A §?UD‘1’ OF PYRUVATE META‘EOLESM
IN THE TOBACCO PLANT

Thesis for the Degree of M. 5.
MICHiGAN EMTE SNWERSETY

Maria E. Frontera~A3rmat
1956

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EAST LANSING, MICHIGAN

A STUDY OF PYRUVATE METABOLISM IN
THE TOBACCO PLANT

by
Maria E. Frontera-Aymet

AN ABSTRACT

Submitted to the College of Science and Arts, Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree 0!

MASTER OF SCIENCE

Department of Chemistry
Year 1956

Approved by

 

Maria E. Frontera-Aymat

The present study was undertaken in an attempt to prove
whether or not the beta-carbon of pyruvate might contribute
to the one-carbon pool and then be a precursor of methyl
groups in the intact tobacco plant.

Pyruvate-3-Clu was fed to three groups of tobacco plants.
The nicotine isolated from them was found to be radioactive.
The nicotine was demethylated and it was found that the radio-
activity in the N-methyl group was not greater than would be
expected if the carbons of the nicotine molecule were randomly
labeled.

From the results obtained in the present study it ap-
pears that the beta-carbon of pyruvate does not contribute

to an appreciable extent to the one-carbon pool in metabolism.

A STUDY OF PYRUVATE METABOLISM IN
THE TOBACCO PLANT

BY

Maria E. Frontera-Aymat

A THESIS

Submitted to the College of Science and Arts, Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Chemistry
1956

AC KN OWLEDGMEN T

The writer wishes to express her sincere
appreciation to Dr. Richard U. Byerrum.for his
counsel and guidance during the completion of

this study.

TABLE OF CONTENTS

PAGE

INTRODUCTION .................................. 1
EXPERIMENTAL AND RESULTS ........................
Growth of Plants ........................
Uptake of Sodium.Pyruvate .................

Administration of the Radioactive Pyruvate ..

\DONMU'L

Isolation and Purification of Nicotine ....
Demethylation of the Nicotine ............. 11
DISCUSSION ...:................................ 1h
SUMMARY .................................... 17

RWERENCES .OOCOOOOOOOOOOOOOO000.00.000.00.0... 18

”PEMIXI OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO... 21

 

INTRODUCTION

The path of carbon during photosynthesis has been a
subject of much interest during the last few years. After
carbon-lb became available it was possible to follow carbon
through the various routes that it takes in the plant on
the way from 002 to sugar and other plant materials. The
method of study used by Calvin gt 3;.(1) has been to feed
a plant some labeled 002 in the light and allow only a very
short time for photosynthesis. By examining the compounds
present in the plant after eXposures to radioactive carbon
dioxide for various times, it has been possible to eluci-
date the first few reactions in the path of carbon during
photosynthesis.

Methyl groups are present in various plant products.
It is interesting to notice that no labeling has been found
in these groups when plants have been allowed to photosyn-
thesize in the presence of labeled 002 for periods up to 30
minutes (2). In view of this finding and in an attempt to

determine how CO goes into methyl groups, carbon-la

2
labeled intermediates in the Goa-methyl pathway have been
fed to intact plants. Using methionine-methyl-C-lh, Byerrum
and Brown (3) demonstrated that the methyl carbon of methi-

onine could be incorporated into the N-methyl group of

nicotine and a direct transfer of the methyl group was postu-
lated. Flokstra (h) also demonstrated the incorporation of
the methyl carbon of methionine into the methoxyl carbon of
lignin in barley. During the same period formate (5) was
found to form the methyl carbon of nicotine and the methoxyl
group of lignin. In an attempt to determine whether the
methyl group of methionine might be transferred by oxidation
and subsequent reduction or directly as a methyl group, Dewey
22.2l- (6) fed methionine doubly labeled with carbon-1h and
deuterium to barley. Direct transmethylation has been shown
to occur since it was observed that the same deuterium to
carbon-1h ratio was present in the methyl group of lignin as
occurred in the methyl group of methionine.

A number of different compounds have been shown to be
methyl precursors in higher plants. Kirkwood and Marion
(7, 10) showed the incorporation of formate into the methyl
groups of choline and hordenine of barley although they were
unable to demonstrate the transfer of choline methyls to
hordenine. However, Byerrum and Wing (8) demonstrated that
the methyl carbons of choline could be transferred to give
the methyl group in nicotine in Nicotiana rustica. Kirkwood
gt 2;. (9) were also able to demonstrate that methionine can
donate methyl groups to the alkaloid ricinine of castor beans.
Seto (11) observed that the methyl group of methionine could
give rise to the methoxyl group of pectin in radish plants.

Glycine betaine serves as a source of labile methyls in the

barley plant, as judged by the transfer of its methyls to

the alkaloids N-methyltyramine and hordenine (12). Sato

(ll) showed the incorporation of the methyl groups of

glycine betaine into the nicotine N-methyl group.' Byerrum,
Hamill and Ball (13) have also demonstrated that the alpha-
carbon of glycine serves as a precursor for the N-methyl
carbon of nicotine in the intact tobacco plant. The incor-
poration of the glycine alpha-carbon into the nicotine methyl
group was at least as rapid as the incorporation of the methyl
group of methionine and over ten times as rapid as the incor-
poration of the carbon of formate. Dewey (1h) fed calcium
glycolate-Z-Clh to tobacco plants and observed the labeling

of the nicotine in the methyl group to be about the same extent
as observed in methionine studies.

The rapid incorporation of formaldehyde into the N-
methyl group of nicotine and the results obtained in previous
studies with methyl precursors in this laboratory led Byerrum
gt 2;. (15) to propose that a two-carbon compound possibly in
equilibrium.with glycine, might be formed in photosynthesis
from.carbon dioxide which could split to give a one—carbon
unit at the oxidation state of formaldehyde. This one-carbon
unit would then give rise to the methyl group..

The formation of a 2-carbon intermediate in the photo-
synthesis of methyl groups, however, is not the only possi-
bility. Vernon and Aronoff (16) suggested that alanine

arises from.phosphoglycerate and that serine and glycine were

 

formed from alanine probably by such a pathway as:

pyruvate —-' alanine -r serine ——>glycine
It was recently demonstrated in this laboratory that the beta-
carbon of serine was incorporated to a large extent into the
N-methyl group of nicotine in tobacco plant metabolism (15).
This finding could indicate that if serine arises directly
from pyruvate, the beta-carbon of pyruvate could contribute
to the one—carbon pool in metabolism. This possibility
seemed of further interest because Arnstein and Keglevic
(l?) have reported that neither alanine nor pyruvate are
efficient serine precursors in the intact rat.

The present study was undertaken in an attempt to prove
whether or not the beta-carbon of pyruvate might contribute
to the one-carbon pool and then be a precursor of methyl
groups in intact photosynthesizing tobacco plants. When
pyruvate-B-Clh was fed to a group of tobacco plants, the nico-
tine isolated from them.was radioactive. However the radio-
activity of the N-methyl group was not greater than would be
expected if the carbons of the nicotine molecule were randomly
labeled. It would therefore appear that the metabolic pathway
suggested by Vernon and Aronoff to occur in plants - the con-
version of phosphoglycerate to serine by way of pyruvate and

alanine - was operating at best only to a minor extent.

EXPERIMENTAL AND RESULTS

Growth of Plants

The tobacco plants used in this investigation were
of a high nicotine strain, Nicotiana rustica L., var.
humilis (3). The seeds were planted in the greenhouse
in flats containing vermiculite.l Within three weeks the
seedlings were transplanted allowing about two or three
inches between each plant. Occasionally tap water was ap-
plied to keep the vermiculite moist and a nutrient solution
containing 1 g of MgSOu.7H20, l g KZHPOh and 5.8 g Ca(N03i2-
hHZO in about four liters of tap water was applied twice a
week. About two or three months were usually required for
the plants to obtain the desired height of about six inches.

To prepare plants for the hydroponic administration of
the sodium pyruvate the plants were removed from.the flats
and the roots were carefully freed from vermiculite first
by shaking and then by soaking and washing with tap water.
The roots of the plants were then soaked in a 0.01 percent

2

Wyandotte detergent germicide for one hour to reduce the

 

1A commercial brand of heat expanded mdca

2W'yandotte detergent germicide, No. 1528, was obtained
from.the wyandotte Chemical Corporation, Wyandotte, Michigan.

bacterial population. After rinsing the roots with tap water
the plants were placed in 125 ml Erlenmeyer flasks containing
1 ml aureomycin solution (1:1000), 6 drops of a 0.1 percent
Wyandotte detergent germicide and 50 ml of an inorganic nu-
trient medium prepared by diluting with two parts of water,
one part of the stock solution. The composition of the stock

solution is asfollows:

2610 mg oa(N03)2.uH20
500 mg KCl

756 mg MgSOu.7H20
500 mg (nah)zsoh
5.6 mg FeClB.6H20
500 mg K2HP0u

2 liters Distilled water

Plants were fed the radioactive material in a special
fume hood to prevent any health hazard from radioactivity.
Artificial light was used in the experimental work. The
source of light consisted of two 36-inch, 30-watt fluores-
cent tubes, placed about lh inches above the tops of the
plants. The lights were left on for twelve hours a day and
:nutrient solution was added to the flask when required to
keep the volume constant. Twice a day, when the lights were
'turned on and when the lights were turned off, a stream.of

<xxygen was passed through the nutrient solution for two min-

iltes to provide aeration for the roots and to prevent wilting.

 

Uptake of Sodium.Pyruvate

Before the administration of radioactive sodium.pyruvate
it was necessary to ascertain the extent of uptake of the
pyruvate by the plant and to determine whether the pyruvate
was destroyed by microorganisms on the roots.

The most sensitive method for the determination of
pyruvic acid depends upon its precipitation with nitrophenyl—
hydrazines (18). Whereas h-nitrophenylhydrazine may be used,
2,u-dinitrophenylhydrazine was preferred because its solution
is stable and it reacts more rapidly with the pyruvate (18).
The 2,h-dinitrophenylhydrazine reagent was prepared by grinding
100 mg of the phenylhydrazine with 100 ml of hydrochloric
acid, prepared by diluting one part concentrated HCl with 5
parts of water. The solution was filtered and kept in the re-

frigerator while not in use. The determinations were carried
out as follows: a three ml aliquot of sample Solution was
placed in a water bath for ten minutes at 25°C. One ml of
2,h-dinitrophenylhydrazine reagent was added and the solution
was mixed. After five minutes, eight ml of ethyl acetate

were added and a stream of nitrogen was bubbled through the
mixture for two minutes. The lower aqueous layer was removed
and discarded. Exactly 6 ml of a h percent solution of
sodium.carbonate were added to the solvent mixture, and
nitrogen was rapidly passed through the solution again for

two minutes. After the two phases had separated, 5 ml were

‘withdrawn from the lower layer. To this solution five ml

of a six percent sodium.hydroxide solution were added, mixed
and allowed to stand for five minutes. The optical density

of the solution was determined in a Beckman Spectrophotometer
Model B, at h20 mu and 520 mu, 5 to 10 minutes after the addi-
tion of the alkali.

A standard curve was made by using various standard
sodium pyruvate solutions containing from 5 to 20 micrograms
per milliliter. Determinations were carried out as explained
above. Optical density was plotted against concentration and
a straight line curve was obtained.

To determine the uptake of sodium pyruvate by the tobacco
plants, four plants were prepared as described previously.
Then 2 m1 of sodium.pyruvate solution (1 mg/ml) were added
to each flask. Four other flasks were prepared with the same
solutions but, instead of plants, contained six root pieces
of about one-centimeter each. Four other flasks containing
the 50 ml nutrient solution, one ml aureomycin and six drops
of the 0.1 percent Wyandotte detergent germicide were used
as controls. All twelve flasks were put in the hood and the
lights were left on for twelve hours a day. After u8 hours,
the plants were removed from the nutrient solution. The
roots were washed with a stream of distilled water and the
washings were collected in the respective flasks. The content
of each flask was filtered through Whatman number one paper.

The filtrate was collected in 100 ml volumetric flasks and

made up to the mark with distilled water. Three ml of each
flask were analyzed for the presence of pyruvic acid as ex-
plained before. It was found that 90 percent of the pyruvate
had been absorbed by the intact plants during the NB hours.
No significant decrease in the original amount of pyruvate
was indicated in the analysis of the solution which had con-
tained root fragments or in the control solutions. Due to
the rapid absorption of pyruvate by the plants a growing
period of seven days was used for the administration of the

radioactive pyruvate.

Administration of the Radioactive Pyruvate

1.5 mg of sodium pyruvate containing 106 counts per
minute as Cl"L were fed to each plant.3 The molar quantity
of pyruvate was calculated to be equivalent to the amounts
of methionine and other methyl precursors used in previous
studies in this laboratory. However, the amount of radio-

activity was ten times more than the amount used by previous

workers.

Isolation and Purification of Nicotine

Seven days after the administration of the radioactive

material the plants were removed from the flasks. The roots

 

fijobtained from the Nuclear Instrument and Chemical
Corporation, Chicago, Ill.

10

were washed under the tap and blotted with a cheese cloth.
The plants were then cut with scissors into very small

pieces that were dried as soon as possible under two infra-
red lamps. The dried material was ground in a mortar with

20 percent of its weight of calcium hydroxide. The resulting
powder was transferred to a Kjeldahl flask and steam dis-
tilled into a flask containing 6 m1 of 6N H01, until the
distillate was clear upon the addition of two drops of
silicotungstic acid (12 g per 100 ml water). Usually from
one to two liters of distillate were collected. The dis-
tillate was concentrated in ygggg to a volume of about ten
milliliters. Following the procedure of Smith (19), the
concentrate was made alkaline and azeotropically distilled
twice to purify the nicotine. Every time the distillate was
collected in hydrochloric acid as described above. The

final distillate was concentrated in 35229 to dryness. One
ml of distilled water was added to the nicotine hydrochloride
and this solution was again concentrated.inflggggg to dryness.
The salt was dissolved in one milliliter of water, and then
the flask was washed out with small amounts of methanol until
'bhe total volume was about 20 ml. An equal amount of methanol
saturated with picric acid was then added to precipitate the
nicotine as the dipicrate. After the mixture had stood for
about 30 minutes, the nicotine dipicrate was filtered off,

‘washed with methanol and recrystallized from hot water. The

11

precipitate was left overnight in a vacuum desiccator and
the next day a determination of melting point was run for
purity. The final product melted at 223°-22h°0 as compared
with the recorded value of 22h°c (20).

The dipicrate was ground in a mortar and then plated
in a tared aluminum dish for counting. The samples were
weighed, counted in a windowless flow counter and corrected
for self absorption. (For calculations see Appendix I,
page 21). The results are presented in Table I. It may
be seen that after feeding pyruvate«-3-Cl’4 to tobacco plants,

the nicotine isolated from them was radioactive.

Demethylation of the Nicotine

As the nicotine was found to be radioactive, it was of
interest to determine how much of the radioactivity was in
the methyl group. The method used was that suggested by
Pregl (21) for the determination of methylimino groups.
Brown's modification (3) of the procedure was used in order
to recover the methyl carbon in a suitable form for counting.
The procedure consisted of the conversion of the alkylimino
substance in the presence of hydriodic acid into the quater-
nary ammonium salt, which on heating was decomposed with
separation of the alkyl iodide. The liberated methyl iodide
was made to react with triethylamine to yield triethylmethyl

ammonium iodide. The demethylation apparatus was similar to

that described by Pregl.

12

In order to obtain the nicotine in a more soluble form
about 200 mg of the nicotine dipicrate were dissolved in
sodium hydroxide and the nicotine recovered by azeotropic
distillation through a Hidmer column. The distillate was
collected in 2 ml of 6N hydrochloric acid and was concen-
trated to dryness 1g 35239. The final concentration of the
solution was done in the demethylating flask. To the nico-
tine hydrochloride in the demethylating flask were added
hS mg NHhI, 2 drops of 5 percent gold chloride solution,
and 3 ml of hydriodic acid, (specific gravity 1.5). The gas
washing vessel contained 0.75 ml of a 5 percent cadmium sul-
fate solution and 0.75 ml of a 5 percent sodium thiosulfate
solution as described by Pregl to remove iodine and hydrogen
iodide.. The receiver contained a 5 percent ethanolic solution
of triethylamine which was cooled in a solid carbon dioxide
methyl cellosolve bath. After the apparatus was assembled
a slow stream of nitrogen was passed through the side arm
and the reaction flask was heated in a cupric oxide bath.

The temperature was raised to 200°C in 20 to 25.minutes, then
slowly to 350-360°C and held there for NS minutes. The heat

was removed and the apparatus was flushed with nitrogen
until the reaction flask had cooled. The receiving tube was
then taken off, corked tightly, shaken and allowed to stand

overnight at room.temperature. The next day the excess
alcohol and triethylamine were evaporated in a steam bath
and then the last traces were removed in a vacuum desiccator.

The white triethylmethyl ammonium iodide was dissolved

13

in less than one ml of absolute ethanol and was plated in
tared aluminum dishes for counting. The samples were dried
in a vacuum desiccator and counted in the flow counter. The

results of these counts are also presented in Table I.

TABLE I

LOCATION OF RADIOACTIVITY IN THE NICOTINE MOLECULE
AFTER THE ADMINISTRATION OF PYRUVATE-Beclh

 

 

 

Number Maximum.Specific Activity
Trial of (Counts per minute per millimole)
Plants
Nicotine Triethylmethyl Percent

Dipicrate Ammonium Iodide Recovered

 

1 16 l.h3 x 103 81 6.
2 6 5.93 x lO2 37. e.
3 23 1.22 x 103 50.

 

The results (Table I) show that less than 10 percent
of the radioactivity of nicotine from pyruvate-3-C11+ fed

plants was located in the N-methyl group.

DISCUSSION

From the results obtained in the present study it
appears that the beta-carbon of pyruvate does not contribute
to any appreciable extent to the one-carbon pool in metabo-
lism.

This finding is in contrast with Vernon and Aronoff's
suggestion that serine and glycine arise from phosphoglycer-
ate by the following pathway:

phosphoglycerate-——rpyruvate ——9alanine.——9serine-—+glycine
If this pathway were operating appreciably in the intact
tobacco plant, the beta-carbon of pyruvate would correspond
to the beta-carbon of serine and a larger amount of radio-
activity would have been found in the N-methyl group of
nicotine than was observed experimentally. This assumption
is based on the findings of Byerrum gt 31. (15) who have
showed that the beta-carbon of serine contributes to the
one-carbon pool in metabolism and that it is incorporated to
a large extent in the N-methyl group of nicotine. Thus it
appears probable that serine does not arise directly from
pyruvate in the intact photosynthesizing tobacco plant. The
results are also in disagreement with those of Newburg and
Burris (22) who suggested that alanine arises from pyruvate

without rupture of C-0 bonds and that carbon-2 of pyruvate

15

becomes carbon-l of glycine. If this were the case, the
beta-carbon of pyruvate would correspond to the alpha-
carbon of glycine, and it has been shown by Byerrum 33 31.
(13) that the alpha carbon of glycine is a good methyl
precursor in the tobacco plant. There is, however, a sig-
nificant difference in the method of study followed in each
case that would possibly explain the difference in results.
Vernon and Aronoff used soybean leaves in the light, Newburg
and Burris employed tobacco leaves in the dark, and in the
presmm study, intact tobacco plants were used in the light.
It is possible that different metabolic pathways exist in
each case.

One suggestion for the metabolic pathway involved in
the incorporation of pyruvate-R-Clu into nicotine in the
intact tobacco plant is that pyruvate is completely oxi-
dized to 002 and H20 by way of the Krebs cycle and that then
this CO2 is metabolized in photosynthesis. Under these con—
ditions the radioactivity would be randomly distributed
within the nicotine molecule. The results show that about
5 percent of the radioactivity of nicotine from pyruvate-341i+
fed plants was in the methyl group. Since nicotine contains
10 carbons, one would expect 10 percent of the total radio-
activity to be in the methyl group if random labeling had
resulted. The counts of triethylmethylammonium iodide

were so low that great counting accuracy was not possible.

16

The present results may therefore actually indicate a random
distribution of the beta-carbon of pyruvate in the nicotine
molecule.

The idea of random distribution does not discard the
possibility of finding most of the radioactivity in some
definite location in any one of the rings of the nicotine
molecule. The fact that less than the theoretical 10 percent
labeling expected for random distribution was found in the
N-methyl group gives a point for speculation. Another pos-
sible metabolic pathway may be the conversion of pyruvate
to glutamate. Calvin gt g1. (23) fed labeled pyruvate to
algae and found that glutamate had over two times as much
radioactivity as any other single product. Pyruvate-3-Clh
may be metabolized to alpha ketoglutarate by way of the
Krebs cycle, and then by transamination be converted to glu-
tamic acid labeled in the gamma position. The glutamic acid
may cyclize and form the pyrrolidine ring labeled in the-
three or four position. This'proposition is speculation and
needs further study. Nothing can be concluded definitely
since the nicotine in the present study was not degraded
beyond demethylation. It is interesting to point out that
Dewey (2h) fed labeled ornithine-Z-Clu to tobacco plants
and obtained labeling in the two and five positions of the
pyrrolidine ring of nicotine. The interconversion of orni-

thine and glutamic acid has not been shown to occur in plants

but it has been demonstrated in animal metabolism (25).

17

SUMMARY

Pyruvate labeled with carbon-1h in the beta-carbon
was administered to tobacco plants and was further metabo-
lized. The nicotine isolated from the tobacco plants was
found to possess radioactivity. Demethylation experiments

showed that about 5 percent of the radioactivity of the
nicotine was in the N-methyl group.

l.

9.

10.

11.

18

REFERENCES

Calvin, M. The Path of Carbon in Photosynthesis. J. Chem.
Eda, .2_é.’ 639 (19149). '

Benson, A. A., Bassham, J. A., Calvin, M., Goodale,T..C.,.Haas,
V.A. and Stepka, H. The Path of Carbon in Photosynthesis.

V. Paper Chromatography and Radioautography of the

Products. J. Am. Chem. Soc.,'Z§, 1710 (1950).

Brown, A. S., and Byerrum, R. U. The Origin of the
Methyl Carbon of Nicotine Formed by Nicotiana rustica L.
J. Am. Chem. Soc., 7a, 1523 (1952). ““"‘

Byerrum, R. U. and Flokstra, J. H. Possible Origins of
the Methoxyl Carbon of Lignin in Barley Hordeum vulgare.
Federation Proc., 1;, 193 (1952).

Byerrum, R. U., Flokstra, J. H., Dewey, L. J., and Ball,
0. D. Incorporation of Formate and the Methyl Group of
Methionine into Methoxyl Groups of Lignin. J. Biol.
Chem.,l§;Q, 633 (195A).

Byerrum, R. U., Dewey, L. J., and Ball, C. D. Metabolic
Intermolecular Transfer of Methyl Groups in Hordeum
vulgare. Federation Proc.,ulg, 186 (1953).

Kirkwood, S. and Marion, L. The Origin of the Methyl
Groups of Hordenine and Choline. Can. J. Chem., 2 ,

30 (1951).

Byerrum, R. U., and Wing, R. E. The Role of Choline in
Some Metabolic Reactions of Nicotiana rustica. J. Biol.

Chem.,‘§Q§, 637 (1953)-

Kirkwood, S. and Dubeck, M. The Origin of the 0- and N-
Methyl Groups of the Alkaloid Ricinine. J. Biol. Chem.

Matchet, T. J., and Marion, L. The Role of Methionine in
the Formation of the N-Methyl Groups of the Alkaloid
Hordenine. Can. J. Chem.,'2l, u88 (1953).

Sato, 0. 3. Methyl Group Synthesis in Plant Metabolism.
-Ph. D. thesis, Michigan State University, East Lansing,

(1955).

12.

13.

15.

16.

17.

18.

19.

20.

21.

22.

23.

19

Sribney, M. and Kirkwood, S. The Role of Betaine in
Plant Methylation. Can. J. Chem., 22, 918 (l95h).

Byerrum, R. U., Hamill, R. D. and Ball, 0. D. The
Incorporation of Glycine into Nicotine in Tobacco
Plant Metabolism. J. Biol. Chem., 210, 6&5 (1951).

Byerrum, R. U., Dewey, L. J., Hamill, R. L., and Ball,
0. D. The Utilization of Glycolic Acid for Methyl
Grogg)Synthesis in Tobacco. J. Biol. Chem., 219, 3R5

l9 .

Byerrum, R. U., Ringler, R. L., Hamill, R. L., and Ball,
0. D. Serine and Formaldehyde as Metabolic Precursors
for the Nicotine N-methyl Group. J. Biol. Chem., glé,
371 (1956).

Vernon, L. P. and Aronoff. 8. Amino Acids Formed During
Shogt;Term.Photosynthesis. Arch. Biochem., 29, 179
19 0 . ‘-

Arnstein, H. R. and Keglevic, D. A Comparison of Alanine
and Glucose as Precursors of Serine and Glycine. Bio-
Chemo Jo, _6_2_, 199 (1956).

Snell, F. D. and Snell, C. T. Colorimetric Methods of
Analysis, Vol. III, Third Edition. D. Van Nostrand
Company Inc., New York, 1953. pp. 353-356.

Smith, C. R. Separation of Nicotine from Related
Alkaloids by Aqueous Distillation. Ind. Eng. Chem.,

All. 251 (19112).

Henry, T. A. The Plant Alkaloids. The Blakiston 00.,
Philadelphia, 19h9, p. 37.

Pregl, F. Quantitative Organic Microanalysis. Third.
E . Edn., The Blakiston 00., Philadelphia, 1937, p.
lréfi" 190 o

Newbergh, R. W. and Burris, R. H. Effects of Inhibitors
on the Photosynthetic Fixation of C02. Arch. Biochem.
and Biophys., g2, 98 (195A).

Milhaud, G., Benson, . A., and Calvin, M. Metaboli m
of Pyruvic acid-2-C and Hydroxy-pyruvic acid-2-C
in Algae. J. Biol. Chem., 218. 599 (1956).

20

2h. Dewey, L. J. Studies on the Biosynthesis of Nicotine
and Lignin. Ph. D. thesis, Michigan State University,
East Lansing (195k).

25. Stetten, M. R. Mechanism of the Conversion of Ornithine
into Proline and Glutamic Acid.in vivo. J. Biol. Chem.,

122. L199 (1951).

21

APPENDIX I

The formula used in correcting the observed count

for self-absorption was

Am = Co ' M
b

where Am maximum specific activity (counts/minute/milli-
mole)

Co 8 observed count (counts/minute)

molecular weight of compound

W = weight of sample counted (milligrams)

fraction of maximum activity at the sample
thickness used (T)....obtained from self-

absorption curve

Sample calculation:
Nicotine dipicrate...Co = 33 c.p.m., W e 36 mg, M = 620,
T g 1207 mg/CMZ

‘ x 620 a 3 . .
Am his 1.115 x lO c.p m /mM

 

 

VITA

The writer was born in Puerto Rico and received her**
secondary education at Mayaguez High School in Mayaguez,“
Puerto Rico. In August, l9u7, She entered the College of
Agriculture and Mechanic Arts of the University of Puerto
Rico, and was graduated in May, 1951 with a Bachelor of
Science Degree. Since graduation she has been working as
a chemistry instructor for the same institution. On June,
1955 she was granted a leave of absence for advanced studies
at Michigan State University. After graduation the writer

will return to her teaching position in Puerto Rico.

CHEMISTRY UBRADate Due
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A study of pyruvate metabolism
in thetobacco plant.

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MICHIGAN STATE UNIV

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