A STUB! Of RIBOSS HH8AB0LISM

in m

fomec© m m ?

ifer
fhowa# Griffith,

Submitted to the School for Advanced Graduate Studio# of
Michigan State University of Agriculture and Applied
Science In partial fulfillment of the retirements
for the degree of

Boofoa of fnmrnmt
Department of Chemistry

19$&

ProQuest Number: 10008594

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X*7*5vi&
Tfaesuthor wishes to express M i m #t sincere thanks
to Br. R. IT. Byemua whose interest, patience end counsel
greatly facilitated the ^lapletlm of this study. He else
wishes to thank JPr. Pei-Hsing l«/u and other members of the
CheR&stry Department for their helpful suggestions.
The author wishes to express his appreciation to the
Department of Chemistry, the
Energy Commiesion an^
the National Xnstitules of Health for tiiair timely financial
aid during this study*

tm
ifift ®Sro3Elt05lP IIBWP O03PR ^TOPEi© jsSK-j,
;1/yO Tfl Klill060*lft^ JBSP&JWMSI
and received M s secondary edneatien at the Minneola High
School.

Ho attended Dodge 01% Junior College, Bodge City,

Kansas for i«o years.

HO transferred % Kansas State College,

Manhattan, Kansas and m s awarded the Bachelor of Science
Degree end the Haster of Science Degree in Mixing industry In

1952 and 195k, respectively.
He entered the Graduate School of Michigan State University
in 195k and served tw> years as a Gradn&te Assistant. He m i
awarded a national Xajrtltatee of Health fellowship for- & m
year. He m s appointed a Special Bradnaie Research Assistant
nnder a national fastihate# of Health p m
of his graduate studios.

ill

for the remainder

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Page
IHTROIXrCTIOH........................

....

......
& m results..

1
3

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

10

Preparation of Plants.. ............................. 10
Isolation and Purification of H i e o t i n e 11
Demethylationof N
i
c
o
t
i
n
e
;
;
12
Results. ........................................lit
Determination of Kadioactivity................................ 15
Parity of D-Ribose-l~C14 .....
16
Intake of Mlibose from Nutrient S o l u t i o n . . 16
17
Results ...
Metabolism of -MUtbose-l^14 by Tobacco Plants................. 18
...
19
Results.
Methyl&tion Studies l&th B-Ribose-i-C1
4
21
Results*.......
22
Formation of Glycine and Serine in Detached Tobacco Plant
Leaves
...
22
Vacuum Infiltration of D~Ribose~l-C14 into Detached Leaves. 2h
Isolation and Purification“of Serine and Glycine...•••..... 25
Degradation of G
l
y
c
i
n
e
.
27
Degradation of-Serine...................................... 29
Results........
30
DISCUSSION...............

33

Synthesis of Nicotine from B-Ribese•••••••••.....
Synthesis of Serine and Glycine from B-Ribose-in-Detached Leaves..
......
SUWIT..............
LITERATURE CITED.....

33
36
Ii2

.......... *...........

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

vii

U3
U8

LIST OF TABLES
m B

Page

I Composition of the Stock NutrientSolution*.....*......
11 Bemethyl&tion of Nicotine dipicrate.....**..*.......
111 Recovery of C14 from Methyl-C14-nicotinedipicrate........

11
ZLU
IS

IF Location of Radioactivity after Feeding Tobacco Plants B-Ribose-l-C14..... ..*....
......... 20
V Location of Radioactivity in Nicotine from Tobacco plants
Fed D-Ribos©-l«*C14............... ........ ..... .
VI Metabolism of L-Ribose-l-G14 by Detached Leaves

....

VII Distribution of C*4 in Serine.......
mi

Distribution of C14 in Glycine.......**.**............

viii

23
31
32
32

J<39f OP PU30SB8

m

Page
X«

of 01yeiae and SwS$ti8:In FXsntys*#*••»•«•••*• * 37

ix

DiTKQKJCTIOU

INl’RGJDOt.TIOH
The transfer of methyl groups was established as a reaction in
Hiring organisms by du Tigneaud at al. (X) in 19bG. Since this tine
much attention has been £sensed on the transfer and biosynthesis of
the one carbon unit* Organic acids, amino acids, and other types of
compounds hare bead shown to be precursors of methyl groups in plants
and animals (1-16).
She only carbon source necessary for growth in pest higher plants
is carbon dioxide.. However, no evidence has been reported that carbon
dioxide can be reduced directly to a methyl group. Also, no compound
formed during the photosynthetic cycle has been shown to be a methyl
group precursor. A pathway by which methyl groups may be formed from
carbon dioxide in pleats is yet to be demonstrated.
Previous work in this laboratory has shown that the nicotine methyl
group may be synthesised by transmethylation reactions and by reduction
of a one-carbon intermediate believed to be an active or bound form of
formaldehyde. The Haet±ve one-carbon unit* was synthesised from the
alpha carbon of glycine, the alpha carbon of glycolic acid, and the
beta carbon of serine, these compounds are labeled rapidly during the
photosynthesis of C**Qa and are believed to arise from a common
precursor formed in the photoaynthetic cycle.
This study was undertaken to demonstrate a pathway for the
formation of glycine and serine from the photosynthetic cycle.

2

the formation of methyl groups from photosynthetic carbon dioxide
could be explained with this information. D-Ribose-C1*4 was adminis­
tered to tobacco plants and detached leaves of tobacco plants.
Nicotine, glycine and serine were isolated and degraded, the dis­
tribution of C1* in these molecules was determined.

HISTORICAL

3

HISTOEICAIj
The transfer of the methionine methyl carbon to an oxygen or
nitrogen atom of another metabolite has been reported from several
laboratories (1-8)» The methionine methyl group, labeled with C14,
m s incorporated into nicotine in tobacco plants (2)* All the C*4 m s
located in the methyl group of the nicotine* Dewey et ^1. (3), using
methionine containing C14 and deuterium in the methyl group, showed
the transmethylation reaction occurred with no intermediate oxidation
and reduction during the transfer* Sato et al. (h) supported these
results by showing that the methyl group of methionine m s transferred
intact to form the methyl esters of pectinic acid in radish plants.
Also, methionine m s shown to be a methyl donor in the formation of
lignin of tobacco plants (5), rieinine of castor beans (6), and
hordenine of barley (?) • Glycine betaine and choline are metabolites
in plants ’
which contain three E-methyl groups. The methyl groups of
glycine betaine were introduced into nicotine to an extent approxi­
mately equal to that of the methionine methyl group (9). Glycine
betaine has been shown to be a methyl donor for the formation of
N-methyltyramin© and hordenine of barley plants (10). The methyl
groups of cholinewere not incorporated into the barley plant alkaloids
or rieinine from the castor bean although the methionine methyl carbon
was utilized to form choline methyl groups (6,11). Byerrum et al. (9)
has shown that choline methyl groups were precursors of the N-raethyl

k

group in nicotine and incorporated to an extant similar to that of
the methyl carbons of methionine and glycine betaine.
Several metabolites labeled with C1* have been administered to
plants in an attempt to establish the origin of methyl groups and the
mechanism of their transfer and redaction. The alpha carbon of glycine
(12)* the alpha carbon of glycolic acid (13)* and the beta carbon of
serine (1U) ware shown to be utilised by tobacco plants for the form­
ation of the nicotine methyl group. They were incorporated to an
extent similar to that of the methyl groups of methionine* glycine
betaine and choline. These carbon atoms were shown also to be
incorporated into the methoxyl groups of lignin from tobacco plants
(15) and methyl esters of pettinie acid from radish plants (16) •
It is significant that glycine* glycolic acid and serin® contribute to
the one-carbon pool and* also* are among the first products to be
labeled when plants photosynthesis© CX4Qa (17-22).
The hypothesis that the one-carbon unit of glycine, serine and
glycolic acid was transferred at the oxidation state of formic acid or
formaldehyde has been tested. Formic acid was shown to be metabolised
and reduced to the methyl group of nicotine from tobacco plants (2)*
pectinic acid from radish plants (16)* hordenine from barley plants (7)*
rieinine from castor beans (6)* and lignin from tobacco plants (5).
The incorporation into nicotine was only one-tenth the extent as that
of the methionine methyl- carbon. Formic acid did not appear to be an
intermediate in the transfer of one carbon units in tobacco plants
because it was a very poor precursor of methyl groups. Byerrum et al.

5

(Hi) fed tobacco plants forraaldehyde-C14• The methyl group of nicotine
contained three to four times more C14 than the nicotine from corres­
ponding experiments with glycine-2-C14, serin©-3-,
,
C14, or glycolic
acid-2-C14• These data supported the hypothesis that the one carbon
unit is transferred at the formaldehyde oxidation state# The role of
foxmldehyde in methyl group synthesis appears to be limited to the
transport mechanism rather than asa .primary source of methyl carbons
since free formaldehyde is not a normal constituent in plants.
A pathway for formation of one-carbon units or methyl groups from
photosynthetic carbon dioxide is not known* No evidence has been
reported that carbon dioxide can be reduced directly to contribute to
the one-carbon unit pool* Gulp (23) and Ganz et a^. (2k) administered
C14QS and NaHC14GS|to tobacco plants and isolated nicotine containing
C14* Gulp found that the N-methyl group contained only 13$ of the
radioactive carbon in the molecule. If random labeling occurred, one
would expect 10$ of the C14 in the methyl group as there are ten carbon
atoms in nicotine* Dubeck and Kirkwood (6) found no G14 in the 0- or
N-methyl group© of rieinine when barley plants were allowed to metabolize
C140s. Kuzin and Merenova (25) administered radioactive carbon dioxide
to excised tobacco plant leaves and found all the C14 to be in the
methyl group. They believed that carbon dioxide could be incorporated
specifically into the methyl group. However, Dawson (26) and Tso and
Jeffrey (27) have shown that the main site of nicotine synthesis is
in the root although the transfer of methyl groups to the nicotine ring

6

system my occur in the leares. Thus, the only probable location of
0M would have been the methyl group.
fhe itotoaynthetie fixation of eofbon dioxide by plants has been
steadied extensively in recent years. The first stable product in
which C&* appears during photosynthesis with G140a is 3-phosphoglyoeric
acid (28). ffce photosynthetic fixation reaction has been shown to be
a chemical combination of ribulos© diphosphate and carbon dioxide to
form two molecules of 3^hoi^hoglyceric acid (2?«*32). Ichihara and
Qreenberg (33) obtained emyraes from rat liver which formed serine from
3-<<phosphogiyceric acid Via phospkohydroxypyruvie acid and phosphoserime•
Also* Vernon

Arneff (17) sftftd Hewbtirgb end Burris 0U) proposed that

a similar type of reaction occurred in plants. They postdated the
coiwersion of alanine to serine without araptura of any oaxbon«*carbon
DQEttlS* ^IISUP'

WMEtM* QOwwAwliQ m- jlSSJPo^ CUF@$b CWVw010H OX

re^iratory Sftfbsn dioxide into methyl groups. Frcntera-«iymt (3£)
fed tobacco plant® pyruvic acidN^44* $he isolated radioactire
nicotine but only 6£ of the &** in the molecule was located in the
methyl group. In contrast, the beta carbon of serine was incorporated
specifically into: the methyl p t f of nicotine (1U). fclbert and
Galley 04} studied Ci4Gjj fixation -by etiolated wheat plants. Alanine
and 3-ph0S£h0g3yesric acid accumulated large amounts of C** during
the initial period of photosynthesis* Serine and glycine were not
labeled substantially during the first 20 hours of photosynthesis.
The incorporation of C14 into glycine and serins coincided with the

7

foraation of cholortJplaste and activation of glycolic acid oxidase,
those data indicated that serine did not arise from a tries* or other
three carton eojocund i t m the phoiosynthetic cycle.
Glycolic acid, serin#, and glycine appear In be interrelated in
plant Rietafcolism (IS,!?)* Other studies hare demnetrated the intereeinrsrston of serin© and glycine {38*39). folbert .and Cohen (II) hare
sheen that barley and wheat leaves m m m t the two carbon atom of
glycolic acid directly into glyain© and the catexyl and alpha, carbons
of serine.. the beta carbon of serin© was derived in part from the
idpha carbon of glycolic acid, the aMwlatien of C34 in glycolic
4

acid (20*22) and glyein© and serine (17*20) during short tine photo*
synthesis has been observed*
fhe interrelationship of glycine, serine and glycolic acid has
bean established. However, the formation of'these jnetabolites from
the photosynthetic cycle is umertain. Galvin and co**tec©rs (21,28)
have obssrvsd a msrfeed imrcaS© in glycolic acid forwtdMsB daring photo*
synthesis in the abates of carte- dioxide. Also, the carbon atom
of glycolic acid and the alpha and beta, carbons of 3-phosphogLyceric
acid m m miiforB&y labeled when plants photosynthesized carbon dioxide*
C*4 (37,ho).. It la believed that glycolic acid is related to the
photosyntheiic cycle and glycine and serine are forte from glycolic
acid.
Smith and Gons&lus (hi) discovered m ern&m, isocitrase, that
cleaved isooitric acid to gpyoxylie acid and succinic acid, the emayra©
was purified from extracts of j». jSSSSEBe Lemberg and Krebs (U2)

8

extended these studies and proposed a new metabolic cycle to occur in
conjunction with the citric acid cycle, the glyoxyl&te cycle consists
of the cleavage of isocitric acid to glyoxylic acid and succinic acid
and the condensation of acetate and glyoxylic &cid to malic acid,
the latter reaction is catalysed by the enzyme malate synthetase (U3).
Recently Beavers and Komberg (Uh,h5) demonstrated the presence of
these enzymes in castor bean plants. The importance of this pathway
for glycolic acid formation is not known. The distribution of C1* in
glycolate during photosynthesis with C140a does not negate this
mechanism.
Wilson and Galvin (29) have attributed glycolic acid formation in
plants to a cleavage of the glycolyl~transkeiolase cos^lex. This
enzyme catalyses the transfer of a glycolyl group to an appropriate
aldehyde acceptor to form a ketose (1*6-48). The formation of pentose
phosphates in plants are believed to occur primarily by this pathway
(28,1*9)• The formation of xylulose~5-phosphate from fructose-6phosphate and glyceraldehyde-3-phosphate and the formation of ribose5-phosplmte and sylulose-5-phosphata from sedoheptulose-7-phosphate
and glyceraldehyde-3-phosphate involves a transketolase enzyme which
transfers a glycolyl fragment. During

tosynthesis the utilisation

of pentose phosphates is rapid. The forward reaction is predominant
due to the constant depletion of pentose phosphates which are necessary
for fixing carbon dioxide. If the reverse reaction ms forced, the
glycolyl~enzyme complex and glyceraldehyde-3-phosphate would compete
forerythrose-U-phosphat®. The erythrose-l*-phosphate pool Is very small

(m% chmaiitogi&pliiciaiydetectable)and eojipeiitlnn for the maaOX
quantity of 1;etro#a phosphate will tooroaoo the mean lifetime of the
0 ^ 1 ^ - m x p m complex. Wilson and Calvin (29) Illuminated plants In
the absence of carbon dioxide and observed a rapid acoumttlation of
pentose phosphates. After a short period glycolic acid concentration
increased and the pentose phosphate concentration decreased* IBm
increase in glycolic mid mas believed to be das to tbs Increase in
gXycolyX~emzym© concentration and its sobsecjuent decomposition to
glycolic acid and free enzyme, fhis mechanism is consistent nith tbs
available information concerning glycolic acid formation*
Qreemberg and Sassenmth (50) investigated the significance of
Ibis metabolic pathway in rats.* They fed. rats ribose-l-C** and
ambin©se-a*G14* Benzoic acid mas ingested along with the pentoses
and glycine m » isolated from urine as hippurin acid. Ho C14 was
detected in the hippurlc acid in either ecxperiinsnt. Weissbach and
Horeeker (19), using a soluble extract from spinach loaves, reported
tbs conversion of 1-0^ribose^^osphaie to glycine, the glycine
was labeled predominantly in the alpha carbon.

EXPERXMEHBLL A m RESULTS

AND BKS0&TS
of Flanta
I5h« tobacco plants used

in this starifcr were Nicotians rustica 1.

var* taaiXus, a strain with a high nicotine content . The seeds were
planted in flats ctmtaining vermteulite* a commercial non^raitrient,
supporting material* After two weeks the seedlings ware transplanted
to insure sufficient separation in the .flats* the plants were fed
twice a week with a mtrient solution composed of 5*3 g, 0a(N0s)a*l4iy>,
1 g. HgSO**?H20 and 1 g* KjjHFO,* in

h liters of water* Additional water

was administered as respired* The plants were grown in the green
house until a height of 6 inches was attained, the- growing period m s

about 90 days.
To prepare plants for hydroponic adidnistation of the radio**
active compound, they were removed imm the flats and the roots rinsed
carefully with tap water to remove the adhering vermiculit©. The roots
were soaked in a 0*1 percent solution of a detergent germicide
(Detergent germdjside No* 1528 mnnfhctured by %andotts Chemical Co.,
Wyandotte, Michigan) for m hour with occasional agitation. The roots
were rinsed free of germicide with tap water and placed in 125 ml*
Erlenmeyer flasks containing 50 ml. of an inorganic nutrient solution.
The nutrient solution m s a It3 dilution of the stock solution whose
con^osition is shown in Table I. The nutrient solution was saturated

11

tMM 1
gq ^ osttion

MS*&m
Calcium nitrate
Potassium chloride
Ferric chloride

op

1000 ml,
1 g.
250 mg.
' 2 mg,

stock

w s m r sc&raraj

JSagaecdnjo sulfate
Anmioniwm sulfate
Potassium dihydrogen
: phosphate

250 mg.
250 mg.
250 mg.

with oxygen .gag to provide aerobic conditions tor the roots, Three
of detergent germ&eide m s added to each
flask to protect against possible microbial destruction of the radio­
active compound during the feeding period*
the plants were grown in a laboratory fume hood following the
adainistration of E-ribose-l-G1*. Artificial lights were need daring
the administration period. Two 36 inch 30 mbt fluorescent tabes and
one 100 watt incandescent bulb m s placed Ik inches above the plants,
The light intensity at the top of the plants m s about 200 footcandles.
The plants were illmiainabed 12 hours per day.

Nicotine ms.isolated, from tobacco plants following the adminis­
tration of B-ribose-l-C1*. The plants were removed from the mtrieat
solution and the roots rinsed with tap water. The plants were cut into
ftfna.il pieces and dried under a heat lamp at 80° C, for 6 hours,
The dacy plant material m s g m m d with a mall mortar and pestle and
transferred to a micro-Kjeldahl flask containing four drops of 1 per­
cent 0, E, antifb&m solution (0, I, antifbam 60, General Electric Co.,

12

Sehmeciedy* I* X.}* Tbe nicotine m s separated by steam distillation
*ad the distillate ©ollmted In 1 aBU ©f 6 M HG1* The stem distil­

lation m e continued until no additional nicotine m m in the distillate,
the- distillate m e tested with silleotungstie acid Which feme a white*
inmluble salt with nicotine, The distillate me, concentrated irvvacao
to a email volume and the idLcohiiie thurified by two eaeeesaive awe©**
treble distillations from a basic solution into 1. ml*. #t 6 M HG1 using
a ^idmar atlnm (J>1)« Aftfsr puriflent!on* ties water and. meeae hydro4"
©ajuorauc acia me i m e i ift m jiij ana, sresianai. nicotine nyaroeniorici©
JifcttaTt

a*

Wutwlfc

drift T^^imnnmfn W T ~ * irli

rfSwt

v*irfaii rt i t nv

*&awfc*4i

-V« rh *ti lit

't

-

J

rhlrt*f liinaed irffr rr>

dissolved in a small ^panfcfiy ©f water and methanol. A saturated
solution ©f p ic ric #©1^ in

was added in eseesss and the nicotine

cipicratd aj,
f©u©ct to erystaii*;©©*. *n© jprsei^3iate was
J 'J I a iM tm

rifcl rfifc

ah' * t

< M*1
* * #gfc a©

tAb^ri*.

M k M M M ia

* t 48:

■

flV ilti'd rik

yiasejrita^.faae from m te f • ■ The
A reported mine is

-*f*-,rrifcili%J*1k'M. * * i tm <1* -ML. ©1 j f L

A

coiiibcvBu
i* k . r ih T

r'

A J l.

aim
«ta-k*fc4al

point m i 225>-227° 0 *

221 ° 0 . (5 2 ).

Hicctin© isolated £tm plants

m m radioactive*

The methyl group of nicotine m m isolated as. methyltriethylai^niuis
iodide by the procedure of JMgpL (51) as modified by M m

and

Byerrum (2). The qoatenary aimaoniUin salt was counted to determine the
quantity of €** in the methyl group*
An additional modification of the deraethyiation procedure m s used*
Brown and Byerrura chose to demethylate nicotine hydrochloride because
the diplcrate derivative is extremely insoluble in acid solvents.
In this study only email quantities of nicotine were isolated from

13

plants and it vas deelrablete eliminate unnecessary transfers of the
nte^ine.

W m m w * $ the

d^ethylatton

m$

carried ©at on the

nicotine dipicraie.

VMdttyr to 60 mg* of nicotine dipierate, h$ mg. amiwoniuia iodide*

t drops 5 percent gold chloride and 3 Am of bydriedie acid (sp. gr.
of 1.7} m m placed in the reaction vessel end the vessel attached to
the demethylation train {£)* the gee washing vessel contained 1.5 ml.
cadmium sulfhte*«SDdium bhiosulfate solution which removed iodine end
hydroiodic aeixfc f&em the gee stream. The delivery tube m s placed
below the surfsee of a 5 percent eolation of trist^lamine in ethanol
in the receiving tube

nee cooled to *|0 C. in. a methyl

cellosolve-*solM carbon dioxide bath. nitrogen gee m s passed slowly
through the demethylaiion 'train

the entire operation# -

the reaction flask ear placed in a capper oxide bath and heated
to 20G& C. in 20 to 25 mimtes. $$»* tei^eratur© m s then raised slowly
to. 350 to 3&Q0 0. and allowed to remain at -this temperature for b&
minutes* The heating element was m s i l and sweeping with nitrogen
gas m s continued for 15 nimtes. Bis delivery tube m s rinsed with
ethanol and the linsings placed in the receiving flask.
The solution in the receiving flask m s mixed* stoppered and
allowed to stand for 12 hours-. After standing, the solution m s
evaporated to a mail volume on a steam bath with a slow stream of
air directed on the liquid surface. The remaining alcohol and tri~
ethylamine were removed in a vacuum desiccator. The ^suiting

lit

Mthyltrt«t3ty5*»aswri»Bi ladtde, a white crystalline solid, w u tranaf erred epaaMtaitTely to a tarad alMifnum plamhet for determination
of weight and e*» content.
Results
s
a@8ssaSR

ftm data «ani in fable IX illustrate that demethylation of
nicotine dipierat® is feasible m i yields are similar to those obtained
i m m nicotine bydraehloride (2).

fh® reliability of this procedure

is illustrated further in fable XXX* Eadioaotive nicotine containing
C** bi&y in the methyl granp (lU) sts demethylaied end the specific
activity of the

iodide m s essentially ®<p&l to

the specific activity of the original nicotine dipierate.

i&itf xx
IMSfMMtTM 0F HXGOfXHB IXPXGSR&fE.
Nicotine
^<&$p£®WKt®

Jteteyltrlefthyl-

^imM Aibi
mg.

S3 .S
<
5*|
7•?
Q
£

IT id
7 ^

30.0

6.3

Percent
Held

fll
$0

15

■BiiB.it rry

x m a** m m mkthjl-c ^Maconas dipicbme
Kaadtaum Specific Activity

pisa? liiiw pea? miiiSBOX©/

Jr

itafejJtMM*

.■ ■riff

u J n .V

o lfe A ^ I

M®1^1tr±etfcyl"

Hicotlll© diplomat©
■IB.B.BBlAiilW W m n

|||BB|*»»4qfm jnfri^dft
iijjlliiiiiW iiiilBIILiiii.KJu|i|iw K >B>»i|WM I*l*B' f 1■H iBN.—

1*.0 x 10°
2.5

A

x

iibi iB

n

■■»!—

..■>

3 .9 * 1 0 °

30 °

2.5

of the f>ai

■i^a shown in

#10

* 'I©*

appendix.

All ©tmnts were msde with m Iteleai^hlcago Model X92X sealer
(Unclear Instruments and Chemieai aarporatioa, Chicago 1G, BJLinois)
and a fmcerlsb Model BQ^X6 pt&p^imaX m m eoonter (fraeerlab, Inc.,
Boston 10,

. She eemiing spates m e kO percent efficient

as determined bp a Katioml W m w m of Standards

sample.

■the substances to be counted, with the ©Keeptien of D~ribose**l*-Cx*,
were ground in a small agate mortar' or dissolved in ethanol and placed
in ata&ana planchets. f m material m e districted in the planehebs
mad the surface mad© m smooth m possible before coaling* the activity
of the

m s corrected to "infinite thichness,f by reference to

a BstS^Oa self absorption carve. D-°ribose-K;14 m s counted as an
infinitely thin layer.
Badioactivlty on chromatograms m s located and estimated with a
form chromatograph scanner (Forro Scientific Company, Evanston, Illinois)

16

coupled with a &golear-Ghieagp Model X62G& wfcemeter and a Model AW
Eateriiri©--Angus

&*$&&

ammeter.

' the nicotine dipicmte and ia»th$itris^

iodide samples

w«r« corrected la 10$ |**rceai efficiency of the courting syaiasu fh©
jgMUS!to activity of the other

m m reported ulthaut correcting

fw the efficiency of the cotmiiag system.

from Ifuelear Xrsir*n»enis ftp** GhiaBical
Cerj^ratior, CJbieago M§ 31Mmia} w m ca^hroraatiogr^had with authentic
IJ-rlbose ttthftilatter saturated |fo©nol and propienie acid^butanol-water
solvent® (5W* Ite % valuer at the pentose corresponded with the
radioactive ares on the ajfe^ai^^g^ajEGa* M© other s^dioactive area was
detected, iffth the chx*0i5a1&0g^pfet strip eoanteif'*

Several studies in this laboratory (£,9,12,13,114,35) hare shorn
that tobacco plants can Absorb various nutrients through their rest
systems from nutrient solution* Before attainistratlon of J^ribose-a-C1*,
It nas necessary to ascertain the absorption mte of ribose and, also*
to determine if microorgaiiisms altered the atfbose molecule before
absorption occurred.
A colorimetric method of analysis m s used to estimate ribose in
the nutrient solutions (55)# A ca^pound with an absorption maximum at
660 mu is formed by a reaction of pentoses, ferric chloride and orcinol

17

to strong acid solution.

Three sou

®£ m m & m m

stitofctaa containing

fcetoeen h4& mg. of jwiose was added to 3 *&- of X percent ©retool
to 0.1 percent ggrrtg chloride dissolved to concentrated hydrochloric
aeto. the misstep© m e heated tor 30 mtouttos to a boHtog eater bato,
essled to m m Imgmmmm' tod the epMtol density determined mlto a
Beckman Model B apectropi«>tometer. a ettotod reference ear*© mas
prepared matog toaw fwititiee of &*tm®m*

Stone the mtotoat «©to»

tion contained several toorpnto compounds that mi$it interfere daring
the datermijmtion, toe eolations were treated with 0.1 gnu Bowsx 50
tod 0.1 g®. Sowax 1 resins (Bow Chemical Company, Midland, Michigan)
before determining the residual rtoose.
Fifty ml. .of nutrient solution, $ drops of detergent germcide
and U mg. of B-ribose m m placed' to 12 flasks. tobacco plants m m
placed to it flasks, k mere inoculated with six root fragments 1 cm. to
length and toe foar rmtotog Hacks received no plant material.
After

36

hours toe nutrient solutions mere deionised and toe B-ribose

mas determined.

Eeaalijff
Ho loss of riboee mas detected to toe control solutions and solu­
tions containing root fragments. The nutrient solution to which plants
mere gram contained a© ribo®@. These results indicate a complete
and rapid absorption of ribose

tem toe nutrient solutions and no

detectable microbial destruction occurred under these conditions.

Pentose phosphates participate in most metabolic reactions of the
pentoses in plants * Th© chemleal literature contains m report concerntag the presence of a pentose kinase in plants* Therefore, it wo#
desirable to establish Aether nboe© m s metabolised after being
absorbed hgr tobacco plants* Also, it m s of interest to determine th©
general distribution of C14 In plant substances after feeding B-rfbose1-C*4 and whether m added carbon .source in the nutrient solution wild
dearease oxidation of the rlbos© by the plants*

tm tobacco plants m m prepared for hydroponic administration of
ribos© as pmriously described* Two mg, of IMH&oee-l^*4 (1 a. 10®
counts per minute) wB-ff placed in tbs nutrient solution of each plant
and 10 mg, of B*glueoa© m s added to tbs imirient solution of on© plant*
The plants were enclosed In ©eparat© g$am containers and carbon dioxide
free air m s passed through th© containers. The respiratory carbon
dioxide in th© exhaust .air stream m s collected in. a m s mshiag bottle
containing § ml, of $«X H HaOH, Th© plants were allowed to metabolise
the ribose for 36 hoars with llghttfuring the first and last 3$ hear
periods.

Following th© feeding period, the plants were removed from the
flask®, dried and groond with a mortar and pestle. The radioactivity
©f the plant material was determined on a flow counter and corrected
for self absorption. Th© C14 remaining in th© nutrient solution was
d«*rawi»«i ty pUaim * W i U •wwafc of «i, U<s*ld in a planchet and

V

tbs m b m

"With the aid of a hast lamp. The respiratory

cartes dioxide m s collected as barium carbonate and counted ae each.
After determinfag the ;radt©aotiviiy In the dry plant Material,
starch m s isolated % the method of Saesid f&ji* (56). This aieifaod
employs an acid alcohol and a eater extraction. the insoluble residue
from the starch preparation mats alee analysed for radioactivity.
The residue consisted of cell sail constituents and part of the plant
proteins.

heaalts
The distribution of radioactivity after feeding Hie plants is
shown in Table XV. About 85 percent of the radioactivity m s recovered
in these experiments* this discrepancy might be due to backscattering
of the beta particle foxing the counting of the ribose-1—C-k**
B-ribose-l-C*4 mft counted as an infinitely thin layer in aluminum
planehets. *3hea counting samples at «infinite thinness,11 many particles
are reflected into the sms&bivs volume of the counting ehas&er. this
effect causes the observed w m & m of counts to be higher than the
actual value* A H the other plant fractions sere counted under con­
ditions where backaeattering could not occur and m m corrected to
"Infinite thiekness11 with a self absorption curve.
Tolbert and Galley (36 ) have shorn that carbon dioxide fixation
m s stimulated 1d m leaves were sprayed with a rlbose solution* This
indicated that ribose could be phosphoaylated and utilised by plants.
These studies show that ribose can be metabolised by plants*

20

vim.v. jy
tfmtim or BADic&cEnrcrr m m

wmma

TO16.CG0 KAHTS MtlBOSB-l-C**

Mgattasai&wasaasis^ ^
Wmfsmfc C** E®^r©r»d
ecavmmia
. fed , ,

"

-----»gaS wtf'”
Carbon dioxide Solution Manorial starch

StabahaMses*

iwtiboee-i-e**

11

66

O.k

39

D^iboae-l-c1*
pltjig Elwtose

M

If

0.2

25

Ribokibase has bean purified from animal tissue (57) «ad in probably
present In plant tissue also. Recently, Niesh (58) has shown that
« M t plants metabolise arabinoae, presumably, by synthesis of the
pentose^S-phosphate initially.
A very striking result was that about one-fifth of the C1* appears
as respiratory carbon dlrad.de. She plants apparently absorb and oxidise
the rfboae vary rapidly. The metabolic rate farther emphasised by the
incorporation of a large «pastity of the C1* into the Insoluble
constituents. Starch contained only a small fraction of the activity.
However the quantity of starch present was very mall aa would be
expected when plants are grown in an atmosphere limiting in carbon
dioxid©.
She y w M t y of C®* remaining in the nutrient eolation of the
ri.^. fed ribaae only was probably a result of root fragments and
material accreted f*o» the plants as it was shown previously that rihose
is absorbed from the nutrient solution in 36 hours. The nutrient

21

solution In which glucose m s added contained almost one-half the
radioactivity originally fed. Although no definite conclusions may
he gained with the limited information obtained, it appear® that the
rate of ribose absorption was considerably slower in the presence of
glucose. Glucose had no sparing effect on the oxidation of ribose as
the <psntitles of C1* respired in the two experiments were similar.
Studies with P-ftibose-l-C
These experiments were undertaken to study the synthesis of methyl
groups from ribose in plants. The formation of glycolic acid from
pentose phosphates and the subsequent conversion of the alpha carbon
of glycolic acid into methyl groups has been postulated to be a pathway
for synthesis of the one carbon unit in plants. The methyl group of
nicotine was shorn to be synthesized from the alpha carbon of glycolic
acid by Byerrum et uL. (13)• If this pathway does exist the one carbon
of ribose would be incorporated into the methyl group of nicotine in
tobacco plants. Wilson and Galvin (29) have shown that plants growing
in the absence of carbon dioxide produce greater amounts of glycolic
acid. In these studies the plants were grown in an atmosphere in which
carbon dioxide concentration was minimized in order to increase the
concentration of gLyeolic acid.
The plants were prepared as described previously. Two mg. of
D-ribose-l-C14 with known activity and 10 mg. D-glucos© m s dissolved
in the nutrient solutions. The plants were placed in a 10 inch desiccator

22

and carfeon dioxide free ©is? passed throu# the desiccator at the rate
of 0*2 liters per minute. Following the growing period the plants
were harvested and nicotine isolated and demethylated as described in
previous options of this report.

MSSM
Hicotine isolated fro® plants fed B-ribose-l-C14 m s radioactive*
Ste results* presented in fable V, show that only a very mall part
of the o*4 m s

incorporated into the methyl group of nicotine, the

distribution of d14 indicates that a rapid and, possibly^ a specific
synthesis of the nicotine ring system from- ribose occurs, and that the
rate of synthesis of methyl groups is relatively slow.

Previous work in this laboratory has shown that the alpha carbon
of glycine and the beta carbon of serine may be converted into the
methyl group of nicotine in tobacco plants (12,lU). It has been postu­
lated that these amino acids might be formed from a two carbon compound
(glycsolaldehyde) which is formed in the photosynthetic cycle (19,29).
Tolbert and Cohen (18) and Sehou jt

(3f) have shown that glycine

and serine may be formed from glycolic acid in plants. Thus, the
formation of glycine, serine or glycolic acid from the photosynthetic
cycle would establish a pathway in plants for the synthesis of methyl
group® from c&fbon ditaxid©.

23

§f
1 P

LOCATE® OP MDIQAC5IPITI IN NICOTINE BE0W TOBACCO PLANTS FED B-RIBOSE-l-C14

■+2

•ti

0*» # fvy•
v0 0~ vO

jH

«
o
H

«

*

s §

W H M
CO -XfJ#

%

*

H

3

M
04

H
-a
a

04 c j ea
a

m

&
rH

9k
Q
rH

*»
O
«-4

M
<VI

H
CH4}

H

-4

OA

&
H

O

O'

4
H

3

!**■ ■ts» rA <M
•a

0

fc*
Q
H

e-

K
o

«

3
o

A A

CL

9.

e*
3

3

vOf
t

K
NO#
CM

cm

2k

The extremely rapid metabolism of ribose by tobacco plants limits
the value of the hydroponic method, of feeding plants for studying methyl
group synthesis, the absorption of ribose from the nutrient solution
m s very slow as compared, to the metabolic transformations of the pentose
and a rapid randomisation of the C14 occurred, that is, the C14 is
distributed to all carbons of the ribose. The initial reaction® of
D-ribose-l-CX4 are masked by similar reactions of uniformly labeled
ribose* Thus, the compounds forced from ribose would be nearly uniformly
labeled* A method m s desired in which the pentose could be introduced
rapidly into tobacco plants and the plants allowed to metabolise for
short periods. The vacuum infiltration method was employed in these
experiments ($9)*
Vacuum Infiltration of P-Bibose~l->C14 into Detached Leaves
Leaves from three month old tobacco plants were immersed in an
aqueous solution containing 2 rag. B-ribose-l-C14 (8 x 10® counts per
minute} per ml. The pressure above the solution was reduced to 30 ram.
mercury to remove the air from the intercellular spaces of the leaves.
Aft®? one minute, air was allowed to enter the chamber and the solution
surrounding the leaves was drawn into the intercellular spaces. The
leaves were weighed before and after infiltration to determine the
wei^it of infiltrated D-ribose~l-C14. The leaves weighed approximately
0.6 g. each and the amount of infiltrated solution m s about 50 percent
of the weight of the original leaf* The stems of the leaves wbre placed
between two pieces of moistened filter paper during the period for
metabolism*

Several loaves were placed in a light proof container to study
metabolism of pentoses in the absence of light and several were
iTtadnated with fluorescent tubes with a li^ii intensity of about 200
ftotamfUNU

Following a three hoar period fbr metabolism the leaves

ware harvested# cat into small pleces and placed in boiling 80 percent
ethanol. the Mixture was cooled end homogenised in a ground glass,
raster driven hoiw*geai*er. the homogsnate was filtered with ^hat^aan No. 2
filter paper to m s m m cell debris and insoluble substances. The
filtrate was diluted to 10 ml. with water end .0*8 mg. carrier glycine

w!tQ

MtnHhjri ' s u a w a ^ l ;« M a

'StCIClOCl#
n

rntj^ in >4

The radioactivity of free ribose remaining in the leaves was
estimated, by
the

an aliquot of the e&rfciact wflftd determining

in the ribose area with a chromatograph strip counter.

The

resolving solvent was t*a®yl alcohol and acetate buffer, pi £.6, (do)
and amllne^phthalic acid reagent (&L) was need to identify the ribose
on the cdu^eww^fOgpam-*

glycine andserim m m isolated from leaf extracts fey me**
dimensional paper ehromtographic methods. The resolving solvents were
einployed in the following order*
(1) t"*syl alcohol saturated with 0*1 M sodium acetate buffer,
pi 5 A (60)*
(2) Phenol saturated with an aqueous solution of 3.? percent sodium
’ dihydrogen phosphate and 6*3 percent sodium citrate (62).
(3) pyridine and water (65*35) (62).

26

Phenol and salts were C, P. grade and the organic solvents were
redistilled before using.
The areas of the chromatograms containing the amino acids were
eluted with water and placed on another sheet of filter paper for
resolution with the succeeding solvent* Authentic glycine and serine
was placed on all chromatograms to identify the amino acid areas
following resolution*
Approximately 1 ml. of the 80 percent ethanol extract was applied
in contiguous droplets to an area of 1 x 20 cm. on a sheet (30 x UO cm.)
of Whatman Ho* 1 filter paper. The extract was applied on a line
5 cm. from the narrower edge of the chromatogram. The solutions were
applied with micropipettes and a stream of warm air was used to
facilitate evaporation of the solvent. The chromatograms were stapled
in the form of a cylinder in such a manner that the edges did not touch.
This self supporting cylindrical chromatogram was placed upright in a
15 x U5 cm. glass jar that contained 50 ml* of the resolving solvent.
The j&rs were sealed and the solvent allowed to ascend until the solvent
front was 8-10 cm. from the top of the filter paper. Resolution was
complete in 20 to U8 hours at room temperature.
The chromatograms were removed from the jars and placed in a
forced draft oven and dried for one hour at 200 F• The amino acid
areas were located by cutting the strips from the chromatogram that
contained known samples of glycine and serine and spraying with ninhydrin solution (62). The corresponding areas that contained glycine
and serine from the plant extracts were removed from the chromatograms.

27

An&ra acids were ©inted tmm the paper with mt«r, A filter paper
^

fastened to m e end of the paper strip «ad the end of the

wiek m e immersed in mier*

the elution m e accoi^>lish@d by capillary

snavarnmt of water through the filter paper* the elution m e contijmed
until 2 iqX* of elaahe m e collected* Glycine and serin© solutions

m m placed m another filter paper sheet for purification with the
succeeding solvent • ffee eluaie containing ^iyeine and serine from
the final ebromtogram m s evaporated to 1 ml, on the steam hath*

Party $$* serine m 20 mg. glycine m s dissolved in the solutions as
A<id4

carrier*
The parity m s established by recrystallizing the amine acids

until their pacific activity m s constant- Glycine and serin© were
obtained in crystalline form by adding 0*1 ml, pyridine and sufficient
acetone to Cause precipitation in 1 ml* of adeems amino mid solution*
fhs precipitate m s collected on a filter m d transferred to a planehet
to determine the specific activity. Tm recrystallisations mr®
sufficient to yield essentially pure amino acids*

Glycine m s degraded lay the ninhydria method (63) idiich Is
summarised belows

00

Ten ®g* of glycine m s dissolved in £ mi, of mter and the solu­
tion placed in one side of a tm compartment reaction vessel.

One hoadvwt ag, ■citric acid and 150 rag* ninhydrin were placed in the
ether coa^ortraent* The vease1 m s swept with nitrogen gas for five
aiBUtes «** * g*» wfcafeiag bottle containing $ «£U of 0.1 M sodium
hydroxide m s attached* The reactant® vara mixed by tilting the vessel
in such a reamer that the glycine solution flowed into the ninhydrin
and citric acid ndxture* The reaction m e allowed to proceed at 100° C.
for 25 minutes* Mitrogan '.gas was passed slowly* through the system
daring the reaction period.
Vhen the reaction m e complete, the carbon dioxide collected in
the gas washing bottle was precipitated as barium carbonate. The yield
of carbon dioxide from the carboxyl carbon was 80-85 percent of the
theoretical value*
The formaldehyde romedning in the reaction vessel m e distilled
in vacuo* Five mi. of water m e added to the residue in. the reaction
vessel and %lm solution concentrated to dryness again* The vacuum
distillations were performed at 50® C* Three ml* of (.8 percent
dtmedoa (5,5*-diH^hylcyclohe!mn©dione-*l,3) in 95 percent ethanol m s
added to the combined distillate. The foimldehyde and dimadon solu­
tion m s adjusted to pH 5*6 with acetic acid and allowed to stand for
2k hours* The dtemdtm derivative of the formaldehyde m s collected on
a filter and washed with

5 ml. of cold water containing one drop of 1

M acetic acid* The melting point m s 188-189° 0* The yield of
formldehyde from th© alpha eaifcon of glycine m s 25-35 percent of
the theoretical yield*

29

Bmim mm de&paded by modifying theproeedare proposed hgr
ironoff (iit)

0
001?
V**

s^*c*h

ilinatmted in the ©qjmticm below*

£
30fif?
wywB
ohd

10110

3° c*

®b® oxidation of sarins with periodic acid m m reported by Hicolet
and Shinn (6U) to proceed s&os&y and usually to take 10 to 2k hoars to
complete. Also, the oxidation of glyoxylic acid, a product formed
from the initial oxidation of serine* m s reported to be insignificant
daring this reaction period (kO$6k) • 3ft m s observed in this study
that serine m s oxidised rapidly with periodic acid, the reaction m s
complete in about 30 stoute® at vmm teuperatare* m e n two ecpivalent®
of periodic acid were used* the mess# periodate oxidised the glyoxylic
acid rapidly* Both oxidations were collets in one hoar.

fwmfy mg, serine and 39.1 rag. periodic acid were dissolved in
$ a£U of water and the reaction mixture allowed to stand for one hoar
at room tm^srature (about

•&*}*

solution containing foimaldehyd©

and gjyaxylie acid m s adjusted to pH 8*5 with sodium hyte»&d« and the
formaldehyde removed fey two successive vacuum distillations, fhe
formaldehyde m m isolated as described in the preceding section, the
reaction flaUk eentaining' the residual sodium glyoxylat© m s swept with
nitrogen gas for five mdnutas and a gas washing bVttto containing 0*1
N sodium hydroxM# m s attached. Hitrogm gas mm passed slowly through
the system during the following procedures. Five ml* of 0.5 K

30

cerate solution (G. F. Ssnith Coi^any, Sotaflbus, Ohio) m s
thrombi a side aria* The reaction m s allowed to continue for
U> minutes at 30°C. Following the reaction period the gas washing
bottle m s recharged with

$ j&, 0*1 h

sodium hydroxide end the reaction

te^emtnre increased to 100® C. for fire minutes. The reaction
mixture m s allowed to cool and sweeping with nitrogen gas continued
for 10 minutes. The carbon dioxide collected in the m s washing bottle
m s precipitated with barium chloride solution and the barium carbonate
collected on a filter, the dimedtm derivative of formaldehyde and
barium carbonate samples

mm

weighed and the radioactivity determined.

The yield of the formaldehyde from the beta carbon of serine m s
60-70 percent of the theoretical value and barium carbonate from' the
alpha and carboxyl carbons m s obtained in 70-80 percent of theoretical
yields.

The data ia Table fl sheer that ribose m s absorbed into the cells
of the leaves

£sm the

Intercellular spaces and rapidly metabolised

daring the three hear period. -the metabolic rate decreased la the
absence of li#t.

Approximately Jf percent of the B-ribose-l-C1'*

remained In the leasee which were not illuminated whereas only 18 per­
cent was recovered from the illnmlnatad leaves.
COyoine and serine isolated from the illuminated leaves m s radio­
active.

The glycine from leaves fed in the absence of light did not

contain detectable qaantittes of CM , however, the serine was radioactive.

31

i m e vi
METABOLISM OF D-RIBOSE-l-C14 BT DETACHED LEAVES
C14 Infiltrated
into Leaves
(cpm)

C14 Recovered
as Free Ribose
(cpm)

Experiment

Humber
of Leaves

Dark

it (2.S3 g.)

9.2 x 10e

3.1* x 10®

Light

It (2.78 g.)

10.1 x 10s

1.8 x 10s

Each figure is the average of two experiments. Less than five percent
variation in individual experiments occurred.
The location of the C14 in serine from the light and dark experi­
ments is similar although the serine from illuminated leaves contained
more total activity. Serine ms isolated from 5.2 ml. of the illumi­
nated leaf extract and k M ml. of the extract from leaves grown in the
dark. Also, a greater <$oantiiy of ribose m s metabolised in the leaves
which were illuminated. Mien this information is considered, it can be
seen that about the same percent of C1* from the ribose metabolised
m s incorporated into serine in either light or dark* These results
are interpreted in greater detail in the Discussion part of this thesis.

Light

3.8it

1.12

2.$9

DISTMEUTIOH OF CM IH SERIHE

32

DISCUSSION

33

Hieoblm synthesized in tobacco plants which were fed B~rlbose-*
X^CJi4 tun radioactive. The methyl group obtained only a. small part
of tbs C14 of the nicotine molecule. About seven percent of theC14
m e In tbs methyl group of nicotine from pirate which. m m harvested
three and seven days after administration of the radioactive compound.
Warn the plants were harvested, two days after administration of the
ribose, the nicotine methyl group contained three percent of the C*4.
The shorter metabolism periods did not result in a large decrease in
the incorporation of the C94 into tbs nicotine molecule.
The transfer of methyl groups to nicotine in tobacco plants
appears to be a relatively slow metabolic reaction (2,65). lest© (65)
has observed that the radioactivity of nicotine increased linearly up
to five days When tobacco plants rare fed ms1^l~Cl4^tMenine. Ho
further increase In 014 content m s noted when the period for metabolism
m s sadended be three weeks. In contrast, the metabolic transformations
of pentoses in plants appear to be m&rmely rapid.

Calvin and co~

workers (28,29,39,66) have studied e**0m incorporated into the pentose
phosphates and related mietebolites. Changes in C14 concentration rare
detected after a period of leas than one second. It m s noted In the
present study that about twenty percent of the C14, from D-*rlbose-lH314
administered to plants, m s retired in 36 hours and a large part of

3U

the Ci* was incorporated into the insoluble cell wall substances. The
rapid rate and great varied of the metabolic reactions of pentoses
and their phosphate esters precludes the possibility that a large
quantity of the pentose would be utilized for methyl fproup synthesis •
Several pathways have been suggested for the formation of one
carbon units from pentose phosphates. The initial step in these postu­
lated metabolic pathways is the synthesis of a two or three carbon
compound which then is converted to glycine* serine or glycolic acid.
Glycine, serine and glycolic acid are interrelated in plant metabolism
and appear t© be synthesized from a common precursor (18,3?)* The
dilution of C14 when serine-3-G14 m s converted to nicotine in tobacco
plants was about 2000. The alpha carbons of glycine and glycolic acid
are ©©nverted into the nicotine methyl group to a similar extent.
Assuming ribose m s converted to glycine, serine, or glycolic acid an
additional large dilution of the C14 would be expected. Thus, the
dilution of C14 in converting D-ribose-l-C14 to the nicotine methyl
group would be 2000 times the dilution in converting ribos© to serine,
glycine and/or glycolic acid.
The C14 from D-ribos© appears to be incorporated into nicotine
at a rate greater than several other G14 labeled compounds which were
tested. Dewey (6?) and Lamberts (68) have studied incorporation of
ornithine and glutamic acid into the pyrrolidine ring of nicotine.
The C14 was not rapidly incorporated into nicotine when tobacco plants
were fed the C^-labeled amino acids under conditions similar to those
used in the present studies.

3S

The C14 content of nicotine from plants fed D-ribose-l-C14 was
similar in experiments in ifoich plants ’were allowed to metabolize two,
three, and seven days following the administration of labeled pentose.
These results Indicate a rapid and possibly specific synthesis of the
nicotine ring system. Since the rate of incorporation of C14 into the
pyrrolidine ring was slow when an immediate precursor, such as glutamic
acid, was fed, the C14 from ]>»ribose-l-C14 may have been incorporated
into the pyridine ring of the nicotine. Although these observations are
without experimental proof, the suggestions appear tenable.
Frontera-Aymat (35) administered pyruvic acid-3-C14 to tobacco
plants and found the nicotine to be radioactive. The methyl group
contained about six percent of the total activity of the nicotine.
Lamberts (68) has shown that glutamic acid-2-C14 is incorporated into
nicotine. About ten percent of the activity was in the pyridine ring.
All radioactive compounds administered to plants and incorporated
in the nicotine ring system were related to intermediates in glycolysis
or the tricarboxylic acid cycle. The results suggest that a compound
such as acetate or possibly a pentose derivative can be converted
rapidly into nicotine in the tobacco plant.
Tory little information is available concerning the biosynthesis
of the nicotine molecule. Ornithine and glutamic acid have been shown
to form the pyrrolidine ring of nicotine (6?,68). The synthesis of the
pyridine ring is still unsolved. The formation of nicotinic acid in
animals and bacteria has been shown to arise in part from tryptophan.
However, the formation of the pyridine ring from tryptophan catabolism

36

does not appear to be an important pathway in plant metabolism (69).
These observations may be of importance in the elucidation of the bio­
synthesis of the pyridine ring of nicotine.
Synthesis er Serine and Glycine from D-Rlbose in Detached leaves
Several metabolic pathways for serin© and glycine synthesis from
D-ribose-l-C14 are shown in Figure 1. The colored dots adjacent to the
carbon atoms denote the major forward metabolic pathway for these atoms.
Reactions which have not been sufficiently established are indicated bydotted lines.
The C14 distribution in serine and glycine synthesised from
D-ribose-l-G14 cannot be accounted for by any single known metabolic
pathway. The C14 distribution suggests that at least two metabolic
pathways are utilised for serine synthesis. On© possible explanation
of the data would be for a portion of the serine to be synthesized from
3-phosphoglyceric acid (reaction E, Figure 1). Glycine and additional
serine could be synthesized from glyoixylie acid produced in the
glymylate cycle (reaction F, Figure 1). Endogenous C14Qa is assumed
to be fixed primarily by condensation with phosphoenolpyruvic acid
(reaction D, Figure 1).
Glycine, glycolic acid, or glyo:xylic acid synthesized from D-ribosel-C14 would be labeled in the alpha carbon atoms. Synthesis of serine
and glycine from glycolic acid has been demonstrated in plants (18,37).
The C14 content of the beta carbon would be less than, or at best
equal to, the C*4 content of the alpha carbon of serine if it were

37

HOf*OH

0-0

H

O *HI

o
o

r *h
4

1

o-o
50

as

jo

4SJg g SviOd
0-0-00*0 M

c

8
S
0
0

Figure !♦ Biosynthesis of glycine and serine in plants

•H
§
O

38

Synthesized only from glycolic acid or glycine since dilution of the
one carbon unit would be expected (reaction C, Figure 1) (18). However,
partial randomization of the C14 in the alpha and beta carbons of
serine does indicate that serin© was synthesized primarily from & two
carbon precursor.
Glycine 3ms been shown to be synthesized from glycolic or glyoxylic
acids in plants (18,37)* The synthesis of glycine from serine, as m s
postulated to occur in animal metabolism (33), appears to be of minor
significance in tobacco plants. The specific activities of the carboxyl
and alpha carbons of glycine and serin© were not identical as would be
expected if glycine were synthesized from serine.
The two carbon precursor of glycine and serine is believed to be
produced in the glyoxylate cycle* This postulate is based on two lines
of reasoning. First, the C14 of the one carbon of ribose cannot b©
incorporated into the two, three, or four carbons of pentose phosphates
by known reactions of glycolysis or the photosynthetic cycle. Therefore,
C24 in the carboxyl carbon of glycine or serin© must originate from
endogenous G14Ga. The reaction of Ci403 and ribulos© diphosphate
(reaction B, Figure 1) would yield only 3-^phosphoglyceric acid-l-C24.
Serine synthesized from 3-phosphoglyceric acid would contain a greater
percentage of the C14 in the carboxyl carbon than glycine synthesized
from a two carbon precursor. If C1403 m s metabolized by fixation with
phosphoenolpyruvic acid, glyoxylic acid synthesized from it, would
contain C24 in the carboxyl carbon. Glycine and serine formed from

39

the glyexylie acid then would contain equal quantities of C14 in “
tie
carboxyl carbon.
The dilution of C*4 in the serin© carboxyl carbon probably occurred
by a direct conversion of 3-phosphoglyceric acid to serine. The fraction
of serine formed from 3-phosphoglyceric acid which is labeled only in
the beta carbon would increase the specific activity of the serine beta
carbon. The fixation reaction of carbon dioxide and phosphoesnolpyruvic
acid is firmly established in plant metabolism (70) and appears to be
particularly significant during dark carbon dioxide fixation (71)*
Secondly, the distribution of C14 in serine from the dark metabol­
ism of D-ribose-l-C14 also provides support for the postulate that the
two carbon precursor of glycine and serin© is synthesised in the giyoxylate cycle. The percentage of C14 in the beta carbon of serine was
greater and the C14 content of the carboxyl carbon m s less than the
corresponding carbons of serin© synthesised during light metabolism.
These data indicate that serin© synthesised from a two carbon precursor
contributes a smaller part of the total serin© synthesised. This result
is consistent with previous observations on light and dark metabolism.
State and Burris (72) and Benson and Galvin (21) noted that glycolic
acid disappeared from plant tissues when the plants were placed in the
dark. Other reports Indicate that glycine is not synthesised or synthe­
sised very slowly during dark metabolism in plants (71,73)* Glycine-C14
m s not detected when D-ribose-I-C14 was metabolised in the nonlllaminated leaves. Therefore, only a small quantity of serine could

ho

have been synthesized from a two carbon precursor* The C14 distribution
of serine formed in leaves during dark metabolism of D-ribose-l-C14
would be expected to contain C14 primarily in the beta carbon and only
a very small part of the C14 would appear in the carboxyl carbon*
It may be seen that data obtained from degradation of serine supports
this hypothesis*
Unfortunately, the specific activity of glycine and serine from
light metabolism could not be calculated since the total amounts of
glycine and serine in the intact plants was not determined* if the
specific activity of the carboxyl and alpha carbons of serine were
greater than the corresponding glycine carbon atoms the hypothesis that
serine was not synthesized exclusively from glycine or other two carbon
precursors would be established*
The mechanisms for glycine and serine synthesis postulated from
the results of this study are consistent with data reported from other
laboratories* Newburgh and Burris (3h) administered pyruvic acid-2-C14
to plants and isolated glycine containing essentially all of the C14
in the carboxyl carbon atom. Hhen plants were allowed to photosynthesis©
Ci40a for 15 and 50 seconds, C14 was incorporated into the carboxyl
carbon of serine and to a lesser extent into the alpha and beta carbons
of serine and both carbons of glycine (17). Nyc and Zabin (7U)
administered pyruvic acid-3-C14 to rats and the labeling distribution
in glycine and serine was similar to that found in this study. Data
from reports in which HC140CK, acetate-l-C14, acetate-2-G14 and

ill

pyruvic aeid~2-«Cl* were administered to plants, animals, or micro­
organisms also support this mechanism for glycine anil serine synthesis

the conversion of 3»*riboe©*l*C** to glycine labeled in the alpha
carbon and serine labeled in the beta carbon demonstrated on© pathway
by which methyl groups msy be synthesized in plants. It was stated
previously that glycin©-2*€14 (IS) and serine~3~Cx4 (lb) were metabolised
by plants and the 8** incorporated into the methyl group of nicotine.
It was suggested that the majority of glycine and serine was synthesised
from gtyoxylie acid tfeich was produced in the glyoxylate cycle.
Additional experiments should be performed to further establish the
biogenesis of these aadne acids. It weald be of interest to administer
radioactive isocitric acid to plants and study the labeling distribution
in glycine, serine and nicotine.

smmj

k2

m m m
1* Tobacco plants absorbed D-ritcse-l-C14 from mi aqueous mitrient
solution. About

20

percent of the c*4 m s respired as 0 **0 * during

a 36 hear growing period* Approximately one-third of the C14 m e
located in the mter- end 60 percent eilmnol-insolubl© constituents*
2* Nicotine isolated from tobacco plant# fed D-ribose-l-G14 m s radio­
active• the K-methyl group of nicotine contained only a small, part
of the C*4. the rat# of incorporation of G14 into the nicotine
molecule suggested that a specific incorporation of a glycolysis
intermediate into the pyridine ring occurred.
3* Tobacco plants leave# were vacuum infiltrated with D-ribose-C14
solution* fhe leave# which were allotted to metabolize in the dart:
synthesised serine-C14 hut no radioactive glycine* Ulnminated
leave# synthesized radioactive glycine and serine*
h* Degradation of glycine and serin© synthesized from D-ribose-l-C14
showed the Ci4 to be located primarily in the alpha and beta carbons
of serine and the alpha carbon of glycine. A mechanism for glycine
and serine %iege»©#i# in plants m e proposed and discussed.
5* The isolation of glycine and serine containing C*4 in the alpha
and beta carbons showed one possible pathway for synthesis of methyl
groups from the photoeynthetic cycle*

LITERATURE CITED

h3

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APPBRKDC

formula used In correcting the observed count to aero wnyplft
thickness mst
s

«■

V

G0

* H

f-rr

ufeer® Am * inajdJmra specific activity (counts/inimte/siilllmole)
0©

observed counts (counts/mtaite)

M « molecular weight of compound
¥ » vsighi of maple counted
b « fraction of
activity at the sample thickness
used (^)^btained from selfabsorption curve..
Sample calculation*

nicotine dlpicr&te *** C0 •« 1&U2 e«pa*t ¥ * 29.0 mg#.* H * 620
f * 10 .2 sag./cm.a, b * 0 J4*0.

A- * J ^ l h r l P L - 1*83 * J©3 e*P.m./milliaole
Efficiency of counting system «
Correction of specific activity to 100$s
JbS2wi|g^S m 1.96 ac 10’
* c .p.m./millimol©