Phosphorylase Activity In Muscle Research Articles

Molecular diagnosis of McArdle disease using whole-exome sequencing.

Whole-exome sequencing (WES) analysis has been used recently as a diagnostic tool for finding molecular defects. In the present study, researchers attempted to analyze molecular defects through WES in a 13-year-old female patient who had not been diagnosed through a conventional genetic approach. DNA was extracted and subjected to WES analysis to identify the genetic defect. A total of 106,728 exons and splicing variants were selected, and synonymous single nucleotide variants (SNVs) and general single nucleotide polymorphisms (SNPs) were filtered out. Finally, nonsynonymous SNVs (c.C415T and c.C389T) of the PYGM gene were identified in nine compound heterozygous mutations. PYGM encodes myophosphorylase and degrades glycogen in the muscle to supply energy to muscle cells. The present study revealed that the patient's father had a c.C389T mutation and the mother had a c.C415T mutation, resulting in A130V and R139W missense mutations, respectively. To the best of our knowledge, the A130V variant in PYGM has not been reported in the common variant databases. All variations of the patient's family detected using WES were verified by Sanger sequencing. Because the patient had compound heterozygous mutations in the PYGM gene, the patient was presumed to exhibit markedly decreased muscle phosphorylase activity. To assess the function of myophosphorylase, an ischemic forearm exercise test was performed. The blood ammonia level sharply increased and the lactate level maintained a flat curve shape similar to the typical pattern of McArdle disease. Therefore, the diagnosis of the patient was confirmed to be McArdle disease, a glycogen storage disease. Through WES analysis, accurate and early diagnosis could be made in the present study. This report describes a novel compound heterozygous mutation of the PYGM gene in a Korean patient.

Phosphoglucomutase (PGM 1) Deficiency: A Novel Defect of Muscle Glycogen Degradation and Synthesis (P07.200)

Objective: Evaluate the cause of exertional muscle fatigue, contractures and rhabdomyolysis in a young woman. Background An 18 year old woman reported lifelong fatigue with modest exercise and episodes of muscle contractures, pain and swelling with marked elevations in serum creatine kinase levels after more vigorous exercise. Design/Methods: Forearm and cycle exercise, muscle glycogen determinations, and relevant enzymatic and genetic evaluation were performed. Results: Ischemic forearm exercise resulted in no increase in lactate, an exaggerated increase in ammonia, rapid contractile fatigue and a finger flexor muscle contracture. Cycle exercise revealed low oxidative capacity and a spontaneous second wind, associated with a 40 beat per minute (bpm) drop in exercise heart rate, from 159 (between minute 5-6) to 119 bpm at minute 14 of sustained exercise with a corresponding fall in perceived exertion, thus mirroring the response heretofore considered diagnostic of McArdle disease. However enzymatic activity of muscle phosphorylase and other known defects of muscle glycogenolysis were within normal limits and muscle glycogen levels were low normal as assessed biochemically (controls = 1.1±0.2%; patient = 0.7%) and with 13-C MRS in contrast to McArdle disease (2.5±0.4 %). Evaluation of other glycolytic enzymes revealed a complete absence of muscle phosphoglucomutase (PGM1) activity, the enzyme that catalyzes the conversion of phosphorylase-derived glucose-1-phosphate to glucose-6-phosphate for metabolism via glycolysis and the reverse reaction for glycogen synthesis from glucose. Genetic analysis revealed that the patient was a compound heterozygote for a nonsense mutation (Q530X in exon 10) and a missense mutation (R422W) in exon 8 of PGM1. Conclusions: This patient has a novel defect in phosphoglucomutase (PGM1) that mimics the clinical and physiological features of impaired muscle glycogenolysis due to muscle phosphorylase deficiency (McArdle disease). The finding of blocked muscle glycogenolysis in combination with low muscle glycogen implies that this enzymatic defect impairs both glycogen degradation and synthesis. Supported by: The Muscular Dystrophy Association and The Giant Tiger Foundation. Disclosure: Dr. Haller has nothing to disclose. Dr. Heinicke has nothing to disclose. Dr. Newby has nothing to disclose. Dr. Wyrick has nothing to disclose. Dr. Dimitrov has nothing to disclose. Dr. Mancias has nothing to disclose. Dr. Cohen has nothing to disclose.

Confirmation of the efficacy of vitamin B6 supplementation for McArdle disease by follow‐up muscle biopsy

No effective treatment for McArdle disease exists.We report a Japanese patient with McArdle disease who was treated with vitamin B(6) supplementation (60-90 mg/day). After treatment, increased muscle phosphorylase activity was confirmed by follow-up muscle biopsy (3.8 times higher than pretreatment levels). Increased lactate levels were seen on the forearm exercise test, and regular work activities could be resumed. Vitamin B(6) supplementation can enhance residual phosphorylase activity and improve insufficient anaerobic glycolysis of skeletal muscle.

Kei on GSK: a contribution by the 2007 recipient of the Young Scientist Award

EDITORIAL FOCUSKei on GSK: a contribution by the 2007 recipient of the Young Scientist AwardAmira KlipAmira KlipPublished Online:01 Jan 2008https://doi.org/10.1152/ajpendo.00731.2007This is the final version - click for previous versionMoreSectionsPDF (29 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat from time to time, we like to highlight articles authored by winners of the awards offered by the American Physiological Society. The Endocrinology and Metabolism section of APS conferred the 2007 Young Scientist Award to Kei Sakamoto, PhD. Kei is a prolific young scientist who has contributed to the American Journal of Physiology-Endocrinology and Metabolism with numerous publications and by serving on its Editorial Board since January 2007.Kei began his doctoral training at Yokohama City University in his native Japan, but soon transferred to the Joslin Diabetes Center/Harvard Medical School to complete it under the supervision of Dr. Laurie Goodyear. In 2003 he joined the group led by Dr. Dario Alessi at the MRC Protein Phosphorylation Unit of the University of Dundee for further training as a postdoctoral fellow. Since January 2006 he has been a Principal Investigator in the same Unit, and this year he was appointed Head of Molecular Physiology.What is Kei focusing on now? These days it is glycogen metabolism.Muscle glycogen synthesis is stimulated by insulin, and this depends on increased glucose transport and phosphorylation to glucose 6-phosphate, an allosteric activator of glycogen synthase, along with the activation of glycogen synthase by insulin-promoted dephosphorylation of the enzyme on sites Ser641 and Ser645. Insulin achieves the latter by inactivating glycogen synthase kinase-3 (GSK-3). Insulin-stimulated Akt phosphorylates and inactivates both the α- and β-isoforms of GSK-3, thus allowing for a net decrease in glycogen synthase phosphorylation at Ser641/645. Hence, it has been thought that insulin stimulates glycogen synthesis largely by dephosphorylating glycgen synthase through the PI3K-PKB-GSK-3 pathway. The study by Sakamoto and colleagues (1), highlighted below, provides genetic evidence that the insulin-stimulated muscle can synthesize glycogen normally in the absence of this system.The story began with a previous study in which Kei participated as a postodoctoral fellow in the group of Dr. Dario Alessi. McManus, Sakamoto, Alessi, and colleagues created a genetically modified homozygous mouse model in which the two endogenous GSK-3 genes had been eliminated and replaced, through gene knockin (KI), by corresponding genes encoding mutations at the NH2-terminal phosphorylation sites [from serine to alanine residues (2)]. The clear advantage of such a model is the complete replacement of the wild-type kinases with constitutively active forms whose expression is transcriptionally regulated by the wild-type GSK-3 gene promoters.Sakamoto and colleagues [Bouskila et al. (1)] now took advantage of that mouse model to show that, compared with wild-type littermates, muscles from these KI mice have similar glycogen content in the fasted and fed state or following a bolus injection of glucose, despite lack of insulin-stimulated glycogen synthase activity. The muscles from the KI mice also had similar rates of glycogen accumulation compared with controls 1 or 2 h after exposure to insulin in vitro. As well, insulin increased normally both glucose transport and glucose 6-phosphate levels in KI mice. Furthermore, KI mice did not have compensatory alterations in glycogen metabolism muscle phosphorylase activity and responded normally to epinephrine by inhibition of glycogen synthase and stimulation of glycogen phosphorylase. The authors suggest, by extrapolation of the results, that glucose 6-phosphate may be the major regulator of insulin-stimulated glycogen accumulation. This is an intriguing proposition that will likely elicit further direct analysis, a challenge that Kei Sakamoto contemplates, probably along with many of our reader experts in this area.We are pleased to highlight the work of this young scientist and hope that it will inspire young investigators in pursuit of the intricacies of endocrinology and metabolism.REFERENCES1 Bouskila M, Hirshman M, Jensen J, Goodyear L, Sakamoto K. Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle. Am J Physiol Endocrinol Metab (November 6, 2007). doi:10.1152/ajpendo.0407.2007.Google Scholar2 McManus EJ, Sakamoto K, Armit LJ, Ronaldson L, Shapiro N, Marquez R, Alessi DR. Role that phosphorylation of GSK3 plays in insulin and Wnt signalling defined by knockin analysis. EMBO J 24: 1571–1583, 2005.Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: A. Klip, Division of Cell Biology, The Hospital for Sick Children, Toronto, ON, M5G 1X8 Canada (e-mail: [email protected]) Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 294Issue 1January 2008Pages E27-E27 Copyright & PermissionsCopyright © 2008 by American Physiological Societyhttps://doi.org/10.1152/ajpendo.00731.2007PubMed18042667History Published online 1 January 2008 Published in print 1 January 2008 Metrics

Endocrine and metabolic alterations in the mink ( Mustela vison) due to chronic phytoestrogen exposure

Phytoestrogens are natural components of plant-based food items with beneficial health effects. The aim of the present study was to investigate the chronic effects of dietary phytoestrogens, genistein (8 mg kg −1 day −1) and β-sitosterol (50 mg kg −1 day −1), on the weight regulation of the mink ( Mustela vison). The parental generation was exposed from August 2002 to May–June 2003 to either β-sitosterol or genistein, while the kits were exposed through gestation and lactation. Food consumption and body masses were monitored monthly. Plasma lipid, glucose, total protein and hormone (ghrelin, leptin, triiodothyronine and thyroxine) concentrations were measured from the parents in August 2002, January 2003 and at the end of the experiment in May–June 2003 when the kits were 21 days of age. Relative food intake was higher in the β-sitosterol-exposed minks than in the control or genistein minks in September 2002. Plasma leptin and total protein concentrations were lower in the β-sitosterol kits compared to the control kits. Furthermore, plasma ghrelin levels and liver phosphorylase activities of the mink kits were higher due to genistein exposure. In mink kits, exposure to both phytoestrogens reduced the plasma thyroxine concentrations. The kidney glycogen concentrations and the muscle phosphorylase activities of phytoestrogen-treated adult minks were elevated. The results of this study suggest that minks are sensitive to perinatal phytoestrogen exposure.

Additivity of adrenaline and contractions on hormone-sensitive lipase, but not on glycogen phosphorylase, in rat muscle.

Hormone-sensitive lipase (HSL) has been proposed to regulate triacylglycerol (TG) breakdown in skeletal muscle. In muscles with different fibre type compositions the influence on HSL of two major stimuli causing TG mobilization was studied. Incubated soleus and extensor digitorum longus (EDL) muscles from 70 g rats were stimulated by adrenaline (5.5 microm, 6 min) or contractions (200 ms tetani, 1 Hz, 1 min) in maximally effective doses or by both adrenaline and contractions. Hormone-sensitive lipase activity was increased significantly by adrenaline as well as contractions, and the highest activity (P < 0.05) was seen with combined stimulation [Soleus: 0.40 +/- 0.03 (SE) m-unit mg protein(-1) (basal), 0.65 +/- 0.02 (adrenaline), 0.65 +/- 0.03 (contractions), 0.78 +/- 0.03 (adrenaline and contractions); EDL: 0.18 +/- 0.01, 0.30 +/- 0.02, 0.26 +/- 0.02, 0.32 +/- 0.01]. Glycogen phosphorylase activity was always increased more by adrenaline compared with contractions [Soleus: 60 +/- 4 (a/a + b)% vs. 46 +/- 3 (P < 0.05); EDL: 60 +/- 5 vs. 39 +/- 6 (P < 0.05)]. After combined stimulation glycogen phosphorylase activity in soleus [59 +/- 3 (a/a + b)%] was identical to and in EDL [45 +/- 4 (a/a + b)%] smaller (P < 0.05) than the activity after adrenaline only. In slow-twitch oxidative as well as in fast-twitch glycolytic muscle HSL is activated by both adrenaline and contractions. These stimuli are partially additive indicating at least partly different mechanisms of action. Contractions may impair the enhancing effect of adrenaline on glycogen phosphorylase activity in muscle.

Differential response of rat skeletal muscle glycogen metabolism to testosterone and estradiol

Although reports on sex steroids have implicated them as promoting protein synthesis and also providing extra strength to the skeletal muscle, it remains unclear whether sex steroids affect glycogen metabolism to provide energy for skeletal muscle functions, since glycogen metabolism is one of the pathways that provides energy for the skeletal muscle contraction and relaxation cycle. The purpose of the current study was to show that testosterone and estradiol act differentially on skeletal muscles from different regions, differentially with reference to glycogen metabolism. To study this hypothesis, healthy mature male Wistar rats (90-120 days of age, weighing about 180-200 g) were castrated (a bilateral orchidectomy was performed to test the significance of skeletal muscle glycogen metabolism in the absence of testosterone). One group of castrated rats was supplemented with testosterone (100 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards). To test whether estradiol has any effect on male skeletal muscle glycogen metabolism 17beta-estradiol (5 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards) was administered to orchidectomized rats. To test whether these sex steroids have any differential effect on skeletal muscles from different regions, skeletal muscles from the temporal region (temporalis), muscle of mastication (masseter), forearm muscle (triceps and biceps), thigh muscle (vastus lateralis and gracilis), and calf muscle (gastrocnemius and soleus) were considered. Castration enhanced blood glucose levels and decreased glycogen stores in skeletal muscle from head, jaw, forearm, thigh, and leg regions. This was accompanied by diminished activity of glycogen synthetase and enhanced activity of muscle phosphorylase. Following testosterone supplementation to castrated rats, a normal pattern of all these parameters was maintained. Estradiol administration to castrated rats did not bring about any significant alteration in any of the parameters. The data obtained suggest a stimulatory effect of testosterone on skeletal muscle glycogenesis and an inhibitory effect on glycogenolysis. Estradiol did not play any significant role in the skeletal muscle glycogen metabolism of male rats.

Partial activation of muscle phosphorylase by replacement of serine 14 with acidic residues at the site of regulatory phosphorylation.

Phosphorylation of glycogen phosphorylase at residue Ser14 triggers a conformational transition that activates the enzyme. The N-terminus of the protein, in response to phosphorylation, folds into a 310 helix and moves from its location near a cluster of acidic residues on the protein surface to a site at the dimer interface where a pair of arginine residues form charged hydrogen bonds with the phosphoserine. Site-directed mutagenesis was used to replace Ser14 with Asp and Glu residues, analogs of the phosphoserine, that might be expected to participate in ionic interactions with the arginine side chains at the dimer interface. Kinetic analysis of the mutants indicates that substitution of an acidic residue in place of Ser14 at the site of regulatory phosphorylation partially activates the enzyme. The S14D mutant shows a 1.6-fold increase in Vmax, a 10-fold decrease in the apparent dissociation constant for AMP, and a 3-fold decrease in the S0.5 for glucose 1-phosphate. The S14E mutant behaves similarly, showing a 2.2-fold increase in Vmax, a 6-fold decrease in the apparent dissociation constant for AMP, and a 2-fold decrease in the S0.5 for glucose 1-phosphate. The ability of the mutations to enhance binding of AMP and glucose 1-phosphate and to raise catalytic activity suggests that the introduction of a carboxylate side chain at position 14 promotes docking of the N-terminus at the subunit interface and concomitant stabilization of the activated conformation of the enzyme. Like the native enzyme, both mutants show significant activity only in the presence of the activator, AMP. Full activation, analogous to that provided by covalent phosphorylation of the enzyme, likely is not achieved because of differences in the charge and the geometry of ionic interactions at the phosphorylation site.

Cooperative binding is not required for activation of muscle phosphorylase.

Muscle and liver glycogen phosphorylase isozymes differ in their responsiveness to the activating ligand AMP. The muscle enzyme, which supplies glucose in response to strenuous activity, binds AMP cooperatively, and its enzymatic activity becomes greatly enhanced. The liver isozyme regulates the level of blood glucose, and AMP is not the primary activator. In muscle glycogen phosphorylase, the residue proline 48 links two secondary structural elements that bind AMP. This amino acid residue is replaced with a threonine in the liver isozyme; unlike the muscle enzyme, liver binds AMP noncooperatively, and the enzymatic activity is not greatly increased. We have substituted proline 48 in the muscle enzyme with threonine, alanine, and glycine and characterized the recombinant enzymes kinetically and structurally to determine if proline at this position is critical for cooperative AMP binding and activation. Importantly, all of the engineered enzymes were fully activated by phosphorylation, indicating that enzymatic activity was not compromised. Only the mutant enzyme with alanine at position 48 responds like the wild-type enzyme to the presence of AMP, indicating that proline is not absolutely required for full cooperative activation. The substitution of either threonine or glycine at this position, however, creates enzymes that no longer bind AMP cooperatively. The enzyme with threonine at position 48 further mimics the liver enzyme, in that the maximal enzymatic activity is also reduced. Significantly, the glycine substitution caused the enzyme to be fully activated by AMP, although binding was not cooperative. The hyperactivation of the glycine mutant by AMP suggests that the total free energy of activation has decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities of carbohydrate metabolism in spontaneously hypertensive rats.

The present study was performed to investigate as to whether peripheral insulin resistance exists in spontaneously hypertensive rats (SHR). After a 12 h fasting period, SHR had significantly higher serum glucose and higher plasma glucagon values in comparison to normotensive control rats (WKY). There was a tendency for higher serum insulin concentrations as well, but this difference did not reach significance. After oral glucose loading or glucose/insulin administration, serum glucose and insulin levels were also higher in SHR compared to WKY rats. Muscle glycogen and glucose concentrations were identical in fasted SHR and WKY rats. With an oral glucose load or glucose/insulin treatment there was a significant increase in muscle glycogen, whereas glucose values declined in skeletal muscle. Both total (a+b-form) phosphorylase activity as well as the active a-form of the enzyme were similar in skeletal muscle of SHR and WKY rats. Glucose/insulin administration or oral glucose loading induced a considerable reduction of both a+b-form and a-form activities. The decrease in muscle phosphorylase activities was almost identical in both groups of animals. There was also a comparable activity of muscle glycogen synthetase activity in all groups of rats. Despite subtile changes of glucose, glucagon and to a lesser degree insulin levels which would be suggestive of insulin resistance, the data obtained from skeletal muscle argue against peripheral insulin resistance in spontaneously hypertensive rats.

Molecular heterogeneity of McArdle disease

McArdle disease (McKusick 23260) is a rare form of metabolic myopathy due to the lack of glycogen phosphorylase activity in muscle. This disease, genetically transmitted as an autosomal recessive character, is clinically characterized by weakness, cramps and attacks of myoglobinuria. Glycogen phosphorylase isoenzymes (muscle, liver and brain types) show a specific tissue distribution, so that the defect is limited to skeletal muscle.

McArdle Disease: Familial Variety of Muscle Phosphorylase Activity in Two Siblings

AbstractTwo siblings with McArdle disease were studied biochemically and histochemically. Case 1 was a 9‐year‐old girl and case 2 was a 14‐year‐old boy. No elevation of serum lactate after exercise was noticed in both cases. PAS staining in case 1 was markedly increased, whereas in case 2 it was moderately increased. Muscle glycogen content in case 1 was 51 mg/gr muscle and in case 2 it was 38.7 mg/gr (control: 11.2 ± 1.28 mg/gr). Muscle phosphorylase activity was completely absent in case 1 and was about 50% of normal value in case 2, by both determinations of histochemical and direct biochemical methods. SDS‐polyacrylamide gel electrophoresis revealed a little amount of muscle phosphorylase protein in both cases and also no abnormal migration of residual activity of muscle phosphorylase was found on polyacrylamide electrophoresis. These data indicate that occurrence of familial variety of muscle phosphorylase in two siblings was due to difference of muscle phosphorylase activity and also different onset of disease and clinical severity among two cases are depending on the degree of residual amount of the enzyme.

McArdle Disease: Familial Variety of Muscle Phosphorylase Activity in Two Siblings

McArdle Disease: Familial Variety of Muscle Phosphorylase Activity in Two Siblings

Parallel insulin-like actions of human growth hormone and its part sequence hGH 7-13.

The insulin-like effects of human growth hormone and the synthetic part sequence, N alpha-acetyl-hGH 7-13, on glycogen synthase and phosphorylase have been compared in an in vivo system using 16--18-day-old rats. Both the hormone and its part sequences had similar effects, increasing muscle glycogen synthase a activity and decreasing liver phosphorylase a activity, without affecting phosphorylase activity in muscle or synthase activity in liver. Insulin had similar effects, but also increased liver synthase a activity. The effects of all three substances could be abolished by prior treatment of the animals with anti-insulin serum, showing that the effects of growth hormone and its part sequences were insulin-dependent. Both growth hormone and the synthetic peptide increased the binding of insulin to liver plasma membrane. It is concluded that the insulin-like activity of human growth hormone is associated with a region containing residues 7 to 13 of the hormone molecule, and that this activity is insulin-dependent. It is suggested that both growth hormone and the synthetic peptide produce insulin-like activity by enhancing the binding of circulating insulin to its receptor.

Purification and characterization of glycogen phosphorylases from abdominal muscle, heart and integument of the crayfish, Orconectes limosus

1. 1. Phosphorylase a from abdominal muscle and phosphorylase b from abdominal muscle, heart and integument of the crayfish Orconectes limosus have been purified. 2. 2. With the exception of integument phosphorylase, the preparations were homogeneous as judged by SDS-PAGE. 3. 3. Subunit molecular weight of abdominal muscle phosphorylase a and b was found to be 100,000. Both dimeric and tetrameric forms were identified by conventional PAGE. 4. 4. On IEF, all phosphorylases investigated showed microheterogeneity in the range of pH 5.7–6.3. 5. 5. The pH dependence of muscle phosphorylase activity was determined. The optimum pH for phosphorylase a is around pH 6.7, for phosphorylase b around 6.9. 6. 6. NaF does not activate phosphorylase a and b. Inhibition occurs only at concentrations higher than those usually used in crude extracts for inhibition of phosphorylase phosphatase. 7. 7. Kinetic behaviour of the purified enzymes has been compared. The only obvious difference between phosphorylase b from muscle, heart and integument is the higher K m for glycogen of integument phosphorylase. Muscle phosphorylase a and b differed with respect to their K m values for AMP and G-1-P. 8. 8. From the present results it is concluded that, with respect to the tissues investigated, no tissue specific forms of phosphorylase are present in crayfish.

Regulation of muscle phosphorylase activity by carnosine and anserine

Carnosine (β-alanyl-L-histidine) activates rabbit muscle phosphorylase a in the presence and absence of AMP and phosphorylase b in the presence of AMP in a biphasic manner with a maximal activation at about 50mM carnosine and with phosphorylase b showing a greater degree of activation than phosphorylase a . Anserine (β-alanyl-L-N π-methyl-histidine) activates phosphorylase a to a lesser extent than carnosine up to a concentration of 90mM, whereas with phosphorylase b a weak activation below 30mM and a concentration-dependent inhibition above this concentration occurs. These effects are specific for the dipeptides and are not shown by their constituent amino acids. Carnosine and anserine activate phosphorylase a in the presence of the allosteric inhibitors ATP, D-glucose and caffeine, and the inhibition of phosphorylase b by anserine is also observed in the presence of these inhibitors.

Adrenal glucocorticoid permissive regulation of muscle glycogenolysis: action on protein phosphatase(s) and its inhibitor(s).

Adrenal glucocorticoids exert a permissive action on the glycogen phosphorylase cascade. Epinephrine activation of muscle phosphorylase and phosphorylase b kinase is depressed in adrenalectomized rats. Phosphorylase phosphatase activity is increased by steroid lack, and normal epinephrine inhibition of the enzyme does not occur. Phosphorylase b kinase phosphatase activity is also increased; epinephrine, however, does not inhibit activity in muscle from normal or adrenalectomized rats. Protein phosphatase inhibitor activity is depressed in boiled dialyzed preparations made from adrenalectomized rat muscle. Cortisol resplacement therapy restores protein phosphatase inhibitor activity, decreases increased protein phosphatase activity, and restores normal epinephrine-induced activation of phosphorylase b kinase and phosphorylase and epinephrine inhibition of phosphorylase phosphatase.

Reversal of phosphorylase activation in muscle despite continued contractile activity.

During studies of the regulation of phosphorylase activity and glycogenolysis in contracting muscle, it was found that conversion of phosphorlyase beta to alpha is transient. Reversal of phosphorylase activation during both continuous and intermittent stimulation in the plantaris might, in part, have been due to development of fatigue. However, a complete reversal of phosphorylase activation was also evident within 5 min in the absence of fatigue in soleus muscles stimulated tetanically with 100-ms-long trains at a rate of 60/min. These muscles showed no significant decline in contractile force. Glycogen breakdown stopped in the soleus when phosphorylase reverted to the beta form, providing evidence that phosphorylase beta was not active. This lack of activity is probably explained by the finding that ATP and AMP concentrations changed little, while glucose 6-phosphate increased. Reversal of phosphorlyase activation soon after the onset of steady-state work may be a mechanism for conserving glycogen when the supply of other substrates is adequate to meet the muscles' energy needs.

McArdle disease: The mystery of reappearing phosphorylase activity in muscle culture—A fetal isoenzyme

To understand the apparently paradoxical appearance of phosphorylase in muscle cultured from patients with McArdle disease, the enzyme in muscle culture was studied immunologically and electrophoretically. Antibody against normal human muscle phosphorylase completely inhibited the enzyme of adult muscle, but it had no effect on phosphorylase activity of muscle cultures from normal individuals or patients with McArdle disease. Also, amounts of antibody that would completely inhibit phosphorylase in mature muscle left about 30% of the activity in muscle obtained from human fetus at four months' gestation. Acrylamide-disc and slab-gel electrophoresis showed a single band of phorphorylase activity in adult muscle and two bands in fetal muscle. This suggested that at four months' gestation, both fetal and mature forms are present but that only the mature isoenzyme is inhibited by the antibody. The enzyme from cultured muscle gave only a single band, with the electrophoretic mobility of the fetal isoenzyme. These data suggest that phosphorylase activity in muscle cultured from patients with McArdle disease is due to a fetal isoenzyme whose genetic control is different from that of the mature enzyme.

McArdle's syndrome. Fine structural changes in muscle

Two cases of McArdle's syndrome are reported. One is a "classical" example; the other is unusual because of the in vitro presence of muscle phosphorylase activity. In the latter case, the electronmicroscopic investigation confirmed the diagnosis. The fine structural changes characteristic of this disease are summarized and it is concluded that histochemical studies alone are insufficient to exclude the diagnosis of McArdl's myopathy.