miles Laboratories
Elkhart, IN

Cell Distribution Center
of the Salk Institute
LaJolla, CA

Kindly provided by
Dr. William Smith
of this department

Sigma Chemical Co.
St. Louis, MO

Synthesis of Conduritol C Epoxide

conduritol B and
conduritol B epoxide

methyl red hydrochloride

m-chloroperoxybezoic acid

Dr. Norman Radin
Department of Biochemistry
University of Michican

J. T. Baker Chemical Co.
Phillipsburg, NJ

Aldrich Chemical Co., Inc.
Milwaukee, WI

METHODS

Synthesis of the Affinity Ligand N—6—Aginoheggnoyl-a—D—

Galactopyranosylggine

The affinity ligand was synthesized according to the
method of Harpaz and Flowers (109), who combined several
well-known organic reactions to prepare an affinity ligand
for the successful purification of coffee bean
a—galactosidase. The synthesis is shown in Figure 6, which

should be helpful in following the steps of the synthesis.

Synthesis of N—Benzyloxycarbonyl~6—Aginohexgnoic Acid. The
synthesis is that of Schwyzer gt_al; (120). The reaction
mixture contained 0.02 moles of 6-aminohexanoic acid in
250 ml of 1.0 N NaOH, 0.25 moles of benzyloxycarbonyl
chloride (Figure 6, Compound 11) dissolved in 250 ml of
diethyl ether, and 250 m1 of 4 N NaOB. The solutions of
aminohexanoic acid and 4 N NaOH were pre—cooled to 4°,

and the three solutions were combined with vigorous
stirring over 15 minutes. The reaction mixture was stirred
for an addtional hour at room temperature. The organic and
aqueous phases separated, and the organic phase was
removed. The aqueous phase was re-extracted three times
with 250 ml of diethyl ether. The aqueous phase was

acidified with 6 N HCI to pH 2.0 and kept overnight at

41

.'- 1.; " will-Ln

' ' ' , ‘ v
1:; ”I. ,) ~<I ( ~ .v
...iku‘.’ J- v. , . 'it .4
11:, w .
.. Y 2! ., ' 2m:

'Ie‘inllrwe' S.-.'f' ,«

' i“ ‘ '.'. ,h‘ ,.
.il " ‘ . 1'3!" I
' ,n ' gnu] .l-u,

,. 1. “I. : ’2‘121
2»! HI '1' . ‘1 H

. , .; I . ,5

'4')“ ‘Hlmtz'lo ":1" (Hum.

JJIII lvrh' N‘I'blifl

army. 2; MIL; é'llt."H}_LII

 

 

 

 

 

 

42

Figure 6. Synthesis of the Affinity Ligand.

The affinity ligand, N~6~Aminohexanoyl—a—Q—
galactopyranosylamine, was synthesized as described in the
Methods section. The principal reactants and products are
as follows:

VI

VII

VIII

IX

6—Aminohexanoic acid

Benzylchloroformate
N—Benzyloxycarbonyl—6—aminohexanoic acid
g—galactose

d—Qfgalactopyranosylamine ammonia complex
Isobutyl chloroformate

Mixed anhydride of III and VI

N—(N—Benzyloxycarbonyl—6—aminohexyl)1x-Qf
galactopyranosylamine

N-6—Aminohexanoy1-d-Qfgalactopyranosylamine

 

 

43

0Q Q
I IC-(CH2)5-NH2 +/C-O-CH2@ II
HO 0:

low CHZOH
HO 0

OH OH IV

0 O

\\ H II
III /C-(CH2)5- N-C-O-CH2@ OH
HO J/NHS

CHZOH
,,0 HO 0
v1 H5C2 c- o c’

v
H3 Cl \ NHz'NHa

O O O

H u u H n
VII H5C2-CIZ-O -C-O-C-(CH2)5-N-C-O-CH2
CH3

CHZOH
HO 0 HCI)

0
VI OH HI |
H H—(CH2)5- -.c 0. -CH2@

CHZOH lHZ/Pd
H0 H0
I

I
IX 0“ H-—c (CH2)5- NH2

 

44

4°. The white product was recrystallized twice from 25

m1 of 0014, and its melting point was compared to the
reported value of 54—550 (120).

Synthesis of (rGalactosylamine Ammonia Complex. The
synthesis of Compound V (Figure 6) is that of Frusch and
Isbell (121). The reaction conditions were anhydrous
because a—galactosylamine, the principal reaction product,
undergoes inversion of configuration to the B-form in
water. The reaction mixture contained 1.0 g of dessicated
NH4CI and 39.6 g of dessicated grgalactose mixed with 200
ml of anhydrous methanol; this was not a solution as
galactose is sparingly soluble in methanol. Anhydrous
ammonia was bubbled into the reaction mixture, which was
gently stirred for eight hours at room temperature. The
product crystallized for four days at 4°. Approximately
10 g of product, one fourth of the reaction mixture, was
filtered and recrystallized from 50 ml of 3% NH40H to
which was added 200 m1 of dry methanol saturated with
ammonia. The product was recrystallized at 4°

overnight. The product was reported to have a melting
point of 95—960 (16), that of B~galactosylamine is
136—1370 (122). The identity of the product being a—

rather than B— was confirmed by 13C—NMR spectroscopy. A

13C—NMR spectrum of the product was recorded on a Bruker

WP-60 Spectrometer by Dr. Hernan Nunez (as are all other
1JC—NMR spectra). The spectrum of B—galactosylamine was

obtained after the product had been dissolved in water for

. . s a!
u
I
l. 4
| .
A
. I .
I I \- l»
I.
l
:I u I
ll
I \I
. .
J. .0
k . I
. ‘I
w . D! n
1
I I
c . c, I
Q n a
(| . v
4 \ Ir .
1.. (P
l v r
.4 4|
, 1i ..
O.
I 6.. 7
I

 

 

45

thirty—four hours.
Synthesis of N-(N-Benzyloxycarbonyl—6—aminohexyl)—a—D—
galactopyranosylamine. The reaction mixture contained 1.50
ml of isobutylchloroformate (Figure 6, Compound VI), 1.58
ml of triethylamine, and 3.06 g of N—benzyloxycarbonyl—
6—aminohexanoic acid (Figure 6, Compound III) dissolved in
19 ml of dry DMF. The reaction mixture was stirred for 25
minutes at —5°. The mixture was filtered, and to the
filtrate (Figure 6, Compound VII) was added 942 mg of
a-galactosylamine (Figure 6, Compound V) and an additional
19 m1 of dry DMF. This latter mixture was stirred in a
stoppered vessel overnight at 4°. The solvents were then
removed by rotary evaporation using a water bath heated to
60°. The product (Figure 6, Compound VIII) was purified
on a column of Iatrobeads (2.5 x 45 cm) eluted with
chloroform—methanol—water (30:10:1, v/v/v), solvent A.
Fractions were collected, and aliquots were spotted on
silica gel 6 plates which were developed in solvent A and
sprayed with orcinol reagent to detect the carbohydrate—
containing material. The main product proved to be
Compound VIII (Figure 6), and those fractions having the
pure product were pooled and the solvents removed by rotary
evaporation.

Synthesis of N—6-Aminohexanoyl-a-D—galactopyranosylamine.
A vacuum flask containing 2.0 g of Compound VIII (Figure

6), 100 mg of Pd on charcoal, and 100 ml of 80% methanol

was attached to both a water aspirator and a cylinder of

 

 

T
'I
.5-
Iviil;
..1 "
I).i I " *
.- . ~ I
.. um
r
’w u“
,v , DIJ
., .
:l; I
.0, Hal '1

u», : :I>'I

,‘IIIII, ,9 A‘
1‘ ., ,I .

1' -. ~II

I 9

I III
.I‘
I .I 11‘
IIvI -'-
. H. ,,
I ;,
.

.
’I
I .III
(' .
~ T
. I'll;

 

I
‘ I
I
_ u
I
.I .
t I,
. I
I

 

 

45

thirty—four hours.
Synthesis of N-(N—Benzyloxycarbonyl—6—aminohexyl)—d«D—
galactopyranosylamine. The reaction mixture contained 1.50
ml of isobutylchloroformate (Figure 6. Compound VI), 1.58
ml of triethylamine, and 3.06 g of N—benzyloxycarbonyl—
6-aminohexanoic acid (Figure 6, Compound III) dissolved in
19 ml of dry DMF. The reaction mixture was stirred for 25
minutes at —5°. The mixture was filtered, and to the
filtrate (Figure 6, Compound VII) was added 942 mg of
d—galactosylamine (Figure 6, Compound V) and an additional
19 ml of dry DMF. This latter mixture was stirred in a
stoppered vessel overnight at 4°. The solvents were then
removed by rotary evaporation using a water bath heated to
60°. The product (Figure 6, Compound VIII) was purified
on a column of Iatrobeads (2.5 x 45 cm) eluted with
chloroform~methanol—water (30:10:l, v/v/v), solvent A.
Fractions were collected, and aliquots were spotted on
silica gel G plates which were developed in solvent A and
sprayed with orcinol reagent to detect the carbohydrate—
containing material. The main product proved to be
Compound VIII (Figure 6), and those fractions having the
pure product were pooled and the solvents removed by rotary
evaporation.

Synthesis of N—6—Aminohexanoyl-o—D—galactopyranosylamine.
A vacuum flask containing 2.0 g of Compound VIII (Figure
6), 100 mg of Pd on charcoal, and 100 ml of 80% methanol

was attached to both a water aspirator and a cylinder of

VI, III}

,
...
. Y.
I.
z

IF

I

t

I"

..

 

 

 

 

46
hydrogen gas. The vacuum flask was alternately evacuated
and flushed with hydrogen gas at least twice to remove all
the air from the flask. The hydrogen gas was let into the
flask, and hydrogenolysis (123) proceeded for four and a
half hours at room temperature and atmospheric pressure
with stirring. The reaction mixture was filtered to remove
the charcoal, and the methanol was removed by rotary
evaporation. A 13C-NMR spectrum indicated that the
product (Figure 6, Compound IX) was pure. Coupled spectra

1 _13

H C) were made of a-galactosylamine,

(
B—galactosylamine, and the product to determine the
coupling constant between the anomeric carbon and its
proton which would indicate the anomerity of the product.
Coupling of the Affinity Ligggg,N—6-Aminohexanoyl-a-D—
galactosylpyranosylggine to Sepharose 4g; The affinity
ligand (Figure 6, Compound IX) was attached to Sepharose 43
by the method of March g£_gl; (124). Approximately 50 ml
of packed beads were washed with 500 ml of water and then
activated by the addition of 7 g of CNBr dissolved in 5 ml
of acetonitrile. The suspension of beads was stirred
gently, kept between pH 11 and 12 by the addition of 5 N
NaOH as needed, and the temperature was maintained at 200
by the addition of ice. The beads were then quickly vacuum
filtered, washed with 300 ml of 0.2 M sodium phosphate, pH
9.5, and mixed with 100 ml of 0.2 M sodium phosphate, pH

9.5, containing 200 mg of the affinity ligand (Figure 6,

Compound IX). This last reaction was kept at 230 for

 

. I ..
I; K V I.
.u r. I
_ . . . / .
. .
I .... .H . H
. i .I . i .. c
I I w. n:
. . .. . .. .
. . I . I.

 
 

47
about 5 minutes and then chilled to 4° by the addition of
ice. Coupling of the ligand to the beads continued for two
days at 4°. The affinity ligand attached to Sepharose 48

will be called the affinity resin.

Enzyme Assays

Enzyme Activity. One unit of enzyme is the amount of

enzyme that hydrolyzes one umol of substrate in one minute.

Assays Using Artificial Substrates

o-Galactosiggse Agplus 8 Activity. This is a modification
of the procedure of Desnick §£_§1J (125) that measures both
enzymes, but it cannot be used to distinguish them. Up to
25 pl of enzyme was put into a 10 x 75 mm test tube and
150111 of 5.0 mM 4—MU-argrgalactoside in Gomori
Citrate-Phosphate pH 4.6 (126) was added to the enzyme.

The enzyme and substrate were incubated at 37° for a

minute or longer as they were previously at 4°, and the
assay was begun by the addition of substrate. The reaction
was carried out at 37° and was terminated by the addition
of 2.35 ml of 0.1 M ethylenediamine. The fluorescence of

the liberated 4-MU was determined in an Aminco J4—7439

which was calibrated with standard 4—MU.

a-Galactosidase A. This assay is based upon the fact that

 

 

A I In
. . ~

 
 

48

crgalactosidase B is an a-N-acety1galactosaminidase and is
competitively inhibited by N—acetyl-Qfgalactosamine. The
assay was performed exactly as the chal A plus B assay
except that the substrate also contained 58.3 mM
N—acetyl—Qfgalactosamine in addition to the 4—MU-a—Gal.
chalactosidase A was inhibited by less than five per cent
under these conditions while (rgalactosidase B was

inhibited by over ninety-five per cent.

ardalactosidase B. cx-Galactosidase B was assayed by the
method of Sung and Sweeley (127). Up to 50 p1 of enzyme
was mixed with 50 pl of 10 mM p-NP1x-GalNAc in 0.1 M Na
Citrate pH 4.3. The enzyme and substrate were incubated at

37° for at least one minute as they were previously at

4°, and the reaction was begun by the addition of
substrate to the enzyme. The reaction was carried out at

37°

and terminated by the addition of 3.0 ml of saturated
sodium tetraborate. The absorbence of the liberated
p-nitrophenol was measured in a Gilford 2400

Spectrophotometer at 410 nm using p-nitrophenol as the

standard.

Non—Quantitative Assay Using a Natural Substrate

To demonstrate that argalactosidase A, but not

crgalactosidase 8, could hydrolyze GbOse Cer, both highly

3

purified enzymes were reacted with bovine GbOseacer

I
I"
4 v-I
v
...

I:
,1..
H. 1

I
. .
-
, .
‘I
I.
I

l
.I.
. .-
HI‘
’ .
r, ‘

49

according to the method of Dean (128). Ten milliunits of
each enzyme was added to 100 nmol of bovine GbOsescer,
5011g of sodium taurocholate, and 50 ul of Gomori
Citrate—Phosphate pH 4.2. The reaction mixtures were
incubated at 37° for one hour when an additional 10
milliunits of the respective enzyme was added to each. The
reactions proceeded for an additional hour and were
terminated by boiling for one minute. The reaction
mixtures were lyophilized, dissolved in chloroform-methanol
2:1, and an aliquot was spotted on a silica gel 6 plate.
The TLC plate was developed in chloroform—methanol-water
(65:25:4, v/v/v). sprayed with 0.5% orcinol in 4 N

H 80 and heated to 1000 to visualize the

2 4'

carbohydrates. Standard sodium taurocholate, GbOseSCer,
LacCer, the expected hydrolysis product, and a blank

reaction mixture were also run on the TLC plate.

Purification of d-Galactosidases A and B from Human Liver

Genergl Methods. chalactosidases A and B were purified
from human liver by a combination of the procedures of Dean
and Sweeley (104,105) and Bishop and Desnick (16).
Approximately one kg of frozen human liver was thawed
overnight in two volumes (w,v) of cold 1 mM PMSF. The
liver was homogenized four times for 30 seconds at top
speed in a Waring Commercial Blendor. The homogenate was

filtered through cheesecloth to remove connective tissue

 

.:.=:II'I:: "4! ”It; .:
. , .JI) I t, I! '.‘ ' :r
.' I I 7 ' 'v :1.
. (I .‘ _ :1;f7 n , ‘1 I
':I I- M v' ':L :r. ';I E . ‘.
. :13 a! .. I." (HIV/"fl“! QVIJI‘QL) 1151
II " ' "‘I';l "v 'I I ). L ii: I I)
. yaw: an; S-Jl’llhii’i 2 'ihi Z; 1 u
.1. 'iI‘IIIl III'IUIU'TI/EIIII n! I:-;':'w:.:1r . ,I
,3”?sz In}: “Jillfl I ' AII Hg" ;-;,. Jun-u'
wI:.vval.ers‘-II..rnnmm! I: :I .I. II'IU. 1 H
/: I III ‘1' I“ Mr». I ,'f»"n.:£ I
It .‘I' I' U!’ ”I u. I"
III} .. .I .' a: f ... ~,-‘.‘1,.'.'
..(I F In 13H)“: ., (.1 , .
‘a'aiv 1‘! 'HIJ "t I;I xfI- Mn. m
2";fo !.I'.,I. .‘ r ) I I «.... ,' ' r,. ‘ .« .
I'I'l' " 11:0 1 ow {I -:n[: , "9 ..:‘-I . I
m H. "1'10 ::.: «-' ,. r'1I1: w I- VII
IdlI I n'-"uII IIIII '[III ' ..

II‘HIIH" m 'T‘IVI! ...I. . I
:III'I I‘IIH Hm I n! w t ; . -
u-‘fi .‘I L'I' I‘ ..l '14); I, J: IL. "......“

..‘l iInII'f'IIHIII .. I i '1. )"v1IIIIu.I ,_.
I‘.’-‘II -’ Mn. .‘HII‘s. as v:

 

50

and then centrifuged at 16,000 X g in a Sorvall RCZ—B for
30 minutes at 4° (as are all steps in the enzyme
purification unless otherwise noted). The pellets were
re-extracted with two volumes of water, recentrifuged. and
the supernatants assayed with 4—MU-a—Gal until 90% of the
activity was solubilized. The supernatants were pooled,
brought to 60% saturation with solid ammonium sulfate, and
centrifuged at 16,000 X g for 30 minutes. The supernatants
were discarded, and the precipitates were resuspended in
500 ll of distilled water. This solution was dialyzed
versus ten liters of 10 nM sodium phosphate pH 6.5 (buffer
A) with two changes of buffer. The dialysate was
centrifuged at 16,000 x g for 30 minutes to remove protein
that precipitated during dialysis, and the pellets were
discarded.

The crgalactosidases were extracted from the
supernatant by stirring with 100 ml of Concanavalin
A-Sepharose for one hour at room temperature. The solution
was re-extracted until greater then 90% of the

~galactosidases had been removed. The beads were removed
from this solution by filtration using a Buchner funnel
with a nylon mesh filter. The filtered beads were washed
with about 50 m1 of buffer A, packed in a column (2 X 20
on). and eluted with 0.1 M a-nethylmannoside in buffer A
until all the enzyme, which was yellow, had been eluted.

The Concanavalin A extract was then applied to a DE—52

column (4.2 x 11 cm) that had been pre-equilibrated with

‘i ...1';

I. ' .p
. H!
, ,
. . 2
n .
u .
‘ ' .1'
. 'II
‘ '1 Ii ”('1'
‘ .l
. I
r. .a I!
_z' ' ' ' ’ .-
‘.l). 'l'. '1 ‘5?
: 1311:
”ll h'v" " .I' '
H" :
, i'hl dull. HI
, ‘‘II ‘1‘! r:
. a-v i' A

.~.. 'imlllt')

H

U .5

‘~‘I;ri,tflll

I H"

g,“

"l i

..ii :—
'i. -
l i'
;('
'III‘ a i-
.n ;
I .’ '
l w 2‘)
‘-l“ .
. H‘):'
il l .‘l
u. t 4
.I 15‘;
V'Ilr‘).ll

I O l
x. ' . -:’
. ' . ..' u'. .
’...sv| :v ,J/~' I'
. ‘4 l ,- .1}; W:—
I I ' I
-;'I/ *t . 'I -.
‘I . ’.' . ’ - . x . “i
l. . .' ). -l- r w.
. A. .in Mr. '
f: 'm 1’ in 1' ' £361"!sz
. . .-:-.:lth n: i 1 (A.
07, - (H, . 'w:ul!:l I.-;
.5 his ".I i ' ."H’, 7fiIIJ’
H I"[l
.' . i 3 "ll
‘_." 1 I ‘ 03!."
I ‘ . -.Z -,\l -I ‘9 \II 1' f f_
2;": IA - l '.‘ H (5"!
z i 'm: -n .- uvzui ..H‘;(

'1 '.:- m .1 )L'«’:.

Hit xiv“.

‘u n :s in“.

 

 

51

buffer A. The column was washed with 200 ml of buffer A to
remove nonbinding proteins. The (rgalactosidases were
eluted with a linear NaCl gradient (4 liters total, 0 to
300 mM) in buffer A. Fractions were collected and assayed
with both 4-MU—a-Gal, 4-MU-a-Gal with GalNAc. and p-NP—
a-GalNAc. Fractions containing only a-galactosidase A were
pooled, and those containing both A and B were pooled. The
two fractions were separately concentrated in an Amicon
Model 52 using a PM 10 membrane to about 20 ml. The
enzymes were then applied to a Sephadex G-150 column (3.5 x
110 cm) and eluted with buffer A at a flow rate of
approximately 20 ml/hour to maximize protein separation.
Fractions were collected, assayed with 4—MU4x—Gal, and the
A280 determined. Fractions having more than 100

milliunits/ml were pooled and concentrated to about 1.0

units/ml of a-Gal A and 5.0 units/ml of a-Gal B.

Affinity Chrogatggggphy of a~Galactosida§e A. The
a—galactosidase A from the G—150 step was diluted with
one-half its volume with 0.15 M NaCl in Gomori
Citrate—Phosphate pH 4.6 (buffer B) and acidified with
dilute HCl to pH 5.0. The enzyme mixture was centrifuged
for 1 minute at 12,000 X g in a Brinkman Eppendorf
Centrifuge Model 5412, and the supernatant fraction was
applied to a column of N-6—Aminohexanoyl—
a-galactopyranosylamine~Sepharose (0.75 x 10 cm).

Fractions of 2 ml were collected. The non—binding proteins

 

..
. .,_—
' ' f' ' II".
. I'I. r I .
I':-;_.'~ '.I . I.i.‘ . - :Afllhij
I .-'- (“I ‘ I I -- " Jiii :. '- 'I l u UNI
I. -. ---'--I_1:I‘1.‘IIII:II v:-“-- -’ 11' 1'. !-'1 .
.1. .'I:I 0. ‘:I.II.~-!I '1 ." --.I::' . 'II
. . - ‘ : , “)5 f. 0" I’I! -. I ‘ l' 1. FHIVFH“
.I )7I‘-. E I I 1'. I- ISIIIII: llllI !. III.-. :..... (I! .l
:. -,.-.' .. _ I 'I I ' IIII 25m I]! I'm ' Mn 2 -‘:..:,1:-., .105
u Jill) 'l:". I‘ I I ' - ' I. ,) I: I (I: l ... I" .
"Ii :II 'I' ) II"!!! '--.'I. I. .. '. ' r I-z' .II I-.: III; I-‘HI H
l“ (I. -' I (. - .i- II'I 'I'E'I Ll‘i‘ I -l-.'
. -:.I ..I ..II‘ ': I'II ' ‘I' * I" IIII'I: Il'
. .u ..I "l‘ ‘ II. i‘_\-:-."I .i‘ IIIIIIIII
'. :- . ' M II'- . l - I . (0!? I fl'tn. .II' ... “I;
I III-- III ‘ H I W. i I -II..'.- = II'; . . .I1- ‘II'In
v. .- - «I . . . ‘I' I7II'II:-I " I'II .
tT-I- Mr: .I'2'..I- 'III'i'I . " I 'I-I ‘ 'Hi II1IIII‘!
.I'I.-.I.' l.'i'l' '-' 'II - I ' --I’-.I!I IH
,. :7 II. I.I".II.II-I'r' "I” ' 1 I .‘ --'.'U Il'l ':-"D
Huh." ‘I: ‘.I I'I .'ll!.|' ' . (qr-I
_I.-:: '1] / .‘1 (I' H?- I‘II ' “Hug-Iver: , H 'II ..
I. .. -.2 II I!.. -' 1m; III II I.-

52
were eluted with buffer C (buffer B adjusted to pH 5.0)

until the A,

280 was zero, after which a linear pH and salt

gradient was begun from buffer C to 0.5 M NaCl in Gomori
Citrate—Phosphate pH 6.0 (buffer 0. 20 ml of each). The
gradient was stopped at pH 5.6, and the a—galactosidase A
was eluted with 0.4 M galactose in buffer D. The fractions
were assayed with 4—MU—a—Ga1. The most active fractions
were pooled and dialyzed versus either buffer A or Gomori
Citrate-Phosphate pH 6.0. The purity of the enzyme was
determined by SDS polyacrylamide gel electrophoresis.
Impurities were removed by either gel filtration on the 3.5
X 110 cm Sephadex G—150 column or rechromatographing on the

affinity resin.

Dual Affinity Chromatography of a—Galactosidase B. The
pooled enzyme from the 0—150 step containing both
a-galactosidase A and B was diluted with half its volume of
buffer B and adjusted to pH 4.7 with dilute “01. Solid
GalNAc was added to the enzyme to make the solution 50 mM
in GalNAc. The enzyme mixture was then applied to the
affinity column (0.75 X 10 cm) that had been
pre-equilibrated with 50 mM GalNAc in buffer B. Fractions
of 2 ml were collected. The non—binding proteins were

was zero. The

eluted with buffer B until the A280

pfl—salt gradient as for the a—galactosidase A was begun,
and the (rgalactosidase A was eluted with 0.4 M galactose

in buffer C. The GalNAc eluted fractions were assayed with

X£I§..

 

53

4—MU-IrGal. The active fractions were pooled, concentrated
to less than 10 ml, and dialyzed versus buffer A to remove
the GalNAc. The enzyme was acidified to pH 4.7 with dilute
BCl, centrifuged to remove precipitated proteins, and
reapplied to the affinity column that had been
pre-equilibrated with buffer B. The column was eluted with
buffer B, and fractions of 2 ml were collected. The A280
and the wgalactosidase activity of the fractions were
closely monitored. Since a—galactosidase B does not
tightly bind to this affinity ligand, not all of the
contaminating proteins can be washed from the column before
the enzyme is eluted. One must exercise Judgment when
deciding to elute the a-galactosidase B. The enzyme should
be eluted after the bulk of non-absorbing proteins have
passed through the column and while the amount of
a—galactosidase B that has non-specifically eluted is
small. The a-galactosidase B was eluted with 0.4 M
galactose in buffer B. The fractions were assayed with
4-MU-aeeal. The most active fractions were pooled and
dialyzed versus either buffer A or Gomori
Citrate-Phosphate, pH 6.0. The purity of the enzyme was

determined by SDS gel electrophoresis.

Gel Electrophoresis. Denaturing polyacrylamide gel
electrophoresis was performed according to the method of
Laemmli (129). Protein bands were generally visualized by

staining with Coomassie Brilliant Blue R, but were stained

. ~I , ‘ ' ..
'I ,' ' ' ~ 4 I z ('I I I III
'I‘ - . ._'. . -I' x.
- I. ‘I I I . _., . .-II .
“s" . .I'i.‘l‘ II I .
. , L ... , . fi’.‘l‘
. -l| . .; l .
I I 1') : .I 'l . .-
-. '! I III 1" I I ‘I!II" ..1
ii' 1'. " ‘ 1'10' I. :II-l
I 'H'.» ‘.l0| I ' .I v: 1, , In
I-':: 'IIstI'III - - I'" ‘ II --
. I - - I '3' IIII:-‘ IV
“1‘:- ' ' l‘ ‘ I .
1 n: .hui- I -1:
‘ . . -' !"" . ’I' ' H I'.~..1
I 1 ) le‘l I'H'IIII' I’.‘ ‘I .‘..'
I'll“! I I'Hc'f I?" HMO]? . I .; -
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54
with alkaline silver reagent (130) when the amount of
protein was small as in assessing the purity of individual
fractions eluted from the affinity resin.
Protein Determination. Protein concentration was
determined in a number of ways: The Lowry method (131)
with BSA as a standard, a modified Lowry (132) using BSA as
a standard, with fluorescamine (133), and determining the
A280 in a Gilford 2400 Spectrophotometer. The last three
methods were used when either the protein concentration or
sample size was low.
Carboxymethylation. The a—galactosidases were
carboxynethylated by the nethod of Gracy (134). The
lyophilized proteins were dissolved at a concentration of
0.5 mg/ml in a solution containing 0.5 M Tris
hydrochloride, 25 mM EDTA, 6 M guanidine hydrochloride, and
8 mM z—mercaptoethanol adjusted to pH 8.5. The mixture was
flushed with N2 to remove oxygen and stirred for one hour
at room temperature. Ten microliters of iodoacetic acid
(10 mg/ml) were added per 100 pg of protein being
carboxymethylated. The mixture was flushed with N2, and
the carboxymethylation continued for twenty minutes in the
dark. The carboxymethylation was terminated by the
addition of 10 ul of 2—mercaptoethanol per 100 ug of
protein. The protein solution was desalted by dialysis in
the dark or by gel filtration on Sephadex G-50.
Pyridethylation. The a—galactosidases were pyridethylated

by the method of Hermodson g£_alJ (135). The proteins were

I
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55

dissolved at up to 30 mg/ml in a solution containing 6.0 M
guanidine hydrochloride, 0.13 M Tris, and 0.10 mg/ml BDTA
adjusted to pH 7.5. A twenty~fold molar excess of DTT
relative to protein sulfhydryl content, which was assumed
to be five per cent of the residues of the proteins, was
added and the mixture was stirred for three hours at room
temperature. A three—fold molar excess of 4—viny1pyridine
relative to DTT was then added and the solution was stirred
for an additional ninety minutes. Alkylation was
terminated by acidification of the protein solution to pH
2.0 with 88% formic acid. The protein solutions were
desalted by either dialysis or gel filtration on Sephadex
G-50.

Trypsin Digestion. The alkylatedcx—galactosidases were

suspended in either 1% NH on or 10 mM sodium phosphate,

4
pH 7.0, at a concentration of 2 to 5 mg/ml, and
TPCK—trypsin was added at a ratio of 50:1 (w,w of protein.
trypsin). The a—galactosidases were digested at room
temperature for 10 hours. The digestion was terminated
either by freezing or lyophilization.

Separation of Peptides by HPLC. All peptide separations
were performed using a Beckman 112 Solvent Delivery System
connected to an Altex 210 Sample Injecter and a Beckman 160
Absorbance Detector which was connected to an Isco-strip
chart recorder. The detector was set at 229 nm by using a

Beckman 229 filter and a Cadmium lamp. The flow rate for

all separations was 1.0 ml/minute. HPLC grade solvents

;.r.
.II/l

 

I I III. ' ‘ I' .W ‘III ‘
i
‘1: I. - III I - I’ I: 'I' I
l
. . ,I II,'~ I I)! IIII I I'III in .
'IIizI
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- II . ‘qu l . IIII: I '» ' , . II I .. (I III I I
'I ,,. III 1’ I.'~I IiII: III'IIIII II I W: .'-I.,.I .

 

 

56
were used, and the solvents were vacuum-filtered to remove
any particles and to de—gas the solvents. The aqueous
solvents were filtered with an HATE 04700 filter, and the
organic solvents were filtered with an FHUP 04700 filter.
TFA was added to the tryptic digest to give a 1*
solution which was injected onto the RP-8 column. The

primary separation utilized a solvent system of H 0 to

2
CascN/z-propanol (3:1, v/v) with 0.1% TBA throughout

(136). The tryptic peptides were separated by starting
with water and having a linear gradient of the organic
phase which increased at one per cent per minute. The
eluate corresponding to peaks on the chromatogram were
collected, and the solvents were removed under a water
aspirator vacuum, but not lyophilized. The peptides were
generally rechromatographed prior to N-terminal sequencing.
The peptides were initially redissolved in 0.1% TFA in
either water or the organic phase used in the primary
separation. but neither of these solvents were very
effective. The best system was to dissolve the peptide in
a small volume of 6 M guanidine HCl which was diluted to
less than 0.5 M guanidine with 0.1% TFA in water prior to
injection. Two systems were used for rechromatographing
the peptides: The first was exactly like the primary
separation except that 0.1% HFBA replaced TFA and there was
an increase of the organic phase of 2% per minute. The

other system was also like the primary separation except

that a uBondapak Phenyl column was used and there was an

.
.
., ,
.. 1,
‘1’. 1 r
l
’ , )‘x.

r/ ).
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57

increase of the organic phase of 2% per minute.

Antibody Production

Polyclonal; Purified human livercx—galactosidase A was
injected at several sites on the back of each of two
rabbits. The first injection contained 20 ug of enzyme in
an emulsion of Freund's Complete Adjuvant. The booster
injections on days 14 and 28 contained 10 ug of enzyme in
Freund's Incomplete Adjuvant. The rabbits were bled on day
42. The specificity of the antibodies was determined by
Dr. Norman C. LeDonne Jr. of this laboratory by
immunoprecipitating [2-3ul—Mannose labelled

a-galactosidase A from Chang liver cells (31).

Monoclonal; Monoclonal antibodies to human liver
a-galactosidase A were induced in mice by a variation of
the method of Galfre g£_glJ (137). Female Swiss white mice
were injected i.p. with 10 pg of partially purified
a—galactosidase A (approximately 450 munits) emulsified
with Freund's Complete Adjuvant. The first booster
injection was two weeks later with 10 pg of enzyme in
Freund's Incomplete Adjuvant. The second booster was like
the first booster and followed it by at least two weeks.
Three days after the second booster injection the mouse was
sacrificed by cervical dislocation. Blood from a heart

puncture was saved for determination of anti—o-

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58

galactosidase A antibodies. The spleen was aseptically
removed, washed with DMEM containing 20 mM HBPBS, then cut,
and teased into a single cell suspension. The cells were
centrifuged at 1000 X g for 10 minutes at room temperature
(all subsequent steps are at room temperature unless
otherwise noted). The RBC's were lysed by gently
resuspending the cell pellet in 5 ml of 0.2% NaCl for 30
seconds, then mixed with 5 ml of 1.6% NaCl and then with 10
ml of DMEM with HEPES. The cells were recentrifuged for 10
minutes at 1000 x g, resuspended in DMBM with HBPES, and an
aliquot was counted in a hemocytometer to determine cell
number.

Approximately 1—5 X 107 mouse spleen cells were

mixed with 1-5 x 106

(a 10:1 mixture) hypoxanthine

guanine phosphoribosyl transferase-negative SP2/0-Ag14
mouse myeloma cells. The SP—Z cells had been grown in 10%
FCS in ONE! containing 100p g/ml of both penicillin and
streptomycin at 37° in a 7% COZ-water saturated
atmosphere. The mixture of cells was centrifuged at 1000 x
g for 10 minutes, the supernatant removed, and 1.0 ml of
fusion medium, PEG, DMSO, DMEM, (7:1:12, w/v/v), was added
at 37°. The cell pellet was gently disrupted and mixed
with the tube in the palm of the hand to keep the fusion
warm for one minute. One ml of DMEM with HEPES was added
and mixed gently for one minute, after which 2 ml of DMEM

with HBPES were added and mixed for one minute. Four ml of

RT medium [DIEM containing 10% (v,v) FCS, 10% (v,v) horse

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59

serum, 10% (v,v) NCTC 109 media, 2 mM glutamine, 100 “M
hypoxanthine, 16 pH thymidine, 3 pH glycine, 100 mg/l
penicillin, and 100 mg/l streptomycin] were added and mixed
for 3 minutes, then 8 ml of HT media were added and mixed
for an additional 3 minutes. The fusion mixture was
centrifuged at 500 x g for 10 minutes. The cell pellet was
resuspended in 50 ml of HT media, and 0.1 ml were added to
the wells of five 96-well Costar plates.

The plates were incubated at 37° in a 7% 00 -water

2
saturated atmosphere. After two days 0.1 ml of HAT media
(HT media containing 1.011H aminopterin) was added to each
well. This media was removed two days later when an
additional 0.1 ml of HAT media was added. The hybridona
cells were grown until the media became acidic (yellow),
and an aliquot was pipetted into a 10 x 75 mm dispo tube to
test for anticx—Gal A antibodies.

Heat-killed Staphylococcus aureus cells were washed 3
times with one volume of 0.1 M sodium phosphate, pH 8.0
(buffer 8), containing 0.5% Triton x—100 and 10 mg/ml BSA.
The g: aureus was resuspended in one volume of buffer E.
Rabbit anti-mouse IgG (Miles) was added to the washed g;
aureus (1:20, v/v), and it was bound by incubating at 37°
for 5 minutes. The mixture was centrifuged and resuspended
in one volume of buffer E. 100 ul of the S; aureus rabbit
anti—mouse IgG complex was added to the media from each of

the clones and incubated for 5 minutes at 37°. The tubes

were centrifuged, and the supernatants were removed.

n‘ I 'l
.
' " . J nu
.
5" , .I
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ll 1 VIP ‘ l. .
. .1
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60

Approximately 1 milliunit of partially purified
a-galactosidase A in Gomori Citrate-Phosphate pH 6.0 was
added to the pellet which was vortexed, incubated at 370
for 10 minutes, centrifuged, and the supernatant removed.
The pellet containing the S; aureus-rabbit anti—mouse mouse
anti-human liver chal A antibody was resuspended in 25 ul
of Gomori Citrate-Phospate pH 4.6 and assayed with 4-HU-a
-Gal. Pre—immune and immune mouse sera were used for

negative and positive controls, respectively.
Synthesis of Conduritol C Epoxide

Conduritol C tetraacetate was synthesized by the
procedure of Stegelmeier (138) and converted to conduritol
C epoxide by the method of Radin and Vunnam (139). Mr.
James Grove and Dr. Fumito Matsuura of this laboratory
performed the first four steps of the synthesis; 1
performed the last three steps. Refer to Figure 7 for

details of the synthesis.

Synthesis of 5,6-dibrogo-2—cyclohexene—l,4—gione. The
reaction mixture contained 43.2 g of benzoquinone dissolved
in 600 ml of ice cold CCl4 to which was added over a

period of 30 minutes 20 ml of Br in 600 ml of CCl4

2
with stirring. The reaction continued for 30 minutes at
40 when 100 g of sodium sulfate was added with stirring.

The reaction mixture was filtered, and warmed to room

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61

Figure 7. Synthesis of Conduritol C Epoxide.

Conduritol C Epoxide was synthesized as described in the
Methods section, and the reactions of the synthesis are
illustrated here. The principal reactants and products are
as follows:

I Benzoquinone

lI 5,6—dibromo—2—cyclohexene~1,4—dione

III 5c,6t—dibromo-2—cyclohexene—lr,4t—diol

IV 1r,4t—diacetoxy—2t,30—dibromo—5-cyclohexene

V 1r,2c.3c.4t—tetraacetoxy—5c,6t—dibromo—cyclohexane
V1 Conduritol C Tetraacetate

VII Conduritol C

VIII Conduritol C Epoxide

 

 

62

 

 

OAC H
IV . < . III
Br Br
CAc OH
A) KMnO4
B) A020
OAc OAc
AcO .. Br Zn AcO
V . ——9 VI
AcO . Br AcO 5
OAc OAc
Triethylaminel
HO
0" m-chloroperoxybenzoic OH
VIII acid H0
HO 6 VII
HO O HO .

OH

63

temperature. The crystals were dissolved by the addition
of 500 .1 of 0614, and the excess Br2 was extracted

with 1.5 l of water. The solvents were renoved by rotary
evaporation, and the product (Figure 7, Compound 11) was
recrystallized four times from C014. The product was
analyzed by infrared and ease spectrosetry.

gynthesis of 5c,6t-dibrogg;g-cyclohexene-lr14t—giol. The
reaction nixture contained 26.8 g of Conpound 11 (Figure 7)
dissolved in 600 ll of diethyl ether to which was slowly
added 9.5 g of sodiu- borohydride dissolved in 150 ll of
water. The reaction proceeded for 140 linutes until the
solution becale alnost colorless. The reaction nixture was
transferred to a separatory funnel, and the ether phase was
reloved. The aqueous phase was extracted three tines with
100 ll of diethyl ether. Solid sodiun sulfate was added to
the combined ether phases, which were then filtered, and
the filtrate was evaporated by rotary evaporation. The
product was recrystallized fro. acetone—pentane (2:1, v/v)
and analyzed by infrared and I388 spectrometry.

Synthesis of ir44t-digcetoxy-gt,3c-dibroggr5-cyclohexene.
The reaction mixture contained 15.4 g of Compound III
(Figure 7) dissolved in 150 .1 of pyridine to which was
added 150 ll of acetic anhydride with stirring on an ice
bath. The reaction was stirred overnight at room
tenperature. and 300 ll of ice water was then added with
stirring. The Iixture was transferred to a separatory

funnel and extracted three tines with 200 ml of CHC13.

64
The CBC].3 extracts were pooled and washed three times
with 100 ml of each of the following in order: 6 N HCl,
saturated sodium bicarbonate. and water. Solid anhydrous
sodium sulfate was added to the CHCl3 layer, which was
then filtered and the solvents removed by rotary
evaporation. The oily product was dissolved in 150 m1 of
ethanol which was allowed to stand for three days at room
temperature. Crystals developed which were collected by
filtering. The filtrate was concentrated to 50 ml by
rotary evaporation and allowed to stand at roon temperature
for additional crystallization. The crystals were washed
with cold ethanol. This entire procedure was repeated with
14.6 g of Compound 111 (Figure 7). The product was
analyzed by infrared and mass spectrometry.
Synthesis of 1rLgcg30,4t-tetrggcetoxy-5c,6t-dibro;g_
cyclohexane. The reaction mixture contained 3.65 g of
Compound IV (Figure 7) dissolved in 125 ml of absolute
ethanol to which was added 3 g of MgSO4 in 20 ml of
water. This solution was cooled on an ice bath, and 3.2 g
of KunO4 in 200 ml of water was added dropwise over a
period of three hours with stirring at 4°. After 90
winutes an additional 3 g of MgSO4 in 20 ml of water and
125 ml of absolute ethanol, both of which were pre—cooled,
were added to the reaction mixture. The reaction continued
an additional 3 hours when 100 g of activated charcoal were

added, and the reaction continued overnight at 4°. The

mixture was then filtered and the activated charcoal was

 

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65 1

washed three times with 20 ml of ethanol. The ethanol
washings and the filtrate were combined, and the solvents

were removed by rotary evaporation. The intermediate

I
I
l
1
i
3

product was dried overnight over P20 Acetylation of

5.
this intermediate was performed as in the conversion of
Compound III to IV in Figure 7 with crystallization of the
product performed at 4°. The product was analyzed by an
infrared spectrum, mass spectrometry, and by comparing its
melting point to the literature value of 1440 (138).

Synthesis of Conduritol C Tetraacetate. The reaction

mixture contained 1.5 g of Compound V (Figure 7) dissolved

 

in 70 ml of diethyl ether, 3.0 g of powdered zinc, and 3.0
ml of glacial acetic acid. The reaction mixture was
stirred for four hours at room temperature. The mixture
was filtered, the zinc was washed with 40 ml of diethyl
ether, and the ether washings were pooled. The ether
fraction was washed once with 50 ml of saturated sodium
bicarbonate, dried over sodium sulfate in a vacuum
dessicator, and the ether removed by rotary evaporation.
The product was identified by obtaining an infrared
spectrum, a mass spectrum, and a melting point which was
compared to the reported value of 91—920 (143).

Synthesis of Conduritol C . The reaction mixture contained
600 mg of Compound VI (Figure 7) dissolved in 30 ml of
methanol—water (7:3, v/v) and 2.12 ml of triethylamine.

The reaction was stirred overnight at room temperature, and

the solvents were then removed by rotary evaporation. The

-:. :" U .: 7‘ :' [in i ' ' ' 'Iiii'5b'fl
I: ll . ..I.I . . ..‘nI .z. I..II':Il--I I--'u
U . IV?" - I - I' l'l

:' _::;- .t- _. :.-..'. . -.‘-- -"’I--In-~IIII air”

94; I. h=-.- .' .3.: a .Iu I airy " '~ . I- nl' nnnw.mu)

a . '1.'.qm(w :d :u .VIJamuul.hua J.hn .m:"v:nna DHIIWfII

:dcf; II] I:' ~;:.' .-' -II-I.'.'EI.-I vII -.IJ F'II-w 'Jiliii'ulfl

qu-.,w .n; . ' L-.:~- ' 'uTIInInnu In etiqu1nqz

bah .. D II qu:-:I V HHNIHMU3 I: . - I lu:~':Iuw aunt; m

3.:. [din .orIIr ..a«*~ AIM! . - q . " .'..u- a I xn3t'Ih in :m l)? n:

anw .in37:m nnz‘er; unI .n nu :.!-: .znnlg in In

e1uJiIm efiI sznwaumeJ mOCI " ?v1rfl 1001 4“: F'.'ch

I;h “EH Io m ”L nirw banana and n2:* "H1 .uu:o)l" 'L

.mn'v Gui heLUUu nwcw aBfllflLfi» Iunto -p' has .I-an

.nUIhum n-III'IIHIIa I: In H". IIIII nuv h-udenv- ."uW u) I.”

1H1: . 'l “flail“?! IIIIIIDOP. 'Iu'x’u Ir-r ID .OJIHIUH'HWH-

:"I 7 I!Olli;'-I‘.: l.-.‘=()'l 'lfJ I’I';','um-I’I IIIIF'i HI! I)": - ' I
hunt-III “L nnjnrh7un In h IIIILwh. =nu I'uan-

a:.. ..w- ‘w .. lg {Hilli‘lm .2 Ixnn mIIII'rvue :” nu h .m:r]1 uhqr

. ”50 re 'u nthv uuijI- -. w' tnxhumci

huu.n-~u: n1u111m nniidn-I LI ' -v.I1nuLa :4 etch-luv;

in 'm Uh nI huthszn If n1un.1; l/ nunvqm

)) I) ma (Hm
'n!flfi§¥fl)3[11 1o Im ;. L bun II=~ .5. I -"¥hW‘iUflLfl“m
but. . . I.'In'I-I-Im')1 mun . In HI'IIII‘IZIVU I»’9"“- If: .zr‘m II-I: IJH'I'I IIII

nut .nn-Yuwoqux“ Ttn‘nr [a Le”nmn l u: afnnvluh ”6'

:3

66

reaction was repeated until the melting point of the
product was the same as the literature value of 148—1510
(140). The removal of acetyl groups was also monitored by
low resolution 1H—NMR spectra which were made by Mr.

Royal Truman of the MSU Department of Chemistry on a Varian
T-60 NMR Spectrometer. The identity of the product was
also confirmed by a mass spectrum of the TMS derivative and
an infrared spectrum, which were compared to the respective
spectra of conduritol B. The purity of the product was
determined by gas chromatography of the TMS derivative
(144). The CO was a Hewlett-Packard 5840 A Gas
Chromatograph with a six foot long 3% SE—30 column. The CO
was programmed for a linear increase in temperature of 20
per minute starting at 140°. in order to determine the
stereochemistry of the product, high resolution coupled and

decoupled 1

H—NMR spectra of the product were made by Dr.
Klaas Hallenga and associates of the M.S.U. Department of
Chemistry on a Bruker WM—250 Spectrometer.

Synthesis of Conduritol C Epoxide. The reaction mixture

 

contained 121 mg of Compound VII (Figure 7) and 221 mg of
m-chloroperoxybenzoic acid suspended in 15 ml of methanol.
The suspension was stirred for five days at room
temperature. The solvents were removed by rotary
evaporation, and the chloro—compounds were extracted from
the flask with 10 ml of diethyl ether. The product was
applied to an Iatrobeads column (2 X 23 cm) and eluted with

ethanol-ethyl acetate—water (2:8:1, v/v/v), solvent B.

 

67

Fractions containing the epoxide were identified by
spotting an aliquot on a Fisher Redi-Plate, developing the
plate in solvent B, and spraying the plate with methyl red
reagent (141). Those fractions having only the principal
product were pooled and the solvents removed. The
conduritol C epoxide was recrystallized from 1 ml of
methanol with 5 ml of absolute ethanol at —80°. The
purity of the product was determined by gas chromatography
of the TMS derivative as described above for conduritol C.
The identity of the product was established by comparing
its melting point to the literature values of 135-1370
(140) and 1450 (142). a mass spectrum of the TMS
derivative which was compared with the TMS derivative of
conduritol B epoxide, and by inhibition of pure human
liver a-galactosidase A. The stereochemistry of the
product was determined by high resolution 1H~NMR as

described above for conduritol C.

Purification of Commercially Tritiated Conduritol C
Epoxide. A sample of 112 mg of conduritol C epoxide was
labelled by the Hilzbach method with tritium gas by the
Amersham Corp. The solvents were removed under a stream of
nitrogen. The product was suspended in solvent B and
applied to a column of Iatrobeads (2 X 20 cm) and eluted
with solvent B. Aliquots of the fractions were counted for
radioactivity as well as checked for epoxide as described

above. The radioactive fractions that also contained the

 

67

Fractions containing the epoxide were identified by
spotting an aliquot on a Fisher Redi-Plate, developing the
plate in solvent B, and spraying the plate with methyl red
reagent (141). Those fractions having only the principal
product were pooled and the solvents removed. The
conduritol C epoxide was recrystallized from 1 ml of
methanol with 5 ml of absolute ethanol at -80°. The
purity of the product was determined by gas chromatography
of the TIS derivative as described above for conduritol C.
The identity of the product was established by comparing
its melting point to the literature values of 135-1370
(140) and 145° (142), a mass spectrum of the TMS
derivative which was compared with the TMS derivative of
conduritol B epoxide, and by inhibition of pure human
liver a—galactosidase A. The stereochemistry of the

1

product was determined by high resolution u—NMR as

described above for conduritol C.

Purification of Coggercially Tritiated Conduritol C
Epoxide. A sample of 112 mg of conduritol C epoxide was
labelled by the wilzbach method with tritium gas by the
Amersham Corp. The solvents were removed under a stream of
nitrogen. The product was suspended in solvent B and
applied to a column of Iatrobeads (2 x 20 cm) and eluted
with solvent B. Aliquots of the fractions were counted for
radioactivity as well as checked for epoxide as described

above. The radioactive fractions that also contained the

 

 

I , I I}: . . "
- I. :I' J: 9.: (-
2w! IIIII'I-IIIII .I .I 2* *5 I , , 's . .‘ .I- - 5.1:)
.‘F'IIII"(§ ‘1 ‘ '{rIO IIII‘II-I. " ‘ 3. . .II.‘ .‘I;:‘I‘_:I:‘ I
I'll " I 3m I . I1 I III i It If I: )i.’ 3 .I
:m . muz? U'ii'lbI I! ~I .f H' .qI J l "uJDPUH
“I It '» Iavnhiv i “I In H ‘ :jLw Iinlwiu"
_ -‘:-III m I was." '-I .~'I‘ '- ‘2, ~)--uI.;- :~ rm .. .2) ‘ JI'VC-‘ltj ~11: ii. .‘ ’.-I.‘I
. ' JIL. I I IEIH L ~': Avrtnn it: on! it
-7.x",.II;»II-, 1. unit-45's: .‘if‘rfi 1".‘I‘. .4 an: 1,. (”I n. ,y ~,_l
v I d U!I :I 7.: HI ‘ . l.' I -I:
1 n) I I '01 .I m i; (L h Ur

.I’r.II!:;I . 'I it) IIH: . 'i 'i.. , I”) an. I) - . * .

I' . ’; Iz'ImeI'II:-..;' . )3..- ~I:I_I._.9.' ‘[.[I ..‘
IiI’H/l . .‘IIII ' I .. I‘ .I5 “HI!" Ii“I“

I I ' 1 ' '2“

J Inif1u3‘. I) I‘I;.. .'2'I . : .I'Htih I'I :iI:.'I‘- ..I.“

"'1 IfltI“"!T-' 1.. l'DIfII ‘W'v'omw. '9'!“'Iv I"“ "MI. .11‘( I .IIIIZ":~.:.'I.I

um .I III'I'JiOk rII l):I::II"H(.II: um I 3'] I ,')tgi:;;u

D’DY'II; 3m. III“) I): 1‘, 1;) mm... II: 7.. {-u, :I; ;, I. --.~.I. I

:.I«.’\ I‘m) ‘.IIII"I'$:-'[I ': .-.I .eIIII‘mI . .‘1 ‘fltI'.lIIi'. 121‘:-

-I.:.'*I’29b .:.-. obixouu 1::7 .rI'LI'I: . I: .‘w II I; \I‘.I= .2.

h
M
~¢
I

':;57 .I.II“.‘iTflU'I we

68

epoxide were pooled, and the solvents were removed under a
stream of nitrogen. The material was then dissolved in
water, applied to a G—25 column (1 X 115 cm), and eluted
with water. Fractions were collected and assessed for both
radioactivity and epoxide. The radio—chemical purity of
the material was assessed by TLC using unlabelled CCE as a
standard and scanning the TLC plate with a Berthold LB 2760
TLC Scanner. The approximate concentration of the 3H—CCE
was determined by chromatographing an aliquot of the
material along with known amounts of unlabelled CCE on TLC
and spraying with methyl red reagent. The specific

radioactivity was determined by counting an aliquot of the

material by scintillatiion counting.

F
I
I
f

 

 

VII’

 

RESULTS

Synthesis of the Affinity Ligand. The synthesis of the
affinity ligand (Figure 6, Compound Ix) was
straight—forward as all the reactions were facile. The
melting point of synthetic N—benzyloxycarbonyl—6—amino
hexanoic acid (Figure 6, Compound 111) was 53.5—54.5o
compared to the literature value of 54—550 (119). The
melting point of the a—galactosylamine—ammonia complex

(Figure 6, Compound V) was 94—980 compared to the

 

literature value of 95—960 (16); the reported melting
point of B—galactosylamine is 136—1370 (122). Neither

the melting points nor spectra of the other intermediates
in the synthesis of the affinity ligand were available to
verify the identity of the respective compounds. However,
the N-(N—benzyloxycarbonyl—S—aminohexyl)—a-Q_
-galactopyranosylamine Compound VIII (Figure 6) had a
migration on thin—layer chromatography, relative to the
minor products, that was similar to that found by Bishop
and Desnick (16). The purity of the affinity ligand
Compound 1x (Figure 6), was determined by 13C~NMR
spectroscopy (Figure 8). There were twelve peaks, as
expected in the spectrum: peaks 2 through 7 were from the
sugar, the others were from the spacer arm derived from
13C

6—aminohexanoic acid. The —NMR spectra of d— and

B—galactosylamine were also obtained (Figure 9) and

69

95!. ’c .3." :I.'< SIil' .IJI1133I.I'II.II.=. "'

\

:IIIIIIIIIIIII’I ‘.. -:1I]f I,

vII'l uli I 513’”-

u. Itobaw uJ: I‘ :5 H: m:" 'h'.uT1?
.IIII'JI'II 'J I.’-I:III'su‘lxolzx..-I: ‘I’I )l'VlJ 1 I 71100 9011!“!!!

U. .. _ .
:i in <;..:. -:..w I'-: Mr! rInn: ..9 'I'IIIIII'II III II- .IIIstsn

‘ii'li .Ii‘i .' ME I‘\( 1r (IIIIII'

"Ty/"FIVI‘J‘IL 9“] [J 59566:“);-

XHiUMUJ :ILtman II'mI.. .:IIII' su1 *- '

,I I .quc gnIIIun

mm. c L'wnqmnw ‘si It -nw It LHH‘IN~ .h quxIr-
LUIJldm “fl: nuuw ~v: ;.r‘ ' I:: J nxuarzuIIi

2,1 "~II ,-:.l'I.-'III:II~IIII;~II' II; .I. III.

I.I-. I" l‘ 1'.I I . III . I ;. 'I'Ii-ImI II. . III-I‘m; vni Hhm 6.11
I)!" '1‘ l I ' ’I 1"} :Iiii - !- I'l’lzII 13:. ..3 {U r!‘ I I ‘ I H
~I ‘1 .I'.. III 'I'

-- HI.'~:‘,‘ 1:1 «I =11. IMDI ad‘- '(‘lI'i‘flv

:II.." 2 'II'I'OIJYII'HI ‘ I '1!

'~: I?) "I'l'l,-‘l'i,' '1 , . ..IIuIFIIVezIIII'IL'I.I‘III'II.I.!'3

'1”! 41 -.-‘.iiil"l'i .IIIII IIIIH IH .II-: "-I-In

I IImI' 9m» II.‘ .e' .1100

(I 'iUll \ PI

IIIII.II'I '[IlIHI‘II w.) .I'..I:' II u.III

IMV . "

. I-. III I I I-I .I' ‘I'IH' ['5‘ .’.I IIIIIIIIIIra-II
.er. .I.. 'r' I '-I HHI‘II . . .- .I'I‘" mli“ .‘-:
:'I I -.‘)'I )‘I I I g-I'u I .1qu .I..III . :1: III: . . ... I'i-IngeI

ML: 13 .‘Ji' ': '!'- Iii 'l) " ’

. I’ll-HI" -. ' 'IInI‘I ‘il'lv .. ..'-I uri: .Ih‘yIIIII
. . -l
bur . - 2wuq- Hm! .' ntI n2.“ J.UHUJWMJI

5h“ 1v :BLi) 'nuI.I0u oat: uwuw qumI;.uLanfng u

 

 

 

 

 

70

bN.w~
Hr.wN
HH.on
bw.wm
v¢.H¢
m>.N®
ww.bw
“n.6b
oc.mb
mo.Mb
ve.w>
mm.omH

OHN
HHH

HNMVIOQL‘QOQ

“mun mxaon Haawoawua

mnu no Eng :fi wuwfinm Huofiamno one .mzwom mna.¢m no :ofiuufifinloo a ma lsnuomam
one .mnuomnm mzzIo awnuo Haw was an Nocsz :wchmz .an >5 umumlonuooam

cola: uwxzum a :fl wwafl was manewm way we Espuomqm m22|o a uza .umuws

mo H: m =fi Me com no :oflumnucoocoo a aw U0>Hommfiv was unnum* huficfiumw one

uzwwfia huazfimmm may we aauuumnw «EzIUMH .w mhsmfim

 

Ema
mm om mp 00_ mm_ 09 mt
_ _ _

71

 

 

 

 

 

 

 

 

 

‘

.\ VIII

n

«ML 5

 

72

Figure 9. 13CmNMR spectra of a~ and B~Galactosylamine.

Panel A. a—Galactosylamine, synthesized as described in the
Methods section, was dissolved at 100 mg/ml in water, and
the spectrum was made. This spectrum was a compilation of
257 scans. The chemical shifts in ppm of the major peaks
are:

1 82.96

2 71.44

3 70.80

4 70.66

5 69.54

6 62.73

Panel B. The above sample of a—galactosylamine was scanned
for 34 hours starting two hours after the sample was
dissolved in water. a—Galactactosylamine has been reported
to undergo inversion of configuration to the B-forl shortly
after dissolving in water (121). Peaks X and Y have the same
chemical shifts as listed above for peaks 1 and 5 of
a—galactosylamine. The spectrum was a compilation

of 86,315 scans. The chemical shifts in ppl of the major
peaks are:

87.24
77.61
75.18
73.82
70.66
62.83

monk-oomp-

 

3x 6
2‘

 

Ul

 

 

 

 

 

__J..W Wk

 

l l
l25 IOO 75 50

25

 

 

 

.flifl (5 'I

i
F
}
.’
I

 

74

3

Figure 10. Coupled spectra (1H-1 C) of the affinity

ligand and the galactosylamines

Panel A. 200 mg of the affinity ligand were dissolved in 3
ml of water. The spectrum is a compilation of 18,541
scans. The coupling constant of the anomeric carbon and its
hydrogen was found to be 164.2 Hz, and the chemical shift of
the anomeric carbon was 78.24 ppm. The chemical shifts in
ppm of some of the principal peaks are:

1 83.7

2 75.5

3 72.8

4 68.6

5 65.6

6 62.8

7 26.4

Panel B. Synthetic a—galactosylamine was dissolved in
water. The spectrum is a compilation of 16,755 scans and

lasted seven hours so that most of the o—galactosylamine
had undergone inversion of configuration to the &form.

The coupling constants of the anomeric carbon and its
hydrogen of the a~ and B—galactosylamine are 162.8 and 150.3
Hz, respectively. The chemical shifts. as determined from
these data of the anomeric carbon of the a— and
B—galactosylamine, are 87.17 and 82.95 ppm, respectively.
The chemical shifts in ppm of some of the principal peaks
are:

1 92.2
2 82.2
3 75.4
4 65.7
5 62.8
6 53.2

 

A 78.24(C,)

 

 

|64.2 Hz 3
(Jo—mu)
I 2 6
5
4 7
I l 1
75 5O 25

 

ppm

B 87-|7(C|’3-onomer)

(JC-l,H-l=|50.3Hz

I0

82.95(C,..<_anome,) 5

(Jo-IIH-I=162.8 Hz )\
I

M

l 1
I25 IOO 75 50 25
P Pm

 

 

5:4

76

compared to the spectrum of the affinity ligand with the
hope that the peaks from the sugar of the affinity ligand
would match those of either galactosylamine; they did not.
For example, the chemical shifts of the anomeric carbon,
which is one of the most distinctive for a sugar, were
78.34, 82.96, and 87.24 ppm for the affinity ligand,
orgalactosylamine, and B-galactosylamine, respectively.
Upon consulting with Dr. Hernan Nunez, it was decided that
the chemical shift information was insufficient to
determine the anomeric configuration of the purported

130) were made of

affinity ligand. Coupled spectra (In—
the affinity ligand and of both galactosylamines in order
to determine the coupling constant of the anomeric carbon
and its hydrogen. The size of this coupling constant in
sugars is characteristic of the anomerity of the sugar.

The coupling of the hydrogens of the carbons splits the
individual peaks previously due to each carbon, and the
midpoint of the two new peaks should be extremely close to
the chemical shift of the original peak. The distance in
Hz between the two new peaks represents the coupling
constant. The coupling constants of the anomeric carbon
and its hydrogen for the affinity ligand,
o—galactosylamine, and B~galactosylamine were 164.2, 162.8,
and 150.3 Hz respectively (Figure 10). These data show
that the anomeric configuration of the affinity ligand is
an That the affinity ligand was successfully used in

purifying two a—galactosidases is post facto evidence that

1h“ 1

(

.II‘ ‘ ‘II I) I .II II II '.2 I" .' II :I' III")
t'IL . . I . In. II | "'1 ." .'I 'i .I. "I I .I' .aI 'I
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-:..r.:I 'I III.....- I' .l'Ll" 'II .I,. - ' . 'u. 17.; III-E
u'an ., II.I : 'iI 3 I.-I aI. n Iaw
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II'I'-.’I")I' - .-. * ., .I,_-.-I.. I .nimnivenzv “34
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b«.1041uq II! ‘u u-. n r§IIuH1 IIturuva ‘u‘ anim"a39h
la Ii.-'l -I-' l ‘I' ' I! 'll III.I.II .131”: 11 VIIJI'II.
- ”w n1 ianxmnivenwapInn Uzou ' LI' ounnIE "In-lrn :uJ
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onquIve onus] 120g :1 enunhientosisn—- nw. --_.3

 

 

77

the anomeric configuration of the affinity ligand is a.
Purification of a—Galactosidases A and B from Hugan Liver

This purification scheme was devised in order to
maximize the yield of pure a-galactosidase A rather than
the B enzyme because the A enzyme, but not B, has been
associated with a lysosomal storage disease and is found in
much lower concentration than the B enzyme. Since the A
enzyme is of such low abundance in the liver, which has one
of the higher concentrations of the enzyme of any major
organ, and because human livers are so difficult to obtain.
extra procedures were taken in order to maximize the yield
of the A form even though the additional contaminants would
have to be removed at subsequent steps.

The data from three purifications of human liver
a-galactosidases are summarized in Table 3. It is not
apparent from the table, but the pellets from the initial
16,000 X g centrifugation had to be re-extracted with water
at least twice in order to obtain greater than 90% yield at
the first step. Previously (104,105) a 30—60%

(NH4)2804 cut was made of the first supernatant; here
it was found that the same specific activity of the
o—galactosidases was found in the 0—303 as in the 30-60%

(N64)280 cut so that only a 0—60% (NH4)280

4 4

cut was subsequently made.

The introduction of concanavalin A—Sepharose into the

. -I. 9:1“. g;' In ,- 2' I '.I'IJ; (I! l‘ufli'lll- 93'

I II: . I I I. ' " ' - . III-I ' -I I.. ..I- _, ..I. 'I." l VHF-1

.l ='=!Iln II' iI-II'II..7:-i .‘II-"HII -' I -! - ' I!"
, ‘ - , I I III' I -I It. II III II'I' :Ill' '1 'Il I‘vfl'
I I ' I.“ " .I II' I'.' II 0 I ‘ r- :.':I ' ~ 'I - I

I. 'j',” :1. ilizu’.

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1: IE. '1 '0 .I I I - I I I L‘- .I 'I‘. I
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III IIII III . 2 L'II'l-‘:i . I-Ir : . . I - :‘III: -’ -IH it:
c.'.--II'.' ll'-”D"‘i..'l'l' '.- .. -I'I:r.-.I I-I ...? ‘1’!“
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. I ' l""'lli: '2: . II' in 'HI'I" -|-'I- 'H'I bHI’ISILIWI
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I lift; 'f-ZIIJILUIJI'“ Z.‘.-.'.'II'I)IZ)I II I!\)!'_II'I))|II’I'_ :‘I'I:

 

 

78

Table 3. The purification of human liver a—galactosidases.

These data represent the yields and specific acitivities of
the human liver a—galactosidases at each step of the
purification scheme and are the average of three
purifications of the enzymes. The yield at the affinity
chromatography step represents only the most recent data as
the optimal conditions for affinity chromatography were
only recently determined.

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Step Yield Sp. Act. Fold X a—Gal A
Total 100‘ 0.00101* 1.0* 58
Homogenate
16,000 X g 95* 0.00166* 1.66* ND
supernatant
0—60% 88* 0.00228* 2.26* ND
(NH4)2804
Con A 87* 0.0509‘K 50.4* ND
Extract
DEAE 70* 74
A only 90** 0.140* 139*
B+A 0.0956* 95*
G—150 66* 72
A only 82** 0.495* 490*
B+A 0.370* 370*
Affinity
Chromatography
A only 58** 45.2 77.200**
8 only 37** 4.18 9,850**

*Data based upon the yield of both a—galactosidases.

**Data based upon the fact that the initial mixture of
a—galactosidases is 58% A.

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79

Figure 11. Column chromatography of a-galactosidases A and
B on 03—52.

The concanavalin A-Sepharose extract was applied to a
column of 03-52 (4.05 x 24 cm) pre-equilibrated in buffer
A. The column was washed with 200 ml of buffer A to
removed non-binding proteins. The a~galactosidases were
eluted with a linear NaCl gradient (4 liters total, 0 to
300 nM) in buffer A. Fractions of 15.6 II were collected
and assayed for a—gal A (closed circles), a—gal 8 (open
circles), and crgal A+B (open squares) activities. Protein
ws monitored by A (closed squares). Fractions
containing only a-galactosidase A and those containing A
and B were pooled separately, as indicated, and
concentrated to 25 ml in an Anicon Model 52 for gel
filtration on Sephadex G—150.

 

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purification scheme, as was previously done for the splenic
and placental enzymes (16,17), greatly facilitated the
purification of both enzymes. There was a nearly
quantitative yield of the enzymes and a purification of
twenty—fold at the Con—A step, which also meant that the
enzyme could be more easily managed.

The DE—52 afforded only a two— to three—fold increase
in specific activity of the u—galactosidases, but there was
an apparent separation of the A from the B enzyme. As can
be seen in Figure 11, the first peak of a—galactosidase to
elute from the DEAE contains only (bgalactosidase A
(Fractions 67-124). The second peak of enzyme eluting from
the column hydrolyzes p—NP— -GalNAc and is apparently
wgalactosidase 8. However, a significant amount of the
second peak of a—galactosidase hydrolyzes 4—MU—a—Ga1 in the
presence of 50 mM GalNAc which indicates that half of this
second peak is a—galactosidase A. The presence of so much
wgalactosidase A in a preparation of a—galactosidase B
would surely invalidate any kinetic or substrate
specificity studies.

The two pools of a—galactosidase from the DE—52 were
separately concentrated and applied to a large G—150
column. The sample size was generally about 2% of the
column volume, and the flow rate was 2 ml/hour/Cm2 of the
cross sectional area of the column as was suggested in the
Pharmacia manual for optimizing protein separations. As

can be seen from the data in Table 3, there is about a

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three—fold increase in specific activity of the enzymes and
about a ninety per cent yield at this step. This increase
in specific activity is primarily due to the removal of
high molecular weight proteins (Figure 12). The enzyme
preparation was reddish brown up to this point, and the
fractions from the G—150 column having the a-galactosidases
were light yellow; those with the unwanted proteins were

brown or red.

~
- w-.~._.-

Affinity Chromatography of a—Galactosidase A. The yield of
pure a—galactosidase A from the affinity chromatography
step was initially around 25%, but occasionally as high as
50%. The enzyme must be acidified prior to affinity
chromatography in order for the enzyme to bind to the
affinity resin because the enzyme elutes from the resin at
pH 5.5 or above. Initially the enzyme was acidified to pH
4.7 as Bishop and Desnick (16) used for the splenic enzyme,
but the pl of the human liver enzyme is 4.6 (105). In the
last enzyme purification, the enzyme was acidified to pH
5.0, and the yield at the affinity chromatography step in
both a trial run and a large preparation was 70%.

Affinity Chromatography of wGalactosidase B. As can be
seen in Figure 13, the 50 mM GalNAc prevented the
a—galactosidase B from binding to the affinity resin so
that the enzyme passed through the column with the unwanted
proteins (Fractions 2—12), while the a—galactosidase A was
retained. The a—galactosidase A was eluted with galactose

at pH 6.0 (Fractions 32—40). After the GalNAc was

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83

Figure 12. Column chromatography of a—galactosidase A on
Sephadex G~150.

Those fractions from the DE—52 step containing only
a—galactosidase A were pooled and concentrated to 25 ml.
The enzyme was appled to a Sephadex G—150 column (3.5 X 110
cm) pre—equilibrated in buffer A. The enzyme was eluted
with buffer A at a flow rate of approximately 20 ml per
hour, and fractions of 5.4 ml were collected. Enzyme
activity was determined with 4—MU1x—Gal (closed circles),
and protein was monitored by determining A 80 (open
circles). Fractions containing greater than 100 munits per
ml were pooled and concentrated in the Amicon Model 52 to
approximately 1.0 unit per ml for purification by affinity
chromatography.

 

 

84

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85

Figure 13. Dual affinity chromatographic separation of
a—galactosidases A and B.

The mixture of partially purified crgalactosidases A and B
from the G—150 step was acidified with HCl to pH 4.7, and
solid GalNAc was added so that the concentration of GalNAc
was 50 mM. The enzyme was then applied at 4 to a 0.75 x
10 cm affinity column at that had been pre—equilibrated
with Gomori Citrate~Phosphate pH 4.6, containing 0.15 M
NaCl and 50 mM GalNAc. Fractions of 2 ml were collected.
The balance of the d—galactosidase B was eluted with 15 m1
of the GalNAc buffer. o—Galactosidase activity was
measured with 4—MU—(kGal (solid line), and the A 0

(dashed line) of the fractions was determined. Tfie unbound
proteins were eluted with Gomori Citrate—Phosphate pH 4.6
containing 0.15 M NaCl until the A 80 of the effluent was
zero. The u~galactosidase A was eluted with Gomori
Citrate—Phosphate pH 6.0 containing 0.4 M galactose and 0.5
M NaCl, fractions 31-40. The fractions containing
a—galactosidase B, 2-14. were pooled, concentrated to less
than 10 ml, and were dialyzed versus 10 mM sodium phosphate
pH 6.5 to remove the GalNAc. Thetx—galactosidase B was
then purified by affinity chromatography as described in
the Methods section.

 

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87
dialyzed from the wgalactosidase B, the enzyme was
re—applied to the affinity resin at pH 4.7. The yield of
a—galactosidase B at the affinity chromatography step was

around 80% even though the p1 of the enzyme is 4.5 (104).

Chemical Characterizations of the Purified a—Galactosidases
A and B. Human liver a—galactosidases A and B had
previously been shown to have native molecular weights of
104,000 and 90,000 (105) respectively. 1 have found that
the subunit molecular weights of A and B are 47,800 and
46,800 daltons, respectively, as determined by SDS—gel
electrophoresis (Figure 14). Both a—galactosidases appear
as very diffuse bands in the gel, which is characteristic
of glycoproteins.

Approximately 100 ug of each enzyme was dialyzed
versus water and submitted to Doris Bauer of this
department for amino acid analysis. An additional 101Jg of
a—galactosidase A was submitted to Dr. Al Smith of the
Department of Biochemistry of the University of California
at Davis for another analysis of amino acid composition.
Both analyses of a—galactosidase A gave similar results.
The composition of a—galactosidase A, as determined by Dr.
Smith, and the composition of a—galactosidase B are given
in Table 4 which also has the composition of several other
human lysosomal enzymes. .

Eight nmol of carboxymethylated a-galactosidase A and

approximately 36 nmol of carboxymethylated (egalactosidase

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Figure 14. Molecular weight determination and assessment
of purity of human liver a~galactosidases A and B.

Five 1g of each protein was applied to the respective well,
electrophoresis was performed according to the method of
Laemmli (129), and the gel was stained with Coomassie
Brilliant Blue R. Lane 1 contained the following standard
proteins whose subunit molecular weights X 10 are

listed in the figure in decreasing order: phosphorylase a,
bovine serum albumin, ovalbumin, aldolase, and
a—chymotrypsinogen A. Lanes 2 and 3 contained
a—galactosidase A and B, respectively. The subunit
molecular weights of the a-galactosidases were determined
from a plot of R versus log MW of the standards and are
47,800 and 46,80 for (rgalactosidase A and B,
respectively.

 

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B were submitted for N-terminal sequencing to Dr. Al Smith
of the University of California at Davis Department of
Biochemistry. The results are given in Table 5, which also
includes the N-terminal sequences of other human lysosomal
glycosidases.

Since the N—terminal sequences of both
a-galactosidases are so similar, it was decided to compare
more of the sequence of the two enzymes by sequencing
internal peptides derived from both enzymes. Bach enzyme
was carboxymethylated and digested with trypsin, and the
tryptic peptides were separated by reversed-phase HPLC as
shown in Figures 15 and 16. Several peptides of
a-galactosidase B (Figure 16), but few of a—galactosidase A
(Figure 15) were separated by HPLC.

One peptide of a—galactosidase A was been purified
(Figure 17), but there was not enough material for
N~terminal sequencing. Several peptides of a-galactosidase
B from the chromatogram shown in Figure 16 were repurified
using the HFBA system as described in the Methods section.
These peptides as well as several peaks from Figure 16 that
seemed likely to have a single peptide were submitted to
the Protein Sequencing Facility of the University of
Michigan for N-terminal sequencing. Unfortunately, there
was not enough of any of these peptides for sequencing (50
or more pmol is a reasonable amount). There was enough
a-galactosidase B for two additional primary separations of

tryptic peptides, one of which is shown in Figure 18.

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Tfliere was enough of several of these peptides for
N—terminal sequencing in these two HPLC separations. Some
of the peptides were repurified using the uBondapak Phenyl
column before being submitted for sequencing (Figures 19,
20, and 21 demonstrate three successful peptide
repurifications). Altogether, nine peptides of
a—galactosidase B were sequenced at the University of
Michigan Protein Sequencing Facility, and these sequences
are given in Table 6, which also has the known sequences of
CNBr and tryptic peptides of human placental

i
a—galactosidase A. . fi'

 

Antibody Production

Polyclonal. Rabbit antibodies raised against human liver
a—galactosidase A were successfully employed by others in
this laboratory to determine the in_yiyg_glycoprotein
processing events of a—galactosidase A (31). The
antibodies were found to be monospecific by
immunoprecipitating single bands of precursor and mature
forms of a—galactosidase A from Chang liver cells.
Monoclonal. Over 750 hybridomas derived from the fusion of
mouse spleen cells and SP/z cells were tested for the
production of anti—(kgalactosidase A antibodies. Two
positive clones were found. Unfortunately, a collaborator
left the cells on the bench overnight; the cells lived, but

ceased production of the antibodies.

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Table 6. Peptide Sequences of Human u—Galactosidases
A and B

Placental a—Galactosidase A (27)

Cleaved by CNBr
—(Met)—Ala—Leu~Leu-Lys—Arg—
~(Met)—Ile—Asn—Arg—Gln—Glu-
-(Met)—Leu—Va1—Ile—Gly—Asn—

Digested with trypsin
-Ala—Leu—Gly—Phe—Tyr—
—Cys-Cys—G1u—Ser—A1a-
~Gln—Phe—Ala—Asp—Ile-
-Leu—Cys—Asp—Asn—Leu—
—Leu—Gln—Ala—Asp—Pro—
—Leu-Gln—A1a—Leu—Phe-
—Lys—Leu—Leu—Gln~Asp—
—Ser—Ile-Leu—Asp—Trp—
—Ser—Tyr—Gly—lle—A1a~

 

Liver a-Galactosidase B (Present work)

Digested with Trypsin
—A1a—Gln—Met—A1a—
-Asp—Met—Gly—Tyr—Thr—Tyr—(Len)—(Gly/Asn)—
—Leu—Asp—Asp—Leu—
—Leu—Leu—Ile—Xa-(Val)—
-Met~A1a—Gln—Asp—Gly-xa—Arg
—Ser—Ala—Asp—(Gln)—(Val)—
—Thr-Asp—Met—Pro—Tyr—Arg
-Val-Glu—Tyr—
—Val—Va1—Gln—Asp—Ala—(Glu)—Thr—Phe—A1a—(Glu)—Xa-(Lys)

The identity of the residues in parentheses are not known
with certainty. The Glu's in the last peptide could be
Gln's.

110

Synthesis of Conduritol C Epoxide

 

The principal intermediate in the synthesis of CCE was
found to be the dibromotetraacetate (Figure 7, Compound V)
by its melting point of 1430 (Table 7) and by the
appearance of two bromines in its mass spectrum (data not
shown). The two bromines were removed by powdered zinc.
and the product had a melting point of 89° which
indicated it could be the tetraacetate of conduritol B, C,
or F. An infrared spectrum of the presumed conduritol C
tetraacetate was compared to the spectrum made by
Stegelmeier (138) both of which are shown in Figure 22. and
the two spectra are identical.

The acetyl groups were removed from (Figure 7,
Compound VI) by triethylamine as described by Radin and
Vunnan (139), but the melting point of the product did not
match the literature value of 148—1500 (140). A low
resolution proton NMR spectrum indicated the presence of
acetyl groups (data not shown). The partially
de-acetylated material was treated with additional
triethylamine until the melting point of the product
matched the literature value for conduritol C which
eliminated the possibility that the product could be any
conduritol but C (Table 7). The infrared spectrum of the
presumed conduritol C is similar. but not identical to the

spectrum of conduritol B (Figure 23). The mass spectra of

 

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Table 7. Melting points of intermediates and related
compounds in the synthesis of conduritol C epoxide

Compound Melting Point
Literature Found
0 o
Dibromo 144 (138) 143

tetraacetate (V)

Conduritol A Liquid (143)
tetraacetate
Conduritol B 91—92° (143)
tetraacetate

0 0
Conduritol C 91—92 (143) 89
tetraacetate (VI)
Conduritol D 102-104° (143)
tetraacetate
Conduritol F 92° (143)
tetraacetate
Conduritol A 141—142° (143)
Conduritol B 2050 (143)
Conduritol c (v11) 148—1600 (140) 149—150°
Conduritol E 179—1800 (143)
Conduritol F 103—104° (143)
Conduritol B 157—159° (140)
Epoxide
Conduritol c 135—137° (140) 139—1410
Epoxide Trans (VIII) 145 (142)
Conduritol c 126° (142)

Epoxide C18

The Roman numerals in parentheses refer to the
intermediates in Figure 7 where the reactions of the
synthesis are illustrated.

 

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the TMS derivative of conduritol B and C were also similar,
but not identical (Figure 24). The purity of the product
as determined by GC was 97.8%.

Conduritol C was reacted with m—chloroperoxybenzoic
acid to produce the trans epoxide. The product had a
melting point of 139—1410 compared to the two literature
values of 135-137° (140) and 145° (142) and the 126°

of the cis epoxide (142). The product gave a yellow color

 

upon spraying with the methyl red reagent and had the same
TLC mobility as authentic conduritol B epoxide. The
infrared spectrum of the presumed conduritol C epoxide is
shown in Figure 25 (there was not enough conduritol B
epoxide available for a suitable infrared spectrum to be
made). The mass spectrum of the TMS derivative of the
product was compared to the spectrum of conduritol B
epoxide; these spectra are similar but not identical
(Figure 26). The molecular ion of CCE was found at m/z
450; it had an abundance of 0.06%. The purity of the
product, as determined by GC of the TMS derivative, was
96.9%.

This conduritol C epoxide was used in inhibition
studies of both o—galactosidases, but doubt was cast upon
the identity of the product that had been synthesized
because of the high Ki observed for the inhibition of
o—galactosidase A towards 4—MU—a—Gal (145). Therefore high
resolution 1H—NMR spectra were made of the purported

conduritol C and conduritol C epoxide to verify the

 

 

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117

Figrure 24. Mass spectra of conduritol B and
conduritol C.

The trimethylsilane derivatives of conduritol B atui
the purported conduritol C were formed by the metfliod
of Sweeley §£_al; (144). The samples were analyzed by
GC-MS in the M.S.U. Mass Spectrometry Facility on a
Hewlett—Packard 5985 GC/MS System. The column was I: 6
foot long 4% 88—30, and there was a linear increase in
temperature from 140 to 2000 at 40 per minute.

Panel A. Conduritol B

Panel B. Conduritol C

 

 

 

 

 

 

 

 

 

 

 

 

 

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121

Figure 26. Mass spectra of conduritol B epoxide and
conduritol C epoxide.

The TMS derivatives of conduritol B epoxide and the

purported conduritol C epoxide were formed and
analyzed by GC—MS as described in the legend to Figure

24.
Panel A. conduritol B epoxide

Panel B. conduritol C epoxide

122

 

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123

structures of these two compounds.

1

Peak Assignments in the H-NMR Spectra of Conduritol C.

 

In Figure 27, protons A and B are the olefinic protons, and
the coupling constant of 10.6 Hz is characteristic of a gi§_
double bond. Peaks C, D, E, and F are the other four ring
protons. In the decoupled spectrum, Figure 28, Peak F is
unaffected by the irradiation of either olefinic proton
indicating that peak F is either proton 4 or 5 (Refer to
Figure 31 for the numbering system as well as the
structures of conduritol C and conduritol C epoxide). The
coupling constant of 8.1 Hz appearing in peaks D and F
indicates that proton D is coupled (or adjacent) to proton
F in the molecule, and similarly, the coupling constant of
2.2 Hz appearing in peaks A and D indicates that these
protons are adjacent. Panel F of Figure 28 shows that
Peaks E and F are coupled. These data indicate that there
are two possible circular permutations of the protons on
the ring: ~B-A-D—F-E-C— and -A—B-C-E—F—D-. The first was
chosen because the coupling constant of 2.2 Hz between
peaks A and D indicates a bond angle of 58° between the
two hydrogens, which is consistent with the structure of
conduritol C, and because the protons at carbon four of
simple sugars have the highest upfield chemical shift of
the ring protons which would indicate Peak F would be
proton four.

1

Peak Assignments in the H-NMR Spectra of Conduritol C

 

Epoxide. The interpretation of the high resolution

124

Figure 27. Proton NMR of conduritol C

Approximately 6 mg of presumed conduritol C were dissolved
in D 0, and the sample was scanned in a Bruker WM—250

Spec rometer under the direction of Dr. Klaas Hallenga of
the M.S.U. Department of Chemistry. The spectrum was
interpreted with the assistance of Dr. Kimihiro Kanemitsu
of this laboratory as well as the staff of Dr. Hallenga.
The chemical shifts in ppm and the coupling constants in
Hz are as follows:

H 3 Chem. Shift J1_2 J2_3 13_4 J4_5 55_6 J6_1
1 5.60 10.63 ND

2 5.79 10.50 2.16

3 4.27 2.20 8.15

4 3.64 8.11 2.15

5 4.09 ND ND

6 4.39 ND ND

The peak at 4.75 ppm is due to H20.

'01]:

 

 

 

125

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126

Decoupled proton NMR spectrum of conduritol C

The sample of purported conduritol C used for Figure 27 was
irradiated with an external source of radio—frequency
radiation at each of the respective absorbances listed in
Figure 27.

Panel

Panel

Panel

Panel

A

B

C

D

Irradiation of the proton
Irradiation of the proton
Irradiation of the proton

A normal coupled spectrum

at 5.79

at 5.60

at 4.39

similar

ppm.
FDI-

to Figure 27.

 

 

 

 

”(I

 

128

Figure 28. Continued.

Panel B Irradiation of the proton
Panel F Irradiation of the proton
Panel G Irradiation of the proton
Panel H A normal coupled spectrum

at 4.27

at 4.09

at 3.64

similar

POI.
ppm.
ppm.

to Figure 27.

 

 

‘
%

 

 

 

 

: .
i

 

I i
1 .

 

 

130

1
H—NMR spectrum of the purported conduritol C epoxide was

facilitated by the assignment of the peaks in Figure 27 to
specific protons in conduritol C. by a more distinctive
decoupled spectrum than that of conduritol C, and by very
well—defined peaks in Figure 29. From the decoupled
spectra of CCE, Figure 30, it can be seen that peak A is
coupled with both peaks D and E, that B is coupled with C
and possibly F, that C is coupled with both B and E, D is
coupled with A and F, E is coupled with A and C, and that F
is coupled with D and possibly C. These data indicate a
circular permutation of —A—D—F—B—C—E—. Peaks E and F of
conduritol C were assigned positions 5 and 4, which should
be changed little by the epoxidation, and these two
resemble peaks C and B, respectively, in CCE so that peaks
C and B were tentatively assigned to be protons 5 and 4,
respectively. The distance in ppm between E and F in
conduritol C and between C and E in CCE is 0.45 ppm which
indicates that this assignment is correct. These data
indicate that peaks A through F are protons 3—6—5—2—4—1,
respectively. The coupling constants for peaks A, D, E,
and F are very well—defined and can be used to determine
bond angles between the protons by a plot of the Karplus
equation. The coupling constants of peaks 8 and C are not
as easily interpreted as the others. but since peak C in
CCE is so similar to peak E in conduritol C. and the J3_4

and J4_5 are so similar in both molecules, the

conformation of both molecules around carbons 3, 4, and 5

 

 

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131

Figure 29. Proton NMR of conduritol C epoxide

Approximately 6 mg of purported conduritol C epoxide were
dissolved in D 0. The sample was scanned, and the
spectrum was interpreted as described in the legenci to
Figure 27. The chemical shifts in ppm and the couziling
constants in Hz are as follows:

H # Chem. Shift J1_2 J2_3 J3_4 J4_5 J5_6 .16_1
1 3.27 2.04

2 3.51 2.29

3 4.16 2.40 8.72

4 3.47 8.72 1.61

5 3 92 1.82 3 67

 

 

 

 

 

 

132
B
C
D E
F
A
1 4 n 4 L J J n 1 n A 41 1 1 I
4(2) 3.8

 

 

 

133

Figure 30. Decoupled Proton NMR spectra of conduzfiitol C
epoxide.

The sample of purported conduritol C epoxide used for
Figure 29 was irradiated with an external source of
radio-frequency radiation at each of the respective
absorbances listed in the legend for Figure 29.

 

Panel A Irradiation of the proton at 4.16 ppm.
Panel B Irradiation of the proton at 3.96 ppm.

Panel C A normal coupled spectrum similar to Figure 29.

 

 

[1‘4’

 

 

Figure 30

Panel

Panel

Panel

Panel

continued.

Irradiation

Irradiation

Irradiation

Irradiation

of

of

of

of

the

the

the

the

135

proton
proton
proton

proton

at

at

at

at

.92

.51

.47

.27

ppm.

ppm .

PP”-

FDI-

 

136

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137

are almost identical.
Three—diggnsional Structure of Conduritol C Epoxide.

The J1_2 of 2.04 for CCE is consistent with that for

epoxides. The J2_3 of 2.34 Hz would indicate a bond

angle of either 55 or 1230 according to the Karplus
plot. Since the epoxide is either gig_(up) or trans
relative to the plane of the molecule, and the expected
bond angles would be either 0 or 1500 for the gl§_and
trans configuration, respectively, the epoxide is trans
(down). The J3__4 of 8.72 Hz, the J4_5

J5—6 of 1.7 Hz, and the J6—1 of 3.9 Hz indicate the

of 1.72 Hz, the

respective angles between these pairs of hydrogens are 168,

58, 45, and 118°.

Substrate and Kinetic Studies of o—Galactosidases A and B.

 

Substrate Specificity. To show that the preparation of
a-galactosidase 8 contained no a-galactosidase A, an assay
was performed of both a—galactosidases using the natural
substrate, GbOseacer. The results of this study are

shown in Figure 32, which shows that a—galactosidase A, but
not B purified by the techniques as described here,
hydrolyzes the natural substrate, GbOse3Cer.

Kinetic Characteriggtions. Some of the kinetic paraaeters

of the a-galactosidases were deterained in order to compare

these enzymes to those in the literature and to establish

 

138

Figure 31. Three-dimensional structures of conduritol (Izuld
conduritol C epoxide.

These structures were drawn like those made by Legler
and Herrchen (142) and are consistent with the data of
Figures 27, 28, 29, and 30.

A. Conduritol C

B. Conduritol C Epoxide

139

 

 

an"

140

Figure 32. Hydrolysis of GbOse3Cer by a—galactosidases A
and B.

Ten munits of each a-galactosidases were incubated at 37°
with 100 nmoles of bovine GbOse Cer in a reaction mixture
also containing 50 ug of sodium taurocholate and 50 ul of

Gomori Citrate—Phosphate pH 4.2. The reaction mixtures were
incubated for one hour when an additional 10 munits of the
respective enzyme were added. The reactions proceeded for

an additional hour and were terminated by boiling.
Comparable amounts of the respective reaction mixtures were
spotted on a silica gel 6 plate which was developed in
chloroform—methanol—water (65:25:4, v/v/v), and

sprayed with the orcinol reagent to visualize the
carbohydrates. Lane 1 contained standard LacCer. Lane 2
contained standard bovine GbOse Cer. Lane 3 contained

the products from a reaction mixture that contained
a-galactosidase A. Lane 4 contained the products from a
reaction mixture that contained a—galactosidase 8. Lane 5
contained the products from a reaction mixture that
contained no enzyme. Lane 6 contained standard sodium
taurocholate.

 

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OTC

142
conditions for determining whether or not CCE is a suicide
inhibitor of a—galactosidase A.

The initial velocities at each substrate concentration
were determined using approximately one milliunit of enzyme
and time points of less than ten minutes, and the initial
velocities were calculated by linear regression of two or
more non-zero time points. The kinetic constants were
determined by a combination of the methods of Wilkinson

(146) and Mannervik (147).

(rGalactosidase A. The Km of crgalactosidase A for the
synthetic substrate 4*MUfiX—Gal is 1.83 mM (Figure 33).
Galactose was found to be a competitive inhibitor of
wgalactosidase A with a Ki of 16.7 In (Figure 34).
N—Acetylgalactosamine is not an inhibitor of
wgalactosidase A (Figure 35). Conduritol C epoxide
appears to be a competitive inhibitor of d—galactosidase A
with a Ki of 330 mM (Figure 36). This last value was based
only upon one concentration of CCE due to a very limited
amount of CCE. The Vmax of a—galactosidase A as determined
in the presence or absence of the competitive inhibitors

was 52.9 14.31 units/mg protein.

wfialactosidase B. The Km of crgalactosidase B for the
synthetic substrate 4—MU4x—Gal is 13.10 :_2.45 mM (Figure
37). Galactose was found to be a competitive inhibitor of

wgalactosidase B with a Ki of 27.9 mM (Figure 38).

I

 

 

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.< omwufiwouoadowla
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N—Acetyl-Q—galactosamine was found to be a competitive
inhibitor of o—galactosidase B with a Ki of 1.65 mM (Figure
39). The Vmax of (rgalactosidase B in the presence or

absence of the competitive inhibitors was 13.9 :_1.68.

Conduritol C Epoxide Inhibition of a-Galactosidase A

It was reported to us (148) that conduritol C epoxide
inhibits a—galactosidase A at 37° with a T 1/2 of 210
minutes at a concentration of 10 mM CCE. Later, we were
told that a concentration of at least 100 mM CCE was
required for inactivating splenic a—galactosidase A (149).
Because of the thermal and pH instability of
o—galactosidase A, reaction conditions were determined that
would allow reaction between CCE and the enzyme and yet
maintain enzyme activity in the absence of CCE. A pH of
6.5 and a temperature of 4° were found to be
satisfactory.

Pure o—galactosidase B and partially purified
o-galactosidase A were incubated at 4° at pH 6.5 for
several days in 100 mM CCE, and samples were assayed at
various times for enzyme activity. The results of this
study are shown in Figure 40, and the data are expressed as
100 times the ratio of the amount of product formed in the
presence of CCE (El) relative to the amount of product
formed in the absence of CCB (Eo) and plotted on a
natural log scale. Conduritol C epoxide inhibited both

enzymes in a time—dependent fashion, but the rate of

 

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158

Figure 40. Time~dependent inactivation of a—galactxnsidases
A and B by conduritol C epoxide.

Human liver a—galactosidasesOA (closed circles) and 13 (open
circles) were incubated at 4 in 10 mM sodium phosphate
pH 6.5 containing 100 mM Conduritol C Epoxide for various

times up to 168 hours. a—Galactosidases A and B were
assayed for two minutes with 4—MU—chal and p—NP-orGalNAc
respectively. The data are expressed as 100 times the

ratio of the amount of product formed in the presence of
OCH (8 ) relative to the amount of product formed in the
absence of 008 (E ) and plotted on a natural log scale.
The rates of inacgivation were detegmined by regresgion
analysis and found to be 5.40 X 10 and 1.10 X 10

hr ' mole '1 for d-galactosidase A and B.

respectively. The correlation coefficients for the rates
are .9889 and .6625 for d—galactosidase A and B,
respectively.

 

159

 

 

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160

inactivation of wgalactosidase B, as determined by
regression analysis, was 20.5% that of d—galactosidase A.
This inactivation is characteristic of suicide inhibitors.
The regression line for d—galactosidase A included all time
points, except for the time points from one through six
hours when the data points were quite variable. Since the
inactivation of wgalactosidase B was so slow (The T 1/2
for inactivation would be 26 days), subsequent experiments
with conduritol C epoxide were performed only with
a—galactosidase A.

After galactose was found to be a competitive
inhibitor of a—galactosidase A with a Ki of 16.7 mM, a
series of experiments were performed to determine if CCE is
a suicide inhibitor of d—galactosidase A. Pure
d—galactosidase A was incubated at 4° in 10 mM sodium
phosphate, pH 6.5, containing 1.0 mg/ml BSA to stabilize
the enzyme. Four series of tubes were used in the
experiment: the first had no inhibitor and these were used
as controls, the second contained galactose, the third
contained CCE, and the fourth contained both galactose and
CCE. The concentration of inhibitor was 100 mM in all
cases. Two or more samples were assayed with 4—MU—d—Gal at
each time point for times up to 128 hours (It is important
to note that the concentration of inhibitor was about 15 mM
in the assay mixture). The data are expressed as 100 times
the ratio of the amount of product formed in the presence

of inhibitor (El) relative to the amount of product

 

161

formed in the absence of inhibitor (Eo) and plotted on a
natural log scale as shown in Figure 41. These data show
that the enzyme is inhibited by CBS alone to a similar
extent (T 1/2 of inhibition was 75 hours) as seen in Figure
40, and that the enzyme is inhibited to a similar extent by
galactose and by galactose in combination with 008. These

data show that the time-dependent inactivation of

 

a-galactosidase A by conduritol C epoxide is substantially
reduced (90.7%) by galactose, a competitive inhibitor of
o-galactosidase A. The protection experiment demonstrated
that CCE is a suicide inhibitor of

a—galactosidase A.

Purification of Commercially Tritiated Conduritol C
Epoxide. As can be seen in Figure 42, very little of the
radioactivity, 100 mCi, co—migrated with authentic 008.
The material was purified by column chromatography on
Iatrobeads and gel filtration on 6-25. Those fractions
having the same TLC mobility as CCE were pooled, and the
yield was approximately 27% or 30 mg of CCE. The specific

activity of the material was 2.5 x 109

cpm per mmole or
2.5 X 10° cpm per nmole.

Inactivation of a—Galactosidase A with Tritiated Conduritol

 

Q_Epoxide. Fifteen milliunits of purified crgalactosidase
A (33011g) were incubated at 4° in 2 m1 of 10 mM sodium
phosphate buffer (pH 6.5) containing the 30 mg of tritiated
conduritol C epoxide. Aliquots were removed from the

reaction mixture and assayed with 4—MU—a—Gal to assess the

162

Figure 41. Protection of o~galactosidase A by galactose
from inactivation by conduritol C epoxide.

Human liver d—galactosidase A was incubated at 4° in 10

mM sodium phosphate, pH 6.5. containing 0.5% BSA in the
presence or absence of inhibitors for various times up to
128 hours. The samples were assayed with 4-MU-a-Ga1 for
two minutes. Results are expressed as 100 times the ratio
of the amount of product formed in the presence of
inhibitor (E ) relative to the amount of product formed

in the absence of inhibitor (E ) and plotted on a natural
log scale. One series of tubes contained 100ml galactose
(closed circles), another IOOmM of both galactose and CCB
(open circles), and the other had IOOmM CCE (open squares).
The rates for inactivation were analyzed by regression
analysis, and the rates for inactivation in the presence of
galactose, galactose plus_§CE, and CCE along were found to
be 6. 7 X 10 , 9.03 X 10 , and 9.64 X 10 hr °

mole- '1, respectively. The correlation coefficients for
the rates of inhibition are .1099, .6370, and .9689 for
gal. gal plus CCE, and CCE, respectively.

 

163

 

 

 

 

 

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164 i

Figure 42. Purity determination of tritiated conduritol C
epoxide.

An aliquot of the commercially tritiated conduritol C
epoxide was spotted on a silica gel TLC plate which was
developed in EtOH, EtOAc, water (2:8:1, v/v/v). The plate
was sprayed with methyl red reagent (131) to visualize the
CCE. A scan of the plate with a Berthold LB 2760 TLC
scanner indicated that less than 1% of the radioactivity
migrated with authentic conduritol C epoxide which
indicated the material needed to be further purified.

165
Top —>

 

O

 

 

 

Bottom ——>J,

166

extent of inactivation over 300 hours. Data are expressed
as 100 times the ratio of the amount of product in the
presence of €08 (El) relative to the original amount of
enzyme activity (80) and plotted on a natural log scale
(Figure 43). The data through 180 hours were analyzed by
least squares, and the T 1/2 for the inactivation of
crgalactosidase A was 75 hours. The specific activity of
the radiolabeled enzyme was 15,700 cpm/nmole of monomer,
which indicates that six molecules of CCE reacted per

monomer of crgalactosidase A.

 

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167

Figure 43. Inhibition of a—galactosidase A by tritiated
conduritol C epoxide.

Fifteen units of a—galactosidase A were incubated with 30
mg of tritiated 808 in 2ml of 10 mM sodium phosphate, pH
6.5, buffer at 4 for thirteen days. Aliquots were
removed at various times and assayed with 4-MU4x-Gal. The
data are expressed as 100 times the amount of activity in
the presence of inhibitor (E ) relative to the initial
activity (BO) and are plotted on a natural log scale.

The data through 180 hours were analyzed by regression
analysis, and the regression line is shown. The T 1/2 for
the inactivation of a—galactosidase was 75 hours.

IOO
80

60

040

°/o EI/E

20

IO

168

 

 

 

 

 

O

IOO

Hours

200

 

DISCUSSION

The Purification Procedure. The first few steps of the
enzyme purification were straight-forward, and the yields
were good primarily because very little enzyme was
discarded in order to maximize the yield. These extra
steps included the re-extraction of the pellets from the
first homogenization, extracting with Con-A Sepharose of
some rather gooey material that contained several units of
enzyme, the elution of the 03-52 column with 1.0 M NaCl to
save a few more units of enzyme, and saving the side
fractions from the 03—52 and G-150 columns.

Two procedures that were previously used in the
purification of human liver a—galactosidases (104,105),
hydroxyapatite and ampholyte displacement chromatography,
were eliminated from the purification scheme. In this
study, the yield at the hydroxyapatite chromatography step
was 70 to 80%, but the hydroxyapatite did not remove any
contaminant that could not be removed by affinity
chromatography. Ampholyte displacement was removed from
the purification scheme because the yield at that step was
36%, and the enzyme was about 40% pure (it is my opinion
that the two major impurities could have been removed by
gel filtration on G—150, but the overall yield would have
been 24%).

In one purification, the Con—A Sepharose extract was

169

 

 

170

concentrated and applied to the G-150 column with the
intention of by-passing the DEAE column. Something in the
enzyme preparation bound to the Sephadex causing the beads
to shrink and the column to stop. Apparently the 03—52
removes this component from the enzyme preparation.

The Affigity Resin. The affinity resin first used was
identical to that of Bishop and Desnick (16) which involved
the coupling of the affinity ligand N—6—aminohexanoyl-a—D-
galactopyranosylamine to carboxyhexyl—Sepharose with a
carbodiimide. Initial attempts to purify either
a-galactosidase gave yields of from five to fifty per cent,
but generally less than twenty-five per cent. In addition
the bulk of unwanted proteins bound to the affinity resin
which indicated that there were numerous free carboxyl
groups remaining from the carboxyhexyI-Sepharose. It was
decided to reduce the non—specific interaction of the
affinity resin by attaching the affinity ligand directly to
Sepharose 48 with cyanogen bromide. In the first use of
such an affinity resin, Harpaz and Flowers (109) used a
spacer arm synthesized from two molecules of
6-aminohexanoic acid. It was decided to attach the
affinity ligand with a spacer arm of only one molecule of
6-aminohexanoic acid because Lowe §£_gl; (150) showed that
affinity resins having a spacer arm of from six to eight
methylene groups are much more effective that those with
ten or more methylenes.

The unwanted proteins were eluted from the affinity

 

171

resin with Gomori citrate~phosphate buffer (pH 4.6)

containing 0.15 M NaCl until the A of the effluent was

280
zero before beginning the pH-salt gradient. In one
purification the affinity resin was washed with the pH 4.6
buffer as usual, the pH-salt gradient was run, then the
resin was washed with Gomori citrate—phosphate, pH 6.0.
containing 0.5 M NaCl until the A280 of the effluent was
zero before the enzyme was eluted with galactose. The
yield of enzyme in the galactose eluate was three per
cent. The lost enzyme was found in the fractions from the
pH 6.0 wash; it was found that a pH above 5.5 elutes
o-galactosidase A from the affinity resin.

The yield of crgalactosidase A at the affinity
chromatography step with the new spacer arm was
consistently between twenty—five and fifty per cent. Since
the pl of the liver enzyme is 4.6 (105), it was decided to
acidify the enzyme only to pH 5.0 prior to applying the
enzyme to the affinity resin. In both a trial and a large
purification of o—galactosidase A, the yield was 70%.
Acidification to a slightly higher pH, but below the 5.5 at
which the enzyme non-specifically elutes from the resin,
may improve the yield even more. The acidification of
a-galactosidase B to pH 4.7 does not lead to poor yields of
the enzyme even though its pl is 4.5 (104), The main
precaution about the affinity purification of
crgalactosidase B is that the enzyme activity in addition

to the A of the pH 4.7 eluted fractions should be

280

 

172

monitored as the enzyme can non-specifically bleed off.

The crgalactosidase B should be eluted with Gomori
citrate-phosphate, pH 4.6, containing 0.15 M NaCl and 0.4 M
galactose after the bulk of the unwanted proteins have
eluted but before much of the enzyme has bled off. The
yield of crgalactosidase B at the affinity chromatography
step was generally about 80%.

In another experiment, the Con—A extract was acidified
to pH 4.7 and mixed with the affinity resin. The enzyme
was eluted from the affinity resin, but the yield was only
25%, and the enzyme was very impure. It would be
reasonable for future investigators to apply enzyme
acidified to pH 5.0 or 5.2 from an earlier step in the
purification procedure to the affinity resin. If the yield
is over 70% at that step, the overall yield would be
greatly improved. Such a procedure has been developed for
the purification of human liver a-L-fucosidase by Alhadeff
g£_gl; (15). These investigators purified a-L—fucosidase
from a 100,000 x g supernatant of the total homogenate with
two passes over a comparable fucosylamine affinity resin as
was used in this study.

Qggl Affinity Chrogatography. The separation of the
mixture of o~galactosidases A and B from the DEAE step
presented a challenge as both enzymes have similar
molecular weights (104 and 90 Kd, respectively) and nearly
identical isoelectric points (4.6 and 4.5, respectively).

A variety of unsuccessful attempts were made to completely

 

 

173

resolve them including differential elution from Con
A-Sepharose, re-chromatography on DEAE, chromatography on
hydroxylapatite, and heat inactivation of wgalactosidase
A. It was then thought that since both d—galactosidases
bind to the affinity resin and GalNAc inhibits
a—galactosidase B, but not A, that GalNAc would selectively
elute —galactosidase B from the affinity resin. A
concentration of 50 mM GalNAc was chosen to elute the
a-galactosidase B because 50 mM GalNAc preferentially
inactivates the d—galactosidase B in the assay for
a—galactosidase A. This idea proved to be correct, and
this separation has proven very useful in improving the
yield of both enzymes. The importance of this procedure
was proven in that 79% of the a—galactosidase A that was
sequenced was originally in the A plus B fraction that
eluted from the DEAE column.

Yields of Enzymes. The overall yield of d-galactosidase A
of 58% in the procedure developed here compares favorably
to the 5.6% yield from placenta (17), the 27% yield from
liver (105), and the 31% yield from spleen (16), especially
sinCe the placental and liver enzymes were not pure. The
overall yield of wgalactosidase B of 37% in this procedure
is comparable to the 35% from placenta (17) and the 21%
yield from liver (104), both of which appeared to be pure

by gel electrophoresis.

174

Physical Characterizations of the Purified (rGalactosidgses

The purified a—galactosidase A has a subunit molecular
weight of 47,800 daltons, as determined by SDS-gel
electrophoresis, and this value is similar to the 49,800
daltons reported for the splenic enzyme. Since the native
molecular weight is 104,000 (105), and N-terminal
sequencing yielded a unique sequence, a-galactosidase A
must be a homodimer, which had heretofore only been
postulated.

The subunit molecular weight of a—galactosidase B is
46,800 compared to the 47,700 reported for the placental
enzyme (17). Since the native molecular weight of the
liver enzyme has been reported to be 90,000 (105), and the
N—terminal sequencing gave a single sequence,
o—galactosidase B is composed of homodimers as is the A
enzyme.

The specific activity of o—galactosidase A of 45.2
units/mg of protein, compares favorably with the 31.3
reported for the splenic enzyme (16). The specific
activity of 4.18 units/mg of a-galactosidase B purified
here is comparable to the 4.52 reported for the placental
(17) and somewhat lower than the 6.56 previously reported
for the liver enzyme (104).

The two crgalactosidases had very similar amino acid
compositions (Table 4), but this similarity did not extend
to other human lysosomal enzymes.

The results of N—terminal sequencing were gratifying

175

because 1) some other human lysosomal glycosidases have
been found to have a blocked N—terminus (15,19); 2) the
sequencing gave single sequences for both proteins,
indicating that both proteins were pure and that the
proteins were homodimers so that it would not be necessary
to separate the subunits if they had been heterodimers; and
3) we postulated that the homologous N-terminal region of
the crgalactosidases was either a general marker for all
lysosomal hydrolases or just a region of homology between
two enzymes that have similar functions.

The possibility that the N-terminal sequence found in
the two wgalactosidases exists in other glycosidases and
serves as a signal for glycosylation, peptide cleavage, or
delivering the enzymes to the lysosome was discounted by
the absence of a similar N-terminal sequence in either
placental a—L-fucosidase (25) or B-glucocerebrosidase
(26). To determine if both a-galactosidases have homology
throughout, tryptic peptides of both a—galactosidases were
generated. We were successful at purifying peptides of
a-galactosidase B for N-terminal sequencing, while other
workers obtained those from (rgalactosidase A (27). It can
be seen in Table 6 that none of the tryptic peptides of
a-galactosidase A are identical to those of a—galactosidase
B, which indicates that the region of homology between
these two enzymes is restricted to the N—terminal region.
Kinetic Characteriggtions. The Km of 1.83 mM for the

hydrolysis of the artificial substrate 4-MU-a—Gal by

176

a-galactosidase A is quite similar to the 3.4 mM (103) and
1.55 ms (17) for the placental enzymes, 2.9 mM previously
reported for the liver enzyme (105) and 2.03 mM for the
splenic enzyme (16); of these other determinations only the
splenic enzyme was pure. The Vmax of 63.9 units/mg of
protein is greater than the 46.7 units/mg for the splenic
enzyme (16). The Km of 13.1 mM of o-galactosidase 8
towards the artificial substrate 4-MU-cr6al is identical to
the 13.1 reported for the placental enzyme and higher than
the 6.7 mM previously reported for the liver enzyme (104).
The Vmax of 13.9 for the a—galactosidase B purified here is
less than the 18.9 reported previously for the liver enzyme
(104) which is the only other report of a Vmax for this
enzyme.

Galactose was found, as expected, to be a competitive
inhibitor of both crgalactosidases with a Ki of 16.7 and
27.9 mM for A and B, respectively. Galactose was
previously reported to be a competitive inhibior of
splenic crgalactosidase A with a Ki of 21 mM (16). GalNAc
was found to be a competitive inhibitor of crgalactosidase
B with a Ki of 1.65 mM: o-NP-chalNAc, another artificial
substrate for a-galactosidase B, was previously reported to
be a competitive inhibitor of d-galactosidase B with a Ki
of 2.7 mM (112).

In a non-quantitative hydrolysis of GbOseacer
a-galactosidase A, but not a-galactosidase B, hydrolyzed

the lipid to lactosyl ceramide. This is consistent with

177

the findings of Kusiak g£_§l; (17) who found that
a—galactosidase A is 300 times more effective than
a—galactosidase B in hydrolyzing this lipid. It was
previously reported (104,105) that a—galactosidase B is 38%
as effective aScx-galactosidase A in hydrolyzing
GbOsescer. It is possible that the previous purification
of a—galactosidase B from liver (104) contained some
a—galactosidase A, which would account for the significant
differences in Km, Vmax and specific activity that were
noted above.
iggctivation of a-Galggtosidases A and B by Conduritol C
Epoxide. Conduritol B gi§_epoxide is a structural analogue
of B-Qfglucose. CBE has previously been shown to be a
competitive inhibitor of the lysosomal enzyme
B-glucocerebrosidase (48). C88 (40,42) and a related
compound (43) were shown to be active site directed
reagents and useful in determining the active site
sequences of three non~lysosomal glycosidases. We decided
that if any conduritol epoxide were an inhibitor of either
o—galactosidase, that it would be conduritol C trans
epoxide, and in searching the literature, we found that
coffee bean a~galactosidase was inhibited by CCE (142).

006 was synthesized and was found to inhibit both
a—galactosidases in a time~dependent fashion at 4° and pH
6.5, which is fully two pH units above the pH optimum of
both enzymes (104,105). These data suggested that

conduritol C epoxide is a suicide inhibitor of both

178

enzymes. Since the rate of inactivation of a—galactosidase
B was so slow, further inhibition studies were performed
only on a-galactosidase A.

To verify that conduritol C epoxide is a suicide
inhibitor of a-galactosidase A, an experiment was performed
that showed that galactose, a competitive inhibitor of
a-galactosidase A, blocked the time—dependent inactivation
of a-galactosidase A. Although conduritol B epoxide can be
inferred to be a suicide inhibitor of B—glucocerebrosidase
because CBE has been shown to reduce the ip_gigg_level of
B-galactocerebrosidase in mice (47), this is the first
demonstration of a suicide inhibitor of any lysosomal
glycosidase.

In addition, 008 appeared to be a competitive
inhibitor of wgalactosidase A with a Ki of 330 mM which is
also characteristic of a suicide inhibitor. This Ki is
much higher than the 1-2u M of CBE for
B—glucocerebrosidase (48). This may be due to CCE having a
more rigid conformation than CBE (The proton NMR spectra of
conduritol C and CCE indicate they have very similar
structures, while the spectrum of conduritol B, data not
shown, indicates conduritol B is a less rigid molecule).
Conduritol B epoxide may rapidly change conformations, one
of which may be similar to the B—glucosyl moiety hydrolyzed
by B~glucocerebrosidase. Additionally, it has been shown
the B—glycosidases are inhibited 50 to 200 times more

rapidly with the corresponding conduritol epoxide than are

179

the respective a—glycosidases with their conduritol epoxide
(Reviewed reference 151).

The suicide inhibitor-enzyme mixture is generally
diluted prior to assaying the enzyme, which was done here.
Since the concentration of 008 in the assay mixture was
about 15 mM and the Ki of 003 towards a—galactosidase A was
found to be 330 mM, the 008 should inhibit the
crgalactosidase A about 1.3% in the assay mixture.

There are three lines of evidence that conduritol C
epoxide is a suicide inactivator rather than only
inhibiting enzyme activity: 1) C03 very poorly
inactivated a—galactosidase B, which efficiently hydrolyzes
many of the same water-soluble oligosaccharides as
crgalactosidase A (108), 2) Galactose, a competitive
inhibitor of a—galactosidase A, blocked the time—dependent
inactivation of a-galactosidase A, which indicates the
inactivation occurs at the active site, and 3)
(rGalactosidase A was inactivated by as much as 75% in
these studies, and this would have required 3.3 M CCE in
the assay mixture as calculated from the Ki of 330 mM.

CCE was commercially tritiated, purified, and used to
label 15 milliunits of pure a-galactosidase A. This was
performed in order that a radioactive peptide could be
purified by HPLC, and the peptide sequenced. The protein
was not digested to peptides because of the difficulties
previously mentioned in separatingcx-galactosidase A

peptides. Another difficulty was that the enzyme appeared

180
to be labelled by six molecules of tritiated CCE per
monomer. This may have been due to incomplete dialysis of
the OCR from the protein, the formation of a reactive
compound during tritiation of the CCE, or the formation of
a reactive compound during catalysis that moved from the

active site of the enzyme then reacted with the enzyme.

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10.

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