SYNTHESIS OF NICOTINAMIDE MONONUCLEOTIDE BY HUMAN ERYTHROCYTES IN VITRO*

BY IRWIN G. LEDERt AND PHILIP HANDLER

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

(Received for publication, November 27,195O)

Previous reports have described the synthesis of pyridine nucleotides from added nicotinic acid by human erythrocytes in vitro (2, 3). This synthesis, both in vivo and in vitro, was demonstrated with nicotinic acid but not when its amide was employed (4, 5). In none of these studies was the synthesized material properly identified except as material which could serve as factor V for Hemophilus parain$uenxae, as determined tur- bidimetrically (6), or for Hemophilus in$uenxae, as determined by nitrate reduction (4), or simply by the cellular incorporation of C14-labeled nico- tinic acid (7). Each of the laboratories expressed the amount of material synthesized as diphosphopyridine nucleotide (DPN) and in no case was the amount synthesized in vitro any greater than that already present in normal cells. The present report describes the synthesis of pyridine nu- cleotides from nicotinamide by erythrocytes in vitro, in amounts as much as 10 times the normal pyridine nucleotide concentration, and the identi- fication of the synthesized material as nicotinamide mononucleotide (NMN).

EXPERIMENTAL

General Procedure-Human venous blood was drawn and heparinized or defibrinated with glass beads. The cells were washed four times at 3” with 4 volumes of Ringer-phosphate, pH 7.2, containing 250 mg. per cent glucose, and made up to a hematocrit of about 50 per cent in this medium. To each ml. of cell suspension taken for incubation, 0.1 ml. of a 22 per cent nicotinamide solution was added to give a final concentration of 2 per cent nicotinamide. The cell suspensions were incubated at 37” with only occasional shaking except when indicated. St.erile technique was employed throughout and, in several cases, the sterility of incubated blood

* These studies were supported by a grant-in-aid, RG-91, from the Division of Research Grants and Fellowships, National Institutes of Health, by the Nutrition Foundation, Inc., and the Duke University Research Council. A preliminary report of this work was presented before the American Society of Biological Chemists (1).

t The data herein were taken, in part, from a dissertation submitted by Mr. Irwin G. Leder to the Graduate School of Duke University in partial fulfilment of the re- quirements for the degree of Doctor of Philosophy.

889

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890 NICOTINAMIDE MONONUCLEOTIDE

was checked by plating out on blood agar. A mixture of penicillin (20 units per ml.) and streptomycin (40 units per ml.) may be employed to inhibit bacterial growth without affecting nucleotide synthesis. After incubation, 5 per cent trichloroacetic acid filtrates were prepared or the cells were hemolyzed in water at 80” as described by Axelrod and Elveh- jem (8). In all cases final hematocrits were taken and the results are reported as micromoles of pyridine nucleotide per ml. of packed erythro- cytes. Except when indicated, pyridine nucleotides were determined by the fluorometric procedure developed in these laboratores (9).

In the earliest experiments of the present study it was assumed that, as

TABLE I

Synthesis of Pyridine Nucleotides from Nicotinamide

To 2 ml. aliquots of a suspension of washed erythrocytes in Ringer-phosphate containing 250 mg. per cent glucose, hematocrit 50 per cent, were added 0..2 ml. of suitable saline solutions of nicotinamide and neutralized nicotinic acid to give final concentrations as indicated. After 24 hours of incubation at 37”, total pyridine nucleotides were analyzed fluorometrically.

ubstrate

Nicotinamide Nicotinicacid

mg. per ml.

0

0.5 0.5 1.0 1.0

20.0 20.0

mg. ger ml.

0

0

0.5 0 0.5 0 0.5

Total pyridine nucleotides

phf per ml. cells

0.10 0.23 0.25 0.29 0.31 0.93 0.91

had been found previously, nicotinic acid was the proper substrate. Con- sequently, these experiments were performed as described above, except that nicotinic acid, rather than its amide, was employed in a final con- centration of 1 mg. per ml. of cell suspension. This resulted in a rela- tively low order of synthesis, the apparent pyridine nucleotide content of the red cells increasing from a normal level of about 0.1 PM to 0.13 or 0.15 PM. Since it seemed possible that there might occur an extensive turn- over of intracellular nucleotides, mediated by the known diphosphopyri- dine nucleotidase (DPNase) of red cells (lo), it was decided to repeat these experiments in the presence of 2 per cent nicotinamide, as the latter serves as an effective inhibitor of this DPNase (11, 12). As shown in Table I, this resulted in an 8- to lo-fold increment in apparent pyridine nucleotide as determined by the fluorometric procedure. It was then es- tablished that similar synthesis occurred when nicotinic acid was removed

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I. G. LEDER AND P. HANDLER 891

from the medium. However, this did not eliminate the possibility that, in the presence of this large concentration of commercial nicotinamide,’ there was present a small amount of actual nicotinic acid. This has not been rigorously disproved but seems unlikely in view of the failure of nic- otinic acid to enhance the effect of nicotinamide when the latter was em- ployed at relatively low concentrations.

As shown in Fig. 1 A, synthesis proceeded at a rather constant rate for about 16 hours and maximum values were obtained within 20 hours. To determine the relationship between nicotinamide concentration and the rate of synthesis, a series of runs was made at varying nicotinamide con-

6 12 18 24 HOURS

1 5

nl&ll. 15 20

NICOTINAMIDE

FIG. 1. A, rate of synthesis of pyridine nucleotides by erythrocytes under opti- mum conditions; B, influence of nicotinamide concentration on pyridine nucleotide synthesis by erythrocytes.

centrations with all tubes incubated for 20 hours. The results are shown in Fig. 1, B. Maximum synthesis was obtained at about 2 per cent nic- otinamide. At appreciably higher concentrations synthesis did not occur and extensive hemolysis was observed.

Indentification of Nicotinamide Mononucleotide-The acetone condensa- tion reaction of Huff and Perlzweig (13), which forms the basis of the fluorometric procedure for pyridine nucleotides employed in these studies, is specific for quaternary pyridine derivatives of nicotinamide. Of the derivatives of nicotinamide lvhich are knomn to occur physiologically, the following fulfil the specificity requirements for this assay: Nl-methylnico- tinamide (MN), nicotinamide riboside (NR), NMN, DPN, and triphos- phopyridine nucleotide (TPN). Unlike MN, the compound produced by

1 Merck and Company, Inc., Rahway, New Jersey.

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892 NICOTINAMIDE MONONUCLI 3TIDE

the erythrocytes was not freely permeable; it was absent from the sus- pending medium and could not be removed from the cells by repeated washing with saline. Moreover, the unknown material was active in some of the biologic test systems to be described below, in all of which MN is completely inactive. When the unknown nucleotide was assayed mic- robiologically by employing H. paruinfluenxae (ATCC 9796), as described by Kohn (6), or by means of the yeast apozymase system, according to the procedure of Axelrod and Elvehjem (8), excellent agreement with the results of fluorometric analysis was obtained, as shown in Table II. In the yeast assay a short delay in the onset of active fermentation was some- times observed when compared with flasks containing DPN prepared from yeast, but this delay was often absent. Of the compounds listed above, NR, DPN, and TPN are known to be active in the Hemophilus assay, but, of these, only DPN can function as the cofactor in apozymase fer-

TABLE II

Analysis of Pyridine Nucleotides Synthesized in Vitro

Experiment No. Analytical method Total pyridine nucleotide

PM ger ml. cells

1 Fluorometric. . . . . . . . . . . . . . . . . . . . 0.91 H. parain~%~enzae........................ 0.81

2 Fluorometric............................ 0.81 Yeast apoaymase assay. 0.87

mentation. NMN has never been tested in the Hemopkilus assay. Sam- ples of NMN were prepared from DPN by means of potato pyrophos- phatase2 and were tested in the standard apozymase assay with no attempt made to study the effect of varying the concentration of other cofactors. Under these conditions, NMN exhibited very little activity for approxi- mately 30 minutes and continued to increase in activity during the next 60 minutes, at which time it was as active as an equimolar amount of DPN.

NR was further eliminated by demonstrating that the synthesized nu- cleotide was rapidly split by DPNase liberated from erythrocytes upon hemolysis (3) or that prepared from rat or rabbit brain according to Hand- ler and Klein (la), as these authors have shown that this enzyme does not split NR (14). 0.5 ml. of washed erythrocytes which had previously been incubated with 2 per cent nicotinamide was hemolyzed in 10 ml. of water in each of four tubes as follows: The first tube contained no additions; the second and fourth contained approximately half the number of moles of DPN; the third and fourth contained 1 ml. of rat brain DPNase. The

2 Kindly supplied by Dr. Arthur Kornberg.

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I. G LEDER AND P. HANDLER 893

rapid splitting of the unknown nucleotide and added DPN by both sources of DPNase is illustrated in Fig. 2. In a similar experiment performed with DPNase from rabbit brain less than 0.02 /IM of pyridine nucloetide per ml. of erythrocytes, of an initial value of 0.78 PM, remained after an incubation period of 3 hours.

30 60 90 120 150 MINUTES

FIG. 2. The destruction of pyridine nucleotides by DPNase prepared from human erythrocyte and rat brain. 3 ml. of previously incubated erythrocytes, containing 1.5 MM of pyridine nucleotide per ml., were washed with saline to remove excess nico- tinamide and brought to a hematocrit of 40 per cent. 1 ml. of this cell suspension was added to each of four tubes corresponding to each of the four curves. The cells were hemolyzed with water in a total volume of 10 ml. containing the following ad- ditions: (1) none, (2) 0.35 PM of DPN, (3) 1 ml. of rat brain DPNase, (4) 0.35 NM of DPN plus 1 ml. of rat brain DPNase. The tubes were incubated at 37” and samples were withdrawn at intervals for fluorometric analysis.

The data presented in Table II and the rapid destruction of the syn- thesized nucleotide by brain DPNase indicate that little or no NR ac- cumulated under these conditions. While the possibility exists that NR may have been formed as a transitory intermediate, these results are in general agreement with the suggestion that the physiological synthesis of nucleotides need not necessarily entail a two-step process of nucleoside synthesis with subsequent phosphorylation (15). It is hoped that the

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894 NICOTINAMIDE MONONUCLEOTIDE

question may be resolved by studies of nucleotide synthesis by hemoly- sates now in progress. However, the maximum rate of NMN synthesis obtained to date is of the order of one-third of that routinely seen in in- tact red cells as reported herein.

The ability of the material from incubated cells to serve as TPN was ascertained by inclusion in a proper medium containing isocitrate3 and an active preparation of isocitric dehydrogenase obtained by acetone frac- tionation of pig heart (16). As judged by absorption at 340 rnp, the syn- thesized nucleotide was devoid of TPN activity.

The evidence adduced to this point indicated that the unknown sub stance might indeed be DPN. This raised the question of the source of the adenine portion of the molecule. Human erythrocytes contain an average of about 1 pM of free adenine nucleotide per ml. of packed cells (17), most of which is adenosinetriphosphate (ATP) (18). This amount of adenine corresponds closely with that which would be required to pro- duce the maximum amount of pyridine nucleotide, as DPN, synthesized in our experiments. If such were the case, it would be expected that, after incubation with nicotinamide, the ATP content of these cells should be markedly reduced. A trichloroacetic acid filtrate of properly incu- bated cells was then examined for the 7 minute, acid-hydrolyzable phos- phorus of ATP and adenosinediphosphate (ADP) by a modification of the method of Sacks (19). About half the ATP-ADP was still present after incubation with nicotinamide and the values found did not differ signi- ficantly from those found in cells incubated in the absence of nicotirramide. This did not appear compatible with the possibility of DPN synthesis under these conditions. That the synthesized nucleotide was not DPN was definitely established by incubating the material with crystalline al- cohol dehydrogenase prepared from bakers’ yeast according to Racker (20) and estimating DPN spectrophotometrically as described by Korn- berg (21). The amount of DPN found in this manner accounted for only a small fraction of the apparent pyridine nucleotide synthesized from ni- cotinamide.

Of the derivatives of nicotinamide listed above, all had been eliminated except NMN. It remained to establish the identity of NMN by some direct evidence. This was accomplished by demonstrating t.hat the un- known nucleotide was active as substrate for the enzyme described by Kornberg (21), which forms DPN from ATP and NMN with the splitting out of pyrophosphate. A given volume of incubated blood was added to half the volume of water previously heated to 85” and a filtrate prepared by heating in a water bath at 80” for 5 minutes and homogenizing the mix- ture with a Potter apparatus. The mixture was centrifuged and the su-

3 Kindly supplied by Dr. H. B. Vickery.

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I. G. LEDER AN’D P. HANDLER 895

pernatant taken for analysis. The enzyme preparation employed was either a simple extract of hog liver acetone powder prepared by grinding 1 gm. of acetone powder with 5 ml. of 0.1 M disodium phosphate in a glass mortar, or was partially purified by ammonium sulfate fractionation (21). The glycylglycine4 and ATP6 employed were commercial preparations. ATP, obtained as the free acid, was found to keep well when stored in a desiccator at room temperature. Treatment of the ATP with an ion ex- change resin, as described by Polis and Meyerhof (22), was sometimes em- ployed, but satisfactory results were obtained without pretreatment. The increase in DPN content, as determined by alcohol dehydrogenase assay following incubation in this enzyme system, was taken as a measure of the

TABLE III Fluorometric and Enzymatic Determination of Synthesized Pyridine Nucleotides

Filtrates were prepared from incubated cells by hemolyzing in water at 80” as described in the text. All values have been corrected for the normal nucleotide con- tent of erythrocytes. DPN represents the increment originally present in the fil- trate as determined spectrophotometrically with alcohol dehydrogenase. The DPN measured after incubation with ATP and the Kornberg enzyme (21) is taken to represent DPN + NMN in the original filtrate. NMN values are calculated by difference. The data are expressed as micromoles of nucleotides per ml. of eryth- rocytes.

Fluorometric

(1)

0.78 1.0 0.92 0.92 0.64

DPN NMN + DPN NMN

(2) (3) (31-W

0.06 0.68 0.62 0.20 0.91 0.71 0.14 0.85 0.71 0.23 0.85 0.62 0.15 0.59 0.44

NMN initially present in the blood filtrate. Sufficient filtrate was taken for analysis so that optical density increments of the order of 0.1 to 0.2 pM were obtained. The results shown in Table III indicated that nearly all of the fluorometrically determined pyridine nucleotide could be identi- fied as NMN plus DPN. Between 75 and 95 per cent of the increment of pyridine nucleotides determined enzymatically, as described above, may be accounted for as NMN and the remainder as DPN. Less than 10 per cent of the fluorometrically determined nucleotide remained unaccounted for. In the procedure for fluorometric acetone condensation, NMN pro- duces as much fluorescence as DPN on a molar basis.

It will be recalled that, although NMN stimulated apozymase fermenta- tion only after an extended incubation period, this delay was absent or

4 Nutritional Biochemicals Corporation, Cleveland, Ohio. 6 Bchwarz Laboratories, Inc., New York.

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896 NICOTINAMIDE MONONUCLEOTIDE

very short when filtrates of erythrocytes, which had been previously in- cubated with 2 per cent nicotinamide, were tested in this assay, although most of the pyridine nucleotides contained in this filtrate were NMN. That the DPN-free NMN did ultimately cause active fermentation would indicate that the apozymase used in these studies (23) contained the en- zyme which synthesizes DPN from NMN and ATP. The absence of ATP, which would account for the extended induction period, was not an important factor when blood filtrates were analyzed, since sufficient DPN was present to cause immediate fermentation which would, in turn, provide ATP for further DPN synthesis. This is in agreement with the

TABLE IV E$ects of Washing, Phosphate, and Plasma on Pyridine Nucleotide Synthesis

1 ml. of erythrocytes, prepared as described in the text, was added to each of seven incubation vessels. The tabulated additions were made and all suspensions brought to 2 per cent nicotinamide and 250 mg. per cent glucose. After incubation for 26 hours at 37”, total pyridine nucleotides were determined fluorometrically.

Treatment

Unwashed cells

Ringer-phosphate-washed cells

Saline-washed cells

-

_-

-

Plasma

ml.

1.0 0.85 0.0 1.0 0.85 0.0 0.0

Additions

.laaRinger. 0.1 M phosphate lhosphate

ml.

0.0

0.0

1.0 0.0 0.0 1.0 1.0

ml.

0.0

0.15 0.0 0.0 0.15 0.0 0.0

Total pyridine nucleotides

u&f per ml. cells 0.18 0.33 0.39 0.37 1.0 0.97 0.88

report of Meyerhof and Kaplan (24), which appeared while this paper was in preparation. The data presented by these authors indicate that NMN is at least as active as DPN in catalyzing fermentation by washed brewers’ yeast in the presence of ATP and a limiting concentration of DPN.

Conditions Necessary for NMN Synthesis-The failure of previous work- ers to obtain results comparable with those reported here may be attrib- uted to two factors. In order to obtain maximum synthesis, the cells must be washed prior to incubation and must be incubated in a medium containing inorganic phosphate in excess of that found in plasma. This is illustrated in Table IV. Defibrinated blood was centrifuged at 2000 r.p.m. for 15 minutes at 3” and the plasma removed and saved. Half the volume of packed cells was withdrawn and stored at 3’, while a small por- tion was washed with saline and the remainder with Ringer-phosphate

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I. (3. LEDER AND P. HANDLER 897

solution in the usual manner. Finally, the washed cells were centrifuged and the supernatant solutions removed. The cells were incubated with gentle rocking with additions as illustrated in Table IV. In addition all solutions were made 2 per cent with respect to nicotinamide and 250 mg. per cent with respect to glucose. Cells suspended in plasma were incu- bated under 5 per cent C02-95 per cent 02 and those containing phosphate buffer under pure oxygen. Although the presence of a glycolytic inhibi- tor in plasma has been demonstrated in studies on red cell hemolysates (25), it can be seen that the effectiveness of the washing operation does not seem to lie in the removal of some inhibitor associated with the plasma, The addition of the original plasma to washed cells did not inhibit the syn- thesis of NMN. Nor did the washing operation materially alter the rate of glucose utilization. The rate of disappearance of glucose, determined by the method of Somogyi as modified by Nelson (26), was the same for washed and unwashed cells suspended in Ringer-phosphate medium. The progressive effect of the washing operation was demonstrated by washing defibrinated blood in the usual manner, except that after each washing 2 ml, of cell suspension were removed and all such bloods incubated simul- taneously in the presence of 2 per cent nicotinamide. The resulting syn- thesis, as micromoles of pyridine nucleotide per ml. of erythrocytes, was as follows: no washing 0.12, one washing 0.22, two washings 0.83, three washings 1.1, five washings 1.2, seven washings 1 .l, eleven washings 0.9. Thus, the maximum effect was achieved with approximately the third and fourth washings.

As noted previously, the nature of the pyridine nucleotide synthesized in limited amount from nicotinic acid was never properly established. In the present study, nicotinamide, rather than free acid, was the effective substrate. While this is in keeping with what might ‘be expected from the structures of DPN and NMN, nevertheless, there is, as yet, no ex- planation for the original observations which have been repeatedly con- firmed.

Synthesis did not proceed in the absence of glucose and was inhibited by the usual glycolytic inhibitors. Fluoride, at 0.01 and 0.005 M, arse- nate at 0.04 M, and iodoacetate at 0.001 and 0.0003 M completely pre- vented nucleotide synthesis. The last, however, reacts non-enzymatically at 37” to produce a compound which responds to the acetone condensation procedure for quaternary nicotinamide derivatives. This compound was probably nicotinamide-W-carboxymethyl iodide. Dinitrophenol at 2 X lo4 and 7 X 10e5 M did not inhibit pyridine nucleotide synthesis under our experimental conditions.

Although methylene blue has been observed to accelerate the utilization of glucose (27, 28) and reduction of methemoglobin (29) by erythrocytes,

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898 NICOTINAMIDE MONONUCLEOTIDE

it is of interest that the rate of nucleotide synthesis was not changed by the addition of 0.005 per cent methylene blue.

The incubation of washed erythrocytes with 2 per cent nicotinamide invariably resulted in the appearance of a dark chromogen, while the chro- mogen developed to only a slight extent when the cells were not washed. The chromogen exhibited the absorption spectrum of methemoglobin and bound cyanide, with the characteristic shift in the absorption band. It cannot be stated with certainty whether the chromogen is methemoglobin or some unidentified nicotinamide hemochromogen.

It was thought of interest to determine whether the cozymase content of yeast cells could be increased by incubating them under the conditions employed in these studies with erythrocytes. Pressed bakers’ yeast was washed three times with Ringer-phosphate buffer, pH 7.2, containing 200 mg. per cent glucose, made up in an equal volume of that medium, and incubated for 24 hours in the presence of 2 per cent nicotinamide. A l- to 2-fold increment in pyridine nucleotides fluorometrically determined resulted from a base value of 0.66 PM (0.60 to 0.77) per ml. of packed cells to 1.7 ,LJM (1.2 to 2.3). It is felt that optimum conditions were not em- ployed in these brief studies and that suitable adjustments of the experi- mental conditions might make this procedure of value as a form of pre- treatment in the preparation of DPN. A similar synthesis by brewers’ and bakers’ yeast under different experimental conditions has been re- ported by Lennerstrand (30).

SUMMARY

The incubation of human erythrocytes washed with isotonic saline or Ringer’s solution in a medium containing inorganic phosphate, glucose, and 2 per cent nicotinamide resulted in an 8- to lo-fold increment in cellular pyridine nucleotides of which 75 to 90 per cent was identified as nicotinamide mononucleotide and the remainder as DPN. Optimum syn- thesis occurred only if the cells were washed free of an inhibitor which was not associated with the plasma.

Nicotinamide mononucleotide, in the presence of limiting quantites of DPN, served as a growth factor for Hemophdus paruin$uenzae and sup- ported the fermentation of washed brewers’ yeast.

Synthesis did not occur in the presence of glycolytic inhibitors and was unaffected by dinitrophenol or catalytic quantities of methylene blue.

The incubation of washed bakers’ yeast in Ringer-phosphate medium containing glucose and 2 per cent nicotinamide resulted in a l- to 2-fold increment in apparent pyridine nucleotides.

BIBLIOGRAPHY

1. Leder, I. G., Perlzweig, W. A., and Handler, P., Federation Proc., 9, 397 (1950). 2. Kohn, H. I., and Klein, J. R., J. Biol. Chem., 130,l (1939).

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I. 0. LEDER AND P. HANDLER 899

3. Kohn, H. I., and Klein, J. R., J. Biol. Chem., 136, 685 (1940). 4. Hoagland, C. L., and Ward, S. M., J. Biol. Chem., 146, 115 (1942). 5. Handler, P., and Kohn, H. I., J. Biol. Chem., 150,447 (1943). 6. Kohn, H. I., Klein, J. R., and Dann, W. J., Biochem. J., 33, 1432 (1939). 7. Leifer, E., Hogness, J. R., Roth, L. J., and Langham, W., J. Am. Chem. Sot., 70,

2908 (1948). 8. Axelrod, A. E., and Elvehjem, C. A., J. Biol. Chem., 131, 77 (1939). 9. Perlzweig, W. A., Rosen, F., Robinson, J., and Levitas, N., J. Biol. Chem., 16’7,

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1028 (1939). 17. Rapoport, S., and Guest, G. M., J. BioZ. Chem., 138, 269 (1941). 18. Albaum, H. G., Cayle, T., and Shapiro, A., Federation Proc., 9, 144 (1950). 19. Sacks, J., J. BioZ. Chem., 181, 655 (1949). 20. Racker, E., J. BioZ. Chem., 184, 313 (1950). 21. Kornberg, A., J. BioZ. Chem., 182,779 (1950). 22. Polis, B. D., and Meyerhof, O., J. BioZ. Chem., 169, 389 (1947). 23. Grieg, M. E., J. Pharmacol. and Exp. Therap., 81, 164 (1944). 24. Meyerhof, O., and Kaplan, A., Arch. Biochem., 28, 147 (1950). 25. Christensen, W. R., Plimpton, C. H., and Ball, E. G., J. BioZ. Chem., 180, 791

(1949). 26. Nelson, N., J. BioZ. Chem., 163, 375 (1944). 27. Barron, E. S. G., 6. BioZ. Chem., 81, 445 (1929). 28. Warburg, O., Kubowitz, F., and Christian, W., Biochem. Z., 221, 494 (1930). 29. Gibson, Q. H., Biochem. J., 42,13 (1948). 30. Lennerstrand, A., Ark. Kemi, Mineral. o. Geol., 14 A, No. 16 (1941).

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Irwin G. Leder and Philip HandlerERYTHROCYTES IN VITRO

MONONUCLEOTIDE BY HUMAN SYNTHESIS OF NICOTINAMIDE

1951, 189:889-899.J. Biol. Chem. 

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