Phorylase Has Been Forthcominglately

Home , Adenosine monophosphate, Phosphoglucomutase

GLYCOGEN SYNTHESIS IN MUSCLE LACKING PHOSPHORYLASE* RUDI SCHMID, PHILLIPS W. ROBBINS, AND ROBERT R. TRAUT

THE THORNDIKB MEMORIAL LABORATORY, HARVARD MEDICAL SCHOOL, AND THE ROCKEFELLER INSTITUTE Communicated by Fritz Lipmann, June 15, 1959 Mounting evidence for the catabolic rather than the anabolic function of phosphorylase has been forthcoming lately.'-4 The discovery by Leloir and Cardini6 of a UDPG-linked glycogen synthesis, on the other hand, presented a most suit- able anabolic system where, in contrast to phosphorylase, glycogen synthesis is virtually a one-way reaction. Nevertheless, to find an unambiguous method of assigning intracellular glycogen synthesis to one or the other enzyme appeared to present considerable difficulties. At this juncture, therefore, it was particularly fortunate when a human myopathy was discovered' where muscle biopsy showed that a rather high glycogen content was coupled with very low levels of phosphorylase. It seemed probable that the UDPG-linked enzyme was present and that it was responsible for the high glycogen content. We wish to report here on muscle biopsy assays from such a patient which showed the level of the UDPG-linked glycogen synthesizing enzyme to be within the range of normality, in contrast to the again confirmed absence of phosphorylase. Materials and Methods.-Uridine diphosphate glucose and triphosphopyridine nucleotide were purchased from the Sigma Chemical Company; and 5'-adenosine monophosphate and glucose-i-phosphate were obtained from Schwarz Laboratories. Rabbit liver glycogen was purchased from Mann Research Laboratories and .glucose-6-phosphate dehydrogenase, free of phosphorylase and phosphoglucomutase, was obtained from C. F. Boehringer and Sons. This enzyme was dialyzed overnight against 2 mM Tris, pH 7.5 and stored, frozen, in small batches. Under these conditions, it was found to retain its activity for several weeks. Phosphoglucomu- tase was prepared from rabbit muscle by the method of Najjar7 and phosphorylase b was prepared by the method of Fischer and Krebs.8 Labeled UDPG was prepared as described previously.4 Muscle biopsies were obtained under local or general anaesthesia and were im- mediately chilled in ice. The tissue was weighed, minced, and suspended in 10 volumes of ice-cold distilled water. Disruption was carried out in a small glass Potter-Elvehjem homogenizer. The homogenates were centrifuged for 10 minutes at 10,000 x g and the enzymatic activities in the supernatant fluid determined. Protein concentration was measured by the method of Lowry et al.9 and the specific activities of all enzymes are defined in terms of protein concentration. The enzymatic activities were determined as soon as possible after homogenization. Ali- quots of the homogenate were diluted with glycerophosphate-cysteine, pH 6 (Cori phosphorylase assay), 0.03 M cysteine (phosphorylase kinase), or cold water im- mediately before being assayed. Phosphorylase was determined by the method of Illingworth and Corin0 in the presence of AMP at pH 6. The units are those found at pH 6 and are not corrected to the activity expected at pH 6.8. When the homogenate obtained from the patient's muscle was being assayed, 0.2 ml. of undiluted homogenate and 0.2 ml. of 1236 VOL. 45, 1959 BIOCHEMISTRY: SCHMID, ROBBINS, AND TRAUT 1237 double-strength glycerophosphate-cysteine were used in place of diluted enzyme. It was demonstrated that even under these conditions only about 15 per cent of the glucose-l-phosphate had been converted to glucose-6-phosphate at the end of the incubation period. 10 Phosphorylase was also determined by the method of Wu and Racker." This assay involved a measurement of the rate of TPN reduction when a limiting concentration of phosphorylase was incubated with excess glycogen, inorganic phosphate, phosphoglucomutase, and glucose-6-phosphate dehydrogenase. The incubation mixture contained 50 mM Tris, pH 7.5, 10 mM cysteine, 10 mM sodium phosphate, pH 7.5, 2 mg per ml of glycogen, 0.2 mM TPN, 10 mM MgC12, 1 mM AMP, phosphoglucomutase, and dialyzed glucose-6-phosphate dehydrogenase. Determinations with crystalline phosphorylase b showed that the rate of TPNH formation was directly proportional to phosphorylase concentration over a wide range. Homogenates were diluted with water for this assay. Phosphorylase kinase assays were carried out according to Krebs and Fischer'2 except that Mg-ATP was used in place of Mn-ATP. Crystalline rabbit muscle phosphorylase, itself free of kinase activity, was used as the substrate. The units are those of Krebs and Fischer, one unit representing the conversion of 100 units of phosphorylase b to a in 5 minutes at 300. Phosphoglucomutase was measured by the method of Najjar7 and also by a modification of the spectrophotometric method described above for phosphorylase. In this modification, the inorganic phosphate and phosphoglucomutase were omitted and the glycogen was replaced with 1 mM glucose-i-phosphate. It was shown that the rate of TPN reduction was proportional to phosphoglucomutase concentration. The UDPG-glycogen synthetase activity was determined with glucose-C'4- labeled UDPG by a modification of the method described previously4 (Table 3). The incubation medium was modified by the addition of 0.4 mM glucose-6-phosphate which was shown by Leloir et al. ' to increase the rate of the reaction. The incorporation was determined by the glycogen precipitation method used previously. It was also determined by stopping the reaction with 0.5 ml. of 0.05 M acetic acid, adsorbing the nucleotides with about 20 mg. of Norite A, and plating and counting the charcoal supernatant fluid. The two methods gave the same results for control homogenates and for the homogenate obtained from the patient's muscle. Results.-The levels of phosphorylase and UDPG-glycogen synthetase in three control biopsies and in the biopsy obtained from the patient are summarized in Table 1. In confirmation of the findings of Dreyfus et al.,'4 it was found that normal human muscle contains 1/4 to 1/3 the amount of phosphorylase activity found in rat or rabbit muscle. It is possible that the apparent trace of phosphorylase activity found in the patient's muscle may not be phosphorylase at all, but simply a reflection of nonspecific phosphatase activity in the case of the Cori assay, and the formation of traces of glucose-6-phosphate from sources other than glycogen in the spectrophotometric assay. This possibility was strengthened by the finding that the omission of AMP caused almost no change in the amount of enzyme measured. The data available indicate, to say the least, that the amount of activity was less than 0.5 per cent of normal. Mixing experiments have shown that the extract from the patient's muscle did not contain a phosphorylase inhibitor. Dial- ysis of the extract overnight against 5 mM Tris, 1 mM FDTA pH 7.5, did not re- sult in the appearance of activity. 1238 BIOCHEMISTRY: SCHMID, ROBBINS, AND TRAUT PROC. N. A. S.

TABLE 1 * ENZYmATIC ASSAYS ON MUSCLE BIOPSIES Controls (3) Patient Average Range Phosphorylase, Cori assay, units/mg protein 0.09 22 16-26 Phosphorylase, spectrophotometric, mjtmoles/min/mg protein 0.7 250 170-300 UDPG-glycogen synthetase, mismoles/min/mg protein 10 21 19-22 Phosphoglucomutase, Najjar assay, milli units/mg protein 30 90 70-100 Phosphoglucomutase, spectrophotometric, miumoles/ min/mg protein 36 150 120-190 Phosphorylase kinase units/mg protein 31 28 21-32 * Muscle biopsies were chilled, weighed, and homogenized in 10 vols. of ice-cold distilled water. After cen- trifugation, the enzymatic activities were determined by the methods described in detail in the text. The UDPG-glycogen synthetase is present in normal controls at about the same level found in extracts prepared from rabbit or lamb muscle.'5 As first shown by Leloir et al., 3 glucose-6-phosphate has an unexplained stim- ulatory effect on UDPG-glycogen synthetase. As seen in Table 2, glucose-6- phosphate appears necessary in assays of the synthetase even in the crude system. Experiments to be reported elsewhere show that in purified preparations, glucose- 6-phosphate appears to be an obligatory component of the system. The patient's muscle tissue contained about '/2 the amount of enzyme in the controls. The seem- ingly lower value should reflect a degeneration of the tissue. As seen in Table 1, the extract from the patient's muscle tissue also contained only 1/4 to 1/3 the phosphoglucomutase of control preparations. The damaged state of the muscle in this patient is described in detail by Schmid and Mahler.16 Dreyfus et al.'4' 17 have reported that diseased or damaged muscle contains, in general, lower levels of the glycolytic enzymes.

TABLE 2* UDPG-GLYCOGEN SYNTHETASE ACTIVITY IN THE PRESENCE AND ABSENCE OF GLUCOSE-6-PHOSPHATE Glucose Incorporation into Glycogen cpm/15 min/mg Protein Origin of Muscle No Addition G-6-P, 4 X 10-4 M Normal 1 1390 4000 Normal 2 273 2280 Patient 571 1870 * The homogenates were prepared and assayed in the manner described in the text.

Measurement of phosphorylase kinase activity in controls gave an average specific activity about '/2 as great as that reported for crude rabbit muscle extracts by Krebs and Fischer.'2 This lower activity is not surprising, especially in view of the fact that rabbit muscle phosphorylase b was used as the substrate rather than human muscle phosphorylase b. Surprisingly, the extract from the patient's muscle appeared to have about the same activity as controls. Analogous results were obtained by Nirenberg'8 who found that HeLa cells, although almost devoid of phosphorylase, contained an active phosphorylase kinase system. These findings seem to show the need for an investigation of the distribution and specificity of phosphorylase kinase activity. In a single experiment, it was shown that normal human serum was free of phosphorylase kinase activity. VOL. 45, 1959 BIOCHEMISTRY: SCHMID, ROBBINS, AND TRAUT 1239

Discussion. The abundance of glycogen in these dystrophic muscles in the virtual absence of phosphorylase rather unambiguously connects in vivo glycogen synthesis to UDPG-glycogen synthetase. This enzyme was found in the dystrophic muscles in approximately normal amounts when related to functioning muscle tissue. Judging from the symptomatology of the patient, it would seem that only skeletal muscle phosphorylase is lacking. Whether this defect is hereditary and what its relationship to glycogen storage diseases may be deserves further considera- tion, as does the question of whether the deficiency is caused by an absence of phosphorylase or by the presence of a defective and inactive protein. When reflecting on the difference between catabolic and anabolic pathways, a more general rule seems slowly to emerge. Development in the area of glycogen synthesis and breakdown runs quite parallel to what is happening in fatty acid synthesis and breakdown. In both cases, metabolism and anabolism were attrib- uted for some time to a chain of reversible reactions proceeding in either direction depending on conditions. It seems now that the anabolic process takes place by a more or less irreversible pathway which may require a rather large energy expend- iture. Metabolic energy, it would appear, is treated as a relatively abundant commodity and cellular mechanisms appear not to be designed to save energy. Summary. Muscle biopsies have been obtained from normal human subjects and from a patient with a progressive myopathy whose muscles lack phosphorylase. The phosphorylase level in a homogenate of the patient's muscle was found to be less than 0.5 per cent of normal, while UDPG-glycogen synthetase was approximately 1/2 of normal. The phosphoglucomutase activity of a homogenate of the patient's muscle was about 1/3 that of controls. The lower UDPG-glycogen synthetase and phosphoglucomutase are interpreted in terms of general degeneration of the tissue, while the lack of phosphorylase is considered to be a specific defect. The phosphorylase kinase activity of the homogenate was normal. The enzymatic pattern and the presence of a high concentration of glycogen in the tissue give strong evidence that glycogen is synthesized by way of UDPG. The authors would like to acknowledge the capable technical assistance of Miss Lydia Hammaker. * This work was supported in part by research grants from the National Cancer Institute and the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U. S. Public Health Service, and the National Science Foundation. The following abbreviations have been used: UDPG, uridine diphosphate glucose; AMP, adenosine monophosphate; TPN, triphosphopyridine nucleotide; TPNH, reduced triphosphopyridine nucleotide; Mg-ATP and Mn-ATP, the magnesium and manganous salts respectively of adenosine triphosphate; EDTA, ethylene diamine tetraacetic acid; Tris, tris(hydroxymethyl)- aminomethane hydrochloride. l Sutherland, Jr., E. W., in Enzymes: Units of Biological Structure and Function, ed. 0. H. Gaebler (New York: Academic Press, 1956), p. 536. 2 Villar-Palasi, C., and J. Larner, Biochim. et Biophys. Acta, 30, 449 (1958). 3Robbins, P. W., and F. Lipmann in CIBA Foundation Symposium on the "Regulation of Cell Metabolism" (London: J. A. Churchill, Ltd., 1959), p. 188. 4Robbins, P. W., R. R. Traut, and F. Lipmann, these PROCEEDINGS, 45, 6 (1959). 5 Leloir, L. F., and C. E. Cardini, J. Am. Chem. Soc., 79, 6340 (1957). 6 Schmid, R., and R. Mahler, J. Clin. Invest., 38, 1040 (1959). 7Najjar, V. A., in S. P. Colowick and N. 0. Kaplan, Methods in Enzymology, (New York: Academic Press, Inc., 1955), vol. 1, p. 294. 1240 BIOCHEMISTRY: SOMERVILLE ET AL. PROC. N. A. S.

8 Fischer, E. H., and E. G. Krebs, J. Biol. Chem., 231, 65 (1958). Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 10 Illingworth, B., and G. T. Cori, in Biochemical Preparations (New York: John Wiley and Sons, Inc., 1953), vol. 3, 1. 11 Wu, R., and E. Racker, personal communication. 12 Krebs, E. G., and E. H. Fischer, Biochim. et Piophys. Acta, 20, 150 (1956). 13 Leloir, L. F., J. M. Olavarria, S. H. Goldemberg, and H. Carminatti, Arch. Biochem. Biophys., 81, 508(1959). 14 Dreyfus, J., G. Schapira, F. Schapira, and J. Demos, Clinica Chimica Acta, 1, 434 (1956). 15 Traut, B. R., unpublished experiments. 16 Schmid, R., and R. Mahler, J. Clin. Invest. (in press). 17 Dreyfus, J., G. Schapira, and F. Schapira, J. Clin. Invest., 33, 794 (1954). 18 Nirenberg, M. W., Biochim. et Biophys. Acta, 30, 203 (1958).

HYDROXYMETHYLDEOXYCYTIDYLA TE KINASE FORMATION AFTER BACTERIOPHAGE INFECTION OF ESCHERICHIA COLI* BY RONALD SOMERVILLEt KANEY EBISUZAKI, AND G. ROBERT GREENBERG DEPARTMENT OF BIOLOGICAL CHEMISTRY, THE UNIVERSITY OF MICHIGAN Communicated by Thomas Francis, Jr., June 8, 1959 Flaks and Cohen' have demonstrated that a new enzyme, deoxycytidylate hydroxymethylase, is formed after infection of Escherichia coli B by T-even bacteriophage. This finding opens the way for further investigation of the possible relationship of the DNA2 of infecting phage to the synthesis of specific proteins and of the control of the formation of viral progeny. We have observed the formation of another enzyme not detectable in normal cells. Hydroxymethyldeoxycytidylate kinase, which brings about the conversion of hydroxymethyldeoxycytidylate to the pyrophosphate level, is formed in a manner analogous to the synthesis of dCMP hydroxymethylase. At the same time an almost precipitous fall occurs in the ability of extracts to bring about net phosphorylation of dCMP. These enzymic changes can be considered as factors controlling the nature of the phage DNA. Material and Methods. Bacteriophage T2r was propagated on E. coli R2 in a glycerol-salts medium3 and concentrated and purified by differential centrifuga- tion.4 To prepare normal extracts E. coli R2 was grown aerobically on nutrient broth to 2-5 X 108 cells/ml, chilled, and harvested. The following manipulations were carried out at 0-4°. The washed cell paste was ground with several volumes of alumina, extracted with 0.05 M potassium phosphate buffer at pH 6.8, centrifuged at 25,000 X g and dialyzed overnight. In the experiments reported in Figure 1, the extracts were dialyzed against 0.01 M Tris buffer at pH 7.2, whereas in those experiments described in Tables 1 and 2 0.01 M potassium phosphate at pH 6.8 was used. For preparation of extracts of infected cells, E. coli R2 was grown with rapid circular shaking to 2-5 X 108 cells/ml in nutrient broth. The cells were infected at a multiplicity of about 5 with T2r and after 10 to 12 minutes