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At least 649 records · Page 36Linked to original sources
PubMed1980

Changes in the sarcoplasmic proteins of skeletal muscles induced by experimental steroid myopathy.

The protein content and the relative distribution of cell compartments was investigated after 14 days of dexamethasone treatment in rabbits. The cell fractions were further analysed by SDS-PAGE. It was found that the loss of myofibrillar fraction was greater than that of the sarcoplasmic one. The susceptibility of fast-twitch fibres to steroid myopathy was more marked compared to the slow fibres. The sarcoplasmic proteins showed the following changes: myoglobin and myokinase decreased, while alpha-GPDH and LDH increased in both muscle types. On the other hand, the alterations of TPI, PGK, PGI, PK, ADA, phosphorylase kinase and phosphorylase b, were opposite in the two types of muscle. The results are in agreement with observations of others indicating that glucocorticoid treatment causes a shift in the equilibrium of synthesis and degradation of myofibrillar and sarcoplasmic proteins. The different susceptibility of fast versus slow muscles to steroid myopathy could be due to the activity-dependent regulatory mechanism of metabolic pathways.

Animals↗
PubMed2005

Functional compartmentation of glycogen phosphorylase with creatine kinase and Ca2+ ATPase in skeletal muscle.

This manuscript discusses aspects of functional compartmentation in the regulation of metabolism. The functional consequences of enzymes coupling between creatine kinase, glycogen phosphorylase and sarcoplasmic reticular Ca2+ ATPase is examined. It is proposed that the coupling of creatine kinase and glycogen phosphorylase classifies as a novel class of diazyme complex with an important regulatory role in the inhibition of glycogenolysis at rest. In addition it is suggested that creatine kinase, glycogen phosphorylase and the sarcoplasmic reticular Ca2+ ATPase may couple to form a three-enzyme complex. From a consideration of the structure and chemical catalysis of the putative three-enzyme complex, a novel net reaction for glycogenolysis in the vicinity of the sarcoplasmic reticulum is suggested (Phosphocreatine+Glycogen+H(+)Creatine+Glycogen(n)(-1)+Glucose-1-Phosphate). The three-enzyme complex may also have an important role in inhibiting glycogenolysis at rest as well as improving the efficiency of high-energy phosphate transfer.

Animals↗
PubMed1984

Isolation and characterization of a high molecular weight protein phosphatase from rabbit skeletal muscle.

A high molecular weight protein phosphatase (phosphatase H-II) was isolated from rabbit skeletal muscle. The enzyme had a Mr = 260,000 as determined by gel filtration and possessed two types of subunit, of Mr = 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On ethanol treatment, the enzyme was dissociated to an active species of Mr = 35,000. The purified phosphatase dephosphorylated lysine-rich histone, phosphorylase a, glycogen synthase, and phosphorylase kinase. It dephosphorylated both the alpha- and beta-subunit phosphates of phosphorylase kinase, with a preference for the dephosphorylation of the alpha-subunit phosphate over the beta-subunit phosphate of phosphorylase kinase. The enzyme also dephosphorylated p-nitrophenyl phosphate at alkaline pH. Phosphatase H-II is distinct from the major phosphorylase phosphatase activities in the muscle extracts. Its enzymatic properties closely resemble that of a Mr = 33,500 protein phosphatase (protein phosphatase C-II) isolated from the same tissue. However, despite their similarity of enzymatic properties, the Mr = 35,000 subunit of phosphatase H-II is physically different from phosphatase C-II as revealed by their different sizes on sodium dodecyl sulfate-gel electrophoresis. On trypsin treatment of the enzyme, this subunit is converted to a form which is a similar size to phosphatase C-II.

Animals↗
PubMed1985

The influence of sorbitol on the glycogenolytic pathway.

The following report presents the results of a study conducted on the influence of sorbitol on levels of glycogen, phosphorylase a (E.C. 2.4.1.1.) and phosphorylase kinase (E.C.2.7.1.38) in slices of rat liver. In addition, it presents data concerning the effects of sorbitol on the activities of phosphorylase a and phosphorylase kinase purified from rabbit muscle. The data reveal a limitation in the glycogenolytic process which seems, in part, due to the inactivation of the phosphorylase a and phosphorylase kinase. Gel-filtration techniques showed, in fact, a breakdown of these two oligomeric enzymes into inactive subunits following incubation with sorbitol.

Animals↗
PubMed1989

Phosphorylation of the human papillomavirus type 1 E4 proteins in vivo and in vitro.

Following incubation of HPV 1-induced warts in the presence of [32P] phosphate several of the E4-encoded proteins were found to be radiolabeled. Two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 17K E4 polypeptides had incorporated [32P]phosphate whereas those of 16K were unlabeled. Purified E4 gene products were separated by ion exchange chromatography into a large number of different species, which were of similar size but of different charge due to varying extents of phosphorylated peptides have been isolated and identified. Phosphoserine and phosphothreonine were identified in all 16/17K E4 fractions but not phosphotyrosine. Both HPV 1 E4 16K and 17K fractions were phosphorylated in vitro by cAMP-dependent protein kinase but not by myosin light chain kinase or by phosphorylase kinase. Incubation with cAMP PK gave incorporation of approx. 0.5 mole phosphate/mol of protein indicating that the cAMP-dependent protein kinase site(s) was partially phosphorylated in vivo. This view was supported by the fact that species which were more heavily phosphorylated in vivo incorporated less phosphate after cAMP-dependent protein kinase phosphorylation. HPV 1 E4 was also phosphorylated at serine and threonine residues by a crude cytoplasmic extract prepared from cultured human keratinocytes and cultured human retinoblasts. These results are discussed in the light of the known effects of phosphorylation on the interactions of other keratinocyte-specific proteins.

Cell-Free System↗
PubMed2000

Fetal-onset severe skeletal muscle glycogenosis associated with phosphorylase-b kinase deficiency.

We report on a premature newborn girl delivered after 32 weeks of gestation by cesarean section after sparse limb movements, fetal tachycardia and late heart rate decelerations had suggested fetal distress. Following 1 day of mechanical ventilation, adequate pulmonary gas exchange was achieved by spontaneous breathing. Main symptoms were virtually complete absence of spontaneous movements, increased flexor tonus of the extremities, and hypotonia of the trunk. Inability to suck or swallow required nasogastric gavage feeding. There were no hypoglycemic episodes. Echocardiography revealed normal myocardial function. Creatine kinase was 237 U/I at 2 days of life, declining to normal values thereafter. Muscle biopsy revealed increased glycogen storage with subsarcolemmal glycogen deposits and low phosphorylase-a activity while total phosphorylase was normal after in vitro activation, suggestive of phosphorylase-b kinase deficiency. No mutation was detected in exon 1 of the myophosphorylase gene. No psychomotor development was observed, and the infant died of central apnea at 3 months of age.

Adult↗
PubMed1998

Effector-sensitive cross-linking of phosphorylase b kinase by the novel cross-linker 4-phenyl-1,2,4-triazoline-3,5-dione.

The dienophile 4-phenyl-1,2,4-triazoline-3,5-dione (PTD) was identified as a novel protein cross-linker, and utilized as a conformational probe of phosphorylase b kinase (PhK), a hexadecameric enzyme with the subunit composition (alphabetagammadelta)4. In its reaction with this enzyme, PTD produced five major cross-linked conjugates as resolved by denaturing gel electrophoresis: alphabeta, betagammagamma, alphagamma and a doublet of differently migrating homodimers, betabeta1 and betabeta2. Cross-linking in the presence of six different activators of the kinase targeted to its various subunits caused substantial changes in the amounts of three of the conjugates. The formation of alphagamma was increased by all of the activators but the largest enhancement was caused by exogenous Ca2+/calmodulin. All except one of the activators decreased the amount of betagammagamma formed, with Mg2+ having the greatest effect, and all except two increased the amount of betabeta1, with Mg2+ again having the largest influence. From the overall similarity of the changes in cross-linking by PTD induced by the various activators, we conclude that, even though they are targeted to different sites and subunits, they induce activated conformations of PhK that have certain structural features in common. Regarding the mechanism of cross-linking by PTD, its reaction with a model nucleophile suggests that its initial reaction with a side chain nucleophile of PhK involves a 1,4-conjugate addition to form a urazole adduct, with the secondary cross-linking reaction occurring through an as yet unknown pathway.

Animals↗
PubMed1981

Fatal infantile glycogen storage disease: deficiency of phosphofructokinase and phosphorylase b kinase.

A girl with congenital limb weakness, mental retardation, and corneal ulceration died with respiratory insufficiency at age 4 years. Histochemistry of muscle biopsy showed only nonspecific myopathy, but electronmicroscopy revealed subsarcolemmal and intramyofibrillar accumulation of glycogen. Biochemical studies showed increased glycogen content of muscle with lack of phosphofructokinase. Phosphorylase b kinase activity was about 30% of normal. The relationship of the double enzyme deficiency to this unusual clinical picture is unclear.

Biopsy↗
PubMed1995

Phosphorylation and inactivation of rat heart glycogen synthase by cAMP-dependent and cAMP-independent protein kinases.

The regulation of cardiac muscle glycogen metabolism is not well understood. Previous studies have indicated that heart glycogen synthase is heavily phosphorylated in vivo on multiple sites. Using purified enzymes, we have investigated the effect of phosphorylation of different sites on the activity of rat heart glycogen synthase. A convenient procedure was developed for the purification of rat heart glycogen synthase. The enzyme was phosphorylated by selected kinases, and glycogen synthase activity, extent of phosphorylation, and phosphopeptide maps were analyzed. Rat heart glycogen synthase, purified to apparent homogeneity (M(r) 87,000 on SDS-PAGE), had a specific activity of 18 U/mg protein and had an activity ratio of 0.74 (activity in the absence divided by the activity in the presence of glucose 6-P). cAMP-dependent protein kinase, glycogen synthase kinase 3, Ca2+/calmodulin-dependent protein kinase II, protein kinase C, and phosphorylase kinase phosphorylated the enzyme with a concomitant decrease in the activity ratio to values ranging from 0.1 to 0.4. Casein kinase II phosphorylated but did not inactivate glycogen synthase. Six tryptic phosphopeptides, obtained from heart glycogen synthase phosphorylated by the various kinases, were separated by reverse-phase chromatography. The phosphopeptide(s) obtained with each kinase eluted at the same position(s) as corresponding phosphopeptides obtained from rat skeletal muscle glycogen synthase. The study shows that the pattern of phosphorylation and effects on activity are very similar for cardiac and skeletal muscle glycogen synthase. It is suggested that the well known differences in heart and glycogen metabolism may be due to the interplay of kinases and phosphatases which could lead to different phosphorylation and activity states of glycogen synthase.

Animals↗
PubMed1983

A monoclonal antibody showing cross-reactivity toward three calmodulin-dependent enzymes.

The spleen cells of a Balb/c mouse immunized with purified bovine calmodulin-dependent cyclic nucleotide phosphodiesterase were fused with nonsecreting mouse myeloma cells (P3-X63-Ag8-653). Antibody producing hybridomas were screened by the enzyme-linked immunosorbent assay using purified phosphodiesterase as the antigen. One monoclonal cell line, CR-B1, was found to produce antibodies which showed positive enzyme-linked immunosorbent assay reactions with bovine brain calcineurin and rabbit muscle phosphorylase kinase in addition to phosphodiesterase. The antibody was purified and characterized. It was shown to immunoprecipitate the calmodulin (CaM)-dependent phosphodiesterase and phosphorylase kinase activities but not those of CaM itself, CaM-independent phosphodiesterase and the catalytic unit of cAMP-dependent protein kinase. The immunoprecipitation of phosphodiesterase could be inhibited by calcineurin and phosphorylase kinase. These results suggest that the antibody interacts at a common site on these calmodulin-dependent proteins. The antigenic determinant in phosphodiesterase does not appear to reside in the calmodulin-binding domain of the enzyme since the antibody and phosphodiesterase interaction is not inhibited by calmodulin, and the calmodulin activation of phosphodiesterase is not affected by CR-B1 antibody. It is therefore suggested that the structural similarity among the three calmodulin-dependent proteins extends beyond the calmodulin-binding domains.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1990

Scanning tunneling microscopy of the enzymes of muscle glycogenolysis.

Scanning tunneling microscopy (STM) has been used to examine the structures of the skeletal muscle enzymes phosphorylase and phosphorylase kinase. The interaction of these two proteins represents the last step in the process of signal transduction which results in muscle glycogen being converted into metabolic energy for use in muscle contraction. Phosphorylase b has a molecular weight of 97,000 and the dimer is seen by STM to have dimensions of 11 X 5.7 nm. Phosphorylase b has a tendency to form linear arrays of dimers on the graphite surface used as the support for STM imaging. Phosphorylase kinase is imaged as a butterfly-like object with lateral dimensions of 36 X 27 nm. The molecular thicknesses given by scanning tunneling microscopy for these two non-conducting molecules is significantly less than expected. The height measurement in STM is dependent not only on the surface topology of the object being imaged, but also on the electronic work function of the object compared to that of the graphite surface on which it lies. In addition to the individual proteins, a complex between phosphorylase and phosphorylase kinase has been observed by scanning tunneling microscopy.

Animals↗
PubMed1983

The protein phosphatases involved in cellular regulation. 6. Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles.

Methods were developed for quantifying protein phosphatases-1, 2A, 2B and 2C in cell extracts, and these procedures were exploited to determine their tissue and subcellular distributions. In addition, the contribution of each enzyme to the total protein phosphatase activity in skeletal muscle and liver extracts towards nine proteins involved in the control of glycogen metabolism, glycolysis/gluconeogenesis, fatty acid synthesis and cholesterol synthesis was assessed. Each protein phosphatase was present at significant concentrations in skeletal muscle, heart muscle, liver, brain and adipose tissue, although the relative amounts differed considerably. In skeletal muscle, protein phosphatase-1 was the major enzyme acting on phosphorylase, glycogen synthase and phosphorylase kinase (beta-subunit), and thus was the major protein phosphatase responsible for the inactivation of glycogenolysis and stimulation of glycogen synthesis. This idea was reinforced by the observation that 50% of the protein phosphatase-1 activity was associated with the protein-glycogen complex. In the liver, protein phosphatases-1, 2A and 2C each appear to play a role in the regulation of glycogen metabolism. Protein phosphatase-1 accounted for a significant fraction of the total potential activity towards phosphorylase and glycogen synthase, and was the major phosphorylase kinase (beta-subunit) phosphatase of this tissue. In addition, it was the only protein phosphatase present in the protein-glycogen complex. Protein phosphatase 2A was also a major phosphorylase phosphatase and glycogen synthase phosphatase in this tissue. Protein phosphatase 2C was a significant glycogen synthase phosphatase in the liver, but had negligible activity toward phosphorylase or phosphorylase kinase (beta-subunit). In the absence of Ca2+, protein phosphatase 2A was the major phosphorylase kinase (alpha-subunit) phosphatase and the only inhibitor-1 phosphatase, in skeletal muscle or liver. In the presence of Ca2+, protein phosphatase 2B accounted for most of the activity towards these substrates. Protein phosphatase 2A was the major enzyme acting on L-pyruvate kinase, ATP-citrate lyase and acetyl-CoA carboxylase in rat liver, suggesting an important role in the regulation of glycolysis/gluconeogenesis and fatty acid synthesis. Protein phosphatase 2C was the major enzyme acting on hydroxymethylglutaryl-CoA (HMG-CoA) reductase and HMG-CoA reductase kinase, suggesting an important role in the regulation of cholesterol synthesis. However, the observation that 20% of the protein phosphatase-1 in liver was associated with the microsomal fraction suggests that this enzyme may also be involved in regulating HMG-CoA reductase, which is tightly associated with microsomes. The activity of protein phosphatase-1 in dilute skeletal muscle and liver extracts was just as sensitive to inhibitor-1 and inhibitor-2 as the purified enzyme. In concentrated extracts, higher concentrations of the inhibitor proteins were required and the inhibition was time-dependent...

Animals↗