Phosphorylation of Mcardle Phosphorylase Induces Activity (Human Skeletal Muscle/Protein Kinase) CESARE G

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 activated 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 myophosphorylase 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 25 spectrophotometer. mogenates of McArdle muscle, whether or not they had been Blanks included boiled muscle homogenate, boiled cAMP- dialyzed. Assay mixtures containing boiled muscle, cAMP-Kase Kase, and cAMP-Kase without homogenate. without homogenate, or boiled cAMP-Kase had no phosphor- Immunological and electrophoretic experiments were per- ylase activity. In the absence of supernatant, addition of ATP, formed with both kinases. For experiments with cAMP-Kase, cAMP, or cAMP-Kase (in any order) had no effect on the mon- 50 ,ul ofdialyzed McArdle muscle supernatants were incubated itored absorbance. AMP, in amounts up to 200 times the con- with 40 mM NaOAc/8.5 mM Mg(OAc)2, pH 6.8/2 mM theo- centration that maximally stimulates control human muscle phylline/1 mM dithiothreitol/1 mM ATP/2 ,M cAMP and 90 phosphorylase b, did not induce activity of McArdle muscle picounits of cAMP-Kase in a final volume of 100 ,ul for 15 min phosphorylase. However, when AMP and cAMP-Kase were at 30°C. For experiments with phosphorylase b kinase, dialyzed added to the supernatant, there was, after a delay of 2 min, McArdle supernatant was incubated with up to 100 picounits a small increase in absorbance, and this was accelerated by ad- ofphosphorylase b kinase. In these experiments, the incubation dition ofATP. The maximal effect ofATP as substrate for cAMP- mixture was 0.5 mM ATP/12 mM NaOAc, pH 7.8/8.5 mM Kase occurred at 0.5-1.0 mM . This effect was reduced or absent Mg(OAc)2/l mM dithiothreitol containing 100 picounits of in supernatants that had not been dialyzed before incubation phosphorylase b kinase, incubations were carried out at 30°C with protein kinase. In dialyzed supernatants, generation of for 10 min. In some experiments, antibody to human muscle phosphorylase activity was reproducible. phosphorylase was added to McArdle supernatant, which had In the absence of added AMP, the phosphorylase activity of been preincubated with 40 mM NaOAc/8.5 mM Mg(OAc)2, pH activated McArdle muscle supernatant was 11.6-14.2% of the 6.8/2 mM theophylline/1 mM dithiothreitol/1 mM ATP/2 AM activity in its presence. Pretreatment of the supernatant with cAMP and 80 picounits ofcAMP-Kase at 30°C for 30 min before Norit A to remove endogenous nucleotides eliminated this phosphorylase activity was measured. Control tubes contained phosphorylase activity. In the presence of 0.8 mM AMP, the normal rabbit serum instead of antibody. When the inhibitor phosphorylase activity of McArdle muscle (Table 1) previously protein of cAMP-Kase was used, it was either mixed with incubated with cAMP-Kase was 16-70% of the activity of four cAMP-Kase before assay or added directly to the assay mixture untreated normal human muscles. in stoichiometric amounts before addition of cAMP-Kase. Between 10 and 90 picounits of cAMP-Kase, phosphorylase Flat-bedelectrophoresis was carried outin 3.25% acrylamide/ activity generated in McArdle muscle homogenates was directly 0.12% (N,N'-methylene)bisacrylamide/1% glycogen in a buffer proportional to the concentration of cAMP-Kase (Fig. 1). At a of 48 mM boric acid/i mM EDTA/42 mM Tris base, pH 8.2. constant amount of cAMP-Kase, the activity was proportional Ten-microliter aliquots of supernatant, with or without cAMP- Kase pretreatment, were applied to the gel. Gels were cast in Table 1. Phosphorylase activity in McArdle muscle after cAMP- a GSC-8 casting apparatus (Pharmacia), and electrophoresis was Kase treatment performed in a refrigerated chamber (GE-4 Pharmacia) for 2 hr Patient Activity* at a constant current of50 mA after a 10 min prerun at 10 mA. Phosphorylase activity was demonstrated by a modification of G.D. 14.3 the method of Takeo and Nakamura (28): gels were incubated R.T. 14.7 4.2 at room temperature for 4 hr in a solution containing 2 mM D.Q. glycerophosphate, pH 6.8/2 mM EDTA/10 mM NaF/1 mM S.O. 10.3 2-mercaptoethanol/30 mM glucose-phosphate/2 mM AMP. Control (n = 4) 27.6 (20.2-30.1) Activity bands were visualized by 10-30 min of incubation in * Expressed as (nmol of NADP reduced. per mg of soluble protein) per a freshly prepared dilution (1:30) of iodine (6%)/potassium io- min. Downloaded by guest on September 28, 2021 2690 Biochemistry: Cerri and Willner Proc. Natl. Acad. Sci. USA 78 (1981)

200 0.14 ..I..f ...i. .. . . I . , ., -I. E E t -- .s- tt --- 160 0.12v .. 120- . U.1Ut)1 )I . :. 1 A- c) '_ _ 80~~~~~ -'1- ....i-- .-tt ....- 0.08 !, L ,", 4080/ s ¢ 40 B _-I t i .-- rAn ..-j :T- MI2

20 40 60 80 0.04 _ cAMP-Kase, picounits FIG. 1. Phosphorylase activity was determined spectrophotomet- 0.02 !1 I LON"--! , rically at 3000. Each assay used 18 ,ugofsolubleprotein inthe presence of0.8 mM AMP. Results were linear at 20-90 picounits ofcAMP-Kase. D 80 70 60 50 40 30 20 10 to the protein concentration ofthe homogenate (Fig. 2). When 150 international units ofthe cAMP-Kase inhibitor protein was Time, min added before the addition of cAMP-Kase, the resulting phos- FIG. 3. Spectrophotometric recording ofphosphorylase reaction at phorylase activity was only 20% ofthat induced without the in- 30°C. Each assay cuvette contained 20 ,ul ofactivated McArdle muscle hibitor. Larger amounts of the inhibitor totally blocked acti- supernatant, which represented 16 j.g of soluble protein, the reaction vation of the phosphorylase. When rabbit antibody against mixture, and 0.8 mM AMP, as described in Materials and Methods. A, normal human skeletal muscle phosphorylase was added to Supernatant plus normal rabbit serum at 1:4 (vol/vol); B, supernatant McArdle muscle supernatant [2:1 (vol/vol)] that had been ac- plus antibody against human phosphorylase at 1:2 (vol/vol); C, super- tivated by incubation with cAMP-Kase, the resulting phos- natant plus antibody against human phosphorylase at 1:4 (vol/vol); D, phorylase activity was 50% of that in a control homogenate of blank (cAMP-Kase plus boiled supernatant; overlapping the baseline). the same muscle incubated with nonimmune rabbit serum; an- moval of 30% or more of the radioactive phosphate. Addition tibody at 4:1 completely eliminated activity (Fig. 3). of 40 mM NaF to the dialysis buffer prevented loss of activity. Phosphorylase activity induced in McArdle muscle super- Preincubation of McArdle muscle supernatants with phos- natant by cAMP-Kase and ATP comigrated with normal human phorylase b kinase and MgATP also induced phosphorylase ac- phosphorylase on polyacrylamide gels (Fig. 4). No activity was tivity. The amounts ofphosphorylase activity produced by both seen in the gel if the McArdle muscle was not treated with kinases were comparable (Table 4) but, in contrast to the effect cAMP-Kase or if the cAMP-Kase preincubation mixture alone ofcAMP-Kase, the activity generatedby phosphorylase b kinase was applied to the gel. was independent of addition of 0.8 mM AMP. When [y-32P]ATP was included in the phosphorylation mixture, 32p was incorporated in the protein band that demon- DISCUSSION strated phosphorylase enzyme activity (Table 2). This band contained 7-15% ofthe 32p transferred to protein. In two muscles, We have confirmed earlier observations ofan enzymatically in- the amount ofphosphate incorporated as a function oftime was active protein in the muscle ofsome patients who have McArdle proportional to the amount of phosphorylase activity induced, but the data did not fit a simple linear regression distribution (Table 3). Furthermore, in separate experiments, despite at- tempted control ofexperimental variables, the amount ofphosphate incorporated in the band migrating as myophosphorylase was not constant (compare the 15-min incubation times of patients S.O. and R.T. given in Tables 2 and 3). Dialysis of the phosphorylated protein in the absence of protein phosphatase inhibitor resulted in complete loss of enzyme activity, and re-

* 180 - 160 c 140 0 120 "^ 100 0 80 "I 60 $- 40 M 20 A B C D z 2 4 8A 10o 2 14 16 Protein, mg FIG. 4. Gel stained for phosphorylase activity. A, McArdle muscle supernatant plus cAMP-Kase, B, control supernatant plus cAMP- FIG. 2. Phosphorylase activity induced by 80 picounits of cAMP- Kase; C, McArdle supernatant; D, control supernatantt; E, phosphor- Kase was linear at 2.5-15 mg of soluble protein. Assays were per- ylation mixture containing cAMP-Kase and distilled water rather formed in the presence of 0.8 mM AMP. than supernatant. Downloaded by guest on September 28, 2021 Biochemistry: Cerri and Willner Proc. Natl. Acad. Sci. USA 78 (1981) 2691

Table 2. Phosphorylation ofphosphorylase by cAMP-Kase Table 4. Phosphorylase activity in McArdle muscle after % oftotal incubation with kinases Phosphate protein Protein Phosphorylase b Patient incorporated* radioactivity Patient kinase* kinaset S.O. 6.05 10 G.D. 14.3 ND R.T. 6.67 15 R.T. 14.7 10.0 D.Q. 1.32 7 D.Q. 4.2 3.24 S.O. 10.3 17.4 * Expressed as nmol of 32P incorporated per mg of soluble protein in homogenate. Activities are expressed as nmol ofNADP reduced per mg ofsoluble protein per min. ND, not done. * In the presence of 0.8 mM AMP. disease. We also found the following: (i) The protein is inactive t Without exogenous AMP. in the presence ofhigh concentrations ofAMP. (ii) The protein was activated by cAMP-Kase or phosphorylase b kinase in the appeared when phosphate was removed from the protein. The presence ofATP. (iii) The resulting protein migrated to the same absence of a correlation between phosphate incorporated and position as normal phosphorylase in gel electrophoresis. (iv) The phosphorylase activity induced is not understood. In gel elec- reactivated protein contained 32P after incubation with cAMP- trophoresis, phosphorylase may be separated incompletely Kase and [12P]ATP. (v) Loss of 32P resulted in loss of phos- from other phosphorylatable proteins. Alternatively, phosphorylase activity. (vi) The effect ofcAMP-Kase was blocked by phorylation may influence, and be affected by, the state of as- either its inhibitor or antibody to normal human phosphorylase. sociation ofmonomers, dimers, and tetramers ofMcArdle phos- (vii) Activity generated by cAMP-Kase was dependent on en- phorylase; the same amount ofphosphate bound to mixtures of dogenous or exogenous nucleotides, but activity induced by differently active species could give various phosphorylase ac- phosphorylase kinase was independent of added AMP. tivities. Other glycolytic enzymes, such as phosphofructokinase For unknown reasons, dialysis of supernatants was usually (32), phosphorylase b kinase (33, 34), and glycogen synthetase necessary before phosphorylase activity could be induced; this (34), have more than one phosphorylatable site that which reg- requirement was not studied. Proteins larger than 12,000 dal- ulates activity. Partially phosphorylated intermediate forms of tons, such as the inhibitor protein to cAMP-Kase, would not muscle phosphorylase have been described (16, 17), and par- pass through the membrane we used for dialysis. Small mole- tially phosphorylated inactive isoenzymes may be present in cules, however, could interfere with the action of the kinases. other tissues. However, there is no evidence that human or Glucose 6-phosphate, for example, inhibits activation ofphos- other mammalian myophosphorylase is regulated by phosphorylase by phosphorylase b kinase (29). phorylation of more than one site. Normal human skeletal muscle phosphorylase b is activated Theoretically, the action of cAMP-Kase on McArdle phos- both by AMP and by phosphorylation by phosphorylase b ki- phorylase could be mediated by phosphorylase b kinase. Phos- nase. In mammalian and nonmammalian tissues, forms ofphosphorylase b kinase is a known substrate for cAMP-Kase; its phorylase have been described (16-18, 29) that are less sensitive phosphorylation results in activation ofphosphorylase in normal or insensitive to concentrations ofAMP that maximally activate human muscle (13, 34, 35). However, this explanation seems normal human myophosphorylase. In canine liver (30), human inapplicable to our results for several reaons: (i) McArdle muscle platelets (18, 19), and human leucocytes (31), there is a phos- lacks both phosphorylase b and phosphorylase a activity; (ii) phorylase isozyme that remains inactive at concentrations of phosphorylase b kinase activity is normal in McArdle muscle AMP that maximally activate phosphorylase b. Addition of up (1, 36); (iii) phosphorylase b kinase is activated by Ca2+(13), to 100 mM AMP did not activate McArdle myophosphorylase, which is unlikely to be absent in McArdle muscle; and (iv) phos- suggesting that this enzyme, although it could be activated by phorylase activity found after treatment with cAMP-Kase was, phosphorylase b kinase, is not phosphorylase b. In this sense, by its activity, primarily phosphorylase b, because it was acti- it resembles the AMP-insensitive isoenzyme ofcanine liver and vated 10-fold by 0.8 mM AMP. human leucocytes and platelets. Alternatively, cAMP-Kase may have directly phosphorylated McArdle phosphorylase required treatment under condi- a site on phosphorylase. Tryptic digests ofhuman (37) and rabbit tions appropriate for protein phosphorylation before activity (38) muscle phosphorylase a suggest only one phosphorylated could be demonstrated, and the resulting enzyme activity dis- amino acid sequence, the site normally phosphorylated by phosphorylase b kinase. If this site was phosphorylated by Table 3. Transfer ofphosphate and phosphorylase activity after cAMP-Kase, the resulting enzyme should have been indepen- cAMP-Kase incubation dent of AMP for activity; the site of action of cAMP-Kase is Incubation therefore likely to be different. time, Phosphate The appearance ofphosphorylase activity could be the result Patient min Activity* transferredt ofincubating a structurally abnormal protein with phosphoryl- ating enzymes; a conformational change unrelated to the normal S.O. 6 5.30 6.24 function ofphosphorylase could account for activation. The re- 10 9.65 8.10 lationship ofthese observations to the molecular defect ofphos- 15 13.34 12.50 phorylase in McArdle disease therefore remains to be deter- R.T. 6 8.68 5.10 mined; because of the steric, charge, and substrate amino acid 10 10.61 6.13 constraints to the action of cAMP-Kase, it should be useful to 15 17.37 7.68 reconcile structural studies of this mutant protein with its ca- * Expressed as nmol ofNADP reduced per minpermgofsoluble protein pacity to be phosphorylated and activated. in the homogenate and determined in the presence of0.8 mM AMP. t Expressed as nmol of phosphate incorporated in the protein band We thank Drs. S. DiMauro, A. Gold, and L. P. Rowland for sug- comigratingwith normal human myophosphorylase per mgofsoluble gestions and encouragement in the course ofthis study and in the prep- protein in the homogenate. aration of the manuscript. We are especially grateful to Dr. DiMauro Downloaded by guest on September 28, 2021 2692 Biochemistry: Cerri and Willner Proc. Natl. Acad. Sci. USA 78 (1981)

for providing us with muscle from patients who have McArdle disease 18. Gergely, P., Castle, A. G. & Crawford, N. (1979) Int.J. Biochem. and antibody to human myophosphorylase. C.G.C. is the recipient of 10, 807-814. a postdoctoral fellowship from the Muscular Dystrophy Association and 19. Karpatkin, S. & Langer, R. M. (1969) J. Biol. Chem. 244, J.H.W. is the recipient of a Teacher Investigator Development Award 1953-1955. from the National Institute of Neurological and Communicative Dis- 20. Karpatkin, S., Braun, J. & Charmatz, A. (1972) Biochim. Bio- orders and Stroke. This work was supported by a center grant from the phys. Acta 220, 22-29. Muscular Dystrophy Association. 21. Keely, S. L. (1979) Mol. Pharmacol. 15, 235-245. 22. Krebs, E. G. (1966) Methods Enzymol. 8, 543-553. 1. Mommaerts, W. F. H. M., Illingworth, B., Pearson, C. M., 23. DiMauro, S, Arnold, S., Miranda, A. & Rowland, L. P. (1978) Guillory, R. J. & Seraydarian, K. (1959) Proc. Natl. Acad. Sci. Ann. Neurol. 3, 60-66. USA 45, 791-797. 24. Berger, M., Gibbels, E., Leven, B. & Seiler, D. (1978) Fortschr. 2. Feit, H. & Brooke, M. H. (1976) Neurology 26, 963-967. Neurol. Psychiatr. Ihrer Grenzgeb. 46, 312-326. 3. Bank, W. J., DiMauro, S., Rowland, L. P. & Milestone, R. 25. DiMauro, S., Rowland, L. P. & DiMauro P. M. (1970) Arch. (1972) Trans. Am. Neurol. Assoc. 97, 179-183. Neurol. 23, 534-540. 4. Grunfeld, J. P., Ganeval, D., Chanard, J., Fordeau, M. & Drey- 26. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. fus, J.-C. (1972) N. Engl..J. Med. 286, 1237-1241. (1951) J. Biol. Chem. 193, 265-275. 5. Lowry, 0. H., Schulz,-D. W. & Passoneau, J. V. (1967)J. Biol. 27. Layzer, R. D., Rowland, L. P. & Renney, H. M. (1976) Arch. Chem. 242, 271-280. Neurol. 17, 512-523. 6. Wang, J. H., Shonka, M. L. & Graves, D. J. (1965) Biochemistry 28. Takeo, K. & Nakamura, S. (1972) Arch. Biochem. Biophys. 153, 4, 2296-2301. 1-7. 7. Graves, D. J. & Wang, J. H. (1973) in The Enzymes, ed. Boyer, 29. Vasquez-Baanante, I. & Perez, M. R. (1979) Comp. Biochem. P. D. (Academic, New York), Vol. 7, pp. 435-482. Physiol. 62, 381-388. 8. Wang, J. H., Shonka, M. L. & Graves, D. J. (1965) Biochem. 30. Wosilait, W. & Sutherland, E. W. (1956) J. Biol. Chem. 218, Biophys. Res. Commun. 18, 131-135. 469-482. 9. Madsen, N. B. (1964) Biochem. Biophys. Res. Commun. 15, 31. Yunis, A. A. & Arimura, G. K. (1969) Biochem. Biophys. Res. 390-395. Commun. 33, 119-128. 10. Madsen, N. B. & Shekowsky, S. (1967) J. Biol. Chem. 242, 32. Hofer, H. W. & Sorensen-Ziganek,' B. (1979) Biochem. Biophys. 3301-3307. Res. Commun. 90, 199-203. 11. Morange, M., Garcia-Blanco, F., Vandenbunder, B. & Buc, H. 33. Riley, W. D., Delanse, R. J., Bratwols, G. E. & Krebs, E. G. (1976) Eur. J. Biochem. 65, 553-3. (1968)J. Biol. Chem. 243, 2209-2215. 12. Lowry, 0. H., Schulz, D. W. & Passoneau, J. V. (1964) J. Biol. 34. Soderling, T. R. (1979) Mol. Cell. Endocrinol. 16, 157-179. Chem. 239, 1947-1953. 35. Walsh, D. A., Perkins, J. P. & Brostrom, C. O., Ho, E. S. & 13. Soderling, T. R., Hickenbottom, J. P., Reimann, E. M., Hun- Krebs, E. G. (1971) J. Biol. Chem. 246, 1968-1976. keler, F. L., Walsh, D. A. & Krebs, E. G.(1970)J. Biol; Chem. 36. Pearsons, C. M., Rimer, D. G. & Mommaerts, W. F. H. M. 245, 6317-6328. (1961) Am. J. Med. 30A 503-517. 14. Stull, J. F. & Mayer, S. E. (1971)J. Biol. Chem. 246, 5716-5723. 37. Hughes, R. C., Yunis, A. A., Krebs, E. G. & Fisher, E. G. (1962) 15. Tao, S. H., Huang, F. L., Lynch, A. & Glinsmann, W. H. (1978) J. Biol. Chem. 237, 40-43. Biochem. J. 176, 347-350. 38. Titani, K., Koide, A., Herzmann, J., Ericsson, L. H., Kumar, S., 16. Bot, G., Kovacs, E. F. & Gergely, P. (1974) Biochim. Biophys. Wade, R. D., Walsh, K. A., Neurath, H. & Fischer, E. H. (1977) Acta 370, 70-77. Proc. Nati. Acad. Sci. USA 74, 4762-4766. 17. Gergely, P., Bot, G. & Kovacs, E. F. (1974) Biochim. Biophys. Acta 370, 78-84. Downloaded by guest on September 28, 2021