THE JOURNAL OF RIOLOGIC~L Crrmns~nr Vol. 243, No. 7, Issue of 4pCl 10, pp. 1448-1456, 1968

Printed in U.S.A.

Studies of Nicotinic Acid Metabolism in Astasia Zoqa

II. PATHWAY AKD REGULATION OF NICOTINAMIDE ADENINE DINUCLEOTIDE BIOSYXTHESIS IN CELL-FREE PREPARATIOKS”

(Received for publication, September 18, 1967)

VARDA KAHK AKD J. J. BLUM$

From the Department of Physiology and Pharmacology, Duke University Medical School, Ijurham, North

Carolina 27706

SUMMARY

Cell-free extracts of Astosia longa possess the following enzymes at the indicated relative activities: (a) quinolinic acid phosphoribosyl transferase, 0.5; (b) nicotinic acid mono- nucleotide pyrophosphorylase, 1; (c) nicotinic acid dinucleo- tide pyrophosphorylase, 1; (d) nicotinamide adenine dinu- cleotide synthetase, 0.003; (e) nicotinamide dinucleotide kinase, 1; (f) nicotinamide deamidase, 1; (g) nicotinic acid methyltransferase, 0.0001. The presence of Enzymes a, c, d, and e, in conjunction with data on the intracellular levels of deamido-NMN, deamido-NAD, NAD, and NADP, estab- lishes this sequence of compounds as the pathway of bio- synthesis of pyridine nucleotides in Astasia and indicates that the conversion of deamido-NAD to NAD is a rate-limiting step.

Cell-free extracts also catalyzed the degradation and pyro- phosphorolysis of deamido-NAD and of NAD, and catalyzed an exchange reaction between nicotinic acid and the nico- tinamide moiety of NAD. These reactions, in conjunction with the presence of Enzymes b and f, and with data showing a high rate of turnover of NAD in exponentially growing cultures, indicate the operation of a nicotinic acid salvage pathway in Astasia. The finding that nicotinic acid is an in- hibitor of Enzyme a and that quinolinic acid is an inhibitor of Enzyme b suggests a possible means of control of the relative rates of the biosynthetic and the salvage pathways. The finding that deamido-NAD, present in relatively high concentrations in Astasia, is an inhibitor of Enzyme b sug- gests another possible control mechanism.

Growth in the presence of nicotinic acid or quinolinic acid did not lead to repression of either Enzyme a or b. Nico- tinamide and high concentrations of nicotinic acid inhibited the growth of Astasia and nicotinamide repressed the level of Enzymes a and b.

* This research was supported by Grant 5 ROl I-IL) 01269 from the Xational Institutes of Health.

$ Recipienh of Research Career Development Award K3 GM 2341 from t,he National Institutes of Health.

Studies of the rate of incorporation of labeled nicotinic acid into Astasia (1) showed that the label appeared in deamido- NRIX, deamido-NAD, and NdDP at, specific act,ivities com- patible with deamido-NRIN, and deamido-NAD being inter- mediates in t,he biosynthetic sequence established by Preiss and Handler (2, 3). It was also noted that addit’ion of nicotinic acid to the growth medium increased the level of NAD and NADP in the cell by less than S-fold, thus indicating that Astasia was capable of closely regulating the intracellular level of the pyridine nucleotide coenzymes (I).

Little is known about the control of the pathway of pyridine nucleotide biosynthesis. In adenine-requiring mut’ants of Bacillus subtilis, the level of ATP may control the activity of this biosynthetic pathway not only at the level of NADP synthesis but also because the nicotinic acid mononucleotide pyrophos- phorylase of B. subtilis requires ,ZTP for activity (4). In Escherichia coli, nicotinic acid mononucleotide pyrophosphorylase appears t,o be the rate-limiting step, and is subject to typical repression-depression control (5). In a number of other species (including the ciliated protozoan Tetrahymena), however, pyri- dine nucleotide formation was regulated neither by a repression- depression of the nicotinic acid mononucleotide pyrophosphoryl- ase nor by feedback inhibit,ion of this enzyme by NAD (6).

To study the pathway and the regulation of pgridine com- pounds metabolism in dstasia, it was necessary to show the presence of the enzymes responsible for t,he biosynthetic steps and to establish the levels of activity characteristic of cells grown in the normal chemically defined medium, i.e. in t’he presence of acetate as the sole carbon source. In this study we have invest,igat,ed the effect of exogenous nicotinic acid and quinolinic acid on the level of some key enzymes in the bio- synthetic and salvage pathway of pyridine nucleotide metabo- lism, and have attempt,ed to provide further insight’ into the fact,ors controlling pyridine nuclcotide metabolism in eukaryotic c&.

A stasia longa (Jahn strain) and Euglena gracilis var. bacillaris, Strain SAIL1 (streptomycin bleached) were grown asenically

1448

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at 25” in a modified Cramer-Myers medium (7) in 500-ml Erlenmeyer flasks. The cells were harvested during exponential vrowth at densities of about 200,000 per ml and, unless otherwise b indicat,ed, were washed in 0.1 M Tris-HCl (pH 7.4) containing millimolar reduced glutathione before sonic disruption. Cells were counted with a Coulter counter (Coulter Company, Hialeah, Florida). Cell-free preparations were obtained by sonic dis- ruption as described elsewhere (8).

Enzymatic activities were assayed at 30” in a total volume of 1 ml, with radioactive substrates in all cases, as described in detail in the text. After suitable times of incubation, aliquots were withdrawn, boiled for 1 min, and analyzed by ascending paper chromatography on Whatman No. 1 filter paper or by ion exchange paper chromatography. The percentage of sub- strate converted to product or products was calculated as the sum of counts associated with the latter divided by the total counts found on the paper. Unless otherwise specified, reaction rates were linear for at, least 1 hour and the activities reported refer to the initial rate.

Protein was measured by the method of Lowry et al. (9). Chromatographic Procedures-Reaction products were sepa-

rated by one-dimensional paper chromatography with one or more of the following solvents: A, n-butyl alcohol-water (86: 14, v/v); U, 0.1 M sodium phosphat,e (pH 6.8)-ammonium sulfate- n-propyl alcohol (100:60:2, v/w/v) ; C, 1 M ammonium acct,ate (pH 5.0).ethanol (3 :7, v/v) ; D, isobutyric acid-NHhOH-water (66:1.7:33, v/v/v, pH 3.8); E, pyridine-water (2:1, v/v) (2, 10, 11). For the separation of NADP from X.&D, it was con-

venient to use Reeve Angel grade SR-2 ion exchange paper loaded with Amberlite IRd-400 resin wit,h 0.1 N formic acid as the developing solvent,. When necessary, nicotinic acid nucleotides were differentiated from nicotinamide nucleotides by exposing the dry chromatograms to methyl ethyl ketone-ammonium hydroxide (1: 1, v/v) (12). Nicotinic acid and nicotinamide were identified by the formation of yellow spots after exposure t’o cyanogen bromide and p-aminobenzoic acid (12),

Radioactive Measurements-Radioact,ivity was determined in a Packard Tri-Carb scintillation spectrometer. Paper strips were counted in approximately 10 ml of scintillation fluid (4 g of 2,5- bis-(2,5-tert-but’gl benzosazolyl)-thiophene per liter of toluene) Aqueous samples up to 0.2 ml were counted in 10 ml of a mixture containing 10 parts of the above scintillation fluid and 3 parts of ethanol (v/v).

Xicotinic acid-i-‘%, nicotinamidc 7-‘4C, and quinolinic acid-6- 14(1 were obtained from Nuclear-Chicago and, unless otherwise

specified, were used at specific activities of 8.4 x 106, 2.5 X IO”, and 2.6 x 10” cpm per pmolc, respectively (1.1 X 106 cpm per

PC). Deanlido-1\‘MN-7-14C was prepared by incubating a cell-free

preparation from about, 2 x lo8 cells with 20 pmoles of nicotinic acid-7-l%, 42 pmoles of PP-ribose-P, 42 pmoles of XIgC&, and 750 @moles of Tris (pH 7.4). Deamido-KAD-7-14C: was pre- pared under identical conditions but with the addition of 750 pmoles of ITP. The reaction products were eluted from a Dowvrx-1 (formate) column with a concave formic acid gradient (12) and the formic acid was removed by lyophilization in the presence of 0.1 N HCI. The products were pure as judged by the presence of a single radioactive ultraviolet quenching spot upon paper chromatography in several solvent systems. Their specific acbivity was determiued by use of the cyanide addition

‘;Iw , I*c 30 60 90 120 150

Minutes

FIG. 1. Nicotinic acid mononucleotide pyrophosphorylase and nicotinic acid adenine dinucleotidc pyrophosphorylase activities in cell-free preparations of dstasiu. In Experiment 1, the re- action mixtke contained 10 mpmoles of liicotinic acid-7.14C, 0.56 ~rnole of MPCIV. 0.1 umole of PI’-ribose-I’. 0.75 ornole of Tris- WC1 (pH 7.4), a~~j;lld i cell-free preparation coriespoliding to 3.1 X lo6 cells (945 fig of protein). ATP (1.1 &moles) was added after 60.min incubation when the concentration of deamido-N-\IN in the reaction mixture was 10mS M. The scale for the amount of each product formed is shown on the right-hand ordinate. In Experi- ment 2, the reaction mixture contjained 50 mpmoles of nicotiuic acid-7-‘“C, 2 pmoles of I’P-ribose-P, 2.7 Mmoles of MgCl,, 12.5 pmoles of Tris-HCI (pH 7.41, 5 moles of ATP, and O.Oti ml of a cell-free preparation corresponding to 5 X lo6 cells per ml (1.02 mg of protein per ml). The scale for the amount, of each product formed is shown on the upper kj/-hand ordinate (--j. When STP was omitted from the reaction mixture, only deamido- NRlN was formed (- - -). 1n Experiment 3, the reaction mixture contained 0.32 ,,znole of deamido-NRIN-7-14C, 7.3 pmoles of ATP, 3.9 pmoles of MgC12, 18.1 pmoles of Tris-IlCl (pH T.-k), and 0.29 ml of the same cell-free preparation as used in Experiment 2. The scale for deamido-NAD formation is the ZOUU lcfl-hand ordinate.

complex assay (13). Unlabtled dramido-KMN and deamido- KAD were prepared in the same manner, but with unlabeled nicotinic acid as the reactant.

KAD-7-1X and in some cases tlcamido-N;11~-7-‘4C were isolated from the trichloracetic acid-soluble pool of .lstasia grown in the presence of nicotinic a(G-7-% (1). These com- pounds were purified and their specific activities were determined by the procedures outlined in an accompanying paper (1). T)eamido-N1\IN-7-14C:, deamido-NAD-7.*“C, and NAD-7-‘“C were used at specific activit’ies of 6.9 x lo6 cpm per ~.lmolr, 1.4 x IO5 cpm per Imole, and 7.3 x 10” cpm per pmole, respectively.

Additional Chemicals-IZTP, AXIS, dcamido-NM>, Y-acetyl pyridine-NhD, t~hionicotinamide-NLIJ1, PP.ribose-P, SID, and

NADP were obtained from P-L Laboratories; nicotinic acid methyl ester and 3-acetyl pyridine were from 1Iann; nicotinic

acid-n:-oxide and nicotinic acid were from Pierce Chemical Company (Rockford, Illinois) ; n’-methylnicot~i~~amide, IV- methylnicotSinic acid, and quinolinic a(?d were from Calbiochrm;

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1450 Studies of Nicotinic Acid Metabolism. II Vol. 243, No. 7

TABLE I Reversibility of nicolinic acid mononucleotide pyrophosphorylase

activity in cell-jree preparations of’ Astasia

l)eamido-NMN-7-14C or rricotinic acid-7-‘“C was used at the concentrations indicated. R.eaction mixtures contained from 1.9 t,o 2.7 ~moles of MgC12 and from 9 to 30 pmoles of Tris-HCl (pH 7.4). hliyuot,s of a cell-free preparation corresponding to 1.2 X 10” cells were used in Experiment 1-A and to 0.3 X 10G cells for all other experiments. A and B refer to experiments conducted with aliyuots of the same cell-free preparation. The sodium salts of inorganic phosphate md inorganic pyrophosphate were used where indicated.

Deamido-XMN + nicotinic acid

Experiment no. Deamido-XMr\ Additions

1-A 2-A

3-A

0.55 0.32 0.32

0.28 0.28 0.28 0.28

mA!4

PP.ribose-P, 2.8 PP.ribose-P, 2.8 Nicotinic acid, 0.7

PP-ribuse-??, 5.5 Pl’i, 5.5 Pi, 5.5

TABIJ~ IL

-

b

Activity

rmoles nzcotinic acid /h&x 109

5.0 42.0 42.0

3.0 30.0

110.0 3.0

Reversibility of nicotinic acid monon?~cleotide pyrophosphorylase activity in cell-j”ree preparatio,ns of Astasia

For explanation, see legend to Table I.

Nicotinic acid + PP.ribose-P --t deamido-XMN

Experiment no.

1-B 2-U 3-B

Yicotinic acid - Additions Activity

0.05 0.36 0.3 0.57

0.57

PP-ribose-P, 2.0 PP-ribose-I’ 2 .H PP-ribose-PI 4.5 PP-ribose-P, 4.5 PPi, 4.5 PP-ribose-P, 4.5 Pi, 4.5

42.0 50.0

212.5 60.0

212.5

nicotinic acid and nicotinamide were from Eastman; ribose I- phosphate was from Sigma.

RESULTS

Nicotinic Acid Xononucleotide Pyrophosphorylase of Astasia- We have recently shown that cell-free preparations of Astasia catalyze the conversion of nicotinic acid to deamido-NMN in a PP-ribose-P-dependent reaction (14). Unlike the nicotinic acid mononucleotide pyrophosphorylase from a variety of cell types (6, 15), ATP did not influence the reaction rate of the rlstasia enzyme; in the absence of ATP, nicotinic acid was converted almost quantitatively into deamido-NMN (Fig. I). Enzyme activity was not affected by sonic disruption in the presence of millimolar mercaptoethanol or millimolar reduced glutathione. The initial reaction rate was independent of PP-ribose-P concen- tration from 0.7 to 2.0 mM, and the rate was linear for 3 hours at the higher concentration of PP-ribose-P but for only 1 hour at

the lower concentrat,ion. The K, for nicotinic acid was 5.1 x 10e5 RI at pH 7.4, a value somewhat, larger t,han the K, values of t,hc partially purified nicotinic acid mononucleotide pyrophos- phorylascs of E. coli (16), bovine liver (17), and baker’s yeast (15). The activit,y in vitro of the ,I stasis enzymr ranged from 50 to 120 pmoles of deamido-NMN formed per hour per lo9 cells (equivalent t,o 350 to 600 mfimolea per hour per mg of protein), a rate far in excess of the rate of nicotinic acid incorporation into pyridine nucleotides in viva (I).

Na,kamura, Nishizuka, and Hayaishi (18) observed that t’he rate of deamido-NMN breakdown to nicotinic acid by liver extracts was ATI’-dependent and appeared to be catalyzed by nicotinic acid mononucleotide pyrophosphorylase. Ogasawara and Gholson (19), however, showed that in yeast the nicotinic acid mononucleot,ide pyrophosphorylasc activity was separable from the nicotinic acid mononucleotidase activity. It was important, therefore, to ascertain the rate of hydrolysis of deamido-N&IN in the crude extracts of rlstasiu. Our finding of a very low nicotinic acid mononucleotidase activity (Tables I and II) supports the view that, this activity is independent of the nicotinic acid mononucleotide pyrophosphorylase activity. In the presence of added PP-ribose-I’, the rate of breakdown of deamido-NMN was greatly increased (Tables I and II). The degradation of deamido-NILIS was faster with added pyrophos- phate than with PP.ribose-l’, but was not stimulated by ortho- phosphate, thus suggesting that PP-ribose-1’ increased the rate of dcamido-NRIN breakdown to nicotinic acid because of its own degradation to yield pyrophosphate. Honjo et al. (15) were unable to show the reversibility of the XTP-requiring nicotinic acid mononucleotide pyrophosphorylase of yeast and Imsande and Handler (17) reported that the ,4TP-sensitive enzyme of bovine liver had an equilibrium constant less than 0.01. The reaction as written

Nicotinic acid -j- PP.ribose-P ti deamido-NMM + PP

might, however, be freely reversible when catalyzed by an enzyme not requiring ATP. From the data shown in Tables I and II, it can be seen that the rate of reaction in the forward direction was comparable to the rate of reaction in the reverse direction. At the end of l-hour incubation in the forward direct’ion, theamounts of reactants and products were such that, had t’he reaction reached equilibrium, the value of K would be given by

K = [deamido-NMN] [PPI - 50 x 4500 [nlcotm~c acid] [PP-ribose-PI = 520 X 4450

E+ 0.1

Since the reaction was not at equilibrium, the value of K (at this pH and for these ionic conditiolls) must be greater than 0.1. Similarly, when t,he reaction was run in the reverse direction, it could be computed that the value of K must be greater than 0.2. Furthermore, the inhibitory effect of increasing pyrophosphate concentration on the rate of the forward reaction is consistent with the reaction being reversible.

It has often been observed that end products of a biosynthetic sequence exert feedback inhibition of the first enzyme of the pathway. With respect, to the biosynthesis of NAD, it is known that tryptophan pyrrolase, an enzyme involved in the formation of quinolinic acid, is competitively inhibited by dcamido-NAD and by deamido-NBM (20). Imsande and Handler (17) reported that, deamido-NX% was a competitive inhibitor of bovine liver nicotinic acid mononuclcot’ide pyrophosphorylase,

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and Dietrich and l\Iunoz (21) found that the nicotirramide mononucleotide pyrophosphorylase of rat liver was inhibited by NpllN, NA%D, NADP, 3.acetyl pyridiue nucleotide, thionicotin- amide dinuclcotidc, and tleamido-NXD. The nicotinic acid monouucleot~ide pyrophosphorylase of .lstasia is not inhibited by NA%D or NhDP, but both deamido-NMN and deamido-NAD are inhibitors of this enzyme (Table III). Nicotinamide, :\T- methylnicotinic acid, nicotinuric acid, quinolinic acid, and picolinic acid wvcre weak inhibitors (Table III). The slight inhibition by nicotinamide was entirely accounted for as an isotopic dilution effect resulting from the formation of unlabeled nicotinic acid by the nicotinamidase activity of the cell-free preparation (see below). The possibility that the hydrolysis of deamido-NMN or of dcamido-NAD to nicotinic acid could have led to a similar isotopic dilution effect, was excluded since it was found that the rate of hydrolysis of deamido-NMN (Tables I and II) and deamido-NAD (Fig. 2) proceeded at a rate too low to account, for the inhibition shown in Table III.

The KS value of deamido-NMN as a competitive inhibitor of

Inhibition stwlies on nicolinic acid mononuclenlide

pyrophosphorylase 0,f Axlasia

A summary of the results of several separate experiments is presented. Nicot,inic ncidm7-14C ranged from 50 t,o 500 mpmoles, PP.ribose-I’ from 1.0 to 3.3 prnoles, RlgCls from 1.0 to 5.0 pmoles, Tris-IICl (pII 7.1) from 8.0 t’o 13.0 pmoles, and aliquots of cell- free preparations corresponding to 0.4 to 2.0 X lo6 cells. The rate of nicotinic acid mononucleotide pyrophosphorylase activity in all experiments was linear for at least 45 min and ranged from 30 to 1‘20 pmoles of deamido-NMN per hour per log cells for con- trols. The control activity for each experiment was taken as lOO(;;. The values listed are averages for at least two experiments for each inhibitor.

Addition Concentration

None Deamido-N&IN

Deamido-NAD

NAD

NADP

NhlN 3.Acetgl pyridine-KAD Deamino-NAD Thionicotinnmide-NAD Nicotinamide N-Methglnicotinamide ,VMet,hylnicotinic acid

3-Acetyl pyridine 3-Pyridiue sulfonic acid Nicotinic acid methyl ester Nicotinic acid-o-oxide Nicotinnric acid Quinolinic acid Picolinic acid

0.36 0.72 1.4 0.37 0.50 0.50 5.0 0.5 1.0 3.9 1.0 5.0 0.50 5.0 2.0 0.50 2.0 1 .3 2.8 0.5 2.8 2.8 5.0 5.0

-

--

-

Activity relative to control

YO

100 82 70 55 65 57

100 89 95 95

100 102 90

100 85 90

100 70

100 95

100 95 75 70 80

160

t

Nicotinic Acid

0 40 80 120 160 200 240 Minutes

FIG. 2. Deamido-NAD breakdown in cell-free preparations of Aslasia. Reaction mixtures included 0.39 pmole of deamido- NAD-7-t%, 2.7 Hmoles of MgC12, 12.5 pmoles of Tris-IICI (pH 7.4), 0.5 pmole of nicotinic acid, and 0.3 ml of a cell-free prep- aration corresponding to 0.G X IO6 cells. Labeled deamido- NMN and labeled nicot,inic acid were the only products formed during incubation. The rates of deamido-NAD-7-r4C breakdown under the above conditions or upon the addition of 3.0 Mmoles of PP-ribose-P are represented by clashed and solid lines, respec- tively.

the nicotinic acid mononucleotide l)SrophosphorSlasc of Astasia (with respect to nicotinic acid as substrate) was 2.2 x 10e4 M,

about. 5 times larger than the Ki determined by Imsande and Handler (17) for the bovine liver enzyme. The kinetics of inhibition by deamido-NAD was not studied in detail but the Ki for deamido-NAD is comparable in magnitude to the Ki for deamido-N&IN.

The Nicotinic Acid Adenine Dinucleotide Pyrophosphorylase of Astasia-When cell-free preparations were incubated with nicot,inic acid and PP-ribose-P, deamido-NMN was the only product formed (Fig. I). Upon completion of the reaction, the addition of ATP resulted in the conversion of deamido-NlMN to deamido-NAD. If ATP was included with nicotinic acid and PP- ribose-P at the beginning of an incubation, both deamido-NMN and deamido-NAD were formed, the former at a more rapid rate, as expected if deamido-NMN is a precursor of deamido-NAD (Fig. 1). The activity of nicotinic acid adenine dinucleotide pyrophosphorylase in the same preparation is shown in Fig. 1, Part 3, where the rate of deamido-NAD formation from deamido- NMN and ATP was linear for at least 2 hours.

The K, was 7.5 x 10-j ~1 with respect to deamido-NMN and about 4.0 x 10d4 M with respect to ATP. These values are comparable to the K, values obtained for the partially purified enzyme of E. coli (16).

In similar experiments we found that the properties and specific activities of the nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide pyrophosphorylases of JS. gracilis var. bacillnris, Strain SMLl, were similar to those of the Astasia enzymes.

The stability of deamido-NAD was examined by incubating deamido-NAD-7-14C with cell free preparations of rlstasiu. Deamido-NMiY and nicotinic acid were the only labeled prod- ucts de&ted (Fig. 2). In t,he absence of PP-ribose-P, the rate

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TULLE IV

NAD synthetase activity in cell-free preparations of dstasia

The reaction mixture contained 0.74 pmole of deamido-NAD- 7-l%, 13.3 rmoles of KC1 and of MgC12, and 0.32 ml of cell extract (corresponding to 2.1 X lo6 cells) prepared from cells sonically disrupted in 0.1 M Tris-HCl-1 rnM reduced glutathione (pH 8.2). ATP, (NHd)&Oh, and I,-glutamine were added as indicated.

Additions

/.lVLOlL?S

(NH,),bO y 4 ) 0.8

(NHd)aSO - 4 , 0.8 ATP, 1.8

L-GIut,amine, 1.3 ATP, 1.8

ATP, 1.8

T- NAD synthesized

-

105 min I

240 min

mpmules

None None

0.9 1.4

1.5

0.30

2.3

0.43

TABLE V

Quinolinic acid phosphoribosyl trans,fera.se activity in cell;free preparations of Astasia

Reaction mixtures contained 0.5 pmole of quinolinic acid-6.‘4C, 2.7 pmoles of MgClz, 12.5 pmoles of Tris-HCl (pH 7.4), and 0.15 ml of cell-free extract corresponding to X0,000 cells (120 fig of protein). No radioactive products were detected upon incuba- tion of the reaction mixture itself for times up to 280 min.

Addition

pnzoles

1.0

3.0 3.0 0.2 3.0 1.0

I

Products formed

Deamido-NMS-6-14C Nicotinic acid 6-‘K

of deamido-NAD breakdown was IOK, but in the presence of PP-ribose-P the rate increased considerably, becoming compara-

ble to its rate of synthesis as measured in the same cell free prep- aration. The presence or absence of unlabeled nicotinic acid did not alter the high rate of deamido-NhD dreakdown or the dis- tribution of radioactivity in the products. The increased deamido-EAD cleavage in t’he presence of PEribose-P is thus similar t,o that observed for the cleavage of dcamido-?MN (Table I) and is attributed to the hydrolysis of PP-ribosc-P, forming pyrophosphatc, and t,he pyrophosphorolysis of the deamido-NAD. Pyrophosphorolysis (or hydrolysis) of the deamido-NI\IN thus formed accounts for the production of nicotinic acid from deamido-NAD in the presence of PP- ribose-P (Fig. 2).

Nicotinamide Adenine Dinucleotide Synthetase oj’ A&a&a-The activity of NAD s~nthetasc \yas assayed under conditions sim- ilar to t,hose reported for a partially purified enzyme from E. coli R (22). XAD synthetase activity was much lower (about >ioo) than that of either the nicotinic acid mononucleotide

pyrophosphorylase or the nicotinic acid adenine dinucleotide pyrophosphorylase (Table IV). Some endogenous amide donor was present in the cell-free preparation, but both L-glutamine and ammonium ions could serve as amide donor. The rates of deg- radation and of pyrophoaphorolysis of NAD were comparable to the rates of degradation and pyrophosphorolysis, respectively, of deamido-NAD. The rate of synthesis of NAD in vivo (about 0.1 pmole per hour per log ~11s) was comparable to the act’ivity in vitro of NAD synthesis and may therefore be a rate-limiting step in the synthesis of NAD from nicotinic acid. It should also be noted that the NAD synthetase activity of Astusiu is about 2% of that reported for crude extracts of yeast (3) and about’ 0.2% of that of Escherichia coli (16).

Nicotinamide Adenine Dinucleotide Kinase in Cell-free Prepara- tions of Astasia-NAD kinme was assayed by the synthesis of labeled NADP from labeled SAD. Reaction mixt,ures of 1 ml contained 5.2 pmoles of r\TAD-7-W, 12.5 pmoles of Tris-WC1 (pH 7.4), 2.7 pmoles of MgC$, 4.0 pmoles of ATP, and 0.2 ml of cell-free preparation (corresponding to 1.2 x 10” cells). The rate of synthesis of NADP was about 130 pmoles per hour per log cells, i.e. about the same as the activity of the nicotinic acid mononucleotide pyrophosphorylase. The synthesis of ShDP required ATP.

Quinolinic Acid Phosphoribosy2 Transferase Activity in Cell-free Preparations of Astasia-The PP-ribose-l-dependent, XTP- independent conversion of quinolinic arid to deamido-NM?: has recently been shown in several species (23-27). In some cases, both nicotinic acid mononucleot,ide pyrophosphorylase activity and quinolinic acid phosphoribosgl transferase act’irity were demonstrable in the cell-free preparations (24) but in other cases only the latter activity was detected (23, 25), and nicot’inic acid accumulated in the medium resulting from the hydrolysis of EAD (27). As shown in Table V, dstasia has quinolinic acid phosphoribosyl transferase activity. Generally the rate i:: linear up to 4 hours of incubation and is about half t,hat of the nicot’inic acid mononucleotide pyrophosphorylase. The activity in vitro of this enzyme in Astasia is much higher than that reported for cell-free preparations of Jl~cobacterium (25) but much lorr-er than that of Pseudomonas (26).

The amount of labeled nicotinic acid produced from quinolinic acid-6-14C (with or without added I’P-ribose-1’) was too small for nicotinic acid to have been an intermediate in t,he conversion of quinolinic acid to deamido-NM?\’ (Table V), in agreement wit,h the conclusions of other workers (23). Furthermore, the addi- tion of 0.2 pmole of unlabclcd nicotinic acid did not alter the rat,e of production of deamido-N1\1ru’-6-%, whereas if nicotinic acid had been an intermediate a large apparent inhibition would have been expected. Thus, the decreased rate of dcamido-X11X6- 14C formation observed in the prcscnce of 1 mM nicotinic acid indicates an inhibition of t,he quinolillic acid phosphoribosy1 transferase, in contrast to the bovine liver enzyme, in which nicotinic acid did not inhibit the conversion of quinolinic acid to

deamido-NMN (24). Other Enzymes dctive toward Pgridine-containing Compounds in

Aslasia-In t,he absence of PI’-ribose-I’, nicotinamide is dcam- idated quantitatively to nicotjinic acid (Fig. 3) at a rate compara- ble to the rate of dcamido-NJIN synthesis by nicotinic acid mo~~onuclcotidc (~~-rol~h(lh(~hollc-lrtje. The rate of dparni- dation was not inhibited by 0.5 mRI nicotinic acid, in agreement with the properties of the nicot,inamidasc of To&a cremonis (28). The I<, value for the nicotinarnidasc of Sstasia was 0.5 m&f,

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larger than the values of 1.4 x 10e5 35 for T. cremonis (28) and 1 .O x 1O-5 M for Ehrlich ascites cells (29)) but much smaller than the value of 0.1 M observed for the normal liver enzyme (29).

Nicotinamide mononucleotide pyrophosphorylase, an enzyme implicated in NAD biosynthesis in some species (30), was not demonstrable in cell-free preparations of Astasiu (Fig. 3). The only product detected after incubating labeled nicotinamide and PP-ribose-P was deamido-NMN, arising from the joint activity of the nicotinamidase and nicotinic acid mononucleotide pyro- phosphorylase. From several experiments like that of Fig. 3 it was concluded that the rate of formation of deamido-NMN was limited by the rate of deamidation of nicotinamide.

No evidence was obtained for the conversion of nicotinic acid or nicotinamide to N-ribosylnicotinic acid or N-ribosylnicotin- amide, as would have been expected if ~stasia had an enzyme comparable to the nucleoside phosphorylase of yeast (31).

The possible synthesis of A7-methylnicotinic acid and N-methyl nicotinamide was tested umder conditions similar to those de- scribed by Joshi and Handler (32) and Waller et al. (33). A slow rat’e of synthesis of N-methylnicotinic acid (about 5 Fmoles per hour per 10” cells) was det,ected in the presence of X-adenosyl methionine, but the formation of 5-methylnicotinamide from nicotinamide was not observed.

Exchange reactions, catalyzing (a) NAD $ nicotinic acid-7- 14C * deamido-NSD-7-14C + nicotinamide and (b) deamido- AXiT-7-14C + deamido-NAD pi deamido-NAD-7-14C + deamido-NMN, were also detected in cell-free preparat,ions of Astasia, amounting to about one-tenth, and one-fifth of the activity of nicotinic acid mononuclcotide pyrophosphorylase, for a, and b, respectively. The rates of these reactions are about the same as the rates of NAD degradation and of deamido-NMN and deamido-NAD degradations described above, respectively. Exchange Reaction a has been described for bovine spleen NAD nucleosidase (34). The presence of a similar enzyme in Astasia might account for both Reactions a and 6.

Effect of Growth Conditions on Enzyme Levels-The growth rate of Astasia was not altered by exogenous nicotinic acid up to 0.2

rn~, but was inhibited at higher concentrat’ions. The inhibition was greatest during the first 24 to 28 hours of exposure (Table VI). It has also been reported that 0.8 m&f nicotinic acid inhibits the growth of E. gracilis (35). Nicotinamide completely inhibited the growth of Astasia at 0.16 mar. Pyridine 3-sulfonic acid at 1.5 mlcI does not inhibit’ the growth of Euglena (35) and did not

inhibit the growth of dstasia at 1.5 m&f. Azaserine, an inhibitor of amidation reactions involving glutamine (3), did not inhibit the growth of Astasia at 2.3 mnl. 3-Acetyl pyridine, however, was a potent inhibitor of growth (Table VI), as might be expected from its ability to form inhibitory analogues of NrlD in other systems (36). Growth was not’ inhibited by tryptophan or quinolinic acid at 1.5 m&l, but quinolinic acid at 12 mv caused growth inhibition.

In the experiments shown in Fig. 4, cells were exposed to comparable concentrat’ions of nicotinic acid, nicot,inamide, or quinolinic acid. The uptake of quinolinic acid was about one- tenth the uptake of nicot’inic acid or nicotinamide, although the cells exposed to quinolinic acid were growing at the same rate as cells growing in nicotinic acid. The rates of uptake of nicot,inic acid and nicotinamide during the first 20 hours of exposure were 145 nmmoles per hour per log cells and 120 mpmoles per hour per log cells, respectively, with nicotinamide exerting a much more pronounced inhibition of growth than nicotinic acid. After

200-

0 20 40 60 00 100 120 140 IE Minutes

FIG. 3. Nicotinamidase activity in cell-free preparations of Rstasia. The reaction mixtures contained 0.5 pmole of nico- tinamide-7-W, 1.1 pmoles of MgCl,, 1.5 pmoles of Tris-KC1 (pH 7.4), and 0.2 ml of a cell-free preparation (corresponding to 0.58 X lo6 cells and containing 156 pg of protein). In addition, 2.0 pmoles of PP.ribose-P were added to reaction mixture A.

TABLE VI

Effect of nicotinic acid and related compounds ore growth and enzyme levels of Astasia

Astasia were grown in Erlenmeyer flasks. Compounds were added at zero time at the indicated concentrations. T1 is the time required for the population to increase from its initial cell density of N, cells per ml to 2 No and 7’~ is the time required for the population to increase from 2 No to 4 No. After about 7%hour exposure to the compound, cells were harvested, washed, and sonically disrupted, and the enzymatic activities were assayed. The assay conditions for Experiment A were: 40 mrmoles of nicotinic acid-7-‘G, 1 ,umole of MgClz, G.2 pmoles of Tris-HCl (pH 7.4), 0.8 pmole of PP-ribose-P, and 28.8 Mg of protein; for Experiment B, 0.5 pmole of nicotinic acid-7-l% or quinolinic acid-&‘%, 5.4 Fmoles of h’IgC12, 12.5 pmoles of Tris-HCl (pH 7.4), 3.0 pmoles of PP-ribose-P, and from 19 to 46 ,~g of protein. ND, not determined.

Experi- ment no.

A

B

-

I-

Addition to growth medium

GXKUI- tration of addition

?jone

Nicotinic acid 0.2 NOIlC

Nicotinic acid 0.8 Nicotinic acid 1.6 Nicotinamide 0.16 3.A&y1 0.0084

pyridine Quinolinic acid 12 .O

Doubling times Enzyme levels

T I 2-z

hrs

12 12 266.0 ND 12 12 266.0 NO

9 9 635.0 238.0 14 14 NH ND 39 25 841.0 278.0

150 95.0 63.0 150 411.0 ND

16 16 825.0 397.0 -

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1454 Studies o,f Nicotinic Acid Metabolism. II Vol. 243, No. 7

7 Nicotinic Acid

I I I 0 20 40 60 1 9(

0.6

FIG. 4. Uptake of nicotinic acid, nicotinamide, and quin- olinic acid by dstasia. Cells grew in the presence of 1.6 mix nicotinic acid-7.‘“C, 1.6 mix nicotinamide-7-14C, or 1.2 InM

quinolinic acid-6-l% (specific act,ivities, 8.2 X 104 cpm per pmole, 5.5 X IO4 cpm per pmole, and 3.2 X lo4 cRrn per @mole, respec- tively). Total radioactivity taken up bp the cells was determined after- 1,22,45, and 70 hours of exposure to the labeled compound. The cells rrew at & doubling time of about 30 hours in the Dresence of nicotin& acid or quinolinic acid, but growth was inhibrted 95% in the presence of the nicotinamide.

about 40 hours, there was a decrease in the amount of label in the cells exposed to nicotinic acid or nicotinamide. Bliquots of the supernatant were concentrat’ed by lyophilization and tested by paper chromatogral)hic techniques for leakage of radioactive pyridine nucleotides into the medium. Ko evidence of such leak- age was detected in dstasia, although such leakage had been re- ported for yeast (37). It is possible, ho!\-ever, that nicotinic acid and nicotinamide did leak out of the cells after 40 hours of growth.

Since cell-free preparations of Astasiu have a nicotinamidase activity of about 120 pmoles of nicotinamide deamidated per hour per log cells, some of the nicotinamide enterilng t’he cells may be converted to nicotinic acid. The stronger inhibition of growth by nicotinamidc than by nicotinic acid, however, suggests that nicotinamide per se inhibits growth. The reasons for the in- hibition of growth caused by high nicotinic acid and for the net, decrease in label in the cells after 40 hours of exposure to labeled nicotinic acid or nicotinamide are not known.

Since nicotinic acid mononucleotide pyrophosphoryla,se is the first enzyme in the biosynthetic pat,hway from nicotinic acid to the pyridine nucleotides, its possible repression by exogenous nicotinic acid was investigated. Cells were grown in the presence of nicotinic acid for several generations and the levels of nicotinic acid mononucleotide pyrophosphorylase and of quinolinic acid phosphoribosyl transferase were assayed. At 0.2 mM nicotinic acid, a concentration which does not, inhibit growth, there was no effect on the activity of nicotinic acid mononucleotide pyrophos- phorylase (Table VI). At 1.6 rnnx nicotinic acid, at which growth inhibition occurs, there was a xma,ll incrra,se in the activity of this enzyme. Quinolinic acid also increased the activity of this en- zyme The growth rate of cells exposed to 0.16 mM nicotinamide or 8.4 PM N-acetyl pyridine v-as strongly inhibited, but the level of nicot,inic acid mononuclrotide r)~rophosI)horylase was greatly

reduced only in the cells exposed to the nicotinamide (Table VI). The effects of nicotinamide, quinolinic acid, and nicotinic acid on the levels of quinolinic acid phosphoribosyl transferase were similar to the effects of these compounds on the levels of nicotinic acid mononucleotide pyrol)hosI)hoi~lase. Thus neither quinolinic nor nicotinic acid represses the formation of either of the two enzymes initiating the biosynthesis of KAD from these precursors. Theae results further support, t,he view that nico- tinamide exerts its inhibitory effect on growth prior to its de- amidation to nicotinic acid and could suggest a simple esplana- tion for the inhibitory effect of nicotinamide on growth.

DISCUSSION

Evidence suggesting the operation of a pyridine nucleotide cycle in biological systems has recently been reviewed (38). Bccording to this concept, quinolinic acid, synthesized from either tryptophan or from glycerol and aspartate, reacts mit,h PP- ribose-P to yield deamido-NMN, the first pyridine nucleotide intermediate in the biosynthesis of KSD and n’*2DP. In organisms that are not’ genetically blocked, nicot,inic acid is as- sumed to arise from NLLD via rticotinamide, and the nicotinic acid is “salvaged” by the activity of nicotinic acid mononucleo- tide pyrophosphorylase. The data pres&ed in this paper in- dicate that such a cycle could be operat,ire in Astasia, all or- ganism which does not’ require exogenous nicotinic acid or related compounds fur gun-th.

The finding that ;lstasiu possesses the cnzymc activities re- quired to synthesize deamido-NMN, deamido~KA11, NAD, and NADP supports the inference from isotol)ic labeling rsperiment~s (I) that the biosynthetic pat,hway for the synthesis of NAD and NADP is the same as that established for a \:arietJ- of other or- ganisms (38).

130th quinolinic acid phosphoribosyl transferase and nicotinic acid mononucleotide pyrophoaphorylase act,ivitics were shown in Astasiu, and at comparable activities. Furthermore, nicotinic acid was not an intermediate in the conversion of quinolinic acid to deamido-KMN. It is noteworthy t,hat quinolinic acid slightly inhibited nicotinic acid mononucleotidc pyrophosphorylase activity and that nicotinic acid inhibited quinolinic acid phos- phoribosyl transferase activity. Such reciprocal inhibition may indicate a physiological control of the relative rai es of t,he salvage pathways as compared to the synthesis pathway de nova.

The nicotinic acid mononucleotide pyrophosphorylase of Astusiu does not require ATP (14) and is not, inhibited by NXD or by NADP at concentrations higher than those likely to be present in situ (12). Preparations of this enzyme from several cell t’ypes, for example, E. coli and B. subtilis (6), are also not inhibited by NAT1 and by KADP. In llstnsia, however, deami- do-NMN and deamido-NAD are effective inhibitors of t,hi:: en- zyme, with Ki values of about 2 x lo-* M. Although the con- centration of deamido-KMN in &tasia is much too small for it to inhibit the act,ivity in viva (I), the concentration of deamido- NAD in Astasiu is about 8 X 10-s M (1) and thus could exert effective inhibition of this enzyme.

The rates of deamido-NMN synthesis from either nicotinic acid or quirtolinic acid in czell-free preparations were much greater than the rate of KAD biosynthesis in vivo even when nicotinic acid was supplied to the growth medium. These ob- servations suggest that the activities of nicotinic acid mono- nucleot,ide I’~ro~)hos~)hor~~lase and of quinolinir acid phosphori- hosyl transferase are not rate-limiting with respect to thr

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Issue of April 10, 1968 V. Kahn a,nd J. J. Blum 1455

biosynthesis of NAD. Consistent with this view are the lack of repression of either of these enzyme activities by growth in the presence of either quinolinic acid or nicotinic acid, although re- pression-depression control of the level of nicotinic acid mono- nucleotide pyrophosphorylase has been reported in E. coli (5).

Under the conditions tested, the activities of quinolinic acid phosphoribosyl transferase, nicotinic acid adcnine dinuclcotide pyroyhosphorylase, and NAD kinase were from 50 to 130 pmoles of product formed per hour per 10y cells, while the activity of N-LD synthetase was over IOO-fold smaller. The low activity in vitro of NAD synthetSase is comparable to the rate of N,4D biosynthesis of living dstasia cells and is in agreement with the mn\lch higher concentration of deamido-NAD than of deamido- T\‘hIN in Astasia (I). It appears that NAD synthetase may be the rate-limiting step in the biosynthesis of NAD (from nicotinic or quinolinic acid precursors) in &c&a, a situation different from that found in E. coli (5) or B. subtilis (4).

Nicotinamide, N>‘IN, and the N-methyl deriva,tives of both nicotinic acid and nicotinamide are present in Astasia grown in the presence of nicotinic acid (I). Some enzymatic reactions which could lead to the formation of these compounds have been studied in cell-free preparations. Gholson (38) has suggested that nicotinamide arises only from the degradation of NAD and that the rate of NADase activit,y is importan; for the control of cell metabolism. NADase has also been implicated in the control of NAD levels in Ehrlich ascites tumor cells (39). In cell-fret preparations of Astnsia, about 20 pmoles of nicotinamide were formed from NAD per hour per log cells, a value much greater than the rate of formation in viva of nicotinamide plus A7-methyl- nicotinamide in Astasia growing in the presence of nicotinic acid (1). Cell-free extracts of Astasia also catalyze a rapid pyrophos- phorolysis of NAD and of deamido-NAD, at about equal rates, a result compatible with the observations of Imsande and Handler (40) and of Dahmen, Webb, and Preiss (41) that the pyrophosphorolysis of NAD and deamido-NAD may be cata- lyzed by the same enzyme. Thus the observed rates of degrada- tion of NAD in vitro could account for the rate of appearance in viwo of N&1X and nicotinamide, and could supply ample pre- cursors of AT-methyl derivatives of nicotinamide and nicotinic acid.

N-I\Iethg-hlicatinic acid aud hr-mcthyh~icotinamide may be considered by-producls of the pyridine nucleotide cycle, arising from nicotinic acid and nicotinamide, respectively, and a suitable methyl donor (33). The rate of N-methylnicotinic acid synthesis by cell-free extracts of nstasia, although smaller than the rate of synthesis in viva of .%mcthylnicotinic acid, is comparable to the rate observed in pea seedlin, cr extracts by Joshi and Handler (32) and in cell-free extracts of Ricinus corr~munis L by Waller ef al. (33). In the former case, as with dstasia, no synthesis of N- methylnicotinamide could be shown.

The demonstration of an active nicotinamidase activity in cell-free extracts of Astasia and our inability to show nicotinamide mononucleotide pyrophosphorylase activiby in such extracts argurs against the possibility that nicotinamide could be a pre- cursor of NAD by way of NllN, a pathway of NAD biosynthesis that has been suggested for some cells (30). The presence of a hivh nicotinamidase activity in Astasia strengthens the concept

thlt a pyridinc nllcleotide cycle is operative in this organism (38). The mechanisms by which quinolinic acid and nicotinic acid

inhibit the growth of -1stasia are not, clear. Although neither substance represses the deamido-N&IN-forming enzymes, @no-

linic acid, by the reciprocal inhibition mentioned above, could interfere with the operation of the salvage pathway, perhaps leading to the intracellular accumulation of nicot,inic acid, which inhibits the growth of ;Istasia. The inhibitory effect of nicotinic acid on growth might be explained if quinolinic acid, accumulated because of the inhibition of the quinolinic acid phosphoribosyl transferase, inhibited some step of its biosynthetic pathway which was involved in ot,her aspects of cell metabolism. High aoncen- trations of nicotinic acid are known lo inhibit the formation of NAD in liver (42) but this cannot be the cause of growth in- hibition in Astasia, since concentrations of nicot8inic acid which inhibit growth increase the cell content of NAD (1).

It has been reported that nicotinamide slows cell division in regenerating rat liver and in mammalian cells in tissue culture (43). The observation that nicotinamide is a strong inhibitor of the growth of &asia is therefore not surprising. It is of interest, however, that nicot,inamide represses the level of both enzymes which form deamido-NMN, whereas nicotinic acid represses ncithcr of these enzymes. A possible function of the pyridine nucleotide cycle suggested by these observations is to regulate the level of nicotinic acid mononucleotide pyrophosphorylase and quinolinic acid phosphoribosyl transferase by controlling the intracellular concentration of nicotinamidr.

Acknowledgments-The authors are indebted to Miss Delores Randolph and Mrs. Barbara Kerton for rxcellent technical assist- ance at various stages of this work.

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Varda Kahn and J. J. BlumBIOSYNTHESIS IN CELL-FREE PREPARATIONS

REGULATION OF NICOTINAMIDE ADENINE DINUCLEOTIDE : II. PATHWAY ANDAstasia longaStudies of Nicotinic Acid Metabolism in

1968, 243:1448-1456.J. Biol. Chem. 

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