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Convenient method for enzymic synthesis of 14C-nicotinamide riboside
ANALYTICAL
46, 181-186
BIOCHEMISTRY
(1972)
Convenient Method for Enzymic Synthesis of 14C-Nicotinamide Riboside LUKA Depnrtment
B. KASAROV of
Microbiology, Philadelphia, Received
AND
ALBERT
Hahnemann Pennsylvania July
G. MOAT
Medical 19102
College,
2, 1971
In our laboratory we have been investigating the enzymes involved in the biosynthesis and degradation of NAD by several microorganisms. In order to determine whether nicotinamide riboside plays a role in NAD biosynthesis, W-labeled nicotinamide riboside was required. However, the labeled compound is not commercially available. Nicotinamide riboside can be prepared from nicotinamide mononucleotide (NMN) by hydrolysis with prostatic monoesterase by the method of Kaplan and Stolzenbath (1) but W-NMN is also unavailable from commercial sources. ‘YJ-NMN can be prepared from 14C-NAD by cleaving the pyrophosphate bond with snake venom phosphodiesterase (2). The procedure requires the use of two enzymic preparations, separation of the 14C-NMN from 5’-AMP by Dowex 1 formate column chromatography and final separation of the nicotinamide riboside formed in the second step. The present paper describes a method for the rapid preparation, in high yield, of nicotinamide riboside with essentially 100% radiochemical purity using a crude enzyme preparation from Proteus vulgaris 0X-19. This preparation normally degrades NAD to free nicotinic acid via NMN, nicotinamide riboside and nicotinamide (Kastov and Moat, unpublished data). Because of the heat stability of the pyrophosphatase and 5’-nucleotidase (3), t,he enzymes which degrade the riboside and nicotinamide can be inactivated by heat treatment, providing a preparation that gives a high yield of nicotinnmide riboside. MATERIALS
AND
METHODS
Nicotinamide[carbonyl-‘“Cl adenine dinucleotide was obtained from Amersham/Searle Corp., Des Plaines, Illinois. Unlabeled nicotinamidendenine dinucleotide (NAD) ChromatoPure, Diagnostic Grade, was obtained from Pabst Laboratories, l\lilwaukee, J$7isconsin. Dowex 1-8X Cl(200400 mesh) was purchased from Bio-Rad, Inc., Richmond, California. 181 @ 1972 by
Academic
Pwss,
Inc.
”
182
KASAROV
AND
MOAT
It was convert,ed to the formate form hy washing with 2 N sodium formate until only traces of Cl- were found in the washings. The products of NAD degradation were identified by paper chromatography using four different solvent systems. (a) propanol/water (4: 1)) (b) pyridine/water (2: l), (c) 1-butanol/acetic acid/water (4: 1:2), and (d) 95% ethanol/l M ammonium acet’ate (7:3). All four were adjusted to pH 5.0 with concentrated HCI. The location of the spots was determined under UV light and by chemical procedures described under “Results and Discussion.” Free ribose was located by using the aniline hydrogen oxalate spray reagent (4). A known sample of ribose was used as a standard. The chromatograms were cut into segments along the direction of solvent flow and placed on stainlesssteel planchets. Radioactivity was determined with a Baird-Atomic lowbeta counter system. All samples were counted to a degree of statistical validity. Inorganic phosphate was assayed by the procedure of Fiske and SubbaRow (5). The absorption spectra were determined with a model 11 Cary recording spectrophotometer. Proteus vulgaris OX-19 was grown in a liquid medium composed of 30 gm/liter Trypticase-Soy Broth (BBL) and 3 gm/liter Bacto yeast extract (Difco), pH 7.4. A 10% inoculum from an 18 hr culture was used. Incubation time was 6 hr at 37°C on a rotary shaker. Cells were harvested by centrifugation in a refrigerated Sorvall centrifuge at 7500 rpm, washed twice with cold 0.9% KCl, and frozen at -20”. The yield of cells was 4.5-5.0 gm wet weight/liter. The crude cell-free enzyme preparation was obtained by disruption of the cells in a Biox X-Press. The disrupted cells were extracted with 0.05 M Tris buffer, pH 7.4, using 1.0-1.5 ml/gm cells (wet weight) and the cell debris and unbroken cells removed by centrifugation at 16,000 rpm in the cold. The resulting clear solution (20-25 mg protein/ml) was placed in a boiling water bath for 2 min, cooled immediately in an ice bath, and centrifuged and the clear supernatant used as the enzyme preparation. The protein concentration in the final preparation was 5-6 mg/ml. Protein was measured by the method of Lowry et al. (6). The same preparation was also made by subjecting a cell suspension containing 2 ml Tris buffer (pH 7.4) per gram cells (wet weight) to sonic disruption in a Branson sonifier for six 15 set intervals. Both preparations were comparable in activity. For the preparation of nicotinamide riboside, a reaction mixture was prepared which contained 30 pmoles NAD, 7.5 PCi 14C-NAD, 12 ml 0.25 M Tris hydrochloride buffer, pH 7.4, and 7.5 ml enzyme preparation (total volume 22 ml). (Preliminary experiments indicated that 0.25 ml enzyme preparation containing approximately 1.5 mg protein/ml was sufficient to complete the degradation of 1 pmole NAD in 2-3 hr. Incubation was
ESZY~IIC
SI-STHESIS
Ok
l’C-h-lCOTl?r’AMII)E
RIBOSIDE
183
conductctl at 37°C.’ for 3 hr. In three separate experiments, paper rhromatography indicated that NAD was degraded to nicotinamidc ribositlc, nicotinamicle, and t’ractes of nicotinamide monouuc1eotide.l Four to ten peg cent, of the radioactivity was observed in the nicotinamide. Nicotinamidc appeared to accumulate RS result of nonenzymic degradation of NAD during the incubation period since the control wit.hont enzyme contained the same percentage of nicotinamide (calculated on the basis of radioactivity). ‘“C-Nicotinamide also appears to arise from the commercial preparation of ‘“C-NAD since various batches of the enzymically prepared material have been found to contain from P-1070 of %-nicotinamide. The mixture was deproteinized with cold 607% HClO, after cooling in an ice bath and the precipitated prot,eins were removed immediately by centrifugation in a refrigerated centrifuge for 5 min at 16,000 rpm. The supernatant was neutralized with 40% KOH and, after removal of the KClO, formed, applied to a 12 x 2.8 cm column of Dowex 1-8X formatc (20&400 mesh). The effluent was collected in 10 ml aliquots simultaneously with the application of the material to the top of the resin bed and after washing the column with distilled water. The ‘%-nicotinamidc riboside was followed by measuring the radioactivity in 20 ~1 samples taken from the first IO-12 t,ubes. Usually the bulk of the radioactirr material was found in two consecutive tubes with smaller amounts of radioactivity being found in the next two tubes. Subsequent fractions that contained traces of radioactivity (less than 20 cpm/20 1.~1)were discarded. The radioactive fractions, containing the nicotinamide riboside, were combined and lyophilized or concentrated to a volume of 5-6 ml by flash evaporation and stored in a frozen state until used. It is very important to note that continued washing with distilled water after the ribosicle had passed the column resulted in the appearance of another small radioactive peak (see Fig. 1). Paper chromatographic analysis showed that, this peak contained nicotinamide and also an unident’ified compound that gave a very strong quenching spot under UV light. Separation of the nicotinamide riboside from nicotinamide by a Dowex I formate column can be cxplainccl as follows: The nicotinamide riboside being positively charged, was not retained by the column; it appeared in the effluent after a single column volume had passed through. The fact that the nicotinamide was retarded indicates that it interacted with the resin. The uncharged nicotinamide may have been simply adsorbed to the resin particles, not held by electrostatic attraction. Because of the small amount of nicotinamide present in the mixture the relatively small adsorption capacity of the resin is enough to retard it. 1 Identical results were obtained against 0.015 M Tris-HCl buffer, pH
with eneymic 7.4, containing
preparations 5 mmoles
dialyzed overnight Z-mercaptoethanol.
”
184
KASAROV
3
6
AND
9
MOAT
12
15
I8
21
24
The
first
peak
TUBE NUMBER FIG.
mide
1. Column chromatography riboside ; the second peak
of reaction is nicotinamide.
RESULTS
AND
products.
is nicotina-
DISCUSSION
The absorption spectrum of the nicotinamide riboside produced under these conditions is shown in Fig. 2. The compound exhibits a sharp maximum at 266 nm when measured in 0.1 M sodium phosphate buffer, pH 7.0. Upon treatment with l.OM KCN, pH 11.0, the peak shifts to 325 nm. The nicotinamide riboside was further identified by the following criteria: Paper chromatography of the preparation with each of the solvent systems showed only one quenching spot and no fluorescent spots under UV light. The Rf values corresponded to those shown in the literature (7-9). The radioactivity migrated with the material that displayed quenching under UV light (see Fig. 3). After treatment with a mixture of methyl ethyl ketone and ammonia (1: 1) according to the method of Kodicek and Reddi (lo), the spot displayed a bluish white fluorescence, indicating that the compound was a quaternary nicotinamide derivative. Upon alkaline hydrolysis in 0.1 M NaOH at 100°C for 10 min to cleave the ribosyl pyridinium bond, only nicotinamide and ribose were observed as products. No other products were detected. Paper chromatography using both the propanol/water and pyridine/water solvent systems revealed a UVabsorbing spot corresponding to nicotinamide. After exposure to cyanogen bromide vapors and successively spraying with 2% p-aminobenzoic acid an orange spot developed, confirming that nicotinamide was a UO), product of alkaline hydrolysis. No inorganic phosphate was detected in
ENZTMIC
SYNTHESIS
OP
WAVE
14C-NICOTINAMIDE
LENGTH,
RIBOSIDE
185
“m
FIG. 2. Absorption spectra of nicotinamide riboside prepared as described in the text. The solid line is the spectrum measured in 0.1 M sodium phosphate buffer, pH 7.0. The broken line is the spectrum measured in 1 M KCN, pH 11.0.
the hydrolyzate. On the basis of these criteria, the product was considered to be nicotinamide riboside. The radiochemical purity of the product was greater than 99% (see Fig. 1). The absence of other quenching or fluorescent spots also provided evidence of a high degree of chemical purity despite the use of a crude enzyme preparation. The fact that no purification steps other than negative absorption on Dowex 1 formate are required lends simplicity to the overall procedure. The total yield of radioactive nicotinamide riboside was 75-80s of the original NAD. The yield of
NAri,
Nii
NMN
NAD
FIG. 3. Paper chromatogram of reaction products and distribution of radioactivity (A). Behavior of reference compounds (B). The solvent system was ethanol/ammonium acetate (7:3) adjusted to pH 5.0. NAm = nicotinamide, NMN = nicotinamide mononucleotide, NAD = nicotinamide-adenine dinucleotide, NR = nicotina mide riboside.
186
KASAROV
AND
nicotinamide riboside was calculated specific activity of the nicotinamide starting NAD.
MOAT
using E,,, = 5.7 X lo3 (11). The riboside was equal to that of the
SUMMARY
A method for the preparation of W-nicotinamide riboside from 14CNAD using a crude enzyme preparation from Proteus vulgaris OX-19 has been described. By heating the preparation in boiling water for 2 min, the enzymes that degrade nicotinamide riboside and nicotinamide are inactivated, providing a system that yields W-nicotinamide riboside at 75430% of the original NAD and with a radiochemical purity greater than 99%. ACKNOWLEDGMENTS These investigations Grant GB-8695.
were
supported
in
part
by
National
Science
Foundation
REFERENCES 1. KAPLAN, N. O., AND STOLZENBACH, F. E., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 901. Academic Press, New York, 1957. 2. KAPLAN, N. O., AND STOLZENBACH, F. E., in “Methods in Enzymology,” Vol. III, p. 899. 3. SWARTZ, M. N., KAPLAN, N. O., AND LAMBORG, M. F., J. 13ioZ. Chem. 232, 1051 (1958). 4. HORROCKS, R. H.. AND MANNING, G. B.. Lrcncet 256, 1042 (1949). 5. FISKE, C. H., AND SUBBAROW, Y.. J. Viol. Chem. 81, 629 (1929). 6. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, -4. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 7. BURTON, R. M., AND SAN PIETRO. A., Arch. Biochem. Biophys. 48, 184 (1954). 8. PREISS, J., AND HANDLER, P., J. Biol. Chem. 233, 488 (1958). 9. NISHIZUKA, Y., AND HAYAISHI, O., J. Biol. Ckem. 238, PC483 (1963). 10. KODICEK, E., AND REDDI, K. K., Nature 168, 475 (1951). 11. ROWEN, J. W., AND KORNBERG, A., J. Biol. Chem. 193,497 (1951).
46, 181-186
BIOCHEMISTRY
(1972)
Convenient Method for Enzymic Synthesis of 14C-Nicotinamide Riboside LUKA Depnrtment
B. KASAROV of
Microbiology, Philadelphia, Received
AND
ALBERT
Hahnemann Pennsylvania July
G. MOAT
Medical 19102
College,
2, 1971
In our laboratory we have been investigating the enzymes involved in the biosynthesis and degradation of NAD by several microorganisms. In order to determine whether nicotinamide riboside plays a role in NAD biosynthesis, W-labeled nicotinamide riboside was required. However, the labeled compound is not commercially available. Nicotinamide riboside can be prepared from nicotinamide mononucleotide (NMN) by hydrolysis with prostatic monoesterase by the method of Kaplan and Stolzenbath (1) but W-NMN is also unavailable from commercial sources. ‘YJ-NMN can be prepared from 14C-NAD by cleaving the pyrophosphate bond with snake venom phosphodiesterase (2). The procedure requires the use of two enzymic preparations, separation of the 14C-NMN from 5’-AMP by Dowex 1 formate column chromatography and final separation of the nicotinamide riboside formed in the second step. The present paper describes a method for the rapid preparation, in high yield, of nicotinamide riboside with essentially 100% radiochemical purity using a crude enzyme preparation from Proteus vulgaris 0X-19. This preparation normally degrades NAD to free nicotinic acid via NMN, nicotinamide riboside and nicotinamide (Kastov and Moat, unpublished data). Because of the heat stability of the pyrophosphatase and 5’-nucleotidase (3), t,he enzymes which degrade the riboside and nicotinamide can be inactivated by heat treatment, providing a preparation that gives a high yield of nicotinnmide riboside. MATERIALS
AND
METHODS
Nicotinamide[carbonyl-‘“Cl adenine dinucleotide was obtained from Amersham/Searle Corp., Des Plaines, Illinois. Unlabeled nicotinamidendenine dinucleotide (NAD) ChromatoPure, Diagnostic Grade, was obtained from Pabst Laboratories, l\lilwaukee, J$7isconsin. Dowex 1-8X Cl(200400 mesh) was purchased from Bio-Rad, Inc., Richmond, California. 181 @ 1972 by
Academic
Pwss,
Inc.
”
182
KASAROV
AND
MOAT
It was convert,ed to the formate form hy washing with 2 N sodium formate until only traces of Cl- were found in the washings. The products of NAD degradation were identified by paper chromatography using four different solvent systems. (a) propanol/water (4: 1)) (b) pyridine/water (2: l), (c) 1-butanol/acetic acid/water (4: 1:2), and (d) 95% ethanol/l M ammonium acet’ate (7:3). All four were adjusted to pH 5.0 with concentrated HCI. The location of the spots was determined under UV light and by chemical procedures described under “Results and Discussion.” Free ribose was located by using the aniline hydrogen oxalate spray reagent (4). A known sample of ribose was used as a standard. The chromatograms were cut into segments along the direction of solvent flow and placed on stainlesssteel planchets. Radioactivity was determined with a Baird-Atomic lowbeta counter system. All samples were counted to a degree of statistical validity. Inorganic phosphate was assayed by the procedure of Fiske and SubbaRow (5). The absorption spectra were determined with a model 11 Cary recording spectrophotometer. Proteus vulgaris OX-19 was grown in a liquid medium composed of 30 gm/liter Trypticase-Soy Broth (BBL) and 3 gm/liter Bacto yeast extract (Difco), pH 7.4. A 10% inoculum from an 18 hr culture was used. Incubation time was 6 hr at 37°C on a rotary shaker. Cells were harvested by centrifugation in a refrigerated Sorvall centrifuge at 7500 rpm, washed twice with cold 0.9% KCl, and frozen at -20”. The yield of cells was 4.5-5.0 gm wet weight/liter. The crude cell-free enzyme preparation was obtained by disruption of the cells in a Biox X-Press. The disrupted cells were extracted with 0.05 M Tris buffer, pH 7.4, using 1.0-1.5 ml/gm cells (wet weight) and the cell debris and unbroken cells removed by centrifugation at 16,000 rpm in the cold. The resulting clear solution (20-25 mg protein/ml) was placed in a boiling water bath for 2 min, cooled immediately in an ice bath, and centrifuged and the clear supernatant used as the enzyme preparation. The protein concentration in the final preparation was 5-6 mg/ml. Protein was measured by the method of Lowry et al. (6). The same preparation was also made by subjecting a cell suspension containing 2 ml Tris buffer (pH 7.4) per gram cells (wet weight) to sonic disruption in a Branson sonifier for six 15 set intervals. Both preparations were comparable in activity. For the preparation of nicotinamide riboside, a reaction mixture was prepared which contained 30 pmoles NAD, 7.5 PCi 14C-NAD, 12 ml 0.25 M Tris hydrochloride buffer, pH 7.4, and 7.5 ml enzyme preparation (total volume 22 ml). (Preliminary experiments indicated that 0.25 ml enzyme preparation containing approximately 1.5 mg protein/ml was sufficient to complete the degradation of 1 pmole NAD in 2-3 hr. Incubation was
ESZY~IIC
SI-STHESIS
Ok
l’C-h-lCOTl?r’AMII)E
RIBOSIDE
183
conductctl at 37°C.’ for 3 hr. In three separate experiments, paper rhromatography indicated that NAD was degraded to nicotinamidc ribositlc, nicotinamicle, and t’ractes of nicotinamide monouuc1eotide.l Four to ten peg cent, of the radioactivity was observed in the nicotinamide. Nicotinamidc appeared to accumulate RS result of nonenzymic degradation of NAD during the incubation period since the control wit.hont enzyme contained the same percentage of nicotinamide (calculated on the basis of radioactivity). ‘“C-Nicotinamide also appears to arise from the commercial preparation of ‘“C-NAD since various batches of the enzymically prepared material have been found to contain from P-1070 of %-nicotinamide. The mixture was deproteinized with cold 607% HClO, after cooling in an ice bath and the precipitated prot,eins were removed immediately by centrifugation in a refrigerated centrifuge for 5 min at 16,000 rpm. The supernatant was neutralized with 40% KOH and, after removal of the KClO, formed, applied to a 12 x 2.8 cm column of Dowex 1-8X formatc (20&400 mesh). The effluent was collected in 10 ml aliquots simultaneously with the application of the material to the top of the resin bed and after washing the column with distilled water. The ‘%-nicotinamidc riboside was followed by measuring the radioactivity in 20 ~1 samples taken from the first IO-12 t,ubes. Usually the bulk of the radioactirr material was found in two consecutive tubes with smaller amounts of radioactivity being found in the next two tubes. Subsequent fractions that contained traces of radioactivity (less than 20 cpm/20 1.~1)were discarded. The radioactive fractions, containing the nicotinamide riboside, were combined and lyophilized or concentrated to a volume of 5-6 ml by flash evaporation and stored in a frozen state until used. It is very important to note that continued washing with distilled water after the ribosicle had passed the column resulted in the appearance of another small radioactive peak (see Fig. 1). Paper chromatographic analysis showed that, this peak contained nicotinamide and also an unident’ified compound that gave a very strong quenching spot under UV light. Separation of the nicotinamide riboside from nicotinamide by a Dowex I formate column can be cxplainccl as follows: The nicotinamide riboside being positively charged, was not retained by the column; it appeared in the effluent after a single column volume had passed through. The fact that the nicotinamide was retarded indicates that it interacted with the resin. The uncharged nicotinamide may have been simply adsorbed to the resin particles, not held by electrostatic attraction. Because of the small amount of nicotinamide present in the mixture the relatively small adsorption capacity of the resin is enough to retard it. 1 Identical results were obtained against 0.015 M Tris-HCl buffer, pH
with eneymic 7.4, containing
preparations 5 mmoles
dialyzed overnight Z-mercaptoethanol.
”
184
KASAROV
3
6
AND
9
MOAT
12
15
I8
21
24
The
first
peak
TUBE NUMBER FIG.
mide
1. Column chromatography riboside ; the second peak
of reaction is nicotinamide.
RESULTS
AND
products.
is nicotina-
DISCUSSION
The absorption spectrum of the nicotinamide riboside produced under these conditions is shown in Fig. 2. The compound exhibits a sharp maximum at 266 nm when measured in 0.1 M sodium phosphate buffer, pH 7.0. Upon treatment with l.OM KCN, pH 11.0, the peak shifts to 325 nm. The nicotinamide riboside was further identified by the following criteria: Paper chromatography of the preparation with each of the solvent systems showed only one quenching spot and no fluorescent spots under UV light. The Rf values corresponded to those shown in the literature (7-9). The radioactivity migrated with the material that displayed quenching under UV light (see Fig. 3). After treatment with a mixture of methyl ethyl ketone and ammonia (1: 1) according to the method of Kodicek and Reddi (lo), the spot displayed a bluish white fluorescence, indicating that the compound was a quaternary nicotinamide derivative. Upon alkaline hydrolysis in 0.1 M NaOH at 100°C for 10 min to cleave the ribosyl pyridinium bond, only nicotinamide and ribose were observed as products. No other products were detected. Paper chromatography using both the propanol/water and pyridine/water solvent systems revealed a UVabsorbing spot corresponding to nicotinamide. After exposure to cyanogen bromide vapors and successively spraying with 2% p-aminobenzoic acid an orange spot developed, confirming that nicotinamide was a UO), product of alkaline hydrolysis. No inorganic phosphate was detected in
ENZTMIC
SYNTHESIS
OP
WAVE
14C-NICOTINAMIDE
LENGTH,
RIBOSIDE
185
“m
FIG. 2. Absorption spectra of nicotinamide riboside prepared as described in the text. The solid line is the spectrum measured in 0.1 M sodium phosphate buffer, pH 7.0. The broken line is the spectrum measured in 1 M KCN, pH 11.0.
the hydrolyzate. On the basis of these criteria, the product was considered to be nicotinamide riboside. The radiochemical purity of the product was greater than 99% (see Fig. 1). The absence of other quenching or fluorescent spots also provided evidence of a high degree of chemical purity despite the use of a crude enzyme preparation. The fact that no purification steps other than negative absorption on Dowex 1 formate are required lends simplicity to the overall procedure. The total yield of radioactive nicotinamide riboside was 75-80s of the original NAD. The yield of
NAri,
Nii
NMN
NAD
FIG. 3. Paper chromatogram of reaction products and distribution of radioactivity (A). Behavior of reference compounds (B). The solvent system was ethanol/ammonium acetate (7:3) adjusted to pH 5.0. NAm = nicotinamide, NMN = nicotinamide mononucleotide, NAD = nicotinamide-adenine dinucleotide, NR = nicotina mide riboside.
186
KASAROV
AND
nicotinamide riboside was calculated specific activity of the nicotinamide starting NAD.
MOAT
using E,,, = 5.7 X lo3 (11). The riboside was equal to that of the
SUMMARY
A method for the preparation of W-nicotinamide riboside from 14CNAD using a crude enzyme preparation from Proteus vulgaris OX-19 has been described. By heating the preparation in boiling water for 2 min, the enzymes that degrade nicotinamide riboside and nicotinamide are inactivated, providing a system that yields W-nicotinamide riboside at 75430% of the original NAD and with a radiochemical purity greater than 99%. ACKNOWLEDGMENTS These investigations Grant GB-8695.
were
supported
in
part
by
National
Science
Foundation
REFERENCES 1. KAPLAN, N. O., AND STOLZENBACH, F. E., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 901. Academic Press, New York, 1957. 2. KAPLAN, N. O., AND STOLZENBACH, F. E., in “Methods in Enzymology,” Vol. III, p. 899. 3. SWARTZ, M. N., KAPLAN, N. O., AND LAMBORG, M. F., J. 13ioZ. Chem. 232, 1051 (1958). 4. HORROCKS, R. H.. AND MANNING, G. B.. Lrcncet 256, 1042 (1949). 5. FISKE, C. H., AND SUBBAROW, Y.. J. Viol. Chem. 81, 629 (1929). 6. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, -4. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 7. BURTON, R. M., AND SAN PIETRO. A., Arch. Biochem. Biophys. 48, 184 (1954). 8. PREISS, J., AND HANDLER, P., J. Biol. Chem. 233, 488 (1958). 9. NISHIZUKA, Y., AND HAYAISHI, O., J. Biol. Ckem. 238, PC483 (1963). 10. KODICEK, E., AND REDDI, K. K., Nature 168, 475 (1951). 11. ROWEN, J. W., AND KORNBERG, A., J. Biol. Chem. 193,497 (1951).