METHYL GROUP SYNTHESIS IN PLANT METABOLISM
UTILIZATION OF GLYCINE-BETAINE AS A METHYL GROUP PRECURSOR
II.

THE BIOSYNTHESIS OF PECTIN METHYL ESTERS

By

Clifford S. Sato

A THESIS

Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

ProQuest Number: 10008655

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ACKNOWLEDGMENT
The author wishes to express his sincere appreciation to
Dr. Richard U. Byerrum, i*hose advice and interest together with
his patience and encouragement have greatly facilitated the
completion of this work, and to other members of the Department
of Chemistry for their advice from time to time. Special gratitude
is also due to Dr. E. H. Lucas, Professor of Horticulture, for
his assistance and the use of the greenhouse, and also to
Dr. L. J. Dewey, Postdoctoral Fellow, for supplying the tobacco
plants and the doubly-labeled methionine.
The author also wishes to thanic the Atomic Energy Commission
and the Department of Chemistry of Michigan State University for
their generosity in providing fellowships under which this work
was made possible.
**********
********
******
****

**
*

ABSTRACT
Glycine-betaine and methionine, both of which were labeled in the
methyl group with carbon-14, were administered to tobacco and radish
plants respectively.

Both compounds were shown to be precursors of

methyl groups as shown by the incorporation of the carbon-l4 into the
N-methyl group of the nicotine molecule in the tobacco plant and into
the methyl ester group of pectin molecule in the radish roots.

The

relative radioactivity of the pectin was much greater than that of the
nicotine.
The N-methyl group of nicotine was cleaved and the released methyl
group was counted for radioactivity as the methyltriethylammonium iodide.
Saponification of pectin followed by distillation of the methanol and a
subsequent oxidation of methanol to carbon dioxide was carried out in a
glass apparatus designed in this laboratory.
counted as barium carbonate.

The carbon dioxide was

Essentially all of the radioactivity of

the nicotine was localized in the N-methyl group, whereas about 95 to
95 percent of the radioactivity of pectin was found to be in the methyl
ester.
The pectic acid residue which was obtained by the saponification
of pectin was shown to contain about one percent of the total radio­
activity of the original pectin.

The methoxyl content was found to be

8.6 percent of the radish root pectin.
Attempts were made to show the existence of the choline oxidase
system in Nicotiana rustica.

Homogenates and mitochondrial preparations

-i-

of tobacco leaves were used, but no active enzyme system could be demon­
strated.

cA^-methyl choline was administered to tobacco plants and one

week later, betaine and choline were extracted from the plants and isolated
as the reineckates.

The reineckate salt was removed and the betaine and

choline were obtained as the chlorides, which were separated by twodimensional paper chromatography.

A radioautograph of the chromatogram

revealed two spots corresponding to choline and betaine.

- ii -

VITA

The author was born June 9* 1925 in North Kohala, Hawaii.

He was

graduated in June 19^+5 from Mid-Pacific Institute, which is located in
Honolulu, Hawaii.

After attending the University of Hawaii for three

semesters, he served in the Enlisted Reserve Corps for nine months.

He

was then called into active service in January 21, 19^6, and served in
the United States Army for three years.

He returned to the University

of Hawaii in February 19^9 and was graduated in June 1951 with a Bachelor
of Science Degree.

He enrolled in the Graduate School of Michigan State

University as a Teaching Assistant in Chemistry and held that position
from September 1951 until July 1955*

ln the fall of 1955» he was

appointed a Special Graduate Research Assistant under an Atomic Energy
Commission grant.

He left this position during the summer of 195^ "to

work at the Argonne National Laboratory as a Resident Student Research
Associate.

He returned to Michigan State University in the fall of 195^

to resume his research assistantship, which he held until the completion
of hie graduate program.

TABLE OF CONTENTS
Page
INTRODUCTION ....................................................

1

EX P E R I M E N T A L ....................................................

7

Synthesis of

-methyl betaine ............................

Preparation of the tobacco plants

7

.......................

8

Uptake of betaine from solution .........................

9

Administration of C^-methyl betaine and the
isolation of nicotine ................................. 11
Demethyl at ion of nicotine ................................ 15
Feeding C-^-methyl choline .................................. l4
Detection of C^-methyl betaine .........................

15

Preparation of the radish plants ...........................

17

Uptake of C^^--methyl methionine froms o l u t i o n ............

18

Administration of C^-methyl methionine and
the isolation of pectin ..............................

19

Pectin doubly-labeled with deuteriumand carbon-l4 ......

21

Determination of the methoxyl (methyl ester)
group of p e c t i n .......................................

21

Isolation and determination of pectic acid
obtained from p e c t i n .................................. 27
Preparation of deuteriomethyl-5,5~dinitrobenzoate
for deuterium analysis

.............................

Methoxyl content of radish root pectin ..................

28
29

TABLE OF CONTENTS
(Continued)

Page

DISCUSSION ......................................................
Studies of betaine
Studies on

as amethyl

donor ........................

thebiosynthesis of p e c t i n .......................

50
JO
J2

SUMMARY ..........................................................

jk

REFERENCES ......................................................

55

APPENDIX ......................................................... 58

LIST OF TABLES
Table
I

II

II

Page
Location of Radioactivity in the Nicotine Molecule after
C^-methyl BetaineAdnoiniBtration ..............................

12

Administration of Isotopically Labeled Methionine and
the Isolation ofPectin fromRadish Roots .....................

22

Location of Radioactivity in Pectin after Methionine
Administration ................................................

22

INTRODUCTION

In the present study two aspects of the transmethylation reaction,
the metabolic transfer of an intact methyl group from one compound to
another, were investigated in higher plants.

The first was to establish

whether the methyl groups of glycine-betaine might be transferred to give
the N-methyl group of nicotine and whether choline is oxidized to glycinebetaine^- before transmethylation can occur.

The second was to determine

whether transmethylation of the methionine methyl group might result in
the formation of an O-methyl of methyl esters in pectin.
Du Vigneaud and his coworkers have shown that transmethylation is a
general reaction in animal metabolism (l).

Through two lines of research,

choline was shown to be one of the naturally occurring sources of methyl
groups.

One, in experiments with young rats on a methionine-free diet

supplemented with homocysteine, choline was shown to support growth (2).
Methionine was formed when young rats were fed homocysteine and choline
(2).

The other approach demonstrated that when choline labeled with

deuterium in the methyl groups was fed, creatine and methionine isolated
from the rat contained deuterium labeled methyl groups (4).

On the other

hand, betaine was shown to be more effective than choline in the formation
of methionine (5)«

Whereas choline and also betaine can prevent fatty

infiltration of the liver in rats and can cause methylation of homocysteine
), liver homogenatea prepared from chicks, guinea pigs, and rabbitB,
which do not oxidize choline, are unable to catalyze the formation of

1 Glycine-betaine will be referred to as betaine henceforth.
-1-

methionine from homocyateine and choline (7)-

Diraathylthetin and propio-

thetin, sulfur-containing compounds analogous to betaine and alaninebetaine respectively, when incubated with homocysteine and rat liver
homogenates yielded methionine (8).

The thetins also supported growth of

young rats kept on a choline and methionine-free diet supplemented with
homocysteine (9)*

However, when sulfocholine was used instead of choline,

rats did not grow (10),
It was shown by Dubnoff that under aerobic conditions, rat liver
homogenates and rat liver slices methylated homocysteine to methionine in
the presence of choline or betaine.

Nevertheless, in the absence of oxygen

only betaine served as the methyl donor ( 7 )•

Further evidence that

choline itself does not function as a methyl donor was supplied by experi­
ments with rat liver homogenates incubated with homocysteine and with
choline labeled with nitrogen-15*

Under aerobic conditions,

glycine wae formed but not N^-dimethylaminoethanol.

5-dimethyl-

If choline were the

active methyl donor, N^5-dimethylaminoethanol would have been formed (12),
In 195^» Brown and Byerrum showed that methyl carbon of methionine
could serve as a precursor for N-methyl group of nicotine in the tobacco
plant, thus, suggesting the occurrence of transmethylation reaction in
higher plants (15).

FlokBtra demonstrated that the methyl carbon of

methionine could also serve as a precursor for the methoxyl carbon of
lignin in barley (1^).

By feeding methionine doubly labeled with deuterium

and carbon-lA in the methyl group to barley and then isolating the lignin,
Dewey showed that the deuterium and carbon-l4 of the methoxyl group was
in the same ratio as that in the doubly labeled methionine.
the methyl group of methionine was transferred intact —

-2-

Therefore,

a true trans­

methylation reaction —

and the methylation process did not occur by

oxidation of the methionine methyl group followed by reduction (1 5 ).

In

the case of barley (l6) and castor beans (17)» methionine was demonstrated
to be a methyl donor in the formation of the alkaloids, hordenine of
barley and ricinine of castor beans; choline on the other hand yielded no
methyl group.

Nevertheless, Wing demonstrated that methyl groups of

choline were precursors of the N-methyl group of nicotine (18).
Working with germinating seedlings of chick-pea, Ahmad and Karim
Bhowed that methionine, acetone, and methanol can act as precursors of
the methyl group of choline (19)*

In another study it was suggested that

small amount b of betaine were found in etiolated seedlings of Hordeum and
that the methyl group of betaine should be available for transfer to
hordenine, if betaine is the active donor (20).

Experiments performed on

Beta vulgaris and Atriplex patula by infiltrating choline and betaine
aldehyde into the leaves resulted in a significant increase in the betaine
content of the tissues.

When the leaves were infiltrated with solutions

of choline and then kept in an atmosphere of nitrogen, no significant
increase in betaine was observed (21).
In view of the above discussion, it seemed interesting to compare
the ease with which betaine can offer methyl groups in comparison with
choline for the synthesis of nicotine.

In order to gain further evidence

as to the mechanism of transmethylation and also to explain the fact that
choline may yield methyl groups in the tobacco plant but not in etiolated
barley seedlings, it was decided to study the enzyme system which might
catalyze the oxidation of choline to betaine.

-5“

Although S-methyl, N-methyl, and C-methyl group formation in animals
and plants have been well known, the possibility of an O-methyl formation
in a inethyl ester by means of transmethylation has not been postulated to
date.

Pectin, a methyl ester of polygalacturonic acid, was Belected as a

desirable compound for this particular study.

The selection was based on

the abundance of pectic substances in plants, the ease of its isolation,
and the rapid rate of its formation.

Since a fast-growing plant was

desirable, it was decided to use radish roots.
The term pectin was derived from the Greek
congeal or solidify (22).

meaning to

Because of a great heterogeneity in the

literature with regard to what is meant by "pectin", the term as used in
this work will adhere to the Revised Nomenclature of the Pectic Substances
adopted in 1944 (2^)*

Wide variations in nomenclature probably resulted

from different preparative methods which yielded products of diverse
compositions.
In 1915, Fellenberg discovered the methyl ester group in pectic sub­
stances (26).

Four

acid in pectin, and

years later,Suarez (24) reported the presence ofuronic
Ehrlic.h (2 5 )postulated that galacturonic acid was the

basic building block of pectic substances.

In the ensuing years,

various structures for pectic acid were presented.

Some of these are:

the octa-uronic structure of Smolenski (made up of four digalacturonic
acids) (27)* the cyclic structure (a basic unit made up of four galact­
uronic acid residues linked in a ring with arabinose and galactose as
side chains) of Nanji, Paton, and Ling (28), and the tetragalacturonic
acid structure of Ehrlich (29).

Although most of the earlier workers

were in favor of the cyclic structure, Morell, Baur, and Link in 19^4

-4-

indicated by means of a modified end-group assay that pectic acid was a
linear polymer (5 0 ).
Studies of physical properties of pectic substances and nitropectin
(pectin nitrated with nitric acid) (5 2 , 55 )> ai*d of periodic acid oxida­
tion of polygalacturonic acid (5^)> showed a close resemblance to cellulose.
Exhaustive methylation of pectic acid followed by hydrolysis indicated
that pectic acid consists of pyranose galacturonic acid residues linked
through 1,4-alpha-glycosidic linkages (55)*
Several enzymes found in plants may degrade pectin.

Three pectic

enzymes generally recognized by the early workers were (5^):

proto­

pe ctinase, an enzyme which hydrolyzed protopectin to soluble pectin;
pectinase, an enzyme which hydrolyzed pectic compounds to galacturonic
and smaller polygalacturonic acids; and pectase, an esterase which hydro­
lyzed the methyl ester groups of pectin and pectinic acids.

In 1945*

there was evidence that pectinase could catalyze the hydrolysis of only
those glycosidic bonds adjacent to free carboxyl groups (57).

McCready

and Seegmiller, in 195^* (5®), divided the pectic enzymes into two classes
—

those having esterase activity and those catalyzing the hydrolysis of

glycosidic linkages.

They have shown that purified citrus pectinesterase

does not hydrolyze the ester groups of di- and trigalacturonic acids nor
the half esters of digalacturonic acid, but attacks esters with Degree of
Polymerization Number of ten and greater.

Purified fungal polygalact­

uronase was shown to hydrolyze glycosidic bonds between uronic acids
containing adjacent free carboxyl groups in all polygalacturonic acids.
However, glycosidic bonds between uronic acids, one of which is esterified,
were not hydrolyzed.

Such results tend to indicate the possible importance

-5-

of the pectineeteraBe over the polygalacturonase with respect to the
formation of the polymethyl eaters of large pectic compounds.

Two

questions still remain in the methyl-ester formation of pectin:

whether

the methyl group replaces the proton of the carboxyl group of the galact­
uronic acid residues, or whether methyl ether is first formed with a
galactose residue and then the methylene group is oxidized to form the
ester.

In view of the great significance of one-carbon precursors in

both animal and plant metabolism, the possible origin of the O-methyl in
the formation of the polyesters appeared worthy of investigation.

-6-

EXPERIMENTAL

Synthesis of C^-methyl betaine
In the studies in which betaine was utilized as a possible methyl
group precursor for the N-methyl group of nicotine, it was first necessary
to synthesize CA -methyl betaine.

This synthesis was carried out as

described by Ferger and du Vigneaud (39).

In 32 ml. of 75 percent

ethanol (v/v), 1 6 .8 mM of sodium dimethylglyeinate was dissolved and then
the mixture cooled to -10°C.

One millicurie of C^-methyl iodide

(specific activity of one millicurie per millimole) obtained from Tracerlab, Inc., under allocation from the Atomic Energy Commission, was then
added.

Five minutes later, 1 5 .6 mM of inactive methyl iodide was added.

The reaction vessel was then stoppered and heated at 70° for eighty-five
minutes.

The resulting mixture was then evaporated to a small volume

in vacuo and a freshly prepared saturated aqueous ammonium reineckate
solution was added.

After acidifying the solution to Congo Red paper

with hydrochloric acid, the mixture was allowed to cool overnight in the
refrigerator.

The resulting betaine reineckate was isolated by filtra­

tion, dissolved in 160 ml. of 0.1 N ammonium hydroxide solution, and then
shaken with excess silver oxide until the reineckate color (red) disappeared.
The silver reineckate was collected on a filter and washed.

The combined

filtrate and washing was heated to 60 ° and aerated four hours to remove
the ammonia.

After concentrating the solution to about 80 ml., it was

acidified with hydrochloric acid and the small quantity of silver chloride
which formed waB filtered off.

The precipitate was washed with a small

-7-

quantity of water and the filtrate and washings were combined, evaporated
to dryness, and dissolved in a minimum volume of boiling ethanol.

This

solution was filtered and then allowed to cool overnight in the refri­
gerator to obtain crystals of betaine chloride.

After collecting the

crystals on a filter and evaporating the filtrate to about
second crop of betaine chloride was colleoted.
recrystallized twice from ethanol:
(Anal. Calc, for C5H1202NC 1 S

50

ml., the

The combined product was

1.55 g* of betaine chloride was obtained.

Cl, 25.08#.

Found:

Cl, 25.20#.) (40).

Preparation of the tobacco plants
Seeds of Nicotians rustics L., var. humilis^, a high nicotine strain,
were planted in flats containing vermiculite^ in the greenhouse.

Tap

water was applied to keep the vermiculite moist, and a nutrient solution
containing 1 g. MgS 0 4 «7 H 2 0 , 1 g. KgHPO^, 5*8 g. Ca(N0^
in four liters of tap water was applied twice a week.

dissolved
Within three weeks,

the plants were transplanted allowing two to three inches between plants.
Probably due to seasonal changes, the plants required about two to three
months to attain the desired height of six inches.
For the hydroponic administration of the radioactive material, the
plants were prepared as follows:

the venniculite was washed off under a

2 The seeds were obtained through the courtesy of Dr. N. A. MacRae
of the Canadian Department of Agriculture, Central Experimental
Farm, Ottawa.
5 Vermiculite is a commercially available heat expanded mica.
^ ThiB germicide was obtained from the Wyandotte Chemicals Corp.,
Wyandotte, Mich., through the Michigan State University Department
of Ho rt i culture.

-8-

stream of water; the roots were soaked one hour in a 0.01 percent solution
of Wyandotte detergent germicide No. 1^28^ to reduce the bacterial popu­
lation; the roots were washed with water; and the plant was placed in a
125 ml. Erlenmeyer flask containing 0.5 ml- of aureomycin (1:1000), the
Cl^-methyl betaine, and 50

• of an inorganic nutrient medium.

This

solution was prepared by diluting with three parts of water, one part of
a stock solution which had the following composition:
Ca(N05 )2 *4H20, 2.61 g.j
756 mg .;

KC1, 500mg.;

FeCl^HgO,

500 mg.; K^FC^., $00 mg.

prevent the growth of root microorganisms.

water, 2 1.;

5 .6 mg.; MgSOi^-T^O,

The aureomycin was added to
Oxygen was bubbled through

the nutrient solution twice a day for two minutes to prevent
wilting of the plants and also to provide aeration for the roots.
Nutrient solution was added as required to keep the volume constant.
The plants were grown in the hood illuminated by two 5^”inch, 50-watt
fluorescent tubes and a 100-watt incandescent bulb placed about fourteen
inches above the tops of the plants.

At this level of the plants, the

light intensity was 200 to 2^0 foot-candles.

The lights were left on

twelve hours every day, from eight o'clock in

the morning to eight o'clock

in the evening.

Uptake of betaine from solution
It was demonstrated by Wing (18) that tobacco plants can absorb
choline from aqueous solution through the roots; therefore, it seemed
feasible that betaine might also be absorbed through the roots.

However,

it was necessary to ascertain the extent of uptake of betaine by the plant
and the destruction of betaine by microorganisms outside the plant.

-9-

Twelve 125 ml. Erlenmeyer flasks were set up, each containing 25 nil.
inorganic nutrient medium, 0.5 ml. aureomycin solution (1:1000), and 2.06
mg. betaine chloride.

Plants, the roots of which were soaked in 0.01

percent solution of Wyandotte detergent germicide for one hour, were
placed into four of the flasks; about six root fragments, into another
four; and the remaining four flasks served as controls.

Seventy-two hours

later, the roots of the plants and the root fragments were washed, the
washings added to their respective flasks and the solution in each flask
filtered into separate 50 ml. suction flasks.

The filtrates were evapo­

rated to dryness in vacuo at 50 to 60° and transferred quantitatively
into 15 ml. centrifuge tubes with enough water to make a volume of 5-0 ml.
Betaine was determined by the periodide method of Reifer (4l) in the
following manner.

After dissolving 0.5 g* Bodium chloride in the 5*0 ml.

solution, 0*5 ml. concentrated phosphoric acid was added, followed by 1 ml.
potassium triiodide (2.5 g* iodine, 5*75 g* potassium iodide, and 10 g.
sodium chloride in 100 ml. water).

The mixture was stirred and then kept

for three hours in a salt-ice bath at - 5 to -10°C.

Three layers were

obtained after centrifuging for five minutes at 25 OO revolutions per
minute in a clinical centrifuge.

The top layer, a solution of potassium

triiodide, was removed by suction without disturbing the second layer,
which was phosphoric acid.

The sides of the tube were washed three times

with 5 ml. portions of ice water without centrifuging, and the phosphoric
acid layer was removed at the last washing.
the betaine periodide was then dissolved in

The bottom layer containing

5 ml.

of

95 percent ethanol

and titrated with 0 .0 5 N sodium thiosulfate solution using a starch
indicator.

The amount of betaine present in the unknown was ascertained

-10-

by reference to a standard curve in which milliliters of 0.05 N sodium
thioaulfate had been plotted against a known quantity of betaine chloride
recrystallized three times from ethanol.

The plants absorbed 9 8 .5 percent

of the betaine chloride originally added during the 72-hour period,
whereas the loss of betaine chloride due to microorganisms was negligible
ae judged from the fact that no lose of betaine occurred in flasks inocu­
lated with root fragments as compared to the controls.

Administration of C^-methyl betaine and the isolation of nicotine
Preliminary feeding experiment showed that 2.06 mg. betaine chloride
having a radioactivity of 0.5 x 10^ counts per minute administered per
plant was not radioactive enough to get a significant count in the
nicotine that was isolated from the plants.

Consequently, betaine

chloride having an activity of 1.92 x 105 counts per minute per 2 .0 6 mg.
waB administered to each plant.

Seven days later, the roots of the plants

were washed with distilled water and then blotted with cheesecloth to
remove the excess water.

The plants were then cut into small pieces with

scissors, dried at a temperature below 80° under an infra-red lamp, ground
in a mortar, mixed with one-tenth of their weight of calcium hydroxide,
and then steam-distilled.

The distillate was collected —

until no

precipitate formed with silicotungstic acid (12 g. per 100 ml. water) —
in a flask containing hydrochloric acid and then it was concentrated
in vacuo.

Following the procedure of Smith (^2), the solution was made

alkaline and the nicotine was purified by two successive azeotropic
distillations into hydrochloric acid.

The acid distillate was evaporated

to dryness in vacuo and the resulting nicotine hydrochloride was dissolved

-11-

in a email volume of water.

An equal volume of saturated methanol

solution of picric acid was added, and the mixture was kept in the
refrigerator for half an hour.

The nicotine dipicrate which precipitated

was collected on a filter, washed with cold water, and then re crystal11 zed
from hot water (m.p. 224-225°; recorded value 224°) (45).

After grinding

the nicotine dipicrate in a mortar, it was plated on an aluminum counting
disc (2.85 sq. cm.).

Counting for radioactivity was done with a window-

less flow counter from Tracer Lab. and a Scaling Unit from Nuclear Corp.
using a gas mixture of helium (99*05 percent) and isobutane (0 ,9 5 percent).
By using the formula shown in Appendix I, the maximum specific activity
per millimole of the nicotine dipicrate was calculated.

The results

shown in the second column of Table I indicate that the nicotine from
Cl4—methyl betaine fed plants was radioactive and suggest that betaine
is a precursor for some portion of the nicotine molecule.

TABUS I
LOCATION OF RADIOACTIVITY IN THE NICOTINE MOLECULE AFTER
C 1 -METHYL BETAINE ADMINISTRATION

Experiment No.

Maximum Specific Activity
(counts per minute per millimole)
Nicotine Dipicrate
Quaternary Iodide

1.

(2 6 plants )

1.97 x 10 ?

2 .0 5 x 10?

2.

(5 0 plants)

1 .6 1 x 10?

I .78 x 10?

-12-

Demethylation of nicotine
Since the nicotine isolated from C^-methyl betaine fed plants was
radioactive, it was of interest to ascertain the amount of the total
radioactivity located in the N-methyl group.

The nicotine N-methyl

group was, therefore, cleaved according to the procedure of Pregl (44)
as modified by Brown (45).

This method consisted essentially of treating

the nicotine with ammonium iodide and hydriodic acid to form the quater­
nary ammonium salt and then splitting off methyl iodide at 550 to 5^0°
in the presence of gold chloride.

An ethanol solution of triethylamine,

cooled in a CC^-methylcellosolve bath, was used to absorb the liberated
methyl iodide.

After standing overnight and then evaporating off the

ethanol and excess triethylamine, the methyltriethylammonium iodide which
had formed was obtained as a white solid.

This was counted in the same

way as the nicotine dipicrate, and the maximum specific activity per
millimole of the quaternary iodide was also calculated by using the
formula shown in the appendix.

The results are shown in Table I.

From the figures illustrated in Table I, it can be seen that, within
experimental error, all the radioactivity of the nicotine was recovered
in the quaternary iodide.

It is, therefore, evident that the radioactivity

of the nicotine after feeding C^-methyl betaine was localized in the
methyl group.

These results indicate that the methyl groups of betaine

are precursors for the N-methyl group of nicotine.

-15-

Feeding C^-methyl choline
In an attempt to ascertain whether or not choline might first be
oxidized to betaine before it could yield its methyl groups for nicotine
synthesis, it was decided to feed radioactive choline to tobacco plants
and after a suitable period of time to try to isolate radioactive betaine
from these plants.
Each of fourteen plants —

prepared by cutting off the roots and

allowing the regeneration of new roots in the synthetic nutrient medium
for a period of one week prior to feeding —

was administered 2.0 mg. of

choline chloride having an activity of about 105 counts per minute.

The

choline used in these experiments was isolated as the reineckate from
plants which had previously been fed C^-methyl choline by Wing (18).
The plants were grown in a manner similar to that described earlier.
One week after feeding the radioactive choline, the roots of each
plant were removed from the nutrient Bolution in which they were growing,
washed with water and then blotted with cheesecloth.

The plants were then

cut into small pieces with scissors and dropped into ^>00 ml. of boiling
0.02 N hydrochloric acid solution to inactivate the enzymes.

After cooling,

the mixture was ground in a Waring Blendor for 15 minutes and then filtered
through an extraction thimble.

The thimble containing the residue was

transferred to a Soxhlet extractor and was extracted for seven and onehalf hours using the acidic filtrate obtained above as the extraction sol­
vent.

Twenty-five milliliters of 1 N sodium hydroxide solution was

added to the extract thus obtained and this solution was then concentrated
in vacuo at 50° i*o about 200 ml.

The nicotine was removed during this

concentration as an azeotropic mixture with water.

_l4_

In order to remove

some of the interfering substancee euch as chlorophyll, leaf pigments,
and phospholipids, the c o d e d concentrated extract was washed with
petroleum ether several times.

The aqueous layer was collected and

filtered through a thimble filter.

After washing the residue with water,

the washings were combined with the filtrate and concentrated to about
25 ml.

An equal volume of freshly prepared saturated aqueous ammonium

reineckate solution was added, and the reineckate suspension was cooled
in an ice-bath for thirty minutes.

The reineckate crystals were

collected on a filter, dissolved in acetone, and the pink color removed
as Bilver reineckate by adding excess silver nitrate solution.

After

centrifugation, the clear solution was decanted and acidified with hydro­
chloric acid.

The silver chloride suspension was centrifuged and the

solution was decanted and then evaporated to dryness in vacuo.
residue had a greenish tinge, it was dissolved in 1 ml.

Since the

of water, and

the precipitation with ammonium reineckate was repeated to obtain a
white crystalline residue which was a mixture of choline and betaine
chlorides.

Detection of C^-methyl betaine
A very small amount of water was added to the crystalline residue
described above and the solution was applied —
five lambda each —

three applications of

on one corner of a Whatman Number 1 filter paper.

The mixture of chlorides was than separated by using the principle of
two-dimensional ascending chromatography.

The first Bolvent system was

n-propanol:l N acetic acid = 5:1 (21); and the second, n-butanol:ethanol:
acetic acidswater = 9:1:1x2.

A radioautograph

-15“

was taken by placing a

Kodak X-ray film over the developed chromatogram for three days and then
developing the film in Kodak developer D-19.

There were two epote on the

film which corresponded to those of betaine and choline.
for a standard solution of betaine and choline were:

The Rf values

solvent 1,

betaine O. 5 6 , choline 0.46; and solvent 2, betaine 0.25, choline 0.25*
Dragendorff reagent (55) was used to identify choline and betaine on the
chromatogram.
These results are an indication that choline was oxidized to betaine
in the plant, although it 1 b possible that the methyl groups of choline
were transferred to a precursor of betaine to give the radioactive
betaine isolated.

- 16-

Preparation of the radish planta
Before any feeding of isotopic compounds to the radish plant could
be carried out, a suitable method of raising the plant was required.
Since the radish plant cannot withstand the shock of transplanting in its
third week of growth, it could not be raised in flats containing soil or
vermiculite.

Consequently, attempts were made to culture the plants in

water in the greenhouse.

The Comet variety5 was used for this purpose.

The seedB were soaked in water for about ten minutes and then spread
upon a wet cheesecloth supported by a half-inch-square-mesh wire screen
coated liberally with paraffin.

This was placed in a five-inch-deep

glass container, painted black on the outside, so that the seeds were
about a half inch below the rim of the container.
to the level of the seeds.

Water was then added

In order to encourage the roots to grow

straight down, the water-level was lowered gradually with growth.
third day, the water was replaced with
Hoagland^ (46).

50

On the

percent nutrient solution of

Solution 1 was prepared as follows:

1 ml. of M potassium

nitrate, 2 ml. of M magnesium sulfate, and water were mixed to make 1
liter of solution.

A supplementary solution was prepared as follows:

2.86 g. boric acid, 1.15 &• manganous chloride, 124 mg. zinc sulfate,

51 mg. copper sulfate, and 20 mg. molybdic acid were dissolved in 1 liter
of water.

A 0.5 percent iron tartrate solution was also made.

The

nutrient solution was then prepared by adding 1 ml. of supplementary
solution and 1 m l . of iron tartrate solution to each liter of solution 1.

5 The seeds were obtained from the Ferry-Morse Seed Co., #59476,*
Detroit, Michigan.
6 This solution was made from "Baker's Analyzed" reagent grade
chemicals.

-17-

On the fifth day, the plants were transferred to a porcelain-coated
pan? containing the nutrient solution of Hoagland.

The pan was covered

with a piece of paraffin-coated cardboard in which holes had been punched.
The holes were seven-eighths of an inch in diameter and their centers
were two inches apart.
cotton over it.

The cardboard in turn had a layer of non-absorbent

The radish plants were placed in the holes in such a

manner that the roots were partially immersed in the nutrient solution in
the pan.

The cotton above the cardboard was used as a support for the

plants.

Five milliliters of the "iron" solution was administered twice

a week, and the level of the nutrient solution was maintained at the base
of the tuberous portion of the root.

Under such conditions, a three-week-

old plant had a bulb about half-inch in diameter and an exuberant root
system; the radish was considered ready for the feeding of C^-methyl
methionine.

Uptake of C^-4-methyl methionine from solution
Although it was shown that tobacco plants absorbed methionine through
the roots (l5)» it was not known whether radish plants would behave
similarly.

Consequently, an experiment was carried out as follows:

twelve 100 ml. short-neck graduates —

each containing 40 ml. Hoagland

solution, 0.5 ml. aureomycin (1:1000), and 2.0 mg. DL-raethionine —
divided into three groups of four.

were

The first group contained plants;

the second contained root fragments; and the third was used as control.
Each graduate was wrapped with paper to keep out direct sunlight.

? This pan was 10§-" x l4§-” x

-18-

A week later, the roots of the plants and the root fragments were
washed with water, and the washings were added to their respective
graduates.

Each solution was then filtered, concentrated in vacuo at

40° to a small volume, and then made to a volume of 10 ml.

For each

determination, a 5 ^1- aliquot was taken and the methionine was determined
according to the method of McCarthy and Sullivan (47).
methionine by the plants was complete.

The uptake of

There was essentially no loss of

methionine due to microorganisms in the second group containing root frag­
ments, nor was there any loss in the control.

Administration of C-^-methyl methionine and isolation of pectin
Three-week-old plants were prepared as described earlier, and each
plant was fed 2.0 mg. methionine having an activity of 6.09 x 10^ counts
per minute.

The plants were harvested after a growth period of nine days.

The leaves were cut off and discarded.

The roots were washed, blotted

with cheesecloth, then cut into thin slices and dropped into boiling
95 percent ethanol —

the volume calculated to give a final concentration

of 70 to 80 percent ethanol upon an assumed 95 percent moisture content
of the radish.

Boiling was continued for five minutes to inactivate the

enzymes and then the extract was allowed to stand overnight.

The pink-

colored ethanol extract was separated from the radish root slices by
filtration through four-layers of cheesecloth.

The residue was returned

to the original beaker, washed with 95 percent ethanol, and the mixture
was again filtered through the original cheesecloth.
ethanol was then added to cover the residue.

Enough 95 percent

The ethanol was filtered

off the following day and the radish root residue was thoroughly dried

-19-

in the oven at 100°C.
The isolation and purification of pectin was carried out essentially
as described by Kertesz (48).

After grinding the dried radish root

residue in a mortar, 60 ml. water was added to every gram of residue.
This mixture was put on a shaker for two hours, and was then centrifuged
fifteen minutes at a maximum relative centrifugal force of 1400 x g.
The supernatant was kept in the cold room (4-6°C.), and the residue was
extracted with 0.05 N hydrochloric acid (25-JO ml. per gram of dried
residue) at 80° for two hours and the mixture then centrifuged.

The

residue resulting from the last centrifugation was extracted three more
timeB in a similar manner.

The supernatants were combined, filtered

(Whatman No. 1), and made to 80 percent with ethanol (v/v).

The crude

pectin which precipitated in the alcohol solution was then filtered by
gravity through a flutted filter paper (Whatman No. 12).

The crude pectin

was^dissolved^in water at 80°, cooled to 25 °, and then reprecipitated
from a 55 percent ethanol solution.
repeated two more times.

This method of purification was

The resulting product was then washed with

95 percent ethanol followed by diethyl ether.

The purified pectin was

air-dried to remove excess ether and finally dried over barium oxide in a
vacuum dessicator.

The yield of pectin along with the radioactivity of

the methionine fed per plant is presented in Table II.

After grinding

the pectin in a mortar and plating on an aluminum counting disc, it was
counted with an end-window counter.

The activity was then corrected to

counts per minute per milligram of pectin and also to counts per minute
per 120 mg. of pectin at infinite thinness by UBing the formula shown in
Appendix I.

The latter means of expressing radioactivity was used because

-20-

this was the weight of pectin which was saponified in experiments to be
described below.

The results are listed in Table II.

It is evident from

the data presented that after feeding C^-methyl methionine to radish
plants there was a high incorporation of radioactivity into the pectin
of the roots.

Pectin doubly-labeled with deuterium and carbon-l4
Doubly-labeled methionine was prepared by dissolving l44 mg. deuteriomethyl methionine with 16 mg. C^-methyl methionine in enough water to
make a solution of 200 ml.
counts per minute,

was

third day of feeding —

A total of 5*0 ml., equivalent to 2.4 x ic/*

administered in two doses —
to each three-week-old plant.

the first and the
After a feeding

period of twelve dayB, the plants were harvested and the pectin isolated
as described above.

Since a solid derivative of the doubly-labeled

methanol was deBired, the second and third purification of the pectin
was carried out in 50 percent acetone solution to eliminate any occlusion
of the ethanol.

The resulting pectin was counted in the same way as the

pectin described earlier.

The specific activity is presented in Table II.

Although the radioactivity of the pectin in this experiment was lower
than in the other two, it will be noted in Table II that the radioactivity
of the methionine administered waB also less.

If one takes this fact

into account it may be seen that approximately the same extent of incor­
poration occurred in the third experiment as in the other two.

Detennination of the methoxyl (methyl eBte_r_) group of pectin
Since the methyl ester of pectin was shown to be readily hydrolyzable

-21-

TABLE II
ADMINISTRATION OF ISOTCPICALLY LABELED METHIONINE
AND THE ISOLATION OF PECTIN FROM RADISH ROOTS

Methionine
Labeled With

Experiment No.

Activity Fed
Per Plant

1.

(5 2 plants) C^^-methyl

6.09 x 10^

2.

(5 8 plants)

C l2f-methyl

6.09 x 10^

5.

(40 plants)

C^D^-methyl

2.40 x 104

Radish Roots
Fresh Weight Dry Weight
(gram )
(gram)

Pectin
Isolated
(mg*)

4.2

505.2

198

7.4

802.8

566

13.5

1662.8

89-5

LOCATION OF RADIOACTIVITY IN PECTIN
AFTER METHIONINE ADMINISTRATION

Maximum Specific Activity
Counts Per Minute Per
120 mg.
MeOH from 120 mg. Pectin
(Counted as BaCO^)
Pectin

Percent Activity
in Methoxyl
Group of Pectin

Experiment
No.

1 mg.
Pectin

1.

42.8

5140

4790

95.2

2.

52.5

5880

568 O

94.8

954

908

95.2

5-

7*95

-22-

(49 ) and since methanol does not* form an azeotropic mixture with water, it
was decided to make use of those properties in determining the methoxyl
content of pectin.

Nevertheless, since the liberated methanol could not be

counted as such, a solid derivative had to be made.

Experience showed that

counting carbon-l4 in the form of barium carbonate had several advantages
over the use of solid derivatives of methanol.

With such considerations,

an apparatus was designed as shown in Fig. 1 for converting the liberated
methanol to barium carbonate.
In chamber I, containing 8.4 ml. of water, 120 mg. of pectin was
dispersed by warming and then stirring with a piece of soft iron (encased
in glass, 5 x 2^ mm.) by means of a magnet.

A small amount of antifoam®

was added and the resulting mixture was frozen in a CC^-methylcellosolve
bath.

This was followed with 0.2 ml. methanol —

C^4-IDethanol —

used as carrier for the

and 5*6 ml. of 1 N sodium hydroxide solution.

This mixture

was immediately frozen, and the container then attached to the apparatus
while keeping it in the cooling bath.

In chamber II was placed a piece of

soft iron; chamber III, 100 mg. of potassium iodate; chamber IIIA, 25 ml.
of combustion mixture saturated with potassium iodate (50); chamber IV,
8 ml. of 1 N sodium hydroxide solution (51); and chamber V, 20 ml. of 40
percent sodium hydroxide solution.

Chamber VI, kept in C02"methylcello-

solve bath, acted as a trap to remove water.

The stopcocks were turned as

shown in Fig. I and the system was evacuated with a Cenco-Pressovac 4 pump.
Stopcock E was then closed and COg-free air was then admitted into the
system by turning stopcock A several times.

After re-evacuating the system,

® This is a silicone product, Dow C o m i n g Antifoam A, obtained from
the Dow Corning Corp., Midland, Mich.

-24-

stopcocks A, B, D, and E were closed.
Chamber I was allowed to thaw, and it was gradually heated to 80°
using a water-bath.
constant stirring.

This chamber was then cooled in an ice-bath with
The stirring was to insure complete saponification of

the pectin and also to bring about the complete mixing of the carrier with
the radioactive methanol.

After reheating chamber I to 80°, chamber II

was warmed with a microburner and stopcock A was carefully opened to
initiate the distillation.

When the initial rapid distillation was over,

chamber II was cooled with the C02-methylcellosolve bath while chamber I
was being stirred with a magnetic stirrer as shown in Fig. I.

Distillation

was continued at 20-25° until about 8 ml. was distilled into chamber II.
Careful manipulation of stopcock A was required to prevent bumping.
Chamber II was thawed, followed by a gradual heating to 80° —
fractionation of methanol from water —
a micro-burner.

to effect

while chamber III was warmed with

Then stopcock B was carefully opened.

After the initial

rapid distillation, the solution in chamber II was stirred with the magnetic
stirrer.

When chamber III was cooled to about 25° > the lower half of it

was chilled with the CC^-methylcellosolve bath.

In order to prevent bumping

and to aid fractionation, stopcock B was carefully regulated until 0.5~1
ml. of distillate was collected in chamber III.

The level of the C02-methyl-

cellosolve bath was then raised to the neck of chamber III, stopcock B
was closed, and about 10 ml. of the combustion mixture was allowed to
drip into chamber III.

Stopcock A was turned to admit C02~free air, and

stopcock B was carefully turned to equalize the pressure in chambers II
and III and closed immediately.

Stopcock C was opened and then stopcock

D was turned to allow carbon-dioxide to escape from Chamber III into

-25-

chamber IV.

The C02“*methylcellosolve bath was replaced with an ice-bath

in order to thaw chamber III at a slow rate.
Using a micro-burner, chamber III was carefully heated to drive the
carbon-dioxide into chamber IV.

Heating was continued for about five

minutes after an "apparent" boiling, at which time the evolution of oxygen
from the solid potassium iodate had ceased.

Chamber IV was then connected

to an aspirator and stopcocks A and B were opened to flush out the system
with C02-free air.
ferred to a

The contents of chamber IV were quantitatively trans­

50 ml. centrifuge tube using 10 ml. of boiled distilled water.

Fifteen milliliters of a saturated barium hydroxide solution was added
and after standing for three to four hours, the suspension of barium
carbonate was centrifuged for five minutes at the maximum speed in an
International Clinical Centrifuge.

The residue was washed twice with

20 ml. of boiled distilled water, transferred to a tared filter stick of
medium porosity, and finally dried at 100°C.

The barium carbonate was

ground in a small mortar and then plated on an aluminum disc (2.85 8C1*
by making a slurry using absolute ethanol.

A hot-plate carefully regulated

with a powerstat Was used to evaporate the ethanol after plating.

The

Tracerlab End-window Counter was used to determine the activity of the
barium carbonate.

The maximum specific activity was calculated by using

the formula shown in Appendix I.

The fraction of maximum specific

activity, f, was obtained from the self-abBorption correction curve of
Calvin, et al (52).

The results are given in Table II and are expressed

as counts per minute obtained from 120 mg. cf pectin.
to note that as the yield of pectin increased —
differences —

It is interesting

attributable to seasonal

from Experiment 1 to 5, the maximum specific activity

- 26-

)

decreased respectively.

Therefore, it seems that as the rate of pectin

formation increased, there was a corresponding dilution in the activity
of the pectin —
groups.

probably due to the decreasing pool-size of the C^-methyl

Or another possibility is that the pool of pectin before feeding

began was slightly greater in the third experiment.

Nevertheless, even

with these possible factors, the radioactivity in the methoxyl group of
pectin remained relatively constant.

Isolation and determination of pectic acid obtained from pectin
The sodium pectinate, which was left in chamber I following the
methoxyl determination of pectin, was acidified with hydrochloric acid.
This mixture was centrifuged and the supernatant was decanted.

The

residue was dissolved in 1 N sodium hydroxide solution, heated to
filtered, acidified with hydrochloric acid, and then centrifuged.

60°,
After

discarding the supernatant, the resulting pectic acid wae dispersed in
water by warming and then reprecipitated by making the solution to
percent (v/v) with ethanol.

The pectic acid, which

wsb

60

precipitated as

a flocculent white precipitate, was dispersed in 95 percent ethanol and
then centrifuged.
made to

The residue was rediBperBed in 95 percent ethanol,

50 percent diethyl ether (v/v), filtered, washed with diethyl

ether, air-dried, and then dried over barium oxide in a vacuum dessicator.
When the pectic acid was counted in the same way as the pectin, it
was found to have some radioactivity.

By using the formula shown in

Appendix I, the maximum specific activities of the samples were calculated
to be 0.15-0.74 counts per minute per milligram, which roughly would be
about 1 percent of the radioactivity of the original pectin.

-27-

In this

case, wf M was obtained from the self-absorption correction curve for
pectin.

It is assumed a priori that calculations baaed on such values of

"f" do not introduce appreciable error in the final activities of the
pectic acid.

The total activity of each sample of pectic acid was not

presented in Table II since pectic acid preparations were not obtained
quantitatively from pectin.

Nevertheless, the low activity in the pectic

acid residue indicates that some of the C^-methyl group was incorporated
into the pectic acid molecule.

According to the method of preparation,

it iB highly improbable that the activity is due to unhydrolyzed methyl
esters.

It is possible that some of the methyl groups of methionine were

oxidized to CO 2 which was then used in the photosynthesis of the uronic
acid portion of the pectin.

Preparation of deuteriomethy1-5,5-dinitrobenzoate for deuterium analysis
By employing the apparatus shown in Fig. I and the procedure described
for the saponification of the methyl ester of pectin,

500

mg. of pectin

dispersed in 1 6 ml. of water was hydrolyzed with 9 ml* of 1 N sodium
hydroxide solution.

About 10 ml. of distillate was collected and trans­

ferred to a large test tube (1" x 8").

One-half gram of anhydrous sodium

acetate and 1C ml. of 40 percent sodium hydroxide solution were dissolved
in the aqueous methanol solution and the mixture was chilled to 0° in an
ice-bath.
In order to prepare the deuteriomethyl-5 ,5"dinitrobenzoate, the
method of Lipscomb and Baker (^4) was modified.

About 5 g. of freshly

prepared 5 ,5-dinitrobenzoyl chloride (55) was dissolved in 50 ml. of
anhydrous diethyl ether and then added to the aqueous methanol solution.

-28-

After the mixture was kept In an ice-bath for half an hour with occasional
shaking, it was transferred to a separatory funnel.

The ether layer was

removed and dried in a stream of air, and the residue was recrystallized
from methanol three times to yield deuteriomethyl^j^dinitro benzoate
(m.p. 106-107°j lit. 107°).

The deuterium-containing methyl-5,5“dinitro-

benzoate will be analyzed for deuterium by a commercial analytical
laboratory.

The analysis has not as yet been completed and therefore it

is not possible to state with certainty that the transfer of a methyl
group from methionine to give the methyl esters of pectin was a trans­
methylation reaction.

Methoxyl (methyl ester) content of radish root pectin

Following the procedures described earlier, 120 mg. of pectin was
saponified (without using any methanol as carrier) and the percent
methoxyl group (methyl ester) in radish root pectin was determined.
yield of barium carbonate obtained from the oxidation of the methanol
from the saponification was 65.7 mg.

By using the formula shown in

Appendix II, the methoxyl content of radish pectin was found to be
8.6 percent.

-

29

-

The

DISCUSSION

Studies of* betaine as a methyl donor
The results obtained from the administration of Cl4-.me-t.hyl betaine
to tobacco plants indicated that, within experimental error, all the
radioactivity of the nicotine was localized in the methyl group.

Thus,

it was suggested that the methyl groups of betaine are precursors of the
N-methyl group of nicotine.

Since the betaine used in this work had one

of the three N-methyl groups labeled with carbon-l4 and Bince betaine
yields only one of its three methyl groups in transmethylation, the
probability of yielding the Cl4-m ethyl group in a methyl group transfer
was one out of three.

When this probability was taken into consideration,

betaine was incorporated to a slightly greater extent than methionine (4^)
into the N-methyl group of nicotine.
When betaine was compared with choline (18) as a methyl group
precursor for the N-methyl group of nicotine in Nicotiana rustics, it was
found that the rate of incorporation of the methyl group from betaine was
slightly higher than that from choline.

Nevertheless, it was also demon­

strated (59) that although betaine was a precursor for the N-methyl groups
of the alkaloids tyramine and hordenine in the barley plant, it failed as
a methyl donor in the young castor bean.

In addition, when C 14-methyl

betaine was administered to 11-day-old barley plants (59), it was found
that the choline, which was later isolated from the plant, possessed
radioactivity.

The relatively low specific activity of the choline

appeared more in the magnitude of a transmethylation reaction than a

-50-

direct reduction of the betaine to choline.

The metabolic significance

of such a reaction 1 b not known since choline is not a methyl donor in
the barley seedlings.
If choline had to be oxidized to betaine before it could be a methyl
donor, then the in vivo oxidation of choline to betaine could have occurred
through a choline oxidase system.
administered to tobacco plants.

Consequently, Cl4-aethyl choline was
The resulting choline and betaine which

were isolated from the plants were separated by a two-dimensional chroma­
tography.

When a radioautography of the chromatogram was taken by using

an X-ray film, two spots corresponding to those of choline and betaine
were found when the film was developed.

It is possible that the spot

corresponding to the radioactive betaine was formed by a transmethylation
of the Cl^-Hjethyl group from choline to a precursor of betaine.

Neverthe­

less, such a path for the formation of betaine seemed unlikely.

Therefore,

these results would seem to indicate that choline was probably oxidized
to betaine.
Since the betaine content of leaves infiltrated with choline and
betaine aldehyde was increased (21), it might be assumed that an enzymic
oxidation of choline to betaine could occur stepwise with betaine aldehyde
as the intermediate.

That choline was not a methyl group precursor in

the sprouting barley (16 ) and in the seedlings of Ricinus communis grown
in darkness (1 7 ) could then be due to the lack of enzyme system which
could oxidize choline to betaine.
Preliminary experiments by Cromwell and Rennie (5®) showed that
homogenatee of roots of Beta vulgaris brought about the conversion of
choline to betaine.

Nevertheless, a later report indicated that Buch a

-51-

conversion was brought about by bacterial systems (21).
In this laboratory, work with homogenates and mitochondria from
leaves of Nicotiana rustics was also conducted to attempt to discover a
choline oxidaBe system.

Various cofactors for choline oxidase were tried

in order to follow the enzymic oxidation of choline by manometric method.
Some inhibitors of polyphenol oxidases were also used in an attempt to
suppress the uptake of oxygen by the endogenous substances.

Nevertheless,

no enzymic oxidation of choline by these preparations could be demonstrated.

Studies on the biosynthesis of pectin
In the present study, evidence has been presented which indicates
that the methyl esters of pectin are synthesized by a transmethylation
reaction in which the methyl group of methionine is transferred to a
galacturonic acid unit to yield the ester.

This is the first instance

reported in which a methyl ester has been formed by a transmethylation
reaction.
A study was made on the rate of incorporation of the methyl group
of Cl4 -methyl and cl4~(i6uteriomethyl methionine into the molecule of
radish pectin.

About 95 to 95 percent of the activity of the pectin was

located in the methyl ester.

The dilution factor —

the molar ratio of

the activities of the methionine as to that of the methanol —
550.

was about

This was about a five times greater incorporation than was obtained

for the N-methyl formation of nicotine using methionine as the precursor
(45).

Such a high incorporation of methyl groups into the pectin molecule

tends to indicate that the reaction was a true transmethylation.

Never­

theless, an analysis of the deuterium to carbon-l4 ratio in the methyl

-52-

aster as compared with the deuterium to carbon-14 ratio in the methionine
methyl group must be made as a final proof.

These experiments are in

progress.
The possibility of methyl ether formation with the sixth carbon of
a galactose residue and a subsequent oxidation to an ester does not seem
likely.

Otherwise, it would seem that the residual pectic acid obtained

by saponification of the pectin would have contained an appreciably
greater radioactivity due to some ether groups.

Nevertheless, one could

argue that the rate determining step was the formation of an ether, and
that the subsequent oxidation of the ether to an ester occurred instantly.
Inasmuch

as

galactose is not considered part of a pectin molecule

( 6 , 5 5 » 5 7 ) and since a parallel organic reaction in which an ether is
oxidized to an ester is not known, such an argument does not seem tenable.
Consequently, the formation of the methyl esters of pectin probably occurred
by a direct esterification of the polygalacturonic acid, or by the poly­
merization of galacturonic acids after esterification.

The latter

possibility would be unlikely in view of the fact that very little galact­
uronic acid has been detected in freBh plant tissues (11).

On the other

hand, 15.4 percent of the total pectic substances (on dry matter basis)
(5 6 ) in radish was characterized as pectic acid.

Therefore,

it seemed

most probable that there was an enzyme system which could catalyze the
substitution of the proton with a methyl group to form the methyl esters
of polygalaeturonic acids.

Such a mechanism suggests that the transfer

of the methyl group was effected as the carboniuro ion and that the
in vivo transmethylation reactions resulting in N-methyl and C-methyl
groups were, likewise, due to the carbonium ion transfer.

-

55

’

SUMMARY

1.After the administration of C-^-methyl betaine
plants,

radioactive nicotine was isolated.

to tobacco

Essentially all of the

radioactivity was shown to be in the methyl group.
2.

Tobacco plants when fed choline labeled in the methyl group

with carbon-l4 produced radioactive betaine.

Radioautography of a

two-dimensional chromatogram revealed the radioactivity of the betaine.
5.

Several attempts were made to follow the enzymic oxidation of

choline, but no activity could be demonstrated with homogenates and
mitochondria.
4.

Radish plants produced radioactive pectin when C^-methyl and

C^-deuteriomethyl methionine were administered.

Almost all of the

radioactivity was localized in the methyl group of the ester.

A direct

transfer of the methyl group from a thioether to an ester was postulated.
That transmethylation involves the transfer of the methyl group as a
carbonium ion was also postulated.

-54-

REFERENCES

1.

du Vigneaud, V., Chandler, J„ P., Cohn, M., and Brown, G. B.,
J. Biol. Cham., 1^4, 787 (1940).

2.

du Vigneaud, V., Chandler, J. P., Moyer, A. W., and Keppel, D. M . ,
J. Biol. Chem., 1£L, 57 (1959).

5-

Moyer, A. W. and du Vigneaud, V., J. Biol. Chem., 145, 575 (1942).

4.

Simraonds, S., Cohn, M. , Chandler, J. P., and du Vigneaud, V.,
J. Biol. Chem., l4£, 519 (1945).

5.

Boraook, H. and Dubnoff,

6.

J. W., J. Biol. Chem.,

169 , 247 (1947).

Hirst, E. L. and Jones, J. K. N . , Advances Carbo. Chem., 2, 2^5 (1946).

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-57-

APPENDIX I

The formula uaed in correcting the observed count to zero sample
thickness was:

Op* M
W • f

where ^

= maximum specific activity (counts/minute/taillimole )

C0

= observed count (counts/minute),less background

M

= molecular weight of compound

W

= weight of sample counted

f

= fraction of maximum activity atthe sample

thickness (T),

obtained from self-absorption curve.

Sample calculation:
C0 = 55.8, W = 59.8 mg., M = 620, T = 21.1 mg./cm.2 , f = 0.295

A

=• — PPrA ft.
—
59.8 x 0.295

= 1.97 x 10? counts/minute/mM at infinite
thinness.

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APPENDIX II

The formula used in calculating the methoxyl content of pectin was:

'M

_

B x Wm x 100

-

------------------------

Wb x P
where M
B

= percent of methoxyl in pectin
= weight of barium carbonate

Wm = formula weight of methoxyl (OCH^)
W b = molecular weight of barium carbonate
P

= weight of pectin

Sample' calculations:
B = 65.7 m g •y

u =

Wm - 51.0, Wb ” 197» P * 12° mg.

-x ?l» 0 ,x .100- - 5^5 percent methoxyl in pectin,
197 x 120

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