Enzymatic synthesis and reactions of uridine 5′-(5-thio-α-d -glucopyranosyl pyrophosphate)

ARCHIVES

OF BIOCHEMISTRY

Enzymatic

AND

BIOPHYSICS

171,

721-726

(1975)

Synthesis and Reactions of Uridine Glucopyranosyl Pyrophosphate)’ TERRENCE

Department

L. GRAHAM

of Biochemistry,

AND

Purdue Received

ROY L. WHISTLER

University May

5’-(5Thio-a-D-

Lafayette,

Indiana

47907

19, 1975

Uridine 5’-(5thio-a-nglucopyranosyl pyrophosphate), UDPTG, is an efficient substrate for yeast uridine 5’-(nglucopyranosyl pyrophosphatel, UDPG, pyrophosphorylase. K, for UDPTG with the pyrophosphorylase is 0.2 mM and the analog reacts with a maximal velocity 96% that of UDPG. UDPTG is also a substrate for yeast UDP-gala&se 4-epimerase. Although not a substrate for bovine liver UDPG dehydrogenase, UDPTG is a potent, mixed-type inhibitor with respect to both UDPG and nicotinamide adenine dinucleotide (NAD). UDPTG is synthesized in 30% yield from 5-thio-n-glucopyranose and in 85% yield from 5-thio-a-nglucopyranose l-phosphate by using mixtures of commercially available enzymes. The pK, of the uracil moiety in UDPTG is the same as that in UDPG, and UDPTG appears to be similar to UDPG in the extent of secondary structural order. UDPTG, however, is more highly acid-labile than UDPG.

In previous work (Graham and Whistler, unpublished) we chemically synthesized uridine 5’-(5thio-a-nglucopyranosyl pyrophosphate), UDPTG.2 This analog, containing sulfur as the heteroatom in the n-glucopyranosyl moiety, is a potent activator of glycogen synthetase and has been of value in examining the nature of UDPG cooperativity with glycogen synthetase (Graham and Whistler, unpublished). These results prompted us to investigate the interaction of UDPTG with the other important UDPG-requiring enzymes, UDPG dehydrogenase, UDP-gala&se 4epimerase and UDPG pyrophosphorylase. UDPTG is of unique value in that it is the nearest analog through which the contribution of the pyranose heteroatom to enzymatic behavior can be evaluated. We also report the enzymatic synthesis of UDPTG and further physical characterization of the molecule. PROCEDURE and Materials Qualitative paper chromatography was formed by the descending method on Whatman

1 paper, and preparative paper chromatography by the descending method on Whatman No. 3 MM paper, with the following irrigants: (A) ethanol/O.5 M ammonium acetate, pH 3.8 (5:2, v/v); (B) ethanol 1 M ammonium acetate, pH 7.5 (5:2, v/v) (1); (C) acetone/ethanol (2:1, v/v); (Dt ethanolimethylethyl ketone/0.5 M morpholinium tetraborate, pH 8.6, in 0.01 M EDTA (7:2:3, v/v) (2). Sugars were visualized by spraying with anisaldehyde reagent (3), and inorganic salts were located by spraying with a saturated solution of alizarin in alcohol, followed by 25% ammonium hydroxide (4). Nucleosides, nucleotides and nucleoside Y-(glycopyranosyl pyrophosphates) were visualized by irradiation with short-wavelength ultraviolet light. Ultraviolet spectra were determined on a Beckman DB spectrophotometer and optical rotations were determined on a Perkin-Elmer Model 141 automatic polarimeter. 5-Thio-oglucopyranose was prepared by the method of Nayak and Whistler (5) and 5-thio-a-o glucopyranose l-phosphate by the method of Whistler and Stark (61. UDPG, UMP, UDP, UTP, ATP, NAD (Grade V, yeast), NADH (Grade III, yeast) and 3-phosphoglycerate were obtained from Sigma Chemical Company, St. Louis, MO. Inorganic pyrophosphatase (Type III, yeast, EC 3.6.1.11, hexokinase (Type F-300, sulfate-free from yeast, EC 2.7.1.11, phosphoglucomutase (rabbit muscle, EC 3.7.5.11, UDPG pyrophosphorylase (Type X, yeast, EC 2.7.7.91, UDPG dehydrogenase (Type III, bovine liver, EC 1.1.1.221, UDP-galactose 4-epimerase (Grade III, from galactose-adapted yeast, EC 5.1.3.2),3-phosphoglycerate phosphokinase (Type X,

EXPERIMENTAL

General

Methods

perNo.

’ Journal Paper No. 5886 of the Purdue Agricultural Experiment Station. 2 Abbreviations used: UDPTG, uridine 5’-(5-thioa-n-glucopyranosyl pyrophosphate); UDPG, uridine 5’-(oglucopyranosyl pyrophosphatel. 721 Copyright All rights

0 1975 by Academic Press, of reproduction in any form

Inc. reserved.

722

GRAHAM

yeast, EC 2.7.2.3) and dehydrogenase (yeast, tained from Sigma.

Enzymatic

Synthesis

glyceraldehyde EC 1.2.1.12)

AND

J-phosphate were also ob-

of UDPTG

The enzymatic synthesis of UDPG from nglucase, n-glucose B-phosphate, n-glucose l-phosphate, and nglucose/nfructose mixtures has been established as a useful method for the synthesis of radioactive UDPG species as well as certain UDPG analogs (7). However, certain disadvantages and uncertainties arise from the common use of partially purified enzyme preparations as the source of the enzyme mixture (8, 9). To circumvent such problems, we used mixtures of the commercially available purified enzymes. In addition to eliminating the problems of contamination by endogenous substrates and the enzymatic isomerization (9) or hydrolysis (8) of newly formed UDPG, such a highly flexible component system can be specifically modified to suit the synthesis of any given UDPG analog. Of the four enzymes needed for the synthesis of UDPG from n-glucose, phosphoglucomutase is the most highly sensitive; its activity is, in fact, dramatically affected by several of the cofactors, products and substrates of the other enzymes. UTP, UDP, orthophosphate, pyrophosphate and high concentrations of magnesium are all inhibitors of phosphoglucomutase (10). The incubation mixture reported here for the synthesis of UDPTG from 5thio-nglucase was balanced to provide near optimal conditions for phosphoglucomutase activity but still allow fairly efficient conditions for the other enzymes. In addition, since sulfate is a potent inhibitor of phosphoglucomutase (lo), any sulfate present in the commercial enzyme preparations was removed prior to their use. In the preparation of UDPTG from 5-thio-nglucase, addition of ATP greatly enhanced the formation of the analog. The mechanism of stimulation by ATP is not clear. Thin-layer chromatography indicated that the formation of 5-thio-*glucose 6-phosphate proceeds quickly when UTP is used as a phosphate donor, and work with 5-thio-nglucose l-phosphate had shown that the UDPG pyrophosphorylase reaction was not limiting. Although the reaction of 5-thio-cY-nglucopyranose l-phosphate with phosphoglucomutase to form Sthio-a-nglucopyranose 6phosphate proceeds normally (111, the reverse reaction has not been investigated in detail. Enzymatic synthesis of UDPTG was achieved from 5-thio-cY+glucopyranose l-phosphate using a mixture of commercial UDPG pyrophosphorylase and inorganic pyrophosphatase. A typical incubation mixture contained 10 mM 5-thio-or-nglucopyranose l-phosphate, 15 mM UTP, 15 mM magnesium acetate, 25 units of UDPG pyrophosphorylase and 10 units of inorganic pyrophosphatase in 5 ml of 50 mM

WHISTLER Tris-HCl (pH 7.4). Reaction, at 3O”C, was complete in 2-4 h, and preparative paper chromatography, at 5”C, was immediately carried out on the mixture using irrigants (Al, (B) and (C) sequentially. The yield of UDPTG measured at 262 nm as calculated from starting 5-thio-a-nglucopyranose l-phosphate was 85%. Purified and lyophilized UDPTG was stored frozen at -20°C. UDPG was also synthesized in a similar incubation system in 95% yield. Enzymatic synthesis of UDPTG from 5-thio-n glucopyranose was achieved by using a mixture of commercial hexokinase, phosphoglucomutase, UDPG pyrophosphorylase, and inorganic pyrophosphatase. A typical incubation contained: 5 mM UTP, 10 mM magnesium chloride, 1 mM EDTA, 10 PM glucose 1,6-diphosphate and 2 mM nglucose (or 5thio-nglucopyranose) in 50 mM Tris-HCl, pH 7.4. When 5-thio-nglucose was used as a substrate, 5 mM ATP was also present in the reaction mixture. Normally, separate 5-ml reaction mixtures were run simultaneously, each reaction mixture containing 40 unite of hexokinase, 40 units of phosphoglucomutase, 25 units of UDPG pyrophosphorylase and 10 units of inorganic pyrophosphatase. The phosphoglucomutase preparation was diluted immediately before use in a reagent mixture, kindly suggested by Dr. William Ray, containing 100 mM imidazole, 20 mM Tris-HCl (pH 7.41, 2 mM magnesium chloride and 1 mM EDTA. If the reagent is to be stored for prolonged periods, the magnesium chloride is added to the mixture just before use. Reaction was allowed to proceed for 36 h at 3o”C, and the UDPTG formed was isolated by paper chromatography as described above. The yield of UDPTG, calculated from 5-thio-nglucose, was 30%. The relatively low yield is due to the high lability of UDPTG; the long incubation time was, however, necessary due to the very low rate of formation of 5thio-nglucose l-phosphate in the phosphoglucomutase reaction. The yield for UDPG, with use of much shorter incubation times (3 h), was 90%. Enzymatically synthesized UDPTG was analyzed for purity by using the same procedures employed to examine chemically snythesized UDPTG (Graham and Whistler, unpublished). This included chemical hydrolyses at pH 2 and in 1 N HCl, enzymatic hydrolysis by nucleotide pyrophosphatase, and mobilities of the molecule and its hydrolysates on paper chromatography and paper electrophoresis (Graham and Whistler, unpublished). The enzymatically prepared UDPTG was homogenous by all criteria and free of UDP, UTP, and uridine 5’-(5-thio-a-ngalactopyranosyl pyrophosphate).

Hydrolysis of Sugar 1 -lf’hosphates SThio-a-n-glucopyranose glucopyranose l-phosphate in 0.5 N HCI. To 1.0 ml

l-phosphate and U-D were hydrolyzed at 45°C of 0.5 N HCl at 45°C was

ENZYME

REACTIONS

added 10 ~1 of a 100 mM solution of the sugar lphosphate. At the specified times, hydrolyses were stopped by immersion of the tubes in an ice bath. When all incubations were complete, free orthophosphate was determined by the method of Fiske and SubbaRow (12). A series of unheated controls, containing the sugar l-phosphates and ice-cold 0.5 N HCl were simultaneously analyzed to correct for possible hydrolysis during phosphate determination.

Enzyme

Assay

723

OF UDP-5-THIO-DGLUCOSE

Procedures

UDPG dehydrogenase was assayed by the method of Zalitis et al. (131, and UDPG pyrophosphorylase was assayed by Method II of Hansen et al. (14). UDP-gala&se 4-epimerase activity was monitored by modifications of the methods of Salo et al. (15). Reaction mixtures contained: 2 mM UDPG (or UDPTG), 0.2 mM NAD, 2.5 units of epimerase and 50 mM glycine buffer, pH 8.5, in a total volume of 500 ~1. Controls, containing boiled enzyme, were run simultaneously with the reactions. After an 8-h incubation the mixtures were evaporated to dryness and hydrolyzed in 50 ~1 of 1 N HCl for 10 min at 100°C. The mixtures were cooled to room temperature and deionized by passing through small columns (5 ml1 containing a 1:l mixture of Amberlite IR-45 (OH-) and IR-120 (H+). The effluents were again evaporated to dryness and applied to silica gel thin-layer chromatographic plates (3 x 12 in.) impregnated with sodium acetate (16). Upon irrigation with 1:l chloroform/methanol the following R;s were obtained: glucose, 0.41; gala&se, 0.37; 5-thio-nglucase, 0.42; and 5-thio-ugalactose, 0.37. Sugars were visualized by charring after spraying with 5% sulfuric acid in ethanol. Alternatively, the epimerase reaction mixtures were spotted directly on the thin-layer plates and hydrolyzed on the plates at 100°C for 10 min in a saturated atmosphere of 50% HCl. Before chromatography the plates were air dried to remove residual HCl. UDP-gala&se 4-epimerase activity was also monitored by paper chromatography of the products of the reaction before hydrolysis. Paper chromatographs were eluted with solvent (D) for 40 h. The RUDPG'sof UDPG, UDP-gala&se, UDPTG and uridine 5’-(5-thio-or-c-galactopyranosyl pyrophosphate), UDPT-Gal, were 1.00, 0.89, 0.99 and 0.90 respectively.

and enzymatic hydrolysis products of UDPTG. Here we report the pKa of the uracil function in UDPTG, the optical rotatory properties of UDPTG in water and in 8 M urea, and the relative labilities of D glucose l-phosphate and 5-thio-nglucose l-phosphate to acid hydrolysis. The pKa of the uracil moiety of UDPTG was determined here by the method of Shugar and Fox (17). The spectral titration curve (Fig. 1) yields a value of 9.5 for the pK,. This value is identical to that obtained for UDPG (181, indicating that the ionic environment of the uracil moiety has not been affected by the introduction of sulfur as heteroatom in the nglucose moiety. By use of the method of Hirano (19), the optical rotation of UDPG and UDPTG were compared in water and in 8 M urea. The UDPG preparation displayed a dextrorotatory contribution of 17.9% resulting from secondary structure, while UDPTG showed a dextro-rotatory contribution of 13.7%. The value for UDPTG falls within the percentage of variation seen with different UDPG preparations (19) and suggests that UDPG and UDPTG may not differ in the extent of secondary order. In earlier work (Graham and Whistler, unpublished), it was noted that UDPTG is much more labile to acidic hydrolysis than UDPG. Although the acid lability of the D glucose-phosphate bond in UDPG is six times greater than that in nglucose lphosphate (201, the relative rates of hydrolysis of n-glucose l-phosphate and 5-thio-nglucose l-phosphate may be taken as an

RESULTS

Physical

Properties

of UDPTG

In earlier work (Graham and Whistler, unpublished) we examined the ultravioletabsorbing characteristics of UDPTG and made a auantitative analvsis of the acidic

PH

FIG. 1. Spectral cal densities

were

titration curve for UDPTG. read at 234 nm.

Opti-

724

GRAHAM

AND

indication of the relative labilities of UDPG and UDPTG, assuming similar environments for the sugar components in UDPG and UDPTG. The rates of hydrolysis of a-n-glucose l-phosphate and 5-thion-n-glucose l-phosphate at 45°C in 0.50 N HCl (Fig. 2) are found to be 0.022 and 0.357 pmol/min, respectively. These results indicate that 5-thio-cr-D-glucose l-phosphate is 16 times more labile to acid than natural a-n-glucose l-phosphate. This latter value agrees well with the finding that methyl 5thio-n-xylopyranosides are hydrolyzed lo15 times faster than methyl n-xylopyranosides (21). Hydrolyses of both glycosides and sugar l-phosphates likely occur by similar S&l reactions at the anomeric carbon cz-3.

UDPTG as Substrate for Yeast UDPG Pyrophosphorylase UDPTG is an efficient substrate for yeast UDPG pyrophosphorylase. The K, of the pyrophosphorylase for UDPG, at 25”C, pH 7.8, was determined to be 0.13 mM. This value compares favorably with the previously reported K, for UDPG of 0.09 mM (23). UDPTG (Fig. 3) displays a K, of 0.20 mM with the yeast pyrophosphorylase. In addition, UDPTG reacts with a maximal velocity 96% that of UDPG. Change in the heteroatom of the n-glucose moiety of UDPG seems to have, therefore, relatively little effect on binding or reactivity with yeast UDPG pyrophosphorylase. Although the specificities of calf and human liver UDPG pyrophosphorylase have been investigated in detail (241, the specificity of yeast UDPG pyrophosphorylase

2

4

FIG. 2. Hydrolysis of sugar a-mglucopyranose l-phosphate ranose l-phosphate (b) were acid at 45°C.

12

14

16

18

l-phosphates. 5-Thio(a) and cr-mglucopyhydrolyzed in 0.50 N

WHISTLER

WDPTG.

mM-’

FIO. 3. K, of UDPG pyrophosphorylase for UDPTG. Pyrophosphate concentration was constant at 10 mM.

has not been extensively investigated despite its use in the synthesis of UDPG and UDPG analogs. Yeast UDPG pyrophosphorylase has been used in the synthesis of the UDPG analogs containing pseudo-uridine (251, 5-hydroxyuridine (261, and 2deoxy-Darabinohexose (27), but no kinetic constants are given. The liver enzymes show low specificity for nucleoside diphosphate hexoses, but the relative reaction rates of analogs with respect to UDPG are low, ranging from 0.1 to 4.0% (24). The finding here that UDPTG is an efficient substrate for the yeast enzyme is interesting, but may simply reflect a generally lower specificity of the yeast enzyme for nucleoside diphosphate sugars. UDPTG as Substrate for Yeast UDP-Galactose 4-Epimerase UDPTG serves as a substrate for yeast UDP-galactose 4-epimerase. The product, uridine 5’-(5-thio-cw-D-galactopyranosyl pyrophosphate) has been tentatively identified on the basis of paper chromatography of the whole molecule and thin-layer chromatography of the free sugar, 5-thio-ngalactose, after hydrolysis. Chromatographic mobilities are reported in Experimental Procedure. Interaction of UDPTG with UDPG Dehydrogenase UDPTG does not serve as a substrate for beef liver UDPG dehydrogenase even upon prolonged incubation at concentrations up to 100 times the K, for UDPG. K,‘s of 2.98 x 1O-5 M for UDPG and 2.0 x lop4 M for NAD were obtained with the dehydrogenase preparation. These values compare rea-

ENZYME

REACTIONS

OF

sonably with the respective literature values of 1.3 x low5 and 1.0 x lop4 M (13). One requirement for reactivity with UDPG dehydrogenase is that the uracil moiety is in the nonionized form (18). Since the fia of the uracil moiety is the same in both UDPG and UDPTG, lack of reactivity of UDPTG with the dehydrogenase is not due to changes in the ionization of this group. Possibly lack of reactivity may be due to a proximity effect of the large sulfur heteroatom on the reduction at C6. Though not a substrate, UDPTG is a highly potent inhibitor of the dehydrogenase with respect to UDPG. From the double-reciprocal plot (Fig. 41, the inhibition pattern appears that of a mixed-type inhibitor. Mixed-type inhibition has been seen for UDPG dehydrogenase with 5hydroxy UDPG (26) and UDP-galactosamine (28). In the case of UDP-galactosamine, the inhibition is nearly competitive; however, with 5hydroxy UDPG, the inhibition is mainly noncompetitive, even though the analog is a poor substrate. The noncompetitive inhibition was explained for 5-hydroxy UDPG when it was found that 5hydroxy UDPG is an even more potent mixed-type inhibitor with respect to NAD (24. As shown in Fig. 5, UDPTG is also a potent, mixed-type inhibitor with respect to NAD. This may explain the partially noncompetitive inhibition by UDPTG with respect to UDPG and suggests that UDPTG, like 5-hydroxy UDPG (24), interferes with both UDPG and NAD binding.

:

P x % h c E

-40

-20

0

20 I/MPG.

40

GO

80

100

mM-’

FIG. 4. UDPTG inhibition of UDPG dehydrogenase with respect to UDPG. In addition to 2.0 x 1O-3 M NAD, reaction mixtures contained: (a), 6.92 x 10m5 M UDPTG; (b), 3.46 X 10m5 M UDPTG; and cc), no addition.

725

UDP-5-THIO-DGLUCOSE

5 40 I/NAD.

8.0 mM-’

12.0

FIG. 5. UDPTG inhibition of UDPG dehydrogenase with respect to NAD. In addition to 3.0 x 1OW M UDPG, reaction mixtures contained: (a), 4.0 x 1OF M UDPTG; (b), 3.1 x 1O-5 M UDPTG; (cl, no addition.

DISCUSSION

Kochetkov et al. (29, 301, on the basis of enzyme investigations, have proposed a preferred secondary structure for UDPG in aqueous solution upon which the activities of UDPG-requiring enzymes, especially UDPG dehydrogenase and UDPG epimerase, critically depend. The proposed structure is maintained by hydrogen bonds between the pyranose and pyrimidine rings, producing a compact U-shaped molecule. Changes in either the pyranose ring or pyrimidine ring which disrupt the proposed hydrogen binding are expected to affect the binding or reactivity of UDPG with its enzymes. A great number of analogs have been investigated with UDPG dehydrogenases and UDPG epimerases from a variety of sources (7). In general this analog work supports the importance of the pyranose and pyrimidine rings for activity. Moreover, physical data obtained from comparison of the optical rotation of UDPG in water and in 8 M urea (19) supports the existence of a secondary structure for UDPG in aqueous solution. However, to our knowledge, none of the UDPG analogs examined with the dehydrogenase or epimerase has been physically examined in optical rotation experiments. UDPTG is possibly the nearest known analog of UDPG by which the role of the pyranose heteroatom in enzyme activity of UDPG can be assessed. UDPTG is of spe-

726

GRAHAM

AN D WHISTLER

cial interest in the interpretation of the model of Kochetkov and co-workers (29, 30), since replacement of the oxygen heteroatom by sulfur would be expected to disrupt hydrogen bonding at that center. Optical rotation comparisons, following the work of Hirano (19), suggest that UDPTG and UDPG do not differ significantly in the degree of secondary order. Moreover, UDPTG apparently binds to the UDPG sites of both yeast UDPG epimerase and bovine liver UDPG dehydrogenase. Thus, both physical and enzymatic observations with UDPTG are consistent with the model of Kochetkov and co-workers (29,30). In that model, no intramolecular hydrogen bond is thought to be present at the heteroatom of the pyranose ring. ACKNOWLEDGEMENT

AM

This work was supported 18482 from the National

in part Institutes

by Grant of Health.

No.

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11. CHEN, M., AND WHISTLER, R. L. (1975) Arch. Biochem. Biophys., in press. 12. FISKE, C. H., AND SUBBAROW, Y. (1925) J. Biol. Chem. 66,375-400. 13. ZALITIS, J., URAM, M., BOWSER, A. M., AND FEINGOLD, D. S. (1972) in Methods in Enzymology (Ginsburg, V., ed.), Vol. 28, pp. 430-435, Academic Press, New York. 14. HANSEN, R. G., ALBRECHT, G. J., BASS, S. T., AND SEIFERT, L. L. (1966) in Methods in Enzymology (Neufeld, E. F., and Ginsburg, V., eds.), Vol. 8, pp. 248-253, Academic Press, New York. 15. SALO, W. L., NORDIN, J. H., PETERSON, D. R., BEVILL, R. D., AND KIRKWOOD, S. (1968) Biochim. Biophys. Actu 151,484-492. 16. WEIDEMAN, G., AND FISCHER, W. (1964) HoppeSeyler’s 2. Physiol. Chem. 336, 189-191. 17. SHUGAR, D., AND Fox, J. J. (1952) Biochim. Biophys. Actu 9, 199-218. 18. GOLDBERG, N. D., DAHL, J. L., AND PARKS, R. E. (1963) J. Biol. Chem. 238, 3109-3114. 19. HIRANO, S. (1971) Biochem. Biophys. Res. Commun. 43,1219-1222. 20. CAPUTTO, R., LELOIR, L. F., CARDINI, C. E., AND PALADINI, A. C. (1950) J. Biol. Chem. 184, 333-350. 21. WHISTLER, R. L., AND VAN Es, T. (1963) J. Org. Chem. 28, 2303-2304. 22. KIRBY, A. J., AND WARREN, S. G. (1967) The Organic Chemistry of Phosphorous, p. 207, Elsevier, New York. 23. BERNSTEIN, R. L., AND ROBBINS, P. W. (1965) J. Biol. Chem. 240,391-397. 24. TURNQUIST, R. L., AND HANSEN, R. G. (1973) in The Enzymes (Boyer, P. D., ed.), Vol. 8, pp. 51-71, Academic Press, New York. 25. RABINOWITZ, M., AND GOLDBERG, I. H. (1963) J. Biol. Chem. 238, 1801-1806. 26. ROY-BURMAN, P., ROY-BURMAN, S., AND VISSER, D. W. (1968) J. Biol. Chem. 243, 1692-1697. 27. BIELY, P., AND BAUER, S. (1966) Biochim. Biophys. Acta 121, 213-214. 28. BAUER, C., AND REUTTER, W. (1973) Biochim. Biophys. Actu 293, 11-14. 29. KOCHETKOV, N. K., BUDOWSKY, E. I., AND SHIBAEV, V. N. (1963) Biokhimiya 28, 609-617 (English translation). 30. BUM)WSKY, E. I., DRUSHININA, T. N., ELISEEVA, G. I., GALBRIELYAN, N. D., KOCHETKOV, N. K., NOVIKOVA, M. A., SHIBAEV, V. N., AND ZHADANOV, G. L. (1966) Biochim. Biophys. Acta 122, 213-224.