ENZYMATIC SYNTHESIS OF NICOTINAMIDE MONONUCLEOTIDE*

BY JACK PREISSt AND PHILIP HANDLER

(From the Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina)

(Received for publication, October 20, 1956)

Previous reports from this laboratory have described synthesis of NMNI from nicotinamide by human erythrocytes (1) and hemolysates (2) in vitro. These studies have been extended (3), t’he reactions have been identified, and the responsible enzymes have been partially purified.

EXPERIMENTAL

Materials-ATP, GMP, and DPN were obtained from the Pabst Labo- ratories, adenine and erotic acid from the California Foundation for Biochemical Research, guanosine, inosine, ribose, and hypoxanthine from the Nutritional Biochemicals Corporation, and guanine hydrochloride hydrate from the Distillation Products Industries. IMP and R5P were obtained as barium salts from the Schwarz Laboratories, Inc., and con- verted to their sodium salts by treatment with Na2S04 in dilute acid solu- tion. NMN was prepared from DPN by the procedure of Plaut and Plaut (4) by using potato pyrophosphatase (5). PRPP2 was prepared by the method of Kornberg et al. (6) and purified by the procedure of Remy, Remy, and Buchanan (7). Nicotinamide riboside was prepared by hy- drolyzing NMN with snake venom (8).

MethodsHemolysates were prepared by alternately freezing and thawing freshly collected erythrocytes which had been washed four times in 0.15 M NaCl. 1 volume of 0.5 M buffer (phosphate or Tris), pH 7.4,

* These studies were supported in part by Contract AT-(40.l)-289 between Duke University and the United States Atomic Energy Commission and by Grant RG-91 from the National Institutes of Health.

The data herein are taken from a dissertation by Jack Preiss presented to the Graduate School of Duke University in partial fulfilment of the requirements for the degree of Doctor of Philosophy.

t Predoctoral Fellow of the National Institutes of Health. 1 The following abbreviations are employed: nicotinamide mononucleotide, NMN;

nicotinamide, NAm; nicotinamide riboside, NR; 5-phosphoribosyl 1-pyrophosphate, PRPP; inorganic orthophosphate, Pi; inorganic pyrophosphate, P-P;; ribose 5-phos- phate, R5P; adenosine triphosphate, ATP; diphosphopyridine nucleotide, DPN; adenosine 5’-phosphate, AMP; guanosine 5’-phosphate, GMP; inosine 5’-phosphate, IMP; tris(hydroxymethyl)aminomethane, Tris.

2 An initial sample of PRPP was generously provided by Dr. Arthur Hornberg.

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760 NICOTINAMIDE MONONUCLEOTIDE SYNTHESIS

per 5 volumes of red cells was added and the stroma were removed at 5’ by centrifugation at 3000 r.p.m.

Acetone powder was prepared by homogenizing washed erythrocytes in 5 volumes of acetone at -12”. The insoluble material was collected on a Biichner funnel, twice rehomogenized in acetone, and dried in air. Such powders retained activity for at least 6 months when stored at -10”. Suitable extracts were prepared by stirring 2 gm. of powder with 10 ml. of 0.1 M buffer (phosphate or Tris), pH 7.4, for 10 minutes at 3”. Insoluble material was removed by centrifugation at 8000 r.p.m.

DPN was assayed with alcohol dehydrogenase, essentially by Racker’s procedure (9). NMN was determined by conversion to DPN with DPN pyrophosphorylase (10) and calculated as the increment in DPN due to addition of this enzyme. Total pyridine nucleotides were occasionally estimated fluorometrically (11, 12). Hypoxanthine was determined spec- trophotometrically with xanthine oxidase (13) and AMP with adenylic deaminase according to Kalckar (14). Guanine was assayed at 290 rnp with xanthine oxidase and rat liver guanase (13). PRPP was measured at 295 rnp with erotic pyrophosphorylase plus erotic acid (6). Nucleoside phosphorylase activities were assayed according to Huennekens et al. (15). IMP and GMP were determined spectrophotometrically after column chromatography. Hemoglobin was assayed by the procedure of Wong (16) and total protein according to Warburg and Christian (17). Enzy- matic reactions were stopped by placing the reaction vessel in a water bath at 100” for 2 minutes.

NMN Synthesis by Hemolysates-Table I shows the synthesis of NMN by hemolysates. The substrate mixture NAm + R5P + ATP was con- sistently about 3 times as effective as the mixture NR + ATP. The presence of DPNase necessitated a large concentration of NAm in these incubations and it was not apparent whether NAm or the riboside was the precursor for NMN. However, the higher activity of the R5P-con- taining system suggested that a pathway other than phosphorylation of the riboside was operative. An absolute dependence on Pi was apparent in all experiments, whereas the effect of arsenate was inconsistent. Occa- sional hemolysates exhibited no activity when arsenate was used in place of phosphate and it appears possible that the arsenate effect may reflect arsenolysis of endogenous phosphate esters to produce a suboptimal con- centration of Pi.

NMN Synthesis by Erythrocyte Acetone Powder-Extracts of acetone powder, prepared as described above, were 6 to 7 times as active as an equivalent volume of hemolysate. More than 95 per cent of the pyridine nucleotide synthesized with the substrate mixture of NAm + R5P + ATP proved to be NMN. Synthesis proceeded almost linearly for 20 hours

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J. PREISS AND P. HANDLER 761

when 0.4 pmole of NMN had accumulated per 0.5 ml. of extract. Table II shows that NMN synthesis by such extracts is dependent upon Pi,

Mg++, and R5P and is stimulated by, but not absolutely dependent upon, ATP. Maximal activity was obtained at 0.005 M Pi.

TABLE I

NMN Synthesis by Hemolysates

The reaction mixtures contained 0.5 ml. of hemolysate, 164 pmoles of NAm, 10 pmoles of Mg++, 4 rmoles of ATP, and the ribose source shown in a total volume of 1.0 ml. of the indicated 0.05 M buffer. The reaction vessels were maintained at 35” for 16 hours with occasional shaking.

Substrate

Ribose 5-phosphate, 2 pmoles. .............. Nicotinamide riboside, 0.45 pmole. .........

NMN synthesized

Phosphate

pm&

0.079 0.026

Tris Arsenate

Jmolc ptfZ&

0.008 0.020 0.000 0.012

TABLE II

NMN Synthesis by Extract of Acetone-Powdered Erythrocytes

The complete system, containing NAm 164 pmoles, R5P 2 pmoles, ATP 4 pmoles, Pi 50 pmoles, Mg ++ 5 pmoles, and extract of acetone-powdered human erythrocytes 0.5 ml. in a total volume of 1.0 ml., was maintained at 35” for 5 hours.

System I NMN I system I NMN

Complete NO Pi 5 rmoles Pi 40 “ “

0.146 No Mg++ 0.013* 0.021 “ R5P 0.036 0.124 ” ATP 0. lost 0.142

* At zero time the system contained 0.021 pmole of NMN and in the absence of Mg++ 0.008 pmole of NMN disappeared.

t The effect of ATP varied in individual experiments; occasionally in its absence NMN synthesis was limited to about half that of the complete system.

Pathway of NMN SynthesisThe absolute dependence upon inorganic phosphate and the superiority of NAm + R5P as substrate mixture, as compared with NR + ATP, suggest that NMN synthesis proceeds by a pathway other than direct phosphorylation of the riboside, although Rowen and Kornberg (8) have reported such direct phosphorylation in liver preparations. In an effort to elucidate the pathway of NMN syn- thesis and the role of Pi, Pi3’ was included in a reaction mixture in which NMN was synthesized by an extract of acetone-powdered erythrocytes.

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762 NICOTINAMIDE MONONUCLEOTIDE SYNTHESIS

After incubation, 2.5 pmoles of NMN and 5 pmoles of P-Pi were added as carriers and the deproteinized mixture was placed on a Dowex 2 column. NMN, ATP, and P-Pi were separated by the procedure of Deutsch and Nielsen (18) ; NMN and NAm were eluted from the column with water. Each of these was determined in the usual manner and aliquots were con- centrated, evaporated to dryness, and counted. As shown in Table III, the acid-labile phosphates of ATP completely equilibrated with the Pi during the incubation, as did P-Pi, whereas the specific activity of NMN was less than 7 per cent of that of the Pi.

Thus, despite the absolute dependence upon Pi, the phosphate moiety of NMN does not arise from the Pi of the medium in the system here em-

TABLE III

NMN Synthesis in Presence of Pia2

The reaction mixture, containing extract of acetone-powdered erythrocytes 1.0 ml., NAm 328 pmoles, R5P 4 pmoles, ATP 8 pmoles, Tris, pH 7.4, 100 pmoles, NaF 40 pmoles, and Pi 10 rmoles (5.76 X 106 c.p.m.) in a total volume of 2.0 ml., was incubated at 35’ for 17 hours.

system I Fraction Spedic activity Equilibration

Complete No R5P Complete No R5P Complete No R5P

ATP ‘I

P-Pi I‘

NMN “

C.#.rn. per pmole #e7 CfmL

3.9 x 106 93 3.8 x 106 91 4.2 X lo6 100 3.8 x 106 91 2.7 X 103 7 2.5 X lo3 6

ployed. Further, NMN could not have arisen by direct phosphorylation of the riboside as shown:

ATP + NR ---f NMN + ADP (1)

Klenow (19) has suggested nucleotide formation by reaction between a base and ribose 1,5-diphosphate. In this case the reaction would be

NAm + ribose 1,5-diphosphate + NMN + Pi (2)

Ribose 1,8diphosphate was thought to arise by the concerted action of phosphoglucokinase, which yields glucose 1,6-diphosphate from ATP and glucose l-phosphate (20), and phosphoglucomutase, which yields ribose 1,5-diphosphate from glucose 1,6-diphosphate and ribose l-phos- phate and also catalyzes a phosphoribomutase reaction (21). In such a system the rates of the phosphoglucokinase and hexokinase reactions would determine the specific activity of the phosphate of glucose and ribose mono- and diphosphates, all of which would be in isotopic equilibrium. Although

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J. PREISS AND P. HANDLER 763

this reaction sequence could explain the presence of P32 in the R5P moiety of NMN, the data do not permit a decision as to whether reaction (2) is operative or whether NMN is formed by the reaction

NAm + PRPP -+ NMN + P-Pi (31

in analogy with the systems described for synthesis of pyrimidine and purine nucleotides (22-26). However, the low specific activity of the phosphate of NMN rendered it relatively unlikely that the reactions lead- ing to ribose 1,5-diphosphate formation were proceeding rapidly enough to account for NMN production.

TABLE IV Synthesis of PRPP by Erythrocyte Preparations

The reaction mixture, containing enzyme source 0.5 ml., R5P 4 pmoles, ATP 8 pmoles, and Mg++ 10 rmoles in a total volume of 1.0 ml. of the indicated 0.05 M buffer, pH 7.4, was incubated for 6 hours at 35”.

Enzyme

Hemolysate “

Acetone powder “ “ “ “ “ “ “ “ “ I‘

Buffer

Pi Tris Pi “ “ “ Tris

‘I

system PRPP

Complete “ “

No Mg++ “ R5P “ ATP

Complete +5 pmoles Pi

pmolc 0.491 0.110* 0.752 0.110* 0.060* 0.522 0.033* 0.485

* The PRPP content of the enzyme sources at zero time varied from 0.120 to 0.160 pmole. In all instances in which NMN synthesis was ineffective there was a net loss of PRPP during incubation.

PRPP Synthesis by Erythrocyte Preparations-Table IV demonstrates the ability of erythrocyte hemolysates and acetone powder extracts to synthesize PRPP. Synthesis required Pi, Mg++, and R5P. The excel- lent synthesis in the absence of added ATP presumably indicates the existence of a system capable of generating ATP from R5P. PRPP syn- thesis was linear for about 4 hours; thereafter PRPP remained constant in concentration and was equivalent to about 8 per cent of the initial R5P. The velocity of PRPP synthesis, therefore, was 3 to 4 times tha’ of maximal NMN synthesis observed in these preparations.

NMN Synthesis from PRPP-Table V shows that both hemolysates and acetone powder extracts efficiently utilized PRPP for NMN synthesis. This reaction was Mg++-dependent but essentially unaffected by the absence of Pi. Thus, the Pi requirement originally noted is a property of

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764 NICOTINAMIDE MONONUCLEOTIDE SYNTHESIS

the PRPP-synthesizing system, in agreement with the observations of Korn et al. (26), although the role of Pi in this regard is obscure.

Purification of NMN-Synthesizing Enzyme--The standard assay system employed in following the course of enzyme purification was as follows: 0.5 ml. of enzyme solution was incubated for 5 hours with 164 kmoles of NAm, 0.3 pmole of PRPP, and 5 pmoles of MgClz at pH 7.4 in a total volume of 1.0 ml. NMN was determined in the usual manner.

60 gm. of acetone powder were extracted with 300 ml. of 0.1 M Pi, pH 7.4, for 30 minutes and the insoluble material was discarded. Solid ammonium sulfate was added to 0.46 saturation. After 10 minutes the precipitate was separated by centrifugation and redissolved in a minimal

TABLE V NMN Synthesis from PRPP by Erythrocyte Preparations

The complete system contained NAm 164 pmoles, PRPP 0.36 rmole, Mg++ 5 &moles, and hemolysate or extract of acetone power 0.5 ml. in a total volume of 1.0 ml. of the indicated 0.1 M buffer, pH 7.4. Incubations at 35” were performed for 18 hours with hemolysate and for 5 hours with acetone powder.

EllZylll‘Z I

BllfflX I

NMN

Hemolysate

Acetone powder

Pi Tris Pi Tris

pWdt?

0.063* 0.070 0.092* 0.114t

* The enzyme source provided 0.010 to 0.015 pmole of NMN. t When MC’ was omitted. the final NMN concentration was 0.018 Nmole.

volume of buffer and the precipitation was repeated twice. The final solution was dialyzed overnight against phosphate buffer and then placed in a water bath at 73” and allowed to come to 70”. After 5 minutes at this temperature, the solution was cooled and the insoluble protein was removed by centrifugation and discarded. The supernatant solution, designated “partially purified enzyme” (PPE), was used in the studies described below. PPE was 36.7 times as active as the initial extract and 53 per cent of the initial activity remained at this stage. No activity was lost during the heat treatment which effected a 5-fold purification.

In Table VI the ability of various fractions to synthesize NMN either from R5P + ATP + NAm or NAm + PRPP and also the synthesis of PRPP from R5P + ATP is compared. It will be seen that the heat treat- ment, which effected 5-fold purification of the NMN-synthesizing enzyme, completely inactivated the PRPP-synthesizing system. Since NMN synthesis was then possible only with PRPP and NAm as the substrate

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J. PREISS AND P. HANDLER 765

mixture, this clearly establishes Reaction 3 as the synthetic path in erythro- cytes.

Whereas NMN synthesis from R5P in cruder preparations was 60 per cent inhibited by 0.02 M NaF, synthesis by PPE was unaffected thereby. The effect of fluoride, then, was related entirely to PRPP synthesis, which was found to be 60 to 70 per cent inhibited at the same concentration. The pH optimum for PPE was between 6.8 and 7.6; at pH 6.1 or 8.5, the reac- tion rate was half maximal. Studies of NMN synthesis at varying con- centrations of NAm revealed that K, for NAm is of the order of 0.1 M

and for Mg++ about 0.6 X 1O-4 M. Much of the initial DPNase activity of hemolysates was lost in the preparation of acetone powder and PPE was devoid of such activity nor did it exhibit inorganic pyrophosphatase

TABLE VI

NMN Synthesis by Partially Purified Enzyme Fractions

Enzyme*

NMN synthesis from PRPP

synthesis from

PRPP f NAm NA&)T;sp RsP + Al’P

p?Wllle pmole pm&r

Initial extract.............................. 0.080 0.140 0.30 After ammonium sulfate treatment. 0.184 0.340 1.07

“ heat treatment at 65” for 10 min.. . 0.190 0.020 0.00 “ “ I‘ “ 70” “ 5 “ 0.190 0.007 0.00

* The protein present in each incubation was equivalent to the total protein of 0.5 ml. of extract of acetone powder.

activity. Adenosine, inosine, and guanosine phosphorylase activities were observed in extracts of acetone powder, but, of these, only the latter two were found in PPE.

Enzyme Specijicity-In the course of studies of the specificity of the NMN-synthesizing enzyme it was observed that relatively low concentra- tions of purines and their ribosides were markedly inhibitory of synthesis by hemolysates and extracts of acetone powder (Table VII). Since these preparations exhibit phosphorylase activity for the ribosides of adenine, guanine, and hypoxanthine, it appeared likely that the latter were cleaved to ribose l-phosphate and the respective purines which then competed with NAm for PRPP. Extracts of acetone powder were found to couple rapidly each of the three purines studied with PRPP to form the corresponding nucleotides. However, as shown in Table VIII, PPE retained inosinic and guanylic pyrophosphorylase activity, but adenylic pyrophosphorylase was lost in the purification procedure.

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766 NICOTINAMIDE MONONUCLEOTIDE SYNTHESIS

TABLE VII

Purine Inhibition of NMN Synthesis

The reaction mixture contained extract of acetone-powdered erythrocytes 0.5 ml., NAm 164 pmoles, Mg+f 5 pmoles and either PRPP 0.3 rmole or R5P 2 pmoles $ ATP 4 pmoles in a total volume of 1.0 ml. of 0.05 M Pi, pH 7.4. The incubation time was 5 hours at 35’.

Addition

None ............................ Hypoxanthine. ................... Guanine. ........................ Adenine .......................... Inosine .......................... Guanosine. ...................... Adenosine. ............. ......... Orotic acid ....................... Uracil.......... .............. Cytosine ......................... Nicotinic acid ....................

-

-

pm&s

0 0.4 0.3 0.7 1.0 1.0 1.0 1.0 1.0 1.0

100.0

NMN synthesized

RSP + ATP PRPP

/m3lc

0.138 0.005 0.000 0.003 0.000 0.010 0.021

pmole

0.130 0.000

0.000 0.013 0.020 0.005 0.112 0.110 0.124 0.111

TABLE VIII

Stoichiometry of Nucleotide Syntheses by PPE

0.5 ml. of PPE was incubated with PRPP in the amount shown plus 5 rmoles of Mg++ and 0.05 M phosphate, pH 7.4. Data are not given for NAm which was present in great excess.

Incubation time

hrs.

12

0.25

0.25

3

NMN PRPP Guanine PRPP GMP Hypoxanthine PRPP IMP Adenine PRPP AMP

Initial

/O?df2

0.01 0.33 0.18 0.20 0.00 0.56 0.33 0.00 0.70 0.33 0.00

-

_- pmole /mole

0.17 +0.16 0.12 -0.21 0.00 -0.18 0.00 -0.20 0.16 $0.16 0.24 -0.32 0.00 -0.33 0.29 +0.29

0.26 -0.07 0.00 0.00

-

-

A

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J. PREISS AND P. HANDLER 767

TABLE IX

Separation of NMN and GMP Pyrophosphorylases

GMP pyrophosphorylase activity was assayed by incubating guanine 0.18 pmole, MgC12 10 pmoles, PRPP 0.19 pmole, 0.5 ml. of dialyzed enzyme fraction, and Tris 50 rmoles, pH 7.4, in a total volume of 2.9 ml. at 35” for 15 minutes. NMN pyrophos- phorylase activity was measured by incubating NAm 164 rmoles, PRPP 0.3 pmole, MgClz 5 @moles, 0.5 ml. of dialyzed enzyme fraction, and 50 pmoles of Tris in a total volume of 1.2 ml. at 35” for 5 hours.

Gel Fraction

Alumina C-y

Ca8 (PO4) 2

-

PPE Supernatant fluid 0.05 M PO4 eluate 0.10 ‘I I‘ “

0.20 “ “ “ 0.30 “ “ ‘I

Supernatant fluid 0.05 M PO4 eluate 0.10 ‘I “ I‘ OIJJ “ “ “ 0.30 “ “ “

TABLE X

-

NMN synthesis GMP synthesis

Poole p9d*

0.056 0.18 0.009 0.00 0.026 0.18 0.029 0.18 0.037 0.00 0.038 0.00 0.000 0.17 0.026 0.16 0.022 0.14 0.024 0.00 0.028 0.00

Separation of NMN and IMP Pyrophosphorylases

Assay incubations contained PRPP 0.3 pmole, MgClz 5 pmoles, Tris 50 rmoles, pH 7.4, 0.5 ml. of dialyzed enzyme fraction, and either hypoxanthine 0.17 pmole or NAm 164 bmoles. The incubation times were 5 hours for NMN and 15 minutes for IMP.

Gel

Alumina CT

Ca8 (PO41 2

.-

-

Fraction

PPE Supernatant fluid 0.01 M PO4 eluate 0.05 “ “ “ 0.10 “ I‘ ‘I 0.20 ‘I “ ‘<

0.30 “ “ “ Supernatant fluid 0.05 M PO, eluate 0.10 ‘I ‘I “ 0.20 “ “ “ 0.30 I‘ “ <‘

-

.-

-

NMN synthesis IMP synthesis

pmoie pmolc

0.067 0.17 0.000 0.00 0.017 0.00 0.039 0.17 0.055 0.17 0.055 0.055 0.047 0.014 0.007 0.00 0.043 0.17 0.037 0.14 0.033 0.00 0.037 0.00

-

-

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768 NICOTINAMIDE MONONUCLEOTIDE SYNTHESIS

The relatively slow synthesis of NMN and high K, for NAm, as com- pared with the rapid synthesis of IMP and GMP at low substrate concen- trations, suggested the possibility that NMN synthesis was accomplished by an enzyme which “normally” operates with a different substrate, per- haps guanine or hypoxanthine. However, as shown in Tables IX and X, NMN-synthesizing activity was clearly separated from both GMP- and IMP-synthesizing activity by adsorption of the protein of PPE on calcium phosphate gel or alumina Cy and elution with phosphate buffers of in-

TABLE XI

Pyrophosphorolysis of NMN

The incubation mixtures contained PPE 0.5 ml., MgClz 5 pmoles, Tris 50 pmoles, pH 7.4, either NMN 0.15 rmole (Experiment 1) or 0.20 pmole (Experiment 2), and the indicated amounts of P-Pi and hypoxanthine. The incubation time was 21 hours at 35”. After the reaction had been stopped by heating, purified yeast pyrophos- ohatase (28) was added and the mixture was incubated for 30 minutes before analyz- kg for NMN in the usual manner.

Experiment No. P-Pi Hypoxanthine Final NMN

pmoles pmle ~mole

0 0 0.138 1.0 0 0.095 1.0 0.28 0.085 5.7 0 0.102 5.7 0.28 0.134 0 0.28 0.134 0 0 0.197 0.2 0 0.135 0.2 0.28 0.135 1.0 0 0.124 1.0 0.28 0.117 5.7 0 0.141 0 0.28 0.193

ANMN

jmolc

-0.043 -0.053 -0.036 -0.004 -0.004

-0.062 -0.062 -0.073 -0.080 -0.056 -0.004

creasing concentration. The 0.3 M eluates were practically devoid of IMP- and GMP-synthesizing activity, contained no hemoglobin, and were 550- to 600-fold purified with respect to NMN synthesis, as compared with the original extract of acetone powder. It is noteworthy that the total NMN- synthesizing activity eluted from the gels was 2 to 3 times that of the starting PPE.

Reversibility-As shown in Table XI, in 21 hours PPE catalyzed the disappearance of 40 per cent of NMN initially present when 6 X 10m4 M

P-Pi was added. Higher concentrations of P-Pi did not augment this effect nor did hypoxanthine, added to trap any PRPP formed by IMP synthesis. Indeed, hypoxanthine, at higher concentrations of P-Pi, inhibited the disappearance of NMN.

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J. PREISS AND P. HANDLER 769

DISCUSSION

The data presented indicate the presence in human erythrocytes of a relatively low concentration of an enzyme which catalyzes the reaction

H+ + NAm + PRl’I” ;It NMN+ + P-Pi”

In keeping with the convention employed for those enzymes which syn- thesize pyrimidine nucleotides, it is suggested that this enzyme be desig- nated NMN pyrophosphorylase, even though the demonstration of reversi- bility was not satisfactory and the equilibrium position for this system could not be ascertained. The relatively slow and difficult pyrophos- phorolysis of the purine nucleotides has been discussed by Kornberg et al. (24) and by Korn et al. (26). The failure of hypoxanthine to accelerate the disappearance of NMN is not understood, since the rate of IMP syn- thesis by PPE is more than 20 times that of NMN synthesis; the actual inhibition of NMN disappearance by hypoxanthine at higher concentra- tions of P-Pi is entirely without explanation. Attempts to demonstrate NAm formation, in the reverse reaction, by measurement of the optical density change at 265 m/l. after acidification were technically unsuccessful, since the deproteinized fractions exhibited considerable absorption at this wave length. Thus, there was no positive demonstration that the P-Pi- dependent disappearance of NMN represented reversal of the synthetic reaction.

The slow rate of the reaction and the unphysiological K, for NAm sug- gest that NAm may not be the “normal” substrate for this enzyme. How- ever, the data clearly indicate that the NMN-synthesizing enzyme is distinct from those erythrocyte enzymes that catalyze IMP, AMP, and GMP synthesis; it seems likely that extracts of acetone-powdered erythro- cytes are devoid of synthetic activity for nucleotides of erotic acid, uracil, cytosine, or nicotinic acid, since these compounds do not interfere with NMN synthesis. Moreover, the activity of the isolated enzyme readily accounts for the maximal rate of NMN synthesis by intact erythrocytes or hemolysates. Finally, it must be noted that, in the course of these studies, no DPN synthesis was observed despite the addition of relatively high concentrations of ATP. Neither erythrocytes, hemolysates, nor extracts of acetone-powdered cells exhibit DPN pyrophosphorylase activity. Denstedt and Malkin (27) have also stated that this enzyme is lacking in human erythrocytes. Consequently, the metabolic role of NMN pyro- phosphorylase, and indeed of NMN, in erythrocytes remains to be estab- lished.

SUMMARY

Extracts of acetone-powdered human erythrocytes have been found to synthesize 5-phosphoribosyl 1-pyrophosphate (PRPP) from adenosine

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770 NICOTINAMIDE MONONUCLEOTIDE SYNTHESIS

triphosphate and ribose 5-phosphate and to synthesize nicotinamide mononucleotide, inosine 5’-phosphate, guanosine 5’-phosphate, and adeno- sine 5’-phosphate by the general reaction H+ + base + PRPP + nucleo- tide + inorganic pyrophosphate. The enzyme which catalyzes nicotina- mide mononucleotide synthesis by this reaction has been purified 600-fold and is distinct from the enzymes which catalyze purine nucleotide synthesis. The specificity, reversibility, and metabolic role of this system are dis- cussed.

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Jack Preiss and Philip HandlerNICOTINAMIDE MONONUCLEOTIDE

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