ASPECTS OF NAB BIOSYNTHESIS
IN CASTOR BEAN ENDOSPERM

Thesis for the Degree of M. S. j

iv‘iICSéiGAN STATE SiNSVERSETY .

BQROTHY BOERNER MANN ‘
1973

 

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Michigan Stave
University

 

     
 

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ABSTRACT

ASPECTS OF NICOTINAMIDE ADENINE DINUCLEOTIDE BIOSYNTHESIS
IN CASTOR BEAN ENDOSPERM
By

Dorothy Boerner Mann

In addition to trigonelline and nicotinic acid, a third major
product, C, was synthesized by an enzyme preparation from etiolated
five-day-old castor bean endosperms in the presence of [7-]4C]

2+. [7-]4C] nicotinic

nicotinamide, adenosine 5'-triph05phate, and Mg
acid served equally well as a substrate for Product C formation. When
[8-]4C] adenosine 5'-triphosphate and nonradioactive nicotinic acid

were incubated with Mg2+

and a similar enzyme preparation, a radioactive
product identical to Product C was obtained.

Product C was identified as nicotinamide adenine dinucleotide by
its elution pattern from a Dowex l-X2 formate column, by its Rf in
three paper chromatographic systems, by its ultraviolet spectrum in
the absence and presence of potassium cyanide and in the absence and
presence of yeast alcohol dehydrogenase, and by its alkaline hydrolysis
to radioactive nicotinamide and nicotinic acid.

Reaction characteristics consistent with this identification

were dependence on adenosine 5'-triphosphate and MgZ+, loss of activity

after passage of the enzyme preparation through a Sephadex G—25 column,

Dorothy Boerner Mann

inhibition by nicotinamide adenine dinucleotide, and stimulation by
S-phosphoribosyl-l-pyrophosphate.

The identification of nicotinic acid mononucleotide and nicotinic
acid adenine dinucleotide as reaction products supports the operation

of the Preiss-Handler pathway in castor bean endosperm.

ASPECTS OF NAD BIOSYNTHESIS IN CASTOR BEAN ENDOSPERM

By

Dorothy Boerner Mann

A THESIS

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

MASTER OF SCIENCE

Department of Biochemistry

l973

DEDICATED

to the memory of my father, Ray
to my mother, Rosalie
and to my husband and colleague, David

ii

ACKNOWLEDGMENTS

I am grateful to Dr. Richard U. Byerrum for his guidance and
encouragement during the course of this research. His suggestions and
those of Drs. James L. Fairley and William W. Wells with regard to this
manuscript are appreciated. My deepest gratitude is extended to my
husband, David, whose help and tolerance contributed immeasurably to
the completion of both my research and this thesis. I also wish to

acknowledge the financial assistance from the National Institutes of

Health.

iii

TABLE OF CONTENTS

ACKNOWLEDGMENTS .

LIST OF TABLES

LIST OF FIGURES .

LIST OF ABBREVIATIONS .
LITERATURE REVIEW
MATERIALS AND METHODS .

Radioactive Chemicals

Nonradioactive Chemicals

Scintillation Fluids

Plants

Enzyme Preparation

Enzyme Assays . .

Paper Chromatography

Product C Purification .

Product Determination on Paper Chromatograms
Dowex l -X2 Formate Column Chromatography .

Product Determination on a Dowex l- X2 Formate Column :

Sephadex G- 25 Column Chromatography

Protein Determinations . . .

UV Spectra of Product C and NAD .

Hydrolysis Studies

Recrystallization to Constant Specific Activity

RESULTS AND DISCUSSION
REFERENCES

iv

vi

vii

Table

10.

ll.

12.

LIST OF TABLES

[7-]4C] Nam and [7-]4C] NA as Precursors of
Product C, Product D, and Trigonelline .

NAD Inhibition of Product C and Trigonelline
Synthesis . . . . . .

Requirements for the Synthesis of Product C,
Product D, and Trigonelline

Optimum ATP Concentration for Product C and
Trigonelline Synthesis

Absorption Maxima and Minima of Authentic NAD and
Product C in the Absence and Presence of KCN
and in the Absence and Presence of Yeast Alcohol
Dehydrogenase . . .

PRPP Requirement of QA Phosphoribosyltransferase .

Physiological Characteristics of Etiolated
Castor Bean Seedlings Germinated in Moist
Vermiculite at 30°C .

Co-chromatography of Fractions from the Dowex l
Formate Column in Figure 7 with Standards .

Recrystallization of [7-]4C] NA to Constant
Specific Activity . . . . . . .

Recrystallization of [8-]4C] Trigonelline to
Constant Specific Activity . .

Base Hydrolysis of Standards and of Fractions from the
Dowex l Formate Column of Figure 7

Effect of PRPP and Glutamine on Product C
Synthesis . .

Page

32

34

35

36

44

ST

55

59

6l

62

63

65

Figure

LIST OF FIGURES

NAD Biosynthesis

Time Course of Nam Utilization and NA Formation
(A) and Product C, Product D, and Trigonelline
Formation (B) . .

Separation of the Products from [7- 4C] Nam on
a Dowex l Formate Column .

Separation of the Products from [8-]4C] ATP on
a Dowex l Formate Column . . .

Absorption Spectra in the Absence and Presence
of KCN. (A) NAD; (B) Product C

Absorption Spectra in the Absence and Presence
of Yeast Alcohol Dehydrogenase. (A) NAD;
(8) Product C . . . . . .

Formation of Product C, Product D, and Trigo-
nelline During Germination .

Separation of the Products from [7-]4C] NA on
a Dowex l Formate Column . . .

vi

Page

3T

40

42

46

48

53

57

Nam
NA
QA
NaMN
NaAD
NAD
NADP
NADPH
NMn
NMN
PRPP
ATP
ADPR
FAD

Tris

PP
POPOP
PPO
BBOT

Captan

LIST OF ABBREVIATIONS

nicotinamide

nicotinic acid

quinolinic acid

nicotinic acid mononucleotide

nicotinic acid adenine dinucleotide

nicotinamide adenine dinucleotide (oxidized)
nicotinamide adenine dinucleotide phosphate (oxidized)
nicotinamide adenine dinucleotide phosphate (reduced)
nicotinamide mononucleoside

nicotinamide mononucleotide
S-phosphoribosyl-l-pyrophosphate

adenosine 5'-triph05phate

adenosine 5'-diphosphate ribose

flavin adenine dinucleotide
2-amino-2-(hydroxymethyl)-l,3-propanediol

inorganic phosphate

inorganic pyrophOSphate
l,4-bis-[2-(4-methyl—5-phenyloxazolyl)]-benzene
2,5-diphenyloxazole
2,5-bis-[2-(5-tgrtfbutylbenzoxazolyl)]-thiophene

N-[(trichloromethyl)thio]-4-cyclohexene-l,2-dicarbox-
imide

vii

ADH yeast alcohol dehydrogenase
UV ultraviolet

i.d. internal diameter

viii

LITERATURE REVIEW

Depending on the organism or tissue, the biosynthesis of NAD
proceeds by one of three pathways, or by an integration of these
pathways, as depicted in Figure l. The elucidation of these pathways
and their identification in various tissues, the preferential
utilization of one pathway versus another, and the possible control
mechanisms will be discussed.

The discovery by Kornberg (l) in l948 of NMN adenyltransferase
provided some indication of the route by which NAD was synthesized.
This enzyme, purified from autolysates of ale yeast and extracts of
hog liver acetone powder, catalyzed the reversible synthesis of NAD and
PPi from NMN and ATP; Mg2+ was required for activity (2). Since the
cleavage of NAD by PPi resembled phosphorolysis, Kornberg suggested
that by analogy the term "pyrophOSphorolysis" could be used to describe
the reaction. Thus this enzyme was originally named NAD pyrophosphory-
lase. This terminology was adopted in the earlier papers for naming
some of the enzymes involved in NAD biosynthesis, even though the
direction of the reaction was not that of pyrophosphorolytic cleavage
(3). Experiments with 32P32Pi provided evidence in support of the
pyrophosphorolytic action of this enzyme: the radioactive ATP formed
from NAD and 32P32Pi had the same specific activity as the 32P32Pi;
the NAD and NMN recovered were free of radioactivity; and the radio-

activity in the ATP was located exclusively in the two terminal

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phosphates (4). Differential centrifugation of mouse liver homogenates
resulted in the recovery of 69 to lOl percent of NMN adenyltransferase
activity in the nucleus (5). The lower recoveries were obtained under
conditions known to damage nuclei. When nuclei were intentionally
disrupted by sonic oscillation, most of the enzyme activity remained
unsedimented, even after centrifugation at 130,000xg.

Using hemolysates and acetone powder extracts of human
erythrocytes, Preiss and Handler (6) established that Nam and PRPP
reacted in the presence of Mg2+ to form NMN and PPi; however, the high
Km for Nam, in the order of 0.l M, temporarily excluded this enzyme,
Nam phosphoribosyltransferase (NMN pyrophosphorylase), from the realm
of physiological significance. It was later detected in acetone
powder extracts of Ehrlich ascites cells from mice, but a high
concentration of Nam, 0.l M, was again required for full activity (7).
An enzyme catalyzing the same reaction but with a specific requirement
for ATP was isolated by Dietrich gt_al, (8) from Ehrlich-Lettre ascites
cells grown in mice. These workers reported a Km for Nam of 1.6 x
lo"5 M. Attempts to isolate this enzyme with a similar high-affinity
for Nam from rat liver failed until it was realized that the enzyme
was in an inhibited state in the crude homogenate (9). A protamine
sufate precipitation step in the purification procedure resulted in a
large increase both in specific activity and percent recovery; it was
assumed that the protamine sulfate had removed an inhibitor of Nam
phosphoribosyltransferase. The purified enzyme exhibited a specific
requirement of ATP; its Km for Nam was 2.96 x 10-6 M. In contrast to
NMN adenyltransferase, this enzyme appeared to be restricted to the

cytoplasm. Thus one biosynthetic pathway from Nam + NMN + NAD had

been established (Figure 1). To date, detection of the Dietrich
pathway has been limited to mammalian systems and bacteria (21,15).
Although Preiss and Handler (6) had observed Nam phosphoribosyl-
transferase activity in human erythrocytes, there were several
indications that a pathway other than the one described above was
operative.. They had shown that at low concentrations of NA, but not of
Nam, isolated erythrocytes could synthesize appreciable amounts of NAD,
even though the cells were freely permeable to both substrates (10).
The previously mentioned high Km for Nam exhibited by Nam phospho-
ribosyltransferase and the very low NMN adenyltransferase activity
found in human erythrocytes further suggested an alternate pathway (6).

[14C] NA, glutamine, and

After incubation of human erythrocytes with
glucose, Preiss and Handler (11) were able to detect two NA derivatives
besides NAD. In two classic papers they described the identification
of these intermediates (12) and the characterization of the enzymatic
reactions involved (13). They were able to detect the two inter-
mediates both with defibrinated human erythrocytes and with acetone
powder extracts. These compounds, I and II, were identified as NaMN
and NaAD respectively by paper electrophoresis, acid and alkaline
hydrolysis, as well as a determination of the molar ratio of NA,
ribose, and phosphate. In addition, compound II was converted by
purified NAD synthetase to NAD at the same rate as synthetic NaAD.

Both compounds, I and II, were inactive with alcohol dehydrogenase.

An ig_ylyg time course showed that NaAD was formed before NAD in the

rat. They concluded that NAD synthesis had proceeded via NA + NaMN +
NaAD + NAD (Figure l).

The enzyme responsible for the first step, NA phosphoribosy1-
transferase (NaMN pyrophosphorylase) was partially purified from
erythrocyte acetone powders and yeast autolysates. It catalyzed the
reaction of NA and PRPP to form NaMN and PPi. The yeast enzyme
exhibited an absolute requirement for ATP, even in the presence of
PRPP, whereas the erythrocyte enzyme did not. Synthesis of NaAD and
PPi from NaMN and ATP by NaMN adenyltransferase (NaAD pyr0ph05phory-
lase) was observed with partially purified enzymes from erythrocyte
acetone powders, yeast autolysates, and hog liver. The data indicated
that NA phosphoribosyltransferase was an enzyme entirely distinct
from Nam phosphoribosy1transferase; however, the close association of
NaMN adenyltransferase and NMN adenyltransferase activities during the
purification from hog liver as well as similar distribution of the two
enzymes in subcellular fractions of rat liver suggested that they were
one and the same enzyme catalyzing two analogous reactions. This

latter conclusion was confirmed in yeast and E, coli by Dahmen gt_al,

 

(14), although adenyltransferases Specific for NMN or NaMN were later

found in Lactobacilli (15). The last step in the scheme proposed by

 

Preiss and Handler was the reaction catalyzed by NAD synthetase,
which they partially purified from rat liver and yeast autolysates.
Stoichiometric relationships indicated that NaAD, ATP, and glutamine
had reacted to form NAD, AMP, PPi, and glutamate. The Preiss-Handler
pathway has been demonstrated in microorganisms (16,17), plants (18),
and other mammalian tissues (20-22).

It had been shown earlier by Porcellati (23) that homogenates of
rat kidney, liver, brain, heart, intestine, and muscle could synthesize

Nam from NA in the presence of ATP. Preiss and Handler (12), too,

7

obtained [14C] Nam from [14C] NA in their jn_yiy9_studies with rats;
the labeled Nam did not appear, however, until two hours after the
[14C] NA administration. They felt that the Nam had originated from
NAD degradation. Further light on the mechanism involved was shed

by the work of Langan et_al, (24), who isolated and identified [14C]
NaAD from the livers of mice given injections of [14C] Nam. These
results suggested that Nam had been hydrolyzed to NA before it

became available for NAD synthesis. A cyclic scheme for the degrada-
tion and resynthesis of NAD was first proposed by Sarma §t_§l, (19).
Their scheme incorporated two possible routes of NAD degradation.

The one involved breakdown of NAD to NMN and then to free Nam,
followed by deamidation to NA, which could then be reconverted to NAD.
The other degradative pathway was implicated by their demonstration of
the enzymatic deamidation of NMN by mouse liver homogenates which did
not possess any Nam deamidase activity. Thus NaMN could be formed
directly from NMN, a process involving one enzyme reaction instead of
three. This latter more economical cycle was also very recently
proposed by Friedmann and Garstki (25), who partially purified a

specific NMN deamidase from Propionibacterium shermanii. Joshi and

 

Handler (26) are also credited with having proposed a salvage
pathway for reutilization of Nam which arose from NAD. The degradative
route in their scheme was the action of NAD glycohydrolase to yield
Nam directly from NAD.

The enzymatic reactions responsible for the two types of
degradation to Nam had been detected much earlier. Direct degradation
to Nam was first observed in 1942 by Handler and Klein (27), who

recovered Nam quantitatively from NAD destruction in brain, liver,

kidney, and muscle preparations from rabbits, rats, and dogs. Kornberg
and Lindberg (28) later observed the direct degradation to Nam as well
as degradation to Nam via NMN. They cited the cleavage of NAD at the
pyrophosphate bond by NAD pyrophosphatase as the predominant

mechanism in rabbit kidney; however, they attributed the splitting of
NAD in rabbit brain to the activity of NAD glycohydrolase, which
cleaves at the glycosidic bond between Nam and ADPR. Both the
glycohydrolase and the pyrophosphatase have also been detected in
plants (29,30) and microorganisms (31,32).

The enzyme that completed (or initiated) the proposed pyridine
nucleotide cycle, Nam deamidase, was first observed by Hughes and
Williamson (33) in Lactobacillus arabinosus. Investigators were able
to demonstrate Nam deamidase activity in other microorganisms
(26,34), plants (35,36), and birds (19), but not in mammals. In
addition, Nam served as a better precursor of NAD in mouse liver than
NA (37) and it appeared to be the primary dietary and circulatory
form of the vitamin (38,39). These objections to the Preiss-Handler
pathway were eventually overcome to some degree by Petrack g__gl,
(40), who were able to detect a rat liver microsomal Nam deamidase
which, they believed, had sufficient activity to account for the
calculated maximum rate of 1Q_yiyg_NAD synthesis. It must be pointed
out that the Km for Naniwas still relatively high (7 x 10'2 M), even
after the removal of a lipid inhibitor during the purification (41,42).
Kirchner g__gl, (43) found that in rabbits most of the Nam deamidase
activity resided in the liver. In fact, the second most active
tissue, small intestine, was only one-fifth as active as liver. The

Km reported for their deamidase was also high (4 x 10'2 M). In a

comparative in vivo study of the utilization of Nam and NA as

 

precursors of NAD in mouse liver, Ijichi §t_al, (44) found that NA,

if administered in small doses, was the better precursor; if large
doses were used, however, Nam served as the more efficient precursor.
A time course in which a large dose of [14C] Nam was administered
revealed that radioactivity in the liver disappeared rapidly and did
not begin to increase until one to two hours after injection. The
decrease in radioactivity indicated excretion of [14C] Nam from the
liver. The eventual increase in radioactivity in the liver paralleled
the increase of [14C] NAD. These workers eXplained their data by the
assumption that deamidation had occurred in the gastrointestianl tract
before reabsorption by the liver. By investigating Nam deamidase in
the stomachs of normal rats and germ-free rats, Tanigawa gt_al, (45)
demonstrated Nam deamidase activity in the pars preventricularis of
normal rats and isolated the responsible bacillus; no activity was
detected in the germ-free rats. Since they had also shown that NA was
absorbed by the small intestine, they felt it very probable that Nam
in the diet might be deamidated by microorganisms in the stomach and
then be absorbed by the small intestine to serve as a precursor in the
Preiss-Handler pathway in various tissues.

The importance of the Preiss—Handler pathway in the liver was
eventually realized; evidence accumulated, however, that extrahepatic
tissues might not employ this route of NAD biosynthesis. The data of
Ikeda gt_al, (46) indicated high activity for NA phosphoribosyl-
transferase in rat liver, moderate activity in the kidney, and low
activity in eight other tissues examined. As previously mentioned,

an alternate pathway, described by Dietrich §t_al: (8), had been

10

demonstrated in a variety of tissues. Further evidence in favor of
this latter pathway came from Greenbaum and Pinder (47), who, working
with rat mammary gland extracts, reported Km values which were low for

5 M and 1.5 x 10‘4 M respectively). They

both Nam and NMN (3.5 x 10-
also showed that there was a time lag in the ig_yiyg incorporation of
Nam into liver NAD; no such lag occurred, however, in mammary gland.
The in_yiyg_study undertaken by Collins and Chaykin (21) on the
management of Nam and NA in the mouse did much to clarify the integra-
tion of the Preiss—Handler and Dietrich pathways. After [14C] NA
injections it became evident that the Preiss-Handler pathway was
Operating in the liver. But the dramatic aspect of the results in

[14c] NA

liver was an extensive accumulation of [14C] Nam following
injection. The time course data indicated that the labeled Nam was
released by NAD which had been synthesized from NA. Similar results
were obtained in the intestine. In the spleen, NAD synthesis
correlated with Nam rather than NaAD availability; nevertheless, high
levels of NaAD accumulated and were maintained. This phenomenon was
even more exaggerated in skeletal muscle. Essentially no NAD synthesis
occurred during the time in which NaAD levels were high. Furthermore,
once the NaAD level decreased, only twenty-five percent of the decrease
could be accounted for. It was clear that radioactivity had left the
muscle, and it was assumed that it had exited as NA. These workers
hypothesized that the infusion of NA from the peripheral tissues was
responsible for the prolonged formation of Nam which had occurred

in the liver. Other tissues examined (kidney, ovary, lung, heart,

and brain) contained more radioactive NAD after Nam injections than

after NA injection. A lag was also observed in the appearance of label

11

in NAD in these organs following injection of NA but not Nam. These
observations were compatible with the idea that the role of the

liver was to convert NA to Nam so that it could be used in the
peripheral tissues for NAD synthesis via the Dietrich pathway. It is
difficult to correlate with this hypothesis the data of Deguchi et_al,
(20), which indicated preference for the Preiss-Handler pathway in rat
brain, and that of Lin and Henderson (22) which dubbed NA the more
effective precursor of NAD in perfused rat kidney; the latter group
did obtain compatible data for rat testis (22), liver (48), and blood
(49), however.

In the experiments of Collins and Chaykin (21) just described,
physiological levels of Nam were administered, yet no evidence for
deamidation appeared. Previous studies of Ijichi et_al, (44) and
Langan gt_al, (24) which indicated deamidation ig_yiyg, had been
carried out with nonphysiological levels of Nam. By increasing the
Nam dosage lOO-fold, Collins and Chaykin (21) were able to show that
some deamidation did occur in the intestine, possibly due to bacterial
action, and that deamidation also occurred in the liver. This was
reasonable, since after administration of such a large dose of Nam,
the Nam concentration in the liver could approach the Km of the
deamidase.

The plant picture is not so complicated, perhaps because the
research is not yet as extensive. No one has demonstrated the
Dietrich pathway in plants. It seems that the Preiss-Handler pathway
is of greater importance, a fact which would correlate with the active
Nam deamidase in plants. Although they did not identify any Preiss-

Handler intermediates, Waller gt_al, (50) did demonstrate jg_vivo

12

conversion of Nam to NA, in_vitro and ifl_VlVO conversion of NA to NAD,

 

and 1g_yiyg conversion of NAD to Nam in castor bean seedlings. A
complete pyridine nucleotide cycle via the Preiss-Handler pathway was
later demonstrated in_yiyg_by Godavari and Waygood (18) in detached
wheat leaves and by Ryrie and Scott (35) in detached barley leaves.
These latter workers confirmed Nam deamidase activity in_yitrg.

A logical question at this point is the origin of the Nam which
is utilized in the Dietrich pathway or which is converted to NA for
utilization in the Preiss-Handler pathway. The answer to that question
lies in either the diet or one of the ge_ggyg_pathways. The only
well-defined gg_ngyg_pathway is that from tryptophan (Figure 1). Its
discovery dates to the early treatment of pellagra in humans and
black tongue in dogs, both now established as vitamin deficiency
diseases (51). In 1921 Tanner (52) treated one of his pellagrous
patients with tryptophan and observed: ... the improvement in this
patient's skin condition has surpassed anything I have ever seen in a
case of pellagra in an equal period of time. The biosynthetic
relationship between tryptophan and NA became apparent when Krehl (53)
demonstrated that NA could be replaced by tryptophan in the diet of the
rat. Kinurenine, 3-hydroxykinurenine, and 3-hydroxyanthranilic acid
were eventually established as intermediates in the tryptophan
pathway (54); however, conversion of the latter compound to NA
remained obscure. Ifl_yjtrg_studies by Priest et_al. (55) had shown
that 3-hydroxyanthranilic acid could be converted to GA. Picolinic
acid was the only other product that could be derived enzymatically
from 3-hydroxyanthranilic acid (56). But neither of these two acids

served as an in vitro precursor of NA. The mechanism was finally

l3

elucidated by Nishizuka and Hayaishi (57), who demonstrated that

enzyne preparations from rat liver and kidney catalyzed the conversion
of 3-hydroxyanthrani1ic acid to NaMN in a PRPP dependent reaction.
Prior investigations had failed because microorganisms used in the
bioassay for NA were unable to use the actual product, NaMN, as a
growth factor (56).

Besides mammals, the tryptophan pathway was demonstrated in

Neurospora (58) and yeast (59). The latter organism was unique in that

 

it could utilize the tryptophan pathway under aerobic conditions, but
under anaerobic conditions it exhibited a pathway similar to that found
in bacteria, as described below.

No isotopic evidence could be obtained for the operation of the
tryptophan pathway in plants (60) or bacteria (61), except for the
aerobe Xanthomonas pruni (62). Alternate routes were sought by radio-
active feeding techniques and eventually by ig_yitrg_studies with

radioactive precursors. The first ig_vivo evidence for the identity

 

of NA precursors in E, cglj_came from the work of Ortega and Brown (63),
who demonstrated that the carbon chains of [14C] glycerol and [14C]
succinic acid were efficiently incorporated into NA. Because of the
labeling patterns obtained, they concluded that the carbon skeleton of
NA was probably derived from a three-carbon compound, such as glycerol,
and a four-carbon dicarboxylic acid, so that one of the carboxyl

groups of the dicarboxylic acid would become the carboxyl group of NA.

The results of i vivo experiments with the aerobes Mycobacterium

 

 

tuberculosis (64) and Serratia marcescens (65) were consistent with

 

 

this hypothesis. Crude extracts of the anaerobe Clostridium

 

butylicum were able to use glycerol and aspartic acid as precursors

14

of NA; after fractionation of the extract, however, aspartate, acetyl
CoA, and formate were required (66). More recent work demonstrated
that N-formyl aspartate was probably a more immediate precursor (67).
Consistent data were obtained with anaerobically grown E, 2211’
which incorporated [1,3-14C] glycerol into NA in a tetrahydrofolate
dependent mechanism (68)(Figure 1).

An E. coli mutant which lacked QA phosphoribosyltransferase was

 

conveniently used by Chandler and Gholson (69-72) to obtain an jg_
yiEEQ_QA synthetase system. They showed that glycerol could be
incorporated into QA when ATP was present, but in the absence of ATP,
glycerol was only one sixth to one-third as effective a precursor as
fructose§l,6-diph05phate or glyceraldehyde—3-phosphate (71). It was
later found that dihydroxyacetone phosphate was two to three times
more efficient than glyceraldehyde-3-phosphate (72)(Figure l). FAD was
a required cofactor, which was reasonable in view of the possible
intermediates which had been proposed by Fleeker and Byerrum (73).
The plant work took advantage of the alkaloids which contained
a pyridine or pyridone ring derived from NA or QA (74,75). It had
already been established that the conversion of QA to NaMN occurred
in microorganisms (76) and plants (74) which did not utilize
tryptOphan. Thus if a three- or four—carbon compound could be
incorporated into a pyridine alkaloid such as ricinine or nicotine,
then that compound could most likely be considered a precursor of QA
and therefore of NAD. Jackanicz and Byerrum (77) demonstrated that

carbon 3 of aspartic acid could form positions 2 and 3 of the

 

pyridine ring of nicotine in Nicotiana rustica. Further experiments by

Flecker and Byerrum (73,78) indicated that positions 4, 5, and 6 were

15

derived from glycerol or glyceraldehyde. They proposed that a
non-symmetrical three-carbon compound condensed with aspartic acid

to form QA (73). These results were consistent with those of Yang and
Waller (79), who studied the biosynthesis of the pyridone ring of

ricinine in Ricinus communis (Figure l).

 

Chaykin (56) has summarized the integration of these three major
routes of NAD biosynthesis. In microorganisms it seems likely that
Nam or NA would be the preferred precursor of NAD. If they are absent
and if tryptOphan is present in sufficient amounts, then the gg_g9yg
pathway via tryptophan would be utilized. If neither Nam nor NA is
available, and if the microorganism is one in which the tryptophan
pathway is inoperative, an alternate gg_ggyg pathway would be resorted
to. This theory was supported by experiments which indicated a
repression of the gg_ggy9_pathway in the presence of excess NA (80).

He feels further that the principle of conservation of biosynthetic
activity would apply in mammals as well. Thus the tryptophan pathway
would operate only if the amount of Nam or NA were insufficient to
maintain adequate pyridine nucleotide levels in the cell.

The gg_ggyg_pathway is regarded by Chaykin as the probable
ultimate source of NAD in plants, since plants do not exhibit
nutritional requirements for complex organic molecules. The pyridine
nucleotide cycle via the Preiss-Handler pathway would then operate as
a salvage cycle for reutilization of Nam from nucleotide breakdown.

As would be expected, NAD biosynthesis is subject to control.
Feedback inhibition, in which the end product of a metabolic sequence
inhibits the enzyme catalyzing the first committed step in its

formation, has been documented for NAD biosynthesis via the pyridine

l6

nucleotide cycle (Preiss-Handler pathway), the Dietrich pathway, and

the dg_ngyg_pathways from aspartic acid-glycerol and tryptophan.

Calbreath and Joshi (81) showed that yeast Nam deamidase, the enzyme

catalyzing the first committed step in the pyridine nucleotide cycle,

was inhibited by NAD. The inhibition was competitive, with an

apparent Ki of 7 x 10'4 M. They demonstrated that NAD was an effective

inhibitor of Nam deamidase in several other microorganisms as well.
Both NAD and NADP were shown by Dietrich and Muniz (82) to

inhibit the activity of purified preparations of rat liver Nam

phosphoribosyltransferase. Fifty percent inhibition occurred at

5 x 10’4

M NAD or NADP. These workers later reported that the
inhibition was non—competitive (83).

The DA synthetase system of Chandler and Gholson (71) was also
blocked by NAD. At 3.33 mM NAD, 98 percent inhibition was obtained;
however, slight inhibition could be achieved at 0.5 mM NAD.

Tryptophan pyrrolase, the first enzyme in the tryptophan pathway,
converts tryptophan to formylkinurenine (84). Its inhibition by NAD
was demonstrated both ifl_yiE§g_and ig_yiyg, Wagner (85), using a
partially purified preparation from rat liver, demonstrated that NAD
was a competitive inhibitor of tryptophan pyrrolase; forty-two percent
inhibition was attained with 5 mM NAD. Because this enzyme is under
hormonal control, Yamaguchi gE_gl, (86) found it necessary to use
hypophysectomized animals in their ifl_yiyg_experiments. Nam
administration evoked increased tryptophan pyrrolase activity in
normal rats, but not in the hypophysectomized ones. Furthermore, it

had been reported that NAD levels reached in the livers of hypophy-

sectomized rats in response to Nam injection were twice those of

17

normal rats (87). Thus they felt that if NAD did inhibit tryptophan
pyrrolase 1fl_yiyg, the effect would be very pronounced in hypophy-
sectomized rats injected with Nam. The expected inhibition resulted.
Measurement of actual hepatic NAD concentrations proved that the
tryptophan pyrrolase activity was inversely related to NAD concentra-
tion, rather than to some other effect of Nam administration. Experi-
ments by Cho-Chung and Pitot (88) with highly purified tryptophan
pyrrolase resulted in data slightly different from that of Wagner (85).
Using the same concentrations of tryptophan (0.4 mM) and NAD (5 MM),
Wagner (85) had obtained 42 percent inhibition whereas these workers
obtained 33 percent inhibition. They showed further that at this
tryptophan concentration, NADPH was an extremely potent inhibitor.

The percent inhibition ranged from 52 percent at 0.1 mM NAD to 95
percent at 1.0 mM NAD.

Besides the pituitary gland, both the adrenal glands and the
pancreas have been implicated in the regulation of tryptophan pyrrolase.
The lowering of tryptophan pyrrolase activity by adrenalectomy was
shown by Knox and Auerbach (89). They showed further that treatment
with cortisone, a hormone secreted by the adrenal cortex, could
increase tryptophan pyrrolase activity in normal as well as adrenal-
ectomized rats. On the other hand, Frieden eE_al, (90) later
demonstrated ig_yiErg_that epinephrine, a hormone secreted by the
adrenal medulla, was a potent non-competitive inhibitor of trypt0phan
pyrrolase. At 3 x 10'5 M epinephrine, 71 percent inhibition was
achieved. The previously mentioned lack of induction of tryptophan
pyrrolase in hypophysectomized rats following Nam injection was also

demonstrated in adrenalectomized rats (86,91). Schlor and Frieden (92)

18

showed that insulin increased tryptophan pyrrolase activity three-
to four-fold in normal rats and four- to five-fold in rats fasted for
48 hours prior to injection.

In the discussion of rat liver Nam deamidase, it was mentioned
that removal of a lipid inhibitor during purification lowered the Km
of the enzyme (41,42). This inhibition was subsequently related to
hormonal action as follows. The increased NAD levels in response to
Nam injection in hypophysectomized rats were attributed in part to
higher Nam deamidase activity (93). That this effect was due to a
reduction of the concentration of the inhibitory material was
demonstrated by the fact that the activity of purified Nam deamidase
was twice as great in the presence of crude liver homogenate from
hypophysectomized rats as it was in the presence of homogenates from
normal rats. Furthermore, the Km for Nam of liver homogenates from
hypophysectomized rats was shown to be one-fourth the Km determined
with normal rats. The inhibitor was eventually identified as a
mixture of free fatty acids, which consisted primarily of arachidonic,
linoleic, and oleic acids (42). This composition of the inhibitor
correlated well with the decrease in free fatty acid content known
to occur following hypophysectomy (42).

Chaykin (56) has integrated these two types of control for
mammalian systems with the suggestion that the feedback inhibition
could provide an intracellular control whereas the hormonal effects
could serve as intercellular controls.

The interplay of honmones and NAD biosynthesis was also demonstra-
ted in plants. Waygood gE_gl, (94) showed that chlorosis in floating

3

Elodea leaves was accelerated by NAD at l x 10' M; this effect was

l9

overcome by 5 x 10'4 M benzimidazole, a synthetic analog of kinetin.
NA at 5 x 10-3 M was also a potent chlorotic agent; its effect was
overcome by 4 x 10-4 M benzimidazole as well as by l x 10'4 M kinetin.
This acceleration of chlorosis by NAD and NA was shown to be light
mediated. In wheat leaves, benzimidazole treatment increased the
total Nam nucleotide content by 20 percent after one day (16 hour
phot0period); kinetin effected a 50 percent increase in the same
period. Treatment with the two compounds together resulted in an 80
percent increase. These workers showed further that the incorporation
of [14C] NA and [14C] Nam into NaAD and NADP was greater in leaves
floated on benzimidazole solutions than in leaves floated on water.
There was no apparent effect of benzimidazole on the incorporation of
label into NAD.

Three branches (Figure 1) off the main route of the pyridine
nucleotide cycle are relevant to this thesis. The first is the
reaction of NAD kinase to yield NADP and ADP from NAD and ATP. NADP
formation according to this reaction has been established in fungi (95),
bacteria (96), plants (97), and avian (98) and mammalian (99) tissues.

The second and third offshoots from the cycle are two analogous
reactions, the formation of N-methylnicotinamide from Nam and the
formation of N-methylnicotinic acid (trigonelline) from NA. The
former derivative has long been known as a urinary metabolite,
apparently excreted by carnivores and omnivores, rather than by
herbivores (100,101). In 1951 Cantoni (102) obtained a cell-free
preparation from rat liver which catalyzed the synthesis of N-methyl-
nicotinamide from methionine and Nam in the presence of Mg2+ and ATP.

The ATP requirement is now understood in light of S-adenosylmethionine,

20

the actual methyl donor (103). N-methylnicotinamide formation was

also reported in castor bean sedlings by Waller gE_gl, (50) and in
bean, wheat, and corn seedlings by Stul'nikova (104). It is difficult
to reconcile these results with those obtained in this laboratory (105)
and those reported by Joshi and Handler (106). It was our experience
that in castor bean seedlings Nam was rapidly and completely

deamidated to NA, which was then converted to appreciable amounts of
trigonelline, not N-methylnicotinamide. Waller £3.21: (50) based

their identification of N—methylnicotinamide only on paper chroma-
tography and did not state the degree of separation of trigonelline

and N-methylnicotinamide by the solvent systems employed. Joshi and
Handler (106) reported that pea extracts were able to synthesize
trigonelline, but not N-methylnicotinamide. They demonstrated that
S-adenosylmethionine was also the active donor in this methylation
process. Either technique of species difference (for example, in the
deamidase activity) could account for the conflict of results presented
here. The biosynthetic relationship between NA and trigonelline was
further indicated by an jn_yiyg_time course performed in wheat by
Godavari and Waygood (18). These workers ascribed to trigonelline the
role of detoxification of NA. It has also been shown that trigonelline

can be used as a source of NA for NAD biosynthesis in Torula cremoris

 

(107). This methyl derivative, first detected in the herb Triggnella

 

foenumsgraecum (108), has been isolated from several plant species

 

(109); it is not a metabolic product of NA in mammals (110).

21

It was of interest in this laboratory to investigate the
synthesis of N-methylnicotinamide in plants. The person assigned to
this project, Anne Bosch* (112) found that an enzyme preparation from
etiolated four-day-old castor bean seedlings was able to synthesize
labeled nicotinic acid, trigonelline, and an unidentified compound,

2+. The NA and

Product C, when incubated with [14C] Nam, ATP, and Mg
trigonelline were identified by their pattern of elution from a
Dower l-X2 (formate) column and by paper chromatography; the former
was also recrystallized with nonradioactive standard NA to constant
specific activity. No N-methylnicotinamide formation could be
detected, even when S-adenosylmethionine was added to the reaction
mixture. The synthesis of Product C was dependent on ATP as well as on
some other compound(s). This was indicated by the fact that passage
of the enzyme preparation through a Sephadex G-25 column resulted in
loss of activity. Nam was the only compound tested which could serve
as a substrate in the reaction.

The research presented here was performed in order to establish
the identity of Product C and to characterize the enzymatic reaction(s)
involved. The problem was approached with the hope that Product C

might be some heretofore unknown metabolite of Nam; however, related

pyridine compounds known to occur naturally were also kept in mind.

A

*Participant in the NSF undergraduate research program,
Summer, 1971 .

MATERIALS AND METHODS

Radioactive Chemicals

 

[7-]4C] Nam and [7-]4C] NA were purchased from New England Nuclear
and [8-]4CJ ATP and [6-]4C] QA were purchased from Amersham/Searle
Corporation. [6—]4C] NaMN was synthesized enzymatically from [6-]4C]
QA according to the procedure described by Mann (185). The purity of

all radioactive chemicals was checked in at least two solvent systems.

Nonradioactive Chemicals

 

The nonradioactive chemicals used in this work were purchased as
follows: Nam from Nutritional Biochemicals Corporation; NA from
Aldrich Chemical Company; NMN and NADP from P-L Biochemicals, Inc.;
PRPP (sodium salt), QA, dithioerythritol, and yeast alcohol dehydro-
genase from Sigma Chemical Company; NAD and ATP (sodium salt) from the
latter two companies; Dowex l-X2 (200-400 mesh, chloride form) from
Dow Chemical Company; Sephadex 6-25 from Pharmacia Fine Chemicals,
Inc.; POPOP, PPO, and BBOT from Packard Instrument Company, Inc.; and
Orthocide, 50% Captan, from a local vendor. The author is grateful
to Dr. Richard Hiles for the synthesis of NaAD and to Dr. Thomas
Griffith for the synthesis of trigonelline-HCl. All other chemicals

were reagent grade.

Scintillation Fluids

 

(I) Bray's scintillation fluid was prepared by adding 289.8 g

naphthalene, 28.98 g PPO, and 0.7225 g POPOP to 3.0 kg p-dioxane. (II)
‘ 22

23

This scintillation fluid was prepared by adding 15.16 g BBOT to 3.79

l toluene.

Plants

Etiolated castor bean seedlings, Ricinus communis L., variety Hale,

 

were used for the studies in this thesis. The seeds were the generous
gift of Mr. Walter Domingo, Director of the Oil Seeds Production
Division of the Baker Castor Oil Company, La Mesa, California. After
being washed with distilled water, the seeds were treated with a 10%
solution of Orthocide for 10 min, drained, and planted in moist
vermiculite in aluminum pans, which were then covered tightly with
aluminum foil. Distilled water was used to water the beans except
for the age studies, when tap water was used. On Day 5 the seedlings
were harvested, and only those seedlings which possessed the same
physiological characteristics were used for a given experiment. In
general, the endosperm was firm and weighed between 0.5 and 0.6 g,
the cotyledons were yellow with no pink or only a trace of pink, and

the secondary roots were slightly developed.

Enzyme Preparation

 

The endosperm of each seedling harvested was split longitudinally
with a razor blade so that the cotyledons could be discarded. The
endosperms were placed in an ice bucket, rinsed with distilled water,
blotted dry with paper towels, and weighed. The remaining procedures
were carried out at 4°C. The tissue was ground to a slurry in a
mortar with 2 volumes of 0.05 M potassium phosphate, pH 7.0, containing
0.01 M dithioerythritol. This extract was then squeezed through 4
layers of cheesecloth and centrifuged at 27,000xg for 15 min. The

24

supernatant fraction was used for all the enzyme studies.

Enzyme Assays

 

The standard assay for Product C formation contained the following
reagents at the final concentrations given in a total volume of 150 pl:
66.7 mM potassium phOSphate, pH 7.0; 6.7 mM dithioerythritol; 4.0 mM

ATP; 13.3 mM MgCl 0.10 mM [7-‘4c1 Nam or [7-14c] NA; and 100 pl

2.
(1.5 mg protein) enzyme preparation. There was some variation from
this standard assay, in the earlier experiments. Such deviations as
well as the specific activities of the substrates will be stated for
each experiment in the results section. The reaction mixture was
generally incubated with shaking for 3 hr at room temperature and the
reaction was stopped by heating the test tube in a boiling water

bath for 2 min. After removal of the protein by centrifugation in a
clinical centrifuge, 10 pl was spotted on Whatman No. 1 paper and
developed by descending paper chromatography, usually in Solvent
System I.

NA phosphoribosyltransferase and 0A phosphoribosyltransferase

were assayed as described by Mann (105).

Paper Chromatography

 

Descending chromatography on Whatman No. 1 paper was performed
with four solvent systems: (I) 1-butanol-glacial acetic acid-water
(4—1-2, v/v); (II) 1 M ammonium acetate, pH 5.0, -95% (v/v) aq.
ethanol (3-7, v/v); (III) 60% (v/v) aq. 1-propanol; and (IV) upper
phase of l-butanol-acetone-water (9-1-10, v/v). System IV was
particularly useful for the separation of Nam and NA in the hydrolysis

studies.

25

Product E_Purification

 

Sheets of Whatman No. 1 paper, 46 x 57 cm, were washed for 3 days
with 0.1 M sodium citrate and then rinsed for 3 days with deionized
distilled water. The sheets were air dried and then cut in half
lengthwise. Product C, which had been eluted from a Dowex l formate
column and concentrated on a rotary evaporator, was streaked on this
paper and developed in Solvent System 11. The UV absorbing band
corresponding to Product C was then eluted with deionized distilled
water by means of a spin thimble purchased from the Reeve Angel
Company. This procedure was repeated with Solvent System III. For
the Product C spectral data, a second streaking and developing in
Solvent System III was done. Appropriate volumes were attained after

the spin thimble elutions by concentration on a rotary evaporator.

Product Determination gg_Paper Chromatograms

 

UV absorbing products and standards were detected by visual UV
quenching with a Mineralight UVS-ll Lamp. Radioactive products and
standards were detected on a Model 7201 Radiochromatogram Scanner.
The areas under the peaks obtained by the latter method were cut from
the Chromatograms and placed in counting vials filled with Scintilla-
tion Fluid II and counted for 10 min in a Packard Model 3310 Tri-Carb

Liquid Scintillation Spectrometer.

Dowex l—X2 Formate Column Chromatography
Dowex 1-X2 chloride (200-400 mesh) was first converted to the
hydroxyl form by eluting with 39 volumes 1N NaOH. Complete removal of
chloride was checked by acidifying a sample of the eluant with conc.

HN03 and adding 1% AgNO3. The resin was then washed with deionized

26

distilled water until the pH of the eluant was that of the water. It
was then converted to the formate form by elution with 8 volumes 1N
HCOOH. Complete conversion was indicated when the pH became less
than 2. Finally, the resin was again washed with deionized distilled
water. A deproteinized reaction mixture was placed on a column of
this resin and eluted with a differential HCOOH gradient according to
the procedure of Ijichi eE_gl, (44), as modified by Ryrie and Scott
(35), with the following two exceptions: for convenience, a 25 ml
burette (1.0 cm i.d.) was used and the last reservoir, 4 N HCOONH4,

was eliminated from the gradient.

Product Determination gfl_a_Dowex l-X2 Formate Column

 

 

The fractions eluted from the Dowex 1 formate columns were assayed
for UV absorbance at 260 nm on a Hitachi-Perkin-Elmer Model 139
Spectrophotometer. A Gilson transferator was used to expedite the
measurements. Radioactivity of the fractions was determined by

counting 0.5 ml aliquots in 5.0 m1 Scintillation Fluid A.

Sephadex G-25 Eolumn Chromatography

 

Sephadex G-25, medium grade (50-150 p particle size) was allowed
to swell overnight in 0.05 M potassium phosphate, pH 7.0, and then was
poured to form a column 18 cm x 7 mm i.d. The column was eluted with
approximately 100 ml of the same buffer and calibrated with a 49:1
(v/v) solution of 0.05 M FAD and 0.2% (w/v) blue dextran which had
been filtered. This same column was then used to desalt a castor

bean endosperm enzyme preparation.

27

Protein Determinations

 

The amount of protein in the enzyme preparations was determined

by the method of Lowry 23.21: (111).

UV Spectra 9f Product E_ggg_NAQ

The author is grateful to Dr. Fritz Rottman and Karen Friderici
for the use of the Model 240 Gilford Spectrophotometer. The instrument
was adjusted to zero absorbance against air and the samples were
scanned from 200 to 400 nm.

The incubation mixture for the cyanide complex formation contained
NAD and KCN at the final concentrations of 13.6 pM and 67 mM
respectively. A Product C sample was likewise incubated with KCN.
Spectra were taken before and 10 min after the addition of KCN.

The incubation mixture for the yeast alcohol dehydrogenase assay
contained at the final concentrations given in a total volume of
1.5 ml: 0.55 M ethanol; 13.6 uM NAD; 53 mM Tris, pH 8.7; and 50 ug
yeast alcohol dehydrogenase. The assay was repeated with Product C
in place of NAD. Spectra were taken before and 10 min after the

enzyme was added to the reaction mixture.

Hydrolysis Studies
The samples used in the hydrolysis studies were the concentrated
Fractions #5, #7, and #9 from the Dowex 1 formate column in Figure 7
and authentic [6-14C] NaMN, NaAD, and NAD. They were each adjusted
to a final volume of 350 pl and were made 0.1N in NaOH, with the
exception of Fraction #9. Although this fraction was radiochemically
pure, some compound, possibly residual HCOOH, prevented the sample

from becoming alkaline. The final volume of this sample was 800 pl

28

and the final concentration of NaOH added was 0.28 N. The samples were
heated in a boiling water bath for 10 min and 60 min. Paper chroma-
tography was carried out in all four systems; however, there was only

enough of Sample #9 for spotting in three systems.

Recrystallization Eg_Constant Specific Activity

 

[7-‘4CJ NA and [8-14C] trigonelline from typical incubations were
isolated on a Dowex l formate column. The respective nonradioactive
standards were added to each of the two samples and they were
recrystallized to constant specific activity. After each recrystalli-
zation, a small amount of crystals was dissolved in deionized distilled
water and diluted to volume. Since these~compounds follow Beer's

Law, the ratio of cpm to absorbance is a valid representation of

specific activity.

RESULTS AND DISCUSSION

Initial experiments were performed to further establish the
nature of Product C synthesis. Depending on the activity of the
enzyme preparation, radiochemical yields of Product C as high as 11%
(1.6 nmoles) could be obtained from a 3 hour incubation. On occasion,
there appeared a fourth product, 0, which never exceeded one-third
the amount of Product C formed. A time course (Figure 2) indicated
that Nam was being rapidly deamidated; Products C and 0, however,
continued to be synthesized even after the supply of Nam had been
depleted. This suggested that NA might serve as the substrate for
Products C and D, as it did for trigonelline. Original attempts to
answer this question failed because of excessive streaking of NA in
Solvent System I. This difficulty was overcome by using NA of
higher specific activity so that less sample had to be spotted.

Table 1 shows that NA was as effective as Nam as a precursor of

Product C. In view of the rapid deamidation of Nam as well as the

fact that the reversibility of this deamidation has never been reported,
it was assumed that NA was the more immediate precursor of Product C.

Since 8 mM NAD had been reported to inhibit Nam deamidase in
microorganisms (81), it was of interest to determine the effect of NAD
on Product C formation. In a preliminary set of experiments, almost
identical inhibition of Product C production resulted when either

[7-14C] Nam of [7-14C] NA was used as the substrate. This indicated

29

 

 

30

Figure 2. Time Course of Nam Utilization and NA Formation

(A) and Product C, Product 0, and Trigonelline Formation (B). A large
scale incubation (2.25 ml) was performed as described in the methods
section, except that the ATP concentration was 6.67 mM. [7-140] Nam
(5.03 mC/mmole) at a concentration of 0.103 mM was used as the
substrate. Aliquots (100 pl) were removed at the times indicated and
the results were obtained from Solvent Systems I and IV.

(A) Nam (H)

(A) NA ( o—o)

(B) Product C (0—0)

(B) Product 0 (H)

(B) Porduct C + Product 0 (H)

(B) Trigonelline (H)

 

% Total Radioactivniy

% Total Rodioactivit

31

 

100

a)
O

03
O
T

b
O

N
0
j

 

 

60 120 180 240 300 360
Time (min)

 

Y

<3

01

 

 

 

 

 

so 120 160 2210 ado 360
Time (min)

32

 

«.mH mo.m m.NH ¢N.H ¢.oH oo.H N.oH om.H mstHmsomwch
o.m oe.o . T m.N me.o u n o puauocm
m.m mH.H m.m mo.H m.m NH.H m.m 0N.H u puuuoca

spp>ppeeopeee puseoes spH>Hpeeopeee puseosa spH>HpeeoHeee possess spH>HpeeoHeee poeuoss

_epo» m mmHOEs _ep0p m mmHoEs _ep0H m mm_oEs _epo_ m mmHOEs

 

 

 

HH psmEHsqum H psmepsmaxm HH pswEstaxm H psmepseaxm posuose

 

 

<2 Hee_-pH sez mueH-pH

 

.HH psmepsmaxm sH umm: we: H Empmxm psm>Hom use H psmEHsmexu Ho mpHemms esp spepuo

op umm: we; HH Eepmxm pse>Hom .HH psmepsmaxm LOH cs e use H psmEHseaxm so» s: N we: mEHp soppeuuusH
esp .zs moH.o me: HmHoEE\uE ou.mv <2 flusH-mU mo pesp use :5 moH.o me: HmHoEE\uE mo.mv Eez mosHTNH

Ho suppespsmusou msp HH psmepseexm sH .zs emo.o we; HeHoEE\uE oo.ov <z mueH-~u Ho pesp use :5 mmo.o

me; 33:58:. m.mC Eez 3.3-3 Ho soHpespseusou esp H psmetmsxm E .25 3.0 we: PE 08 5.595588
msp pesp pemoxm .soHpoem muospee esp sH ueuHsommu pesp me; HH use H mpsmepseexu spou sow msuumuose

memme use .wsHHHmsomHsp use .0 puuuosa .u pueuose mo msomszumss me <2 mueH-~H use Eez mueHTNH .H HHm<p

33

that the inhibition was not due solely to inhibition of the deamidase.
(It was evident from the scans of the radiochromatograms that some
inhibition of Nam deamidase had occurred, but it was not quantitated.)
Table 2 presents the inhibition effected by NAD on Product C and
trigonelline fromation when [7-140] NA was used as the substrate. At
1 mM NAD, Product C and trigonelline formation was inhibited by 86.6%
and 22.3 % respectively. Synthesis of the latter compound was more
effectively inhibited (70.9%) by 5 mM NAD. NAD had never been
reported to inhibit trigonelline formation. Since funneling of NA
into trigonelline is a detoxification mechanism (18), the inhibition
by NAD was unexpected. Furthermore, since there is a constant level
of NAD in plants at a particular stage of development (18), and since
trigonelline can serve as a source of NA for NAD synthesis in
Torula cremoris (107), high levels of NAD might be expected to inhibit
the demethylation of trigonelline but not its synthesis. No methyl
donor had been added to the reaction mixture; thus it is possible
that NAD inhibited the synthesis of S-adenosylmethionine rather than
the synthesis of trigonelline itself.

The requirements for the synthesis of Product C are summarized
in Table 3. The reaction was completely dependent on ATP and less
dependent on MgZ+. Trigonelline formation exhibited a lesser

2+. These

requirement for ATP but hardly any requirement for Mg
results indicate the presence of endogenous M92+, since methyl group
transfers from methionine as well as other reactions involving ATP
require the participation of Mg2+ (113). That a high level of ATP was
required for the optimum formation of Product C is evident from Table

4. The highest yields were obtained at 2.67 or 4.00 mM ATP.

34

 

 

 

 

 

 

m.em N.mm u.H H.o em.o Ho.o o.oH

m.oe N.Nm o.m N.o us.o mo.o o.m

w.mm m.mm o.m e.H NN.H Hm.o o.H

N.m m.om e.m m.u me.H mo.H m.o

H.NH m.om- o.m N.oH mm.H mm.H H.o

- . m.oH m.m mm.H om.H o.o
esp—research u puuuosa msermsomHsh o puuuoss msHHHmsomHsH u pouuose stv

sopppepseH e spH>HpaeaHeem Hepap e peseosa maHOEs soppemuueesoe

 

 

.mxemme epeuHHeuu Eose mpHsmes esp Ho emese>e esp use umpsoems meuHe> use

.mpHsmms esp spepuo up umm: me; H Eepmxm psm>Hom .epespmuum esp we uem: me; 25 moH.o Ho soHpeLpsmusoo e
pe HmHoEE\uE ou.mv <z flueHTNH .25 mm.m we: ap< Ho soHpespsmosou esp pesp pamoxm .soppumm muospme esp sH
umupsummu pesp me: mseueuose Hemme ms» .mHmmspsxm esHHHmsomHsp use 0 puuuosa Ho soHpHuHssH a<z .N m4m<k

35

TABLE 3. Requirements for the Synthesis of Product C, Product 0, and
Trigonelline. The data in this table were compiled from several
experiments. All activities are relative to the control of the
particular experiment, which was given a value of 100. The assay
procedure was that described in the methods section, except that in
Experiment I, IV, and V the concentration of ATP was 6.67 mM and in
Experiment II, an equivalent concentration of Tris, pH 7.0, was used
in place of phosphate or in addition to phosphate. The substrates were
[7- 4C] Nam 339.4 mC/mmole) at a concentration of 0.095 mM in Experi-
ment 1, [7-1 C] Nam (13.5 mC/mmole) at a concentration of 0.098 mM in
Experiment 11, and [7-14C] NA (6.60 mC/mmole) at a concentration of
0.102 mM in the remaining experiments.

 

 

Relative Activity
Modification of Assay

 

PFEHUEt—C' PFEHUEE—D Trigonelline

None 100 100 100

(I) — ATP 0 - 19

(I) - Mg2+ 44 - 81

(I) - ATP, - M92+ o - 19

(II) - Phosphate, + Tris 4 22 8

(II) - Phosphate, + Tris, 0 0 0
+ 4 mM ATP

(II) + PhOSphate, + Tris 26 22 28

(III) Boiling of Enzyme 0 0 0

(IV) Sephadex G-25 Treat- 0 — 106

ment of Enzyme

(V) + Supernatant of
Boiled Enzyme 50 - 76

 

36

TABLE 4. Optimum ATP Concentration for Product C and Trigonelline
Synthesis. The data in this table were compiled from two experiments.
The assay procedure was that described in the methods section, except
that the ATP concentration was varied as given in the table below.
[7-14C] NA (6.60 mC/mmole) at a concentration of 0.102 mM was used as
the substrate. Solvent System I was used to obtain the results. The
values for 1.33 - 8.00 mM ATP are the average of the results from
duplicate assays.

 

 

ATP Relative Activity
Concentration

(mM) Product C Trigonelline
0.05 13 34
0.10 24 43
0.20 37 48
1.33 79 96
2.67 100 96
4.00 100 100
5.33 93 99
6.67 93 96

8.00 91 84

 

37

Almost no activity for Product C formation was obtained in Tris
buffer, although about one-fourth the control activity could be
obtained in Tris buffer supplemented with the amount of phosphate in a
regular reaction mixture (Table 3). Observation of the Chromatograms
under UV light revealed that the ATP was completely degraded in the
assays containing Tris buffer, but hardly degraded at all in those
containing phosphate buffer alone. A two-fold increase of ATP in the
reaction mixture did not overcome this effect. Similar results were
obtained for trigonelline formation, which is consistent with its
requirement for ATP under these assay conditions. Product 0 was
detected in the experiments with Tris and phosphate buffers. No
stimulation of its synthesis resulted from the addition of phosphate
to the reaction mixture containing Tris buffer and no activity at all
was obtained in Tris buffer in an assay fortified with the extra ATP.

Boiling of the enzyme preparation for one minute resulted in
complete loss of activity for Product C formation, as did passage of
the enzyme preparation through a Sephadex G-25 column (Table 3). This
latter treatment did not affect trigonelline formation, a result which
is not understood in light of a probable requirement for endogenous
methionine under these conditions. Since desalting of the enzyme
preparation had prevented Product C synthesis, it was expected that
addition of the low molecular weight fraction from the Sephadex G-25
column would restore activity to the desalted protein fraction.
Addition of the supernatant from a boiled enzyme preparation would
likewise be expected to stimUlate the activity of an assay containing
untreated enzyme. The former experiment was tried by Bosch (112), who

found that activity could not be restored. The author of this thesis

38

found that the latter treatment did not stimulate activity either;
in fact, both Product C and trigonelline formation appeared inhibited
by 50% and 24% respectively.

At this point it was desired to obtain Product C in a quantity
sufficient for identification; therefore, a large scale incubation
was performed. Dowex l formate column chromatography of the reaction
mixture revealed a radioactive peak with corresponding UV absorbance
that was eluted after NA (Figure 3). The percentage of radioactivity
in this peak was close to the amount converted to Product C, as
calculated from the results of paper chromatography. The fractions
belonging to this peak were pooled and concentrated on a rotary
evaporator; the sample was then purified by paper chromatography in
Solvent Systems II and III. The radioactivity ran with the UV
absorbance both during and after the purification. Furthermore, the
Rf of the purified product in all four solvent systems was that
expected for Product C.

Alkaline hydrolysis of this sample provided one of the main clues
leading to the identification of Product C: besides UV absorbing
radioactive products, there were UV absorbing products without radio-
activity. This raised the possibility that ATP might be serving as a
substrate in the reaction. That this was indeed the case is
illustrated in Figure 4. When nonradioactive NA and [8-140] ATP were
incubated with an enzyme preparation, a product was formed which was
eluted from a Dowex l formate column in the same position as Product C.
In anticipation of the result, carrier NAD had been added to the
reaction mixture placed on the column, since the enzyme preparation

was not on a large enough scale for endogenous UV absorbing material

39

 

.sopppesm seem so umsHEsmpmu use: xpp>ppueopues use museQLOmue >2 .soppuwm

muospmE msp sH umopsummu we .ss\HE Hm Ho mpes onm e pe psmpueLm :oouz Hewpsmswewpu

e sppz umpuHm use; mpuuuosa esp .25 mmo.o wo soHpeLpswusou e pe mez HmHoEE\uE mm.mv

Eez musHimg use .25 mo.m we; ah< Ho coppespsmusou msp pesp pamuxm .soppumm muosme

esp sH umupsummu me Ls u so» umELomsma me: HHE o.omv coppenuusp mHeum emce— < .sE:Hou
mpeEsos H xw30o e so Eez muuHTNH Eosw mpuuuocm asp Ho soHpeseamm .m msumpm

 

 

 

4O

Aumoooypoa

(Iw 90/9 on x um)

20:85.2 9 .3232 8.8.3

 

 

 

IOOUI z O.N

 

 

 

 

 

.:.

H :08: szd 88.85

 

 

:1
9
----(uuu 092) aouoqmsqv

 

 

H

 

 

41

.soHpoese soeo so uosH5smpmu use; 2pH>HpoeoHues use mosessomse

>3 .soHpomm muospo5 esp sH uosHsommu me .ss\H5 mm mo mpes 30H; e pe psoHuesm

Ioour HeppsosoHeHu e ssz uopon wee; mpoouoso osk .s5:Hoo esp so ueoeHo esopr5

soHpoeos uoNHsHoposoou esp op uouue use; mmHoE: H.ov o<2 use HmmHoE: o.Nv <2 sopsseu

.25 eoH.o mo soHpespsmosoo e pe psmmmso we; <2 o>HpoeoHuesso2 .mpespmsum m>HpoeoHues

osp me uom: me: 25 oh.m eo soHpespsoosoo e pe MoHo55\05 Rm.mv ¢F< mueH1mH pesp

pomoxo .soppoom muospo5 esp sH uosHsomou we um5soeso we; soHpeseosH ss e < .s5oHou
ope5sos H xozoo e so e5< mus—-mg 5ose mpozuoso esp Ho soppeseoom .e msomps

 

42

000.

42p! X de) Mimoooipoa

......(luj 9'0
0
O
O
N

HcoHHooHeHEEmH .3552 5288...

 

 

 

 

 

 

 

 

 

 

‘12
O

— -- - (mu 093) eouquosqv

8

 

NM HVIN @— m
s‘. IIIIIII If\\pll\\\.'l||l’ fi
s
H HH ’cs
H H H
H H __
H H
H _
a — — 1
HH . H _
r .t. H .
o<z H n t
H__
C
(2
C I.
2 fl 2565 a
10001 2 0N6 IOOUI 2.0.0

 

43

to be detected. The UV absorbance from the carrier NAD corresponded
exactly to the radioactivity of the peak. The fractions belonging

to this peak were pooled, concentrated on a rotary evaporator, and
then purified by paper chromatography in Solvent Systems II and III.
When this sample and a similarly purified sample of Product C
synthesized enzymatically from [7-14C] NA were each co-chromatographed
with authentic NAD, there was perfect correspondence of radioactivity
and UV absorbance of the NAD standard in Solvent Systems I, II, and
III. The Product C from [8-14C] ATP was then mixed with the Product C
from [7-14C] NA, and this sample was again co-chromatographed with the
authentic NAD. The radioactive peaks were coincident with each other
and with the UV absorbance in these three systems.

Further evidence that Product C was NAD was obtained from the UV
spectrum of another sample of Product C which had been isolated from a
Dowex l formate column and purified by paper chromatography in Solvent
Systems 11 and III. Authentic NAD and Product C exhibited almost
identical Spectra, both before and after the formation of the cyanide
addition product (Table 5 and Figure 5). The reaction with cyanide
is not specific for NAD; NMn, NMN, NaAD, and NADP also form cyanide
complexes with UV spectral characteristics similar to those of NAD
(114). When Product C was reacted with yeast alcohol dehydrogenase,
additional proof of its identity as NAD was Obtained (Table 5,

Figure 6). In contrast to the maximum at 327 nm resulting from the
cyanide complex, a peak appeared at 340 nm, representing the reduced
Nam moiety of NAD. Since both Nam and adenine contribute to the

260 nm maximum, a decrease, but not complete loss, in absorption at this

wavelength was associated with the formation of the new maxima at

44

TABLE 5. Absorption Maxima and Minima of Authentic NAD and Product C
in the Absence and Presence of KCN and in the Absence and Presence of
Yeast Alcohol Dehydrogenase. The spectra were obtained as described
in the methods section.

 

 

Sample Absorption Maxima (nm) Absorption Minima (nm)
NAD - KCN 260 233
Product C - KCN 260 234
NAD + KCN 261 327 237 290
Product C + KCN 260 327 236 290
NAD - ADH 260 234
Product C - ADH 260 234
NAD + ADH 260 340 234 290
Product C + ADH 260 340 236 290

 

45

Figure 5. Absorption Spectra in the Absence and Presence
of KCN. (A) NAD; (8) Product C. The spectra were obtained as
described in the methods section.

 

46

 

 

 

 

 

 

0*
A
)5
8
3
2
4
oh
+c~
\_—_J
256‘ ‘ ‘2éo‘ ‘ ‘360‘ "7340‘ 7 ‘3éo‘
Wavelength (nm)
or.
E3
02» —c~
>~
8
g r-
O
3
4.
oh
+c~

 

 

 

. . 2 .__s_s1gRyHHH_HHEUGHHHHHstcHH—H—H1flfiyh-

Wavelength (nm)

 

 

47

Figure 6. Absorption Spectra in the Absence and Presence
of Yeast Alcohol Dehydrogenase. (A) NAD; (B) Product C. The spectra
were obtained as described in the methods section.

 

 

Absorbancy

 

 

 

 

 

 

 

 

0.3'
A
1 ea.
0.2T
0.1+
1.
220 260
Wavelength (nm)
L B
0‘5 —ADH
>~
E 04» \
+ADH
0.2»
T
{20 1230 ‘ ‘ 300 ‘ T 3210 ‘ 360' ‘

 

 

Wavelength (nm)

 

 

49

327 nm and 340 nm. Yeast alcohol dehydrogenase is NAD dependent,
although slow rates have been reported for NADP (115,116). The
spectral data taken together with the column and paper chromatographic
data to be presented later preclude the identity of Product C as any
one of the nucleotides mentioned here other than NAD which also form
cyanide complexes or react with yeast alcohol dehydrogenase.

It seemed likely that the NAD was being formed via the Preiss-
Handler pathway, since NA was able to serve as the substrate.
Accordingly, standard assays for NA phosphoribosyltransferase and 0A
phosphoribosyltransferase were performed in order to compare the
products with those formed in a Product C assay. Scans of radio-
chromatograms developed in Solvent Systems I and II from assays of the
former enzyme resulted in a pattern essentially identical to that
obtained when assaying for Product C. Radioactive peaks corresponding
to both Products C and D were evident. The product of 0A phosphori-
bosyltransferase also ran at the Rf of Product 0 in these two systems.
These two enzymes both form the same product, NaMN (Figure 1). Since
the basic difference between the assay for NA phosphoribosyltransferase
and Product C synthetase was the presence of PRPP in the former, it
appeared that ATP was serving to promote the synthesis of PRPP. The
loss of activity after passage of the enzyme preparation through a
Sephadex G-25 column could be partially explained by the loss of the
other substrate for PRPP synthesis, i.e., ribose-S-phosphate. The
assay conditions were probably favorable for PRPP synthesis, since
it has been shown to require Mg2+ in Salmonella typhimurium (117) as
well as Pi in human erythrocytes (118). This could correlate, too,

with the lack of activity in Tris buffer in the absence of phosphate.

50

Furthermore, the PRPP synthesizing capacity would be expected to be
high in castor bean endosperm extracts, since PRPP is also a required
substrate for the rapid RNA and DNA synthesis which occurs in developing
tissues (119).

Since NA was being converted to NAD in the absence of PRPP, one
would expect the same of 0A. This phenomenon, however, was not
observed by Mann (105), who was using castor bean seedlings which were
slightly older physiologically. When seedlings at the same growth
stage as those used for Product C synthesis were assayed for DA
phosphoribosyltransferase activity in the presence and absence of PRPP,
the results in Table 6 were obtained. Essentially no enzymatic
activity occurred in the absence of both PRPP and ATP. PRPP alone was
sufficient to permit almost complete conversion of 0A to NaMN. In
the absence of PRPP, ATP was able to support some synthesis of NaMN
and other nucleotides, but not to the extent that occurred when PRPP
was present either alone or with ATP. 0f the products obtained in the
absence of PRPP, 62% ran at the Rf of NAD in Solvent System I; no
attempt was made to determine if other nucleotides which run at this
Rf were formed.

Since it had been noticed throughout this study that the size of
the seedlings was very critical to the formation of Product C under
these conditions, the enzyme activity was measured over a seven-day
germination period. As can be seen in Figure 7, optimum synthesis
of Product C occurred on Day 3 and good activity was maintained
through Day 5. Product 0 showed the same optimum, but its formation
dropped off a day earlier than that of Product C. Trigonelline

synthesis peaked on Day 4, although good activity was also obtained on

51

TABLE 6. PRPP Requirement of 0A Phosphoribosyltransferase. The assays
were performed according to the procedure of Mann (105), except that
ATP was present at a concentration of 6.0 mM. The incubation time was
3 hr.

 

Omission nmoles Product* % Total Radioactivity
None 59.3 97.2
- PRPP 11.1 18.2
- ATP 53.9 88.4
- PRPP, - ATP 0.1 0.2

 

*NaMN and NaAD and/or NAD

 

52

gal: msZHmsomHfi use 2 IV

a poeuoss HH onlov u poouose .5soomousm esp 5ose um>o5os pos «so; msouonpoo esp

m use N mxeu so .mxemme opeoHHoou 5osp mpHomms esp mo mmesm>e msp mp pspoo

soem .mpHomos esp sHepoo op uom: we; H 5opm2m pso>Hom .opespmsom o>HpoeoHues

msp me uom: me; 25 NoH.o Ho soHpespsmosoo e pe HoHo55\u5 ou.mv <2 mueHlmH

.soppoom muospo5 osp sp uosHsomou me uo5soesoo use; msoHpesoosH use .soHpesHEHoo
ospseu esp—HosomHLH use .o poouose .u poouoss mo soHpe5som .m osompm

 

 

53

 

 

see

 

 

(\i

U18

405 it? 6 Th

101d bin/.114 g/lanpmd salouJu

Age (days)

54

Days 2, 3, and 5. In the course of this research, the synthesis of
trigonelline always accompanied the synthesis of Product C. This
association is evident from the results presented here. By Day 7, NA
was no longer being converted to Product C or trigonelline; a minimal
amount of Product 0 was still being formed though. A single attempt
to obtain Product C with an extract from mature green castor bean leaf
tissue failed. It must be stressed that the seedlings used in the age
study were selected according to physiological as well as chronological
age. The seedlings harvested on any one day were approximately the
same size and exhibited the same physiological characteristics

(Table 7). These results, representing ig_yiE§9_enzymatic activities,

do not necessarily represent ig_vivo NAD synthesis, where substrate

 

and cofactor availability may be controlling factors (115).

In the earlier large scale incubations it was noticed that minor
radioactive products were formed. These compounds, which could be
eluted from a Dowex l formate column, were apparently being masked
in paper chromatography by the major products formed. In order to
obtain a more complete profile of the reaction products, another
large scale incubation of [7-14C] NA of higher specific activity was
carried out. Figure 8 shows that twelve radioactive peaks were eluted
from a Dowex l formate column. The fractions belonging to each of
these peaks were pooled, concentrated on a rotary evaporator, and
adjusted to a volume appropriate for detection of the radioactive
compound(s) by paper chromatography. Chromatography of all twelve
fractions was performed in Solvent Systems I, II, and III, and the
results were compared to those of standards chromatographed in the

same three systems. After this preliminary identification, the

55

TABLE 7. Physiological Characteristics of Etiolated Castor Bean
Seedlings Germinated in Moist Vermiculite at 30°C.

 

 

Day Characteristics

 

2 The seed coat had just cracked open
and the radicle was not over 1.0 cm
long. The average fresh weight was
0.4 g/endosperm.

 

3 The average length of the radicle
was 1.35 cm and the average fresh -- 3
weight was 0.5 g/endospenn.
4 The average length of the radicle (”’T\
was 3.4 cm and the average fresh _,,
weight was 0.5 g/endosperm. Secon- , 4
dary roots were present but not well
developed.
5 The average length of the hypocotyl
was 0.7 cm and the average fresh <::::)‘]
weight was 0.65 g/endosperm. The
endosperm was still firm and there 5
was good secondary root development. 4/(\\A
6 Th ’// \\
e average length of the hypocotyl //’é’l
was 2.7 cm and the average fresh ./{}‘,’ \;\\
weight was 0.8 g/endosperm. The .,,?/ , \
endosperm was soft. 15 \\J

7 The average length of the hypocotyl
was 4.4 cm and the average fresh
weight was 0.7 g/endosperm. The
endosperm was mushy and nearly
consumed.

 

 

 

56

.soHpoeHH soem so umsHEsopou use; 2pH>Hpoe
-oHues use mosessomse >2 .soHpomm muospo5 wsp sH umsHHommu me .Hs\H5 Hm Ho
opes one e pe psoHuesm Ioouz HersosmeeHu e ssz uopoHo use; mpouuoso osH
.ss emo.o so soppespseosoo e pe we; HeHoss\o5 oe.oH <2 moeH-HH esp .ss s sop
Doom pe um5soesoo we: HH5 o.omH soHpeseosH oHeom mmHeH < .s5:Hou ope5HoH

H xozoo e so <2 mueHlmu 5osH mpoeuoss esp Ho soHpeseoom .w mssmHH

 

 
 

57

Mb) hlhllaoogpog

oou on.

558.55 E 8252 8.82...

 

6
N

 

M H
H
«be M. H
H
H
a m H
O I . .
re @ H H
9.00 0 u n .
~03 c. H u .
M W .H H H. I
7H1 . ("I u u u .
_ V .00 +581 2 on H.581 2 ON H H M07509.- Zowd H.509... 8.0.0c
C. 65.87. 32.8

I‘n‘lla‘n‘i‘u I 1
’ )Ke’o

 

-

 

.
-------—a—-——---—————---—-
.

 

---—-_-_- --_.’¥_---______-___ ... _--a’—’

 

8

Q
—— (wuoezl eouoqmtqv

"l

 

 

58

samples were co—chromatographed with the respective standards in these
three systems. Six of the twelve fractions from the column were
identified by this method (Table 8). Fraction #1 contained 16.7% NMN
and 83.3% trigonelline, whereas the other five fractions identified
contained a single radioactive compound: NA, NAD, NaMN, NaAD, and NADP.
The radioactivity of the fractions migrated coincident with the UV
absorbance of the standards for all the samples. In the case of
Fraction #7 where a radioactive standard was employed, the two peaks
were coincident. The elution pattern of these compounds from a Dowex l
formate column is consistent with the column data reported by Ijichi
_E_al, (44) and thus supports their identity.

In this particular incubation, paper chromatographic analysis in
Solvent System I had indicated four peaks with the following percen-
tages of total radioactivity: Product C, 11.9%; Product 0, 4.8%;
trigonelline, 17.9%; and NA, 65.4%. Since the Rf on paper was now
known for each of the twelve radioactive fractions eluted from the
column, it was possible to calculate the percentage composition of the
above four radioactive peaks. The peak on paper considered Product C
was comprised of 95.8% NAD, 3.4% NaAD, and 0.8% NADP. What was being
measured as Product 0 included 27.8% NaMN and 72.2% NMN. The presence
of NMN was not realized until the very end of this study; its
identification was therefore limited to its elution pattern from the
Dowex l formate column and its Rf on paper, as just described. The
identification of NADP was also restricted to these two methods,
since such a small quantity of it was formed. Trigonelline and NA
accounted for 97.7% and 99.3% of the radioactivity on paper which had

been attributed to them. The remaining radioactivity in these two

59

TABLE 8. Co-chromatography of Fractions from the Dowex l Formate
Column in Figure 7 with Standards. The compounds were detected as
described in the methods section.

 

 

Sample Rf of Sample in Solvent Systems

I II III

#1 + Trigonelline .27 .62 .50
#1 + NMN .09 .25 .29
#4 + NA .69 .73 .67

#5 + NAD .04 .16 .17, .29*

#7 + [6-‘4CJ NaMN .10 .21 .32
#9 + NaAD .04 .13 .31
#11 + NADP .02 .04 .26

 

*Two peaks because of streaking; the intensity of the UV absorbance
corresponded to the intensity of the radioactivity.

60

peaks was due to unidentified compounds.

Since [7-14C] NA was the precursor used in this research, it
might be expected that it would have lost radioactivity in the form of
14CO2 and that the above percentages would thus be invalid. This
was checked by incubating the reaction mixture in a vessel equipped
for CO2 collection, as described by Mann (105). Not more than 1.0%

14

of the initial radioactivity was released as CO

2.
Recrystallization to constant specific activity confirmed the
identity of the NA and trigonelline eluted from the Dowex l formate
column (Tables 9 and 10). Since the Product C fraction from the
column was now known to be NAD, its alkaline hydrolysis was repeated
along with that of authentic NAD. The fractions believed to be NaMN
and NaAD were also hydrolyzed and the results compared to those of
standards (Table 11). Fraction #5 from the column yielded both
Nam and NA, and the former was seen to decrease and the latter to
increase from 10 to 60 minutes. The authentic NAD sample behaved
similarly, i.e., both Nam and NA could be detected under UV light; the
NA spot, which was faint at 10 minutes, became darker by 60 minutes.
On the other hand, when Fraction #7 from the column and authentic
[7-14C] NaMN were hydrolyzed, no Nam could be detected and NA accounted
for the only radioactive peak from both the sample and the standard at
10 minutes and at 60 minutes. Likewise only one radioactive peak and
only one UV spot corresponding to NA resulted from the hydrolysis of
Fraction #9 from the column and NaAD respectively. A slight complica-
tion with Fraction #9 occurred in one solvent system, IV, where there

was a small radioactive peak at the origin in addition to the peak for

NA. Standard NaAD exhibited the same behavior in this solvent system.

61

TABLE 9. Recrystallization of [7-140] NA to Constant Specific
Activity. A fraction equivalent to Fraction #4 of the Dowex l
formate column in Figure 7 was combined with authentic NA and
recrystallized from hot water. Specific activities were determined
as described in the methods section.*

 

Crystallization Absorbance260 nm cpm/0.5 ml Ratio of cpm:

(1:20 dilution) absorbance
#1 .58 105 181
#2 .61, .51 105 174
#3 .91, .92 162 178, 175
#4 .48 85 177

 

*These results were obtained by Anne Bosch (112).

62

TABLE 10. Recrystallization of [8-14C] Trigonelline to Constant
Specific Activity. A fraction equivalent to Fraction #1 of the
Dowex 1 formate column in Figure 7 was combined with authentic
trigonelline and recrystallized from hot absolute ethanol. Specific
activities were determined as described in the methods section.

 

 

 

Crystallization Absorbance cpm/0.5 m1 Ratio of cpm:
260 nm
absorbance
1:50 1:100 1:50 1:100
dilution dilution dilution dilution

#1 .770 .385 542 704 1408

#2 .150 .075 104 693 1387

#3 .225, .225 .120 157 698 1308

#4 .162 .084 118 728 1405

 

63

TABLE 11. Base Hydrolysis of Standards and of Fractions from the

Dowex l Formate Column of Figure 7.

described in the methods section.

The samples were hydrolyzed as

 

 

Sample

NAD

Fraction #5

[6-‘401 NaMN

Fraction #7

NaAD

Fraction #9

Product

+ (35%) + (65%)

- + (100%)
— + (100%)

- +

+ (>96%*; 81%1)

min
NEE'
+
+ (95%)

+

(91%)

+

(100%)

+

(100%)

+

+ (>96%*; 82%1)

 

*Solvent Systems II and III

#Solvent System IV

 

64

The important point here is that no Nam was detected and NA was.

In a final characterization of the reaction, PRPP and glutamine
were added to the reaction mixture to see if an increase in Product C
formation could be obtained. As can be seen in Table 12, 40% more
Product C was formed when PRPP was added to the incubation mixture.
Addition of glutamine was, however, without significant effect. When
both PRPP and glutamine were added, the effect-was similar to that of
PRPP alone. Both glutamine and NH4C1 have been found to serve as amide
donors for the NAD synthetase reaction in yeast (120). The effect of
NH4C1 was not tested in this study.

As stated previously, the ATP present in the incubation mixture
could be serving as a PRPP generator. The ATP requirement also
encompasses, of course, the role of actual substrate in the synthesis
of NaAD. Beyond this are its implicit roles in the NA phosphoribosy1-
transferase and NAD synthetase reactions. Mann (121) has shown that if
ATP was not present, almost no activity of the former enzyme could be
obtained in the presence of PRPP with a castor bean endosperm enzyme
preparation which had been passed through a Sephadex G-25 column to
remove endogenous substrates. A requirement of NA phosphoribosyl-
transferase for ATP in addition to PRPP has also been demonstrated in
yeast (13,118) and bovine liver (123,124). Exceptions are the NA
phosphoribosyltransferase found in human erythrocytes (13) and
Astasia longa (125). Stoichiometric utilization of ATP in both the
NA phosphoribosyltransferase agd NAD synthetase reactions has been
established in yeast (120,126).

In conclusion, the results presented here indicate the operation

of the Preiss-Handler pathway in etiolated castor bean endosperm for

65

TABLE 12. Effect of PRPP and Glutamine on Product C Synthesis.
The assays were performed as described in the methods section,
except that the volume was increased by 10 ul; the reagent
concentrations therefore were: 62.4 mM potassium phosphate,

6.2 mM dithioerythritol, 3.8 mM ATP, 12.5 mM M9012, and .096 mM
[7-146] NA (6.60 mC/mmole).

 

 

Addition nmoles Product C % Total
Radioactivity
None 1.62 10.6
PRPP 2.26 14.8
Glutamine 1.56 10.2

PRPP, Glutamine 2.08 A 13.6

 

66

the following reasons: (1) synthesis of NaMN and NaAD, the two
Preiss-Handler intermediates, along with NAD was observed; (2) NA as
well as Nam could serve as the substrate in the biosynthetic pathway;
and (3) an active Nam deamidase was apparent (Figure 2A). The fact
that 3.5% of the total radioactivity (10.1% of the products)
corresponded to NMN does not rule out the operation of the Preiss-
Handler pathway. Prior conversion of NA to Nam would have had to
occur, and this has never been reported. Furthermore, an active
nucleotide pyrophosphatase, which could account for the breakdown
of NAD to NMN, has been demonstrated in plants (29,127).

All the reaction products identified in this investigation were
characterized by paper chromatography in three solvent systems and
by their pattern of elution from a Dowex 1 formate column. In
addition, NA and trigonelline were recrystallized to constant specific
activity and NaMN, NaAD, and NAD were subjected to alkaline hydrolysis.
NAD was further identified by its UV spectrum in the absence and
presence of KCN and in the absence and presence of yeast alcohol
dehydrogenase.

The formation of NAD from Nam or NA under these conditions was

2+, and another compound(s) which was removed by

dependent on ATP, Mg
Sephadex G-25 treatment of the enzyme preparation. The latter may be
explained in light of the PRPP requirement of NA phOSphoribosyl-
transferase and the amide donor requirement of NAD synthetase. NAD

was also shown to inhibit its own biosynthesis.

 

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