Proc. Nati. Acad. Sci. USA Vol. 78, No. 5, pp. 2688-2692, May 1981 Biochemistry Phosphorylation of McArdle phosphorylase induces activity (human skeletal muscle/protein kinase) CESARE G. CERRI AND JOSEPH H. WILLNER Department of Neurology and H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, Columbia University College of Physicians and Surgeons, New York, New York 10032 Communicated by Harry Grundfest, January 7, 1981 ABSTRACT In McArdle disease, myophosphorylase defi- mediate between those of phosphorylases b and a. Karpatkin ciency, enzyme activity is absent but the presence of an altered et al. (19, 20) found that incubation of human platelets with enzyme protein can frequently be demonstrated. We have found MgATP+ resulted in an increase in total phosphorylase activity that phosphorylation of this protein in vitro can result in catalytic and concluded that the data were "consistent with the presence activity. We studied muscle of four patients; all lacked myophos- in human platelets of inactive dimer and monomer species of phorylase activity, but myophosphorylase protein was demon- phosphorylase, which require MgATP for activation." Because strated by immunodiffusion or gel electrophoresis. Incubation of activation of these isozymes was probably due to protein phos- muscle homogenate supernatants with cyclic AMP-dependent pro- phorylation and also because incomplete phosphorylation could tein kinase and ATP resulted in phosphorylase activity. The ac- tivated enzyme comigrated with normal human myophosphory- result in reduced activity, we evaluated the possibility that the lase in gel electrophoresis. Incubation with [y-32P]ATP resulted activity ofphosphorylase in McArdle muscle could be restored in incorporation of 32P into the band possessing phosphorylase by phosphorylation of the inactive phosphorylase protein pres- activity. Activation of phosphorylase by cyclic AMP-dependent ent in some patients. protein kinase was inhibited by antibodies to normal human my- ophosphorylase or by inhibitory protein to cyclic AMP-dependent MATERIALS AND METHODS protein kinase. Incubation of muscle homogenates with phos- EDTA, peak II ofcAMP-Kase from bovine heart, phosphorylase phorylase b kinase and ATP also resulted in phosphorylase activ- b kinase, protein kinase inhibitor (from rabbit skeletal muscle), ity. After the action of cyclic AMP-dependent protein kinase, the glycogen, 2-mercaptoethanol, Na ,B-glycerophosphate, di- resulting activity was similar to that ofphosphorylase b. However, thiothreitol, ATP, theophylline, cAMP, AMP, cysteine, NADP, incubation with phosphorylase kinase resulted in activity similar phosphoglucomutase, glucose-6-phosphate dehydrogenase, and to that ofphosphorylase a. For several reasons, it is not likely that Coomassie brilliant blue were purchased from Sigma. By gel McArdle disease is due to lack ofnormal phosphorylation, but res- electrophoresis, there was no evidence that the cAMP-Kase was toration of activity to the mutant protein by phosphorylation may contaminated by phosphorylase b kinase or that the preparation provide a clue to understanding the mechanism of this genetic of phosphorylase b kinase contained cAMP-Kase; only a single defect. dense band migrated in electrophoresis of either preparation. With histone as substrate, we found no cAMP-Kase activity in McArdle disease is due to genetic lack ofphosphorylase (1,4-a- the phosphorylase b kinase, as determined by the method of D-glucan:orthophosphate a-D glucosyltransferase, EC 2.4.1.1) Keely (21); conversely, the cAMP-Kase preparation was unable activity in skeletal muscle (1). In some patients, the enzyme to activate phosphorylase b, as determined by the method of protein seems to be absent (2). In others, the presence ofphos- Krebs (22). [y-32P]ATP, Aquassure, and Protosol were obtained phorylase can be demonstrated immunologically (3, 4) or by gel from New England Nuclear. Acrylamide and (N,N'-methyl- electrophoresis (2), but the protein lacks catalytic activity. ene)bisacrylamide were purchased from BioRad. All other re- In normal mammalian skeletal muscle, phosphorylase is agents were ofthe highest purity available and were purchased present in at least two differently active states, phosphorylase from Fisher. Antibodies to normal human skeletal muscle phos- a and phosphorylase b (5-7). Phosphorylase b consists of two phorylase, a gift of S. DiMauro, were characterized previously monomers (8) and requires AMP for full expression of activity (23). (9-11). Phosphorylase a, a dimer ofidentical subunits, can also The diagnosis of McArdle disease in the four patients we be activated by AMP at high temperatures and low substrate studied was established by biochemical, histochemical, and concentrations (12) but is usually considered to be active in the clinical criteria (24). Muscle biopsies were performed with in- absence of AMP (7). The conversion from phosphorylase b to formed consent under local anesthesia; specimens for biochem- phosphorylase a depends on phosphorylation of a serine in po- ical study were frozen and stored in liquid nitrogen until sition 14 ofthe amino acid backbone ofthe phosphorylase mono- analyzed. mer and is catalyzed by phosphorylase b kinase, which in turn In all the McArdle muscles we studied, phosphorylase pro- is activated by either Ca + or 3',5'-cyclic AMP (cAMP)-depen- tein was demonstrated by gel electrophoresis. In each of two dent protein kinase (cAMP-Kase) (13, 14). Protein phosphatases muscles also analyzed in Ouchterlony plates, we found cross- dephosphorylate and inactivate phosphorylase a (15), convert- reacting material to antibody against human myophosphorylase. ing it back to phosphorylase b. We did not have access to a patient in whom phosphorylase Gergely and Bot described partially phosphorylated hybrid protein was not identified by these techniques. forms of phosphorylase in rabbit muscle (16, 17) and human Fragments of muscle were pulverized in a mortar cooled to platelets (18). The activities of these isoenzymes were inter- liquid nitrogen temperature. To the powder, we added 6 vol of 60% glycerol (voVvol)/20 mM 2-mercaptoethanol/10 mM The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: cAMP, 3',5'-cyclic AMP; cAMP-Kase, cAMP-depen- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. dent protein kinase. 2688 Downloaded by guest on September 28, 2021 Biochemistry: Cerri and Willner Proc. Natl. Acad. Sci. USA 78 (1981) 2689 EDTA/50 mM NaF/40 mM /3-glycerophosphate (pH 6.8) (glyc- dide (4%) in 7% acetic acid. Some gels were destained and erol/buffer A). The frozen suspension was then transferred with stained with Coomassie brilliant blue [1.25 g in methanoVgla- a spatula to an all-glass homogenizer, diluted 1:1 with buffer A, cial acetic acid/distilled water (227:46:227)]. and homogenized (25). In most experiments, the supernatant To correlate the amount of radioactive phosphate incorpo- obtained after centrifugation at 15,000 rpm for 10 min in a Sor- rated to the amount ofphosphorylase activity induced, ATP in vall refrigerated centrifuge was dialyzed in a membrane that the preincubation mixture was labeled with 100 mCi/mmol (1 excluded proteins larger than 12,000 daltons for 2 hr at 40C Ci = 3.7 X 1010 becquerels) of [y-32P]ATP. Aliquots of the in- against 500 vol of buffer A. Without further treatment, this su- cubation mixture were transferred to a "stop solution" (100 mM pernatant was used for assays. To remove endogenous AMP, NaF/10 mM EDTA) after 6, 10, and 15 min. Subsequently, some homogenates were treated with Norit A (20). Protein was these aliquots were assayed for phosphorylase activity and sub- determined according to Lowry (26). jected to electrophoresis. We also dephosphorylated 32P-la- To monitor phosphorylase activity by a continuous reaction, beled phosphorylase in the McArdle supernatant to by dialysis we used the method of Layzer (27), with minor modifications. against 100 mM imidazole/10 mM theophylline/20 mM 2-mer- The reaction mixture contained 2 mM MgCl2, 1 mM NADP, captoethanol (pH 7.0) at 4°C for 40 min in the absence ofprotein 1% glycogen, 5 units of phosphoglucomutase, and 3 units of phosphatase inhibitor; phosphorylase activities and amounts of glucose-6-phosphate dehydrogenase in 20 mM Na phosphate 2p incorporated were measured before and after dialysis. Gels buffer (pH 7.2). were dried and cut with a sharp razor at 2-mm intervals. Percent The effects of cAMP-Kase and phosphorylase b kinase were of gel protein radioactivity was calculated as the difference be- determined by their direct addition to the reaction mixture. To tween the radioactivity ofthe band migrating with, and staining monitor the effect of cAMP-Kase, we added 100 ,1 of dialyzed as, human phosphorylase and the parallel position in the strip supernatant, 5.0 nmol of cAMP, 0.5 ,mol of ATP, and up to incubated with reaction mixture and no muscle divided by the 200 picounits of cAMP-Kase. After 15 min at 30°C, we then total radioactivity bound to protein in the strip that contained added 0.8 ,mol of AMP to the reaction mixture. The effect of supernatant. phosphorylase b kinase was determined by adding 100 ,ul of dialyzed supernatant, 0.5 ,mol ofATP, and up to 150 picounits RESULTS ofphosphorylase b kinase. The change in absorbance at 340 nm No residual phosphorylase enzyme activity was detected in ho- was measured with a Beckman type