Proc. Nati. Acad. Sci. USA Vol. 74, No. 10, pp. 4160-4162, October 1977 Biochemistry
Action of nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and nicotinamide mononucleotide (enzymic phosphorylation/thin-layer chromatography/sterical restraints in NAD phosphorylation)
ELINOR F. BRUNNGRABER AND ERWIN CHARGAFF* Cell Chemistry Laboratory, The Roosevelt Hospital, New York, New York 10019
Contributed by Erwin Chargaff, June 9, 1977
ABSTRACT The action of the nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and on its 5t-phosphate results in the addition of one phosphate moiety to each of the substrates. Although the proof is not conclusive, it is likely that the phosphate group is transferred to the 3'-hydroxyl of the ribose. This is in contrast to the behavior of the enzyme toward NAD in which only the adenylic acid portion is phosphorylated enzymically.
under UV illumination, after the dried papers or plates had been exposed for 1 hr to an atmosphere of methyl ethyl ketone and ammonia (1:1, vol/vol). Enzymic Phosphate Transfer to NR and NMN. The assay mixture (total volume 0.3 ml) in 0.13 M sodium acetate of pH 5.2 was 133 mM with respect to p-nitrophenylphosphate and about 15 or 30 mM with respect to NR or NMN. In each assay about 35 ,Ag of nucleotide phosphotransferase (step 4 in ref. 4) was used. Incubation at 370 was continued until the maximum of phosphorylation product was obtained. Samples were streaked on paper and developed. The separated zones were eluted with water and the extracts, if necessary, concentrated under reduced pressure.
The nucleotide phosphotransferase of Escherichia coli, an enzyme capable of transferring organically bound phosphoric acid to the 2'- or 3'-hydroxyl of a nucleoside or nucleotide, has been characterized by us before (1, 2). It also was observed that NAD could act as the phosphate acceptor, with the production of NADP (3). Only the ribose of the adenosine moiety of NAD appeared to be phosphorylated, and the NADP produced consisted of 40% of the 2' isomer and of 60% of the 3' isomer. It was of interest to determine why the other ribose molecule of NAD, the one attached to the nicotinamide portion, was protected from being phosphorylated. Was it for configurational reasons, or was the transferase unable to act on ribo or ribophosphate derivatives of nicotinamide? As we show here, the first seems to be the case, since nicotinamide riboside (NR) and mononucleotide are excellent substrates for the phosphotransferase. The experiments used enzyme preparations purified by affinity chromatography (4). COMPOUNDS AND PROCEDURES Compounds. Nicotinamide mononucleotide (NMN), thymidine 3',5'-diphosphate, and p-nitrophenylphosphate were commercial preparations. NR was prepared as described (5). Nucleoside phosphotransferase of carrot (EC 2.7.1.77) was isolated and purified as described (6). The nucleotide phosphotransferase of E. coli was isolated and purified, following the procedure in our recent paper (4). NMN adenylyltransferase (EC 2.7.7.1) was prepared and assayed as described (7). The 5'-ribonucleotide phosphohydrolase of potato (EC 3.1.3.5) was supplied by Miles Laboratories; the 5'-ribonucleotide phosphohydrolase of Crotalus adamanteus venom (EC 3.1.3.5) and the 3'-ribonucleotide phosphohydrolase of rye grass (EC 3.1.3.6) came from Sigma Chemical Co. Analytical. The nicotinamide derivatives were separated either on filter paper (Whatman no. 1) or by thin-layer chromatography on plates (0.1 mm cellulose, MN, polyethyleneimine-impregnated), supplied by Brinkmann. The solvent consisted of 7 volumes of ethanol and 3 volumes of 1 M ammonium acetate, pH 5.5. The quaternary pyridinium compounds were detected (8) by their blue-white fluorescence
RESULTS AND CONCLUDING REMARKS Phosphate transfer to NR and NMN With either NR or NMN used as the acceptor for phosphate transfer by the nucleotide phosphotransferase, one newly formed UV-absorbing spot appeared on the chromatograms. These products, which are designated as (p)NR and (p)NMN, respectively, gave the characteristic fluorescence after being exposed to an atmosphere of methyl ethyl ketone and ammonia. Their rates of formation are compared in Fig. 1. Initially, the nucleotide NMN is slightly more efficient as acceptor than the nucleoside NR, 5 ,mol of (p)NMN being formed in 30 min as compared to 3 ,umol of (p)NR. When the reaction was permitted to go to completion, however, both compounds gave approximately the same yields: about 60% of the acceptor. As shown in Table 1, the analytical determination of phosphorus confirmed the addition of one phosphate moiety to either of the two substrates used. Relative chromatographic mobilities are also included. The spectra of the enzymic phosphorylation products were identical with those of the respective acceptors, NR and NMN, when measured in 0.05 M KH2PO4 of pH 7. In 1 M KCN the characteristic spectral shifts (9) were observed (Fig. 2). Position of transferred phosphate In the case of NR, three hydroxy groups, 2', 3', and 5', are available as acceptor sites. The enzymic product (p)NR differs chromatographically from NMN, and the 5' position thus is excluded. The product (p)NR must, therefore, be the 2'- or the 3'-phosphate or a mixture of both isomers. Liquid chromatography of (p)NR in the Varian instrument revealed only one component, but this is not necessarily conclusive. A tentative decision between the 2' and 3' positions was sought through the
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: NR, nicotinamide riboside; NMN, nicotinamide mononucleotide or nicotinamide ribose 5'-phosphate; (p)NR and (p)NMN, products of enzymic phosphate transfer. * To whom correspondence should be addressed.
4160
Natl. Acad. Sci. USA 74 (1977) BgProc. Brunngraber and Chargaff Biochemistry:
4161
z
Q
z -a
0
z -a
EZ. 240 250 260 270 280 290 300 310 320 330 340 350 360
Wavelength,
nm
FIG. 2. Comparison of ultraviolet spectra of NMN and (p)NMN in 0.05 M KH2PO4 of pH 7.0, in the presence and the absence of 1 M KCN. 0
2
1
3
0 Hours
1
2
3
FIG. 1. Action of E. coli nucleotide phosphotransferase on NR (A) and on NMN (B). (A) 0, 12.5 mM substrate; 0, 25 mM substrate. (B) 0, 14.5 mM substrate; 0, 27 mM substrate. Assay conditions are given in text.
indirect enzymic evidence presented in Table 2. The inability of snake venom 5'-nucleotidase and of potato nucleotidase at pH 8.9 to hydrolyze (p)NR offers additional proof that it is not the 5'-hydroxyl that is phosphorylated by the E. coli phosphotransferase. The resistance of the product to the 3'-nucleotidase of rye grass would seem to indicate that it is a 2'-phosphate. It is, however, possible that the activity of this enzyme is limited to purine and pyrimidine nucleotides. The effect of the nucleoside phosphotransferase of carrot on (p)NR is of some interest. This enzyme, which transfers phosphate principally to the 5' position of the acceptor nucleoside (6), has been shown to do the same with a 2'-nucleotide, but not with a 3'-nucleotide (13). When (p)NR was tested in the presence of phenylphosphate as donor (6), no phosphate transfer was observed, but rather some hydrolysis. The carrot enzyme also acts as a nucleotidase, especially in the absence of an acceptor, and is known to hydrolyze 5'- and 3'-nucleotides, but hardly the 2' isomers (6). As is shown in Table 2, (p)NR and NMN were hydrolyzed to the same extent. We are, therefore, inclined to regard (p)NR as the nicotinamide riboside 3'phosphate, despite the failure of the rye grass nucleotidase to act on it.
In the case of NMN as acceptor, the 2'- and 3'-hydroxyls are available for enzymic phosphate transfer. The product, Table 1. Phosphorus contents and chromatographic mobilities of phosphorylation products
Acceptor and product NR
(p)NR NMN
(p)NMN *
assay,
P content,
Rf*
,4mol/ml
Ag-atoms/ml
Molar ratio, P/NR
3.2 0.65 1.0 0.075
4.50 4.36 7.53 6.87
0 4.45 7.74 14.4
1.02 1.03 2.1
NR in
Determined by thin-layer chromatography, with the mobility of NMN taken as 1.0.
(p)NMN, which again appeared as a single peak in liquid chromatograms, was resistant to the action of snake venom 5'nucleotidase. This is in keeping with previous observations that nucleoside 3',5'-diphosphates are not attacked by the 5'-nucleotidase (1). Although it is not clear whether this enzyme has ever been tested with authentic 2',5'-diphosphates, its ability to dephosphorylate 2'-deoxy-5'-nucleotides shows that a free 2'-hydroxyl is not a prerequisite of activity. The present evidence, therefore, makes it likely that it is the 3'-hydroxyl that is phosphorylated by the E. coli enzyme in both NR and NMN. Concluding remarks We wish to consider two points. The first concerns the fact that, with NAD as the substrate, the nucleotide phosphotransferase phosphorylates only the hydroxyls of the adenosine moiety (3), whereas, as shown here, with free NMN or NR as acceptors it experiences no difficulty in transferring phosphate to a hydroxyl of the nicotinamide riboside. An inspection of a space-filling model of NAD will show that in its extended form the hydroxyls of the ribose attached to nicotinamide are freely accessible, but that it is easy to arrive at configurations-especially when the aglycons are stacked with respect to each other-in which the NMN hydroxyls are sterically protected from the approach of the enzyme in comparison with those of the AMP moiety. Kinetic explanations could also be adduced, but we lack the data. The second point has to do with the biological significance of the phosphorylation discussed here. Only one series of orienting experiments with NMN adenylyltransferase (7) was Table 2. Comparison of hydrolase action on (p)NR and NMN* % hydrolyzed Enzyme (p)NR NMN 100 0 Crotalus 5'-nucleotidase (10) Potato 5'-nucleotidase (11) 77 55 pH 5.0 21 0 pH8.9 0 0 Rye grass 3'-nucleotidase (12) 49 49 Carrot phosphotransferase (6) * The action of the enzymes was followed with 5 mM solutions of (p)NR and NMN under identical conditions, as specified in the cited papers.
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performed, in order to ascertain a possible regulatory effect of the new derivatives on NAD metabolism. When (p)NMN was added to the regular assay mixture, which contained NMN, little effect on the formation of NAD was noted. The preincubation, however, of the assay mixture with the E. coli nucleotide phosphotransferase and a phosphate donor, such as pTp, 3'TMP, 2'-AMP, or 2'-GMP, before the addition of the adenylyltransferase depressed NAD formation to the extent of 20-70% in different experiments. This is obviously a problem that would deserve to be pursued further. This work was supported by research grants from the National Institutes of Health, U.S. Public Health Service, and the National Science Foundation.
Brunngraber, E. F. & Chargaff, E. (1970) Proc. Natl. Acad. Sci. USA 67, 107-112. 2. Brunngraber, E. F. & Chargaff, E. (1973) Biochemistry 12, 1.
3005-3012.
Proc. Nati. Acad. Sci. USA 74 (1977) 3.
Brunngraber, E. F. & Chargaff, E. (1973) Biochemistry 12, 4. Brunngraber, E. F. & Chargaff, E. (1977) Proc. Natl. Acad. Sci. USA 74, 3226-3229. 5. Kaplan, N. 0. & Stolzenbach, F. E. (1957) in Methods in Enzymology, eds. Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York), Vol. 3, pp. 899-905. 6. Brunngraber, E. F. & Chargaff, E. (1967) J. Biol. Chem. 242, 4834-4840. 7. Kornberg, A. (1950) J. Biol. Chem. 182,779-793. 8. Kodicek, E. & Reddi, K. K. (1951) Nature 168,475-477. 9. Colowick, S. P., Kaplan, N. 0. & Ciotti, M. M. (1951) J. Biol. Chem. 191, 447-459. 10. Sulkowski, E., Bjork, W. & Laskowski, M., Sr. (1963) J. Biol. Chem. 238, 2477-2486. 11. Kornberg, A. & Pricer, W. E., Jr. (1950) J. Biol. Chem. 186, 557-567. 12. Shuster, L. & Kaplan, N. 0. (1953) J. Biol. Chem. 201, 5353012-3016.
546. 13. Tunis, M. & Chargaff, E. (1960) Biochim. Biophys. Acta 40, 206-210.
Action of nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and nicotinamide mononucleotide (enzymic phosphorylation/thin-layer chromatography/sterical restraints in NAD phosphorylation)
ELINOR F. BRUNNGRABER AND ERWIN CHARGAFF* Cell Chemistry Laboratory, The Roosevelt Hospital, New York, New York 10019
Contributed by Erwin Chargaff, June 9, 1977
ABSTRACT The action of the nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and on its 5t-phosphate results in the addition of one phosphate moiety to each of the substrates. Although the proof is not conclusive, it is likely that the phosphate group is transferred to the 3'-hydroxyl of the ribose. This is in contrast to the behavior of the enzyme toward NAD in which only the adenylic acid portion is phosphorylated enzymically.
under UV illumination, after the dried papers or plates had been exposed for 1 hr to an atmosphere of methyl ethyl ketone and ammonia (1:1, vol/vol). Enzymic Phosphate Transfer to NR and NMN. The assay mixture (total volume 0.3 ml) in 0.13 M sodium acetate of pH 5.2 was 133 mM with respect to p-nitrophenylphosphate and about 15 or 30 mM with respect to NR or NMN. In each assay about 35 ,Ag of nucleotide phosphotransferase (step 4 in ref. 4) was used. Incubation at 370 was continued until the maximum of phosphorylation product was obtained. Samples were streaked on paper and developed. The separated zones were eluted with water and the extracts, if necessary, concentrated under reduced pressure.
The nucleotide phosphotransferase of Escherichia coli, an enzyme capable of transferring organically bound phosphoric acid to the 2'- or 3'-hydroxyl of a nucleoside or nucleotide, has been characterized by us before (1, 2). It also was observed that NAD could act as the phosphate acceptor, with the production of NADP (3). Only the ribose of the adenosine moiety of NAD appeared to be phosphorylated, and the NADP produced consisted of 40% of the 2' isomer and of 60% of the 3' isomer. It was of interest to determine why the other ribose molecule of NAD, the one attached to the nicotinamide portion, was protected from being phosphorylated. Was it for configurational reasons, or was the transferase unable to act on ribo or ribophosphate derivatives of nicotinamide? As we show here, the first seems to be the case, since nicotinamide riboside (NR) and mononucleotide are excellent substrates for the phosphotransferase. The experiments used enzyme preparations purified by affinity chromatography (4). COMPOUNDS AND PROCEDURES Compounds. Nicotinamide mononucleotide (NMN), thymidine 3',5'-diphosphate, and p-nitrophenylphosphate were commercial preparations. NR was prepared as described (5). Nucleoside phosphotransferase of carrot (EC 2.7.1.77) was isolated and purified as described (6). The nucleotide phosphotransferase of E. coli was isolated and purified, following the procedure in our recent paper (4). NMN adenylyltransferase (EC 2.7.7.1) was prepared and assayed as described (7). The 5'-ribonucleotide phosphohydrolase of potato (EC 3.1.3.5) was supplied by Miles Laboratories; the 5'-ribonucleotide phosphohydrolase of Crotalus adamanteus venom (EC 3.1.3.5) and the 3'-ribonucleotide phosphohydrolase of rye grass (EC 3.1.3.6) came from Sigma Chemical Co. Analytical. The nicotinamide derivatives were separated either on filter paper (Whatman no. 1) or by thin-layer chromatography on plates (0.1 mm cellulose, MN, polyethyleneimine-impregnated), supplied by Brinkmann. The solvent consisted of 7 volumes of ethanol and 3 volumes of 1 M ammonium acetate, pH 5.5. The quaternary pyridinium compounds were detected (8) by their blue-white fluorescence
RESULTS AND CONCLUDING REMARKS Phosphate transfer to NR and NMN With either NR or NMN used as the acceptor for phosphate transfer by the nucleotide phosphotransferase, one newly formed UV-absorbing spot appeared on the chromatograms. These products, which are designated as (p)NR and (p)NMN, respectively, gave the characteristic fluorescence after being exposed to an atmosphere of methyl ethyl ketone and ammonia. Their rates of formation are compared in Fig. 1. Initially, the nucleotide NMN is slightly more efficient as acceptor than the nucleoside NR, 5 ,mol of (p)NMN being formed in 30 min as compared to 3 ,umol of (p)NR. When the reaction was permitted to go to completion, however, both compounds gave approximately the same yields: about 60% of the acceptor. As shown in Table 1, the analytical determination of phosphorus confirmed the addition of one phosphate moiety to either of the two substrates used. Relative chromatographic mobilities are also included. The spectra of the enzymic phosphorylation products were identical with those of the respective acceptors, NR and NMN, when measured in 0.05 M KH2PO4 of pH 7. In 1 M KCN the characteristic spectral shifts (9) were observed (Fig. 2). Position of transferred phosphate In the case of NR, three hydroxy groups, 2', 3', and 5', are available as acceptor sites. The enzymic product (p)NR differs chromatographically from NMN, and the 5' position thus is excluded. The product (p)NR must, therefore, be the 2'- or the 3'-phosphate or a mixture of both isomers. Liquid chromatography of (p)NR in the Varian instrument revealed only one component, but this is not necessarily conclusive. A tentative decision between the 2' and 3' positions was sought through the
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: NR, nicotinamide riboside; NMN, nicotinamide mononucleotide or nicotinamide ribose 5'-phosphate; (p)NR and (p)NMN, products of enzymic phosphate transfer. * To whom correspondence should be addressed.
4160
Natl. Acad. Sci. USA 74 (1977) BgProc. Brunngraber and Chargaff Biochemistry:
4161
z
Q
z -a
0
z -a
EZ. 240 250 260 270 280 290 300 310 320 330 340 350 360
Wavelength,
nm
FIG. 2. Comparison of ultraviolet spectra of NMN and (p)NMN in 0.05 M KH2PO4 of pH 7.0, in the presence and the absence of 1 M KCN. 0
2
1
3
0 Hours
1
2
3
FIG. 1. Action of E. coli nucleotide phosphotransferase on NR (A) and on NMN (B). (A) 0, 12.5 mM substrate; 0, 25 mM substrate. (B) 0, 14.5 mM substrate; 0, 27 mM substrate. Assay conditions are given in text.
indirect enzymic evidence presented in Table 2. The inability of snake venom 5'-nucleotidase and of potato nucleotidase at pH 8.9 to hydrolyze (p)NR offers additional proof that it is not the 5'-hydroxyl that is phosphorylated by the E. coli phosphotransferase. The resistance of the product to the 3'-nucleotidase of rye grass would seem to indicate that it is a 2'-phosphate. It is, however, possible that the activity of this enzyme is limited to purine and pyrimidine nucleotides. The effect of the nucleoside phosphotransferase of carrot on (p)NR is of some interest. This enzyme, which transfers phosphate principally to the 5' position of the acceptor nucleoside (6), has been shown to do the same with a 2'-nucleotide, but not with a 3'-nucleotide (13). When (p)NR was tested in the presence of phenylphosphate as donor (6), no phosphate transfer was observed, but rather some hydrolysis. The carrot enzyme also acts as a nucleotidase, especially in the absence of an acceptor, and is known to hydrolyze 5'- and 3'-nucleotides, but hardly the 2' isomers (6). As is shown in Table 2, (p)NR and NMN were hydrolyzed to the same extent. We are, therefore, inclined to regard (p)NR as the nicotinamide riboside 3'phosphate, despite the failure of the rye grass nucleotidase to act on it.
In the case of NMN as acceptor, the 2'- and 3'-hydroxyls are available for enzymic phosphate transfer. The product, Table 1. Phosphorus contents and chromatographic mobilities of phosphorylation products
Acceptor and product NR
(p)NR NMN
(p)NMN *
assay,
P content,
Rf*
,4mol/ml
Ag-atoms/ml
Molar ratio, P/NR
3.2 0.65 1.0 0.075
4.50 4.36 7.53 6.87
0 4.45 7.74 14.4
1.02 1.03 2.1
NR in
Determined by thin-layer chromatography, with the mobility of NMN taken as 1.0.
(p)NMN, which again appeared as a single peak in liquid chromatograms, was resistant to the action of snake venom 5'nucleotidase. This is in keeping with previous observations that nucleoside 3',5'-diphosphates are not attacked by the 5'-nucleotidase (1). Although it is not clear whether this enzyme has ever been tested with authentic 2',5'-diphosphates, its ability to dephosphorylate 2'-deoxy-5'-nucleotides shows that a free 2'-hydroxyl is not a prerequisite of activity. The present evidence, therefore, makes it likely that it is the 3'-hydroxyl that is phosphorylated by the E. coli enzyme in both NR and NMN. Concluding remarks We wish to consider two points. The first concerns the fact that, with NAD as the substrate, the nucleotide phosphotransferase phosphorylates only the hydroxyls of the adenosine moiety (3), whereas, as shown here, with free NMN or NR as acceptors it experiences no difficulty in transferring phosphate to a hydroxyl of the nicotinamide riboside. An inspection of a space-filling model of NAD will show that in its extended form the hydroxyls of the ribose attached to nicotinamide are freely accessible, but that it is easy to arrive at configurations-especially when the aglycons are stacked with respect to each other-in which the NMN hydroxyls are sterically protected from the approach of the enzyme in comparison with those of the AMP moiety. Kinetic explanations could also be adduced, but we lack the data. The second point has to do with the biological significance of the phosphorylation discussed here. Only one series of orienting experiments with NMN adenylyltransferase (7) was Table 2. Comparison of hydrolase action on (p)NR and NMN* % hydrolyzed Enzyme (p)NR NMN 100 0 Crotalus 5'-nucleotidase (10) Potato 5'-nucleotidase (11) 77 55 pH 5.0 21 0 pH8.9 0 0 Rye grass 3'-nucleotidase (12) 49 49 Carrot phosphotransferase (6) * The action of the enzymes was followed with 5 mM solutions of (p)NR and NMN under identical conditions, as specified in the cited papers.
4162
Biochemistry: Brunngraber and Chargaff
performed, in order to ascertain a possible regulatory effect of the new derivatives on NAD metabolism. When (p)NMN was added to the regular assay mixture, which contained NMN, little effect on the formation of NAD was noted. The preincubation, however, of the assay mixture with the E. coli nucleotide phosphotransferase and a phosphate donor, such as pTp, 3'TMP, 2'-AMP, or 2'-GMP, before the addition of the adenylyltransferase depressed NAD formation to the extent of 20-70% in different experiments. This is obviously a problem that would deserve to be pursued further. This work was supported by research grants from the National Institutes of Health, U.S. Public Health Service, and the National Science Foundation.
Brunngraber, E. F. & Chargaff, E. (1970) Proc. Natl. Acad. Sci. USA 67, 107-112. 2. Brunngraber, E. F. & Chargaff, E. (1973) Biochemistry 12, 1.
3005-3012.
Proc. Nati. Acad. Sci. USA 74 (1977) 3.
Brunngraber, E. F. & Chargaff, E. (1973) Biochemistry 12, 4. Brunngraber, E. F. & Chargaff, E. (1977) Proc. Natl. Acad. Sci. USA 74, 3226-3229. 5. Kaplan, N. 0. & Stolzenbach, F. E. (1957) in Methods in Enzymology, eds. Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York), Vol. 3, pp. 899-905. 6. Brunngraber, E. F. & Chargaff, E. (1967) J. Biol. Chem. 242, 4834-4840. 7. Kornberg, A. (1950) J. Biol. Chem. 182,779-793. 8. Kodicek, E. & Reddi, K. K. (1951) Nature 168,475-477. 9. Colowick, S. P., Kaplan, N. 0. & Ciotti, M. M. (1951) J. Biol. Chem. 191, 447-459. 10. Sulkowski, E., Bjork, W. & Laskowski, M., Sr. (1963) J. Biol. Chem. 238, 2477-2486. 11. Kornberg, A. & Pricer, W. E., Jr. (1950) J. Biol. Chem. 186, 557-567. 12. Shuster, L. & Kaplan, N. 0. (1953) J. Biol. Chem. 201, 5353012-3016.
546. 13. Tunis, M. & Chargaff, E. (1960) Biochim. Biophys. Acta 40, 206-210.
