Kinase Activity In Muscle Research Articles

The effects of different carbon sources on growth, tyrosine kinase activity and gene expression of insulin receptor 1 in GIFT tilapia

A feeding trial was conducted to evaluate the effects of different sugar sources on growth performance, serum biochemistry, immunity, insulin receptor 1 and tyrosine kinase activity of GIFT tilapia. Four hundred and eighty fish with specifications of 31.70±0.68 g were selected and placed in 16 aquariums. The experiment was divided into four treatment with four replicates of thirty fish per tank. After 50 days of feeding, the results were as follows: From the indexes of final average weight, weight gain rate and specific growth rate, the growth performance was sucrose, glucose, starch and dextrin group in turn, and the sucrose group performed best (P<0.05). The activcities of acid phosphotase, alkaline phosphotase, superoxide dismutase and lysozyme in sucrose group were significantly higher than that of the other three groups (P<0.05). The serum glucose level of sucrose group and glucose group is significantly higher than that of dextrin group and starch group (P < 0.05). In terms of liver glycogen level, the content of sucrose group was significantly higher than that of dextrin group and starch group (P < 0.05). In terms of muscle glycogen level, the content of sucrose group was the highest and that of starch group was the lowest. The tyrosine kinase activity of glucose group was the highest and significantly different from dextrin group (P< 0.05). The tyrosine kinase activity in muscle was always higher than that in liver (P < 0.05). The IR-1 mRNA in starch group and dextrin group was significantly higher than that in sucrose group. Different sugar source diets had a significant effect on the expression of IR-1 mRNA in the muscle of tilapia. In conclusion, compared with dextrin and starch, sucrose and glucose can be better utilized by GIFT tilapia and show good growth performance. Key words: GIFT tilapia; Carbohydrates; Growth; Immunity; Insulin Receptor 1; Tyrosine Kinase

Serum Fetuin-A and Insulin Levels in Classic Congenital Adrenal Hyperplasia.

Androgens play a pivotal role in non-reproductive organs such as the kidney, heart, liver, and pancreas. As androgen receptors are expressed in pancreatic and liver cells, excess testosterone can result in hypersecretion of insulin and fetuin-A, a protein produced in the liver. The expression of fetuin-A, a natural inhibitor of tyrosine kinase activity in muscle and liver, leads to insulin resistance. In addition, insulin and fetuin-A levels are thought to be affected by drugs such as glucocorticoids (GCs) and fludrocortisone. However, whether fetuin-A and insulin levels are affected by androgens and GCs in patients with classic congenital adrenal hyperplasia (CAH) is unknown. This cross-sectional study included 56 CAH patients and 70 controls. Analyses were stratified by sex and prepubertal/pubertal status to control for potential changes in serum metabolic/inflammatory markers associated with the production of sex steroids. Fasting blood glucose, insulin, triglyceride, total cholesterol, high density lipoprotein-cholesterol, aspartate aminotransferase, alanine aminotransferase, fetuin-A, and high-sensitivity C-reactive protein (hs-CRP) levels were measured in blood samples. In addition, 17α-hydroxyprogesterone, androstenedione, total testosterone, free testosterone, and dehydroepiandrosterone sulfate levels were measured before medication was administered. Insulin and fetuin-A levels were significantly higher in CAH patients than in controls. The unfavourably high levels of these substances exhibited a positive correlation with total and free testosterone. Regression analysis revealed that fetuin-A and free testosterone were the only independent predictors of the insulin level, while insulin and free testosterone levels significantly predicted the fetuin-A level (R2=42.7% and 59.8%). Differences were also observed in triglyceride and hs-CRP levels between the pubertal and prepubertal groups. We conclude that serum fetuin-A and insulin levels may be associated with androgens in CAH patients.

Abstract 3380: Cancer usurps skeletal muscle as an energy repository

Abstract Cancer cells produce energy through aerobic glycolysis, but contributions of host tissues to cancer energy metabolism is unclear. In this study, we aimed to elucidate the cancer-host energy production relationship, in particular, between cancer energy production and host muscle. During the development and progression of colorectal cancer (CRC), expression of the secreted autophagy-inducing stress protein HMGB1 increased in the muscle of tumor-bearing animals. This effect was associated with decreased expression of pyruvate kinase PKM1 and pyruvate kinase activity in muscle via the HMGB1 receptor RAGE. However, muscle mitochondrial energy production was maintained. In contrast, HMGB1 addition to CRC cells increased lactate fermentation. In the muscle, HMGB1 addition induced autophagy by decreasing levels of active mTOR and increasing autophagy-associated proteins, plasma glutamate and 13C-glutamine incorporation into acetyl-CoA. In a mouse model of colon carcinogenesis, a temporal increase in HMGB1 occurred in serum and colonic mucosa with an increase in autophagy associated with altered plasma free amino acids levels, increased glutamine and decreased PKM1 levels. These differences were abolished by administration of an HMGB1 neutralizing antibody. Similar results were obtained in a mouse xenograft model of human CRC. Taken together, our findings suggest that HMGB1 released during tumorigenesis recruits muscle to supply glutamine to cancer cells as an energy source. Citation Format: Yi Luo, Junya Yoneda, Hitoshi Ohmori, Takamitsu Sasaki, Hiroki Kuniyasu. Cancer usurps skeletal muscle as an energy repository. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3380. doi:10.1158/1538-7445.AM2014-3380

Effects of Glyphosate on Growth Rate, Metabolic Rate and Energy Reserves of Early Juvenile Crayfish, Cherax quadricarinatus M.

Early juveniles of the crayfish Cherax quadricarinatus were exposed for 60 days to 10 and 40 mg/L of pure glyphosate (acid form) in freshwater. Mortality was 33 % at the highest concentration, while no differences in molting were noted among treatments. After the first month of exposure, weight gain was significantly (p < 0.05) reduced in the 40 mg/L group. At the end of the assay, lipid levels in muscle, as well as protein level in both hepatopancreas and muscle were significantly (p < 0.05) reduced. These results suggest long-term utilization of both lipid and protein as main energetic reserves, likely in response to the chronic stress associated with herbicide exposure. Besides, the lower pyruvate kinase activity in muscle suggests a possible metabolic depression in this tissue. The hemolymphatic ASAT:ALAT ratio showed higher levels than the control at the highest glyphosate concentration, indicating possible damage to several tissues.

Cancer Usurps Skeletal Muscle as an Energy Repository

Cancer cells produce energy through aerobic glycolysis, but contributions of host tissues to cancer energy metabolism are unclear. In this study, we aimed to elucidate the cancer-host energy production relationship, in particular, between cancer energy production and host muscle. During the development and progression of colorectal cancer, expression of the secreted autophagy-inducing stress protein HMGB1 increased in the muscle of tumor-bearing animals. This effect was associated with decreased expression of pyruvate kinase PKM1 and pyruvate kinase activity in muscle via the HMGB1 receptor for advanced glycation endproducts (RAGE). However, muscle mitochondrial energy production was maintained. In contrast, HMGB1 addition to colorectal cancer cells increased lactate fermentation. In the muscle, HMGB1 addition induced autophagy by decreasing levels of active mTOR and increasing autophagy-associated proteins, plasma glutamate, and (13)C-glutamine incorporation into acetyl-CoA. In a mouse model of colon carcinogenesis, a temporal increase in HMGB1 occurred in serum and colonic mucosa with an increase in autophagy associated with altered plasma free amino acid levels, increased glutamine, and decreased PKM1 levels. These differences were abolished by administration of an HMGB1 neutralizing antibody. Similar results were obtained in a mouse xenograft model of human colorectal cancer. Taken together, our findings suggest that HMGB1 released during tumorigenesis recruits muscle to supply glutamine to cancer cells as an energy source.

Muscle choline kinase beta defect causes mitochondrial dysfunction and increased mitophagy

Choline kinase is the first step enzyme for phosphatidylcholine (PC) de novo biosynthesis. Loss of choline kinase activity in muscle causes rostrocaudal muscular dystrophy (rmd) in mouse and congenital muscular dystrophy in human, characterized by distinct mitochondrial morphological abnormalities. We performed biochemical and pathological analyses on skeletal muscle mitochondria from rmd mice. No mitochondria were found in the center of muscle fibers, while those located at the periphery of the fibers were significantly enlarged. Muscle mitochondria in rmd mice exhibited significantly decreased PC levels, impaired respiratory chain enzyme activities, decreased mitochondrial ATP synthesis, decreased coenzyme Q and increased superoxide production. Electron microscopy showed the selective autophagic elimination of mitochondria in rmd muscle. Molecular markers of mitophagy, including Parkin, PINK1, LC3, polyubiquitin and p62, were localized to mitochondria of rmd muscle. Quantitative analysis shows that the number of mitochondria in muscle fibers and mitochondrial DNA copy number were decreased. We demonstrated that the genetic defect in choline kinase in muscle results in mitochondrial dysfunction and subsequent mitochondrial loss through enhanced activation of mitophagy. These findings provide a first evidence for a pathomechanistic link between de novo PC biosynthesis and mitochondrial abnormality.

Insulin signaling pathways in a patient with insulin resistance of difficult management - a case report

Insulin signalling pathways were investigated in a 33 year-old woman with immunologic insulin resistance. Her past medical history was remarkable for intermittent use of insulin and allergic reactions to several drugs, and measure of plasma anti-insulin antibodies level corroborated the clinical suspicion of immune mediated insulin resistance (8074 nU/ml - RIA - Ref value: <60). Treatment with several immunosuppressive regimens was tried, however the results were disappointing. Possible subcellular mechanisms of insulin resistance were investigated by performing analysis of insulin receptor and post receptor signaling in skeletal muscle biopsy. The expression of insulin receptor (IR), insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT-4) was evaluated in total extract from muscle tissue by Western blotting. Basal IR, IRS-1 and GLUT-4 expression was detected, however receptor autophosphorylation was not observed. A study of translocation of GLUT-4 to plasma membrane showed that tissue presented low levels of membrane-associated GLUT-4. When in vitro stimulation was undertaken, tissue was capable to be responsive to insulin. Our results suggest that even though IR expression was normally occurring, IR β-subunit tyrosine kinase activity in muscle was down-regulated leading to alterations in insulin post receptor signaling. Consistent with normal insulin receptor and post receptor signaling, our results were compatible with decreased insulin binding to IR probably due to neutralization by anti-insulin antibodies. In conclusion, this patient has immunologic insulin resistance and treatment should be based on immunosuppressive drugs as tolerated.

P-110: Measurement of insulin receptor kinase activity in muscle and erythrocytes of a family with mutations of the insulin receptor gene

P-110: Measurement of insulin receptor kinase activity in muscle and erythrocytes of a family with mutations of the insulin receptor gene

High-fat feeding increases insulin receptor and IRS-1 coimmunoprecipitation with SOCS-3, IKKα/β phosphorylation and decreases PI-3 kinase activity in muscle

Suppressor of cytokine signaling (SOCS) proteins and/or activation of the proinflammatory pathway have been postulated as possible mechanisms that may contribute to skeletal muscle insulin resistance. Thus, the aims of the present investigation were to determine in high-fat-fed skeletal muscle: 1) whether SOCS-3 protein concentration is increased, 2) whether coimmunoprecipitation of SOCS-3 with the insulin receptor-beta subunit and/or IRS-1 is increased, and 3) whether select components of the proinflammatory pathway are altered. Thirty-two male Sprague-Dawley rats were assigned to either control (CON, n = 16) or high-fat-fed (HF, n = 16) dietary groups for 12 wk and then subjected to hind limb perfusions in the presence (n = 8/group) or absence (n = 8/group) of insulin. Insulin-stimulated skeletal muscle 3-MG transport rates and PI-3 kinase activity were greater (P < 0.05) in CON. IRS-1 tyrosine phosphorylation was decreased (P < 0.05), and IRS-1 serine 307 phosphorylation was increased (P < 0.05) in HF. Insulin receptor-beta (IR-beta) subunit coimmunoprecipitation with IRS-1 was reduced in HF. SOCS-3 protein concentration and SOCS-3 coimmunoprecipitation with both the IR-beta subunit and IRS-1 was increased (P < 0.05) in HF. IKKalpha/beta serine phosphorylation was increased (P < 0.05), IkappaBalpha protein concentration was decreased (P < 0.05) and IkappaBalpha serine phosphorylation was increased (P < 0.05) in HF. Increased colocalization of SOCS-3 with both the IR-beta subunit and IRS-1 may provide steric hindrance that prevents IRS-1 from interacting with IR-beta, while increased IKKbeta serine phosphorylation may contribute to increasing IRS-1 serine phosphorylation, both of which independently can have deleterious effects on insulin-stimulated PI-3 kinase activation in high-fat-fed rodent skeletal muscle.

Regulation of muscle protein synthesis during sepsis and inflammation

Prolonged sepsis and exposure to an inflammatory milieu decreases muscle protein synthesis and reduces muscle mass. As a result of its ability to integrate diverse signals, including hormones and nutrients, the mammalian target of rapamycin (mTOR) is a dominant regulator in the translational control of protein synthesis. Under postabsorptive conditions, sepsis decreases mTOR kinase activity in muscle, as evidenced by reduced phosphorylation of both eukaryotic initiation factor (eIF)4E-binding protein (BP)-1 and ribosomal S6 kinase (S6K)1. These sepsis-induced changes, along with the redistribution of eIF4E from the active eIF4E.eIF4G complex to the inactive eIF4E.4E-BP1 complex, are preventable by neutralization of tumor necrosis factor (TNF)-alpha but not by antagonizing glucocorticoid action. Although the ability of mTOR to respond to insulin-like growth factor (IGF)-I is not disrupted by sepsis, the ability of leucine to increase 4E-BP1 and S6K1 phosphorylation is greatly attenuated. This "leucine resistance" results from a cooperative interaction between both TNF-alpha and glucocorticoids. Finally, although septic animals are not IGF-I resistant, the anabolic actions of IGF-I are nonetheless reduced because of the development of growth hormone resistance, which decreases both circulating and muscle IGF-I. Herein, we highlight recent advances in the mTOR signaling network and emphasize their connection to the atrophic response observed in skeletal muscle during sepsis. Although many unanswered questions remain, understanding the cellular basis of the sepsis-induced decrease in translational activity will contribute to the rational development of therapeutic interventions and thereby minimize the debilitating affects of the atrophic response that impairs patient recovery.

S-nitrosation of the insulin receptor, insulin receptor substrate 1, and protein kinase B/Akt: a novel mechanism of insulin resistance.

Evidence demonstrates that exogenous nitric oxide (NO) and the NO produced by inducible nitric oxide synthase (iNOS) can induce insulin resistance in muscle. Here, we investigated whether this insulin resistance could be mediated by S-nitrosation of proteins involved in early steps of the insulin signal transduction pathway. Exogenous NO donated by S-nitrosoglutathione (GSNO) induced in vitro and in vivo S-nitrosation of the insulin receptor beta subunit (IRbeta) and protein kinase B/Akt (Akt) and reduced their kinase activity in muscle. Insulin receptor substrate (IRS)-1 was also rapidly S-nitrosated, and its expression was reduced after chronic GSNO treatment. In two distinct models of insulin resistance associated with enhanced iNOS expression-diet-induced obesity and the ob/ob diabetic mice-we observed enhanced S-nitrosation of IRbeta/IRS-1 and Akt in muscle. Reversal of S-nitrosation of these proteins by reducing iNOS expression yielded an improvement in insulin action in both animal models. Thus, S-nitrosation of proteins involved in insulin signal transduction is a novel molecular mechanism of iNOS-induced insulin resistance.

Early steps of insulin receptor signaling in chicken and rat: apparent refractoriness in chicken muscle

The early steps of insulin receptor (IR) signaling (tyrosine phosphorylation of IR β-subunit, IRS-1 and Shc and PI 3′-kinase activity) have been characterized in two target tissues in the chicken: liver and muscle. The signaling cascade appeared to depend on nutritional status in the liver, but not in muscle (with a possible exception for a minor tyrosine phosphorylation of the 52 kDa Shc isoform). In this study, we compared the responses of the liver and muscle to exogenous insulin (10 or 1000 mU/kg) in chickens and rats. In the liver, IRS-1 and Shc proteins were present in smaller amounts and the regulatory subunit p85 of PI 3′-kinase was present in larger amounts in chickens than in rats. In the basal state (saline injection), the level of tyrosine phosphorylation of IR was lower, and that of Shc higher, in chickens than in rats. PI 3′-kinase activity in chickens was half that in rats. Insulin activated all components of the cascade in a dose-dependent manner in both species. A different pattern was observed in the muscle. In the basal state, the levels of tyrosine phosphorylation of IR and of PI 3′-kinase activity were much higher in chickens than in rats (by factors of 2 and 30, respectively). Insulin strongly activated all components of the cascade in rats (but with no significant increase in the phosphorylation of Shc). No activation was observed in chickens (with only a slight but significant increase in the tyrosine phosphorylation of Shc). The insulin cascade therefore appears to respond normally in chicken liver but to be refractory in chicken muscle. The large amount of p85 and high levels of PI 3′-kinase activity in muscle may contribute to this situation, making chicken muscle an interesting model of insulin resistance.

Induction of Cdk5 activity in rat skeletal muscle after nerve injury.

Cyclin-dependent kinase 5 (Cdk5) was originally identified as a serine/threonine kinase and subsequently demonstrated to play a critical role in the development of CNS. We recently reported the novel function of Cdk5 in the neuregulin signaling pathway during the development of neuromuscular junction (NMJ). Here, we report the regulation of Cdk5 and p35 in rat skeletal muscle after nerve injury. Northern blot analysis revealed that Cdk5 and p35 transcripts were up-regulated in muscle after nerve denervation. The temporal profiles for the regulation of Cdk5 and p35 transcripts were different, suggesting that these changes in gene transcription might be regulated by different mechanism. Our finding on the ability of tetrodotoxin to induce p35 transcript in muscle suggested that electrical activity could regulate p35 expression. In addition to the induction of mRNA expression, the total Cdk5 and p35-associated kinase activity in muscle increased prominently after nerve denervation. Taken together, our findings suggest that Cdk5 and p35 may play important physiological roles in muscle regeneration following nerve injury.

Defective insulin-induced GLUT4 translocation in skeletal muscle of high fat-fed rats is associated with alterations in both Akt/protein kinase B and atypical protein kinase C (zeta/lambda) activities.

The cellular mechanism by which high-fat feeding induces skeletal muscle insulin resistance was investigated in the present study. Insulin-stimulated glucose transport was impaired ( approximately 40-60%) in muscles of high fat-fed rats. Muscle GLUT4 expression was significantly lower in these animals ( approximately 40%, P < 0.05) but only in type IIa-enriched muscle. Insulin stimulated the translocation of GLUT4 to both the plasma membrane and the transverse (T)-tubules in chow-fed rats. In marked contrast, GLUT4 translocation was completely abrogated in the muscle of insulin-stimulated high fat-fed rats. High-fat feeding markedly decreased insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity but not insulin-induced tyrosine phosphorylation of the insulin receptor and IRS proteins in muscle. Impairment of PI 3-kinase function was associated with defective Akt/protein kinase B kinase activity (-40%, P < 0.01) in insulin-stimulated muscle of high fat-fed rats, despite unaltered phosphorylation (Ser473/Thr308) of the enzyme. Interestingly, basal activity of atypical protein kinase C (aPKC) was elevated in muscle of high fat-fed rats compared with chow-fed controls. Whereas insulin induced a twofold increase in aPKC kinase activity in the muscle of chow-fed rats, the hormone failed to further increase the kinase activity in high fat-fed rat muscle. In conclusion, it was found that GLUT4 translocation to both the plasma membrane and the T-tubules is impaired in the muscle of high fat-fed rats. We identified PI 3-kinase as the first step of the insulin signaling pathway to be impaired by high-fat feeding, and this was associated with alterations in both Akt and aPKC kinase activities.

Evaluation of Phosphoenolpyruvate as a Phosphoryl Group Donor for Phosphoproteins in Skeletal Muscle

The possible existence of a phosphoenolpyruvate-dependent protein kinase activity in muscle first reported by Khandelwalet al.(FEBS Lett.162, 127–132, 1983) was further examined in this study. [32P]Phosphoenolpyruvate was used to assay rabbit muscle extract that was first dialyzed and then passed over a gel filtration column. Fractions from the column were assayed for activity (as shown by counts incorporated into an acid-precipitable product), which was observed only when two distinct and resolved fractions were combined. The approximate masses of these two components, as estimated by nondenaturing gel filtration chromatography, were 30 and 160 kDa. The activity showed time and concentration dependence and was inactivated by heat and proteolysis. [γ-32P]ATP could not substitute for [32P]phosphoenolpyruvate in the activity assays, nor was there evidence for the formation of ATP during the assays. Elution of the reaction mixture from a sizing column revealed several radioactive peaks. Other tissues (heart, kidney, liver, lung, spleen, and back skeletal muscle) from rabbit and rat were screened; the total activity was detected in all tissues examined, but was highest in the skeletal muscle of both species, with that from rabbit being twice that from rat.

Low glucose-1, 6-bisphosphate and high fructose-2, 6-bisphosphate concentrations in muscles of patients with glycogenosis types VII and V

The level of glucose-1, 6-bisphosphate, a potent allosteric activator of phosphofructokinase, was markedly decreased in muscles of patients with glycogenosis type VII (muscle phosphofructokinase deficiency) and type V (muscle phosphorylase deficiency). Glucose-1-phosphate kinase activity in muscle was virtually absent in a patient with glycogenosis type VII, whereas it was normal in a patient with type V blycogenosis. Glucose-1-phosphate level was increased in type VII glycogenosis, whereas it was decreased in type V glycogenosis. Another activator of phosphofructokinase, fructose-2, 6-bisphosphate was increased in muscles of patients with both types of glycogenosis although it was much higher in type VII than in type V. This finding may be partly related to the difference of fructose-6-phosphate concentrations. The results suggest that phosphofructokinase would contribute to the major glucose-1-phosphate kinase activity in normal human muscle and would also form a kind of self-activating system.

Glyceraldehyde-3-phosphate dehydrogenase mRNA: Activity and amount in dystrophic hamster muscle

The activity and amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in muscle of young dystrophic hamsters was reduced to approximately half the level found in control animals. No changes in brain or liver enzyme activity were found. Several other glycolytic enzyme activities and creatine kinase activity in muscle were unchanged, except for modest decreases in aldolase and pyruvate kinase. To assess the synthesis of glyceraldehyde-3-phosphate dehydrogenase, the poly(A) + RNA was isolated from muscle polysomes of dystrophic and control animals and its activity was assessed in an mRNA-dependent translation system. The translatability of the mRNA for GAPDH found in the dystrophic muscle preparations also was half of that found in the control muscle preparations. Decreases were also found in the translatability of mRNA for tropomyosin.

Postnatal development of phosphorylase kinase in mouse skeletal muscle

The postnatal development of phosphorylase kinase activity was studied in mouse (strain Ha ICR ) skeletal muscle, and the properties of the enzymes from skeletal muscle of 6- to 10-day-old and adult mice were compared. Phosphorylase kinase activity in skeletal muscle was found to increase more than 12-fold during postnatal development with over 80% of the increase occurring after the second week of life. Comparison of the phosphorylase kinase activity in muscle from 6- to 10-day-old and adult mice showed the two enzymes to have similar kinetic properties, but three lines of evidence indicated that they have different structures: (i) They differed in their cross-reactivity in quantitative immunotitrations using antisera prepared to purified rabbit skeletal muscle phosphorylase kinase. (ii) They differed in their heat lability at 42.5 °C. (iii) They differed in their subunit structure as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The phosphorylase kinase from muscle of 6- to 10-day-old mice had only the α′,β, and γ subunits, while the enzyme from adults showed α,α′,β, and γ subunits. These results indicate that skeletal muscle of adult mice has a different phosphorylase kinase isozyme(s) from that present in muscle of newborn mice.

Ribonucleoprotein complexes of pyruvate kinase from human skeletal muscle.

About 25% of total pyruvate kinase activity in muscle appears in a bound form which is insoluble in water or diluted salt solutions at pH 5.8. That activity is associated with the ribonuc-leoprotein complexes and is soluble at high ionic strength. A procedure is described for the purification and crystallization of this enzyme form herein called pyruvate kinase MB and water soluble form MA.

Phosphorylase kinase mediating the effects of cyclic AMP in muscle

In the classic view of the control of phosphorylase b to a conversion by catecholamines, cyclic AMP acts as the second messenger stimulating the activity of cyclic AMP-dependent protein kinase to covalently modify phosphorylase kinase. Phosphorylation of phosphorylase kinase converts this enzyme from a nonactivated to an activated form with a markedly higher activity at pH 7. There is now considerable evidence that the activity of phosphorylase kinase is also regulated by changes in the Ca 2+ concentration. The activity of both nonactivated and activated phosphorylase kinase is stimulated by Ca 2+ in the range of concentrations that have been reported to occur in the sacroplasm of contracting muscle, with the activated phosphorylase kinase having a lower K a for Ca 2+. Thus there are at least two mechanisms for the regulation of phosphorylase kinase activity in muscle. These mechanisms may act independently or in concert in controlling glycogenolysis stimulated by catecholamines, anoxia, or tetanic electrical stimulation. Additional substrates for phosphorylase kinase besides phosphorylase have recently been shown. Phosphorylase kinase has been reported to phosphorylate a protein in cardiac sarcoplasmic reticulum with an associated increase in Ca 2+ uptake. Several potential artifacts must be considered. Phosphorylase kinase present in crude muscle membrane fractions would be a source of high-affinity Ca 2+ binding sites. Limited tryptic proteolysis of phosphorylase kinase has been reported to alter the structure of the enzyme without a marked reduction in either Ca 2+ binding affinity or phosphorylase b to a converting enzyme activity. Phosphorylase kinase is also a potential source of endogenous kinase activity in crude membrane fractions. Its phosphorylase b to a converting activity is not completely inhibited by ethylene glycol bis (B-aminoethylether)-N,N'-tetraacetic acid (EGTA), which is frequently used to inhibit endogenous phosphorylase kinase activity in these fractions. Thus, studies utilizing crude membrane fractions should assess the contribution of phosphorylase kinase and its proteolytic fragments to any Ca 2+ binding and endogenous kinase activity present in the subcellular fraction. A role for phosphorylase kinase in regulating the inotropic state of muscle has been postulated from the observations that phosphorylase kinase phosphorylated troponin and myosin light chains. The phosphorylated protein needs to be shown to have altered properties in regulating actomyosin ATPase activity. Furthermore, phosphorylation of troponin, myosin light chains, or sarcoplasmic reticulum by phosphorylase kinase has not yet been demonstrated in intact muscle. In mice with the phosphorylase kinase deficiency mutation, the mutant phosphorylase kinase catalyzed the phosphorylation of troponin at a rate of 29%–57% of that of the control strain enzyme, but the mutant enzyme phosphorylated phosphorylase b at a rate of less than 0.2% of normal. Thus, the absence of a myopathy in phosphorylase kinase-deficient mice does not rule out a physiologic role for phosphorylase kinase in covalently modifying the contractile proteins or regulating Ca 2+ flux in muscle.