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PubMed1990

The effect of various aminoglycoside antibiotics on glycogen phosphorylase activity in liver and kidney medulla of rabbit.

Glycogen phosphorylase activity in both liver and kidney medulla of rabbit was stimulated in the presence of caffeine by various aminoglycoside antibiotics in the following rank order: gentamicin greater than neomycin greater than amikacin = kanamycin greater than or equal tobramycin, while streptomycin did not affect the enzyme activity. In contrast, in the presence of AMP, the stimulatory action of antibiotics was not observed. Since in the gentamicin-treated rabbits stimulation of glycogen phosphorylase activity by about 30% in both liver and kidney medulla was accompanied by a decrease of liver glycogen content by about 60% it is likely that a decline in liver glycogen level following antibiotic treatment is due to an increased glycogen phosphorylase activity.

Animals↗
PubMed1990

The effect of ischemia on glycogen phosphorylase activity in rat liver: a quantitative histochemical study.

The effects of ischemia in vitro for 0-60 min at 37 degrees C on glycogen phosphorylase activity in rat liver have been studied under different feeding conditions. Glycogen phosphorylase activity was demonstrated with a recently developed quantitative histochemical method using a semipermeable membrane and the PAS-reaction. The cytophotometrically measured glycogen phosphorylase activity in livers from 24 h-fasted rats was approximately five times the activity in livers from normally fed rats. The activity in periportal areas was about 1.5 times higher than the activity in pericentral areas in livers from starved rats, but more or less evenly distributed in livers from fed rats. Enzyme activity in pericentral areas of livers from 24 h-fasted rats started to decrease after 20 min of ischemia. After 50-60 min of ischemia, the activity was decreased to approximately 25% of the control activity. Livers from normally fed rats showed unchanged activity in periportal and pericentral areas after 10-60 min of ischemia. It has been assumed that the activation of the enzyme was disturbed by ischemia, possibly as a consequence of plasma membrane damage.

Animals↗
PubMed1990

[Base activity of glycogen phosphorylase b in human serum. Study of reference and discrimination values].

Serum glycogen phosphorylase activity as one of the sensitive indicators for the diagnosis of acute heart infarction was determined at 216 healthy probands using a standardized fluorometric procedure with a detection range from 0.03 to 6 nmol/s x l serum. Basal activity was averaged at 1.22 +/- 0.39 nmol/s x l serum with a confidence interval of C.I.95 1.168 ... 1.272. Due to the estimated Gaussian distribution of the individual activity values (n = 216) the reference range was calculated from 0.5 to 2.0 nkat/l (2.5- to 97.5-percentile, respectively), the latter one being equal to the reference value of the glycogen phosphorylase activity in healthy human beings. The reference value was found to be independent of age (16-65 years) and sex. Using the discrimination value for serum glycogen phosphorylase activity at 3 nkat/l serum two groups of patients suffering either from acute myocardial infarction (n = 79) or chronic ischemic heart disease (n = 92) were separated with a diagnostic sensitivity and specificity of 0.91 and 0.93, respectively, of this diagnostic tool.

Adolescent↗
PubMed1975

Purification and properties of rabbit liver phosphorylase phosphatase.

A procedure for the purification of rabbit liver phosphorylase phosphatase is described. The specific activity of the preparation is 2,100 units/mg of protein, representing a 25,000-fold purification. During the initial steps of the purification a large activation of enzyme activity was observed. The molecular weight of the purified enzyme was estimated by Sephadex G-75 chromatography to be 35,000, and by sucrose density ultracentrifugation to be 34,000 (2.9 S). On Na dodecyl-SO4 polyacrylamide disc gel electrophoresis a single component with a molecular weight of 34,000 was observed. The pH optimum is 6.9 to 7.4, and the Km for rabbit muscle phosphorylase alpha is 2 muM. The same procedure is also applicable to the extensive purification of phosphorylase phosphatase from rabbit muscle.

Animals↗
PubMed1985

[Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle].

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.

Animals↗
PubMed1989

The gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin.

Phosphorylase kinase is a Ca2+-regulated, multisubunit enzyme that contains calmodulin as an integral subunit (termed the delta-subunit). Ca2+-dependent activity of the enzyme is thought to be regulated by direct interaction of the delta-subunit with the catalytic subunit (the gamma-subunit) in the holoenzyme complex. In order to systematically search for putative calmodulin (delta-subunit)-binding domain(s) in the gamma-subunit of phosphorylase kinase, a series of 18 overlapping peptides corresponding to the C terminus of the gamma-subunit was chemically synthesized using a tea bag method. The calmodulin-binding activity of each peptide was tested for its ability to inhibit Ca2+/calmodulin-dependent activation of myosin light chain kinase. Data were obtained indicating that two distinct regions in the gamma-subunit, one spanning residues 287-331 (termed domain-N) and the other residues 332-371 (domain-C), are capable of binding calmodulin with nanomolar affinity. Peptides from both of these two domains also inhibited calmodulin-dependent reactivation of denatured gamma-subunit. The interactions of peptides from both domain-N and domain-C with calmodulin were found to be Ca2+-dependent. Dixon plots obtained using mixtures of peptides from domain-N and domain-C indicate that these two domains can bind simultaneously to a single molecule of calmodulin. Multiple contacts between the gamma-subunit and calmodulin (delta-subunit), as indicated by our data, may help to explain why strongly denaturing conditions are required to dissociate these two subunits, whereas complexes of calmodulin with most other target enzymes can be readily dissociated by merely lowering Ca2+ to submicromolar concentrations. Comparison of the sequences of the two calmodulin-binding domains in the gamma-subunit of phosphorylase kinase with corresponding regions in troponin I indicates similarities that may have functional and evolutionary significance.

Amino Acid Sequence↗
PubMed1978

Dextrothyroxine treatment of phosphorylase-kinase deficiency glycogenosis in four boys.

Four boys, aged 2 years 5 months to 3 years 7 months, with large hepatomegaly due to phosphorylase-kinase deficiency glycogenosis, were given a trial of sodium dextrothyroxine (D-T4) at a mean dose of 0.165 mg/kg/day for an average period of 6 months. Phosphorylase-kinase was undetectable in the haemolysates of erythrocytes (3 patients) or in the liver (one patient) before, and still undetectable in the haemolysates of the four patients during treatment, thus pointing to X-linked phosphorylase-kinase deficiency glycogen storage disease (GSD IXb). D-T4 administration resulted in complete normalization of liver size, decrease of serum GOT (p less than 0.02), GPT (p less than 0.05) and triglycerides (p less than 0.01) to normal values, as well as correction of mild asymptomatic hypoglycemia (p less than 0.01). As long as the outcome of type IXb glycogenosis in adult life remains undefined, dextrothyroxine therapy seems an effective means of reducing liver size and correcting part of the biochemical abnormalities of the disease.

Alanine Transaminase↗
PubMed1987

Transient activation of cyclic AMP-dependent protein kinase and phosphorylase during the cardiac cycle in the canine myocardium in situ and the effect of propranolol.

The activity ratio of cyclic AMP-dependent protein kinase and glycogen phosphorylase were determined at various stages of the contraction cycle in the canine heart in situ. Both activity ratios vary transiently throughout the contraction-relaxation cycle, the higher values occurring during systole. The rise in the activity ratio of protein kinase was found to coincide with the maximum in the level of cyclic AMP. Propranolol pretreatment prevents the transient in the level of the nucleotide as well as in the activity ratio of the kinase indicating i) a causal relationship between these changes and ii) a neurohumoral, beat-to-beat regulation by catecholamines. Contrary the activity ratio of phosphorylase retains, in the presence of propranolol, its transient changes during the cardiac cycle, probably caused by a Ca2+-mediated activation of phosphorylase kinase during the contraction process.

Animals↗
PubMed1988

Dose dependent reversal by i.v. glucose administration of the stimulation of rat liver glycogen phosphorylase by epinephrine infusion.

Infusion of 0.5 ml kg-1 min-1 20% glucose i.v. failed to suppress the stimulatory effect of 0.25 microgram kg-1 min-1 epinephrine dissolved in and infused together with the above glucose solution on the activity of liver glycogen phosphorylase in adult male rats. However, 40% glucose solution administered in the same way abolished the effect of the epinephrine infusion completely. An i.v. pulse of 1 g kg-1 glucose in the form of a 40% solution immediately before the infusion of the same dose of epinephrine in saline had only a temporary inhibitory effect on epinephrine induced increase of phosphorylase activity. Finally, i.p. administration of 10 mg kg-1 phentolamine 60 min before epinephrine infusion in saline completely blocked the increasing effect of the latter on both liver phosphorylase and serum glucose, while that of 4 mg propranolol 30 min before the hormone failed to exert any influence. This reconfirmed the well known mediation of the glycogenolytic effect of catecholamines in adult male rats by alpha type adrenergic receptors.

Animals↗
PubMed1985

Phosphorylase phosphatase regulatory subunit. "Western" blotting with immunoglobulins against inhibitor-2 reveals a protein of Mr = 60,000.

Rabbit muscle phosphorylase phosphatase has been isolated in different laboratories as an inactive complex of Mr = 70,000, composed of separate catalytic (Mr = 38,000) and regulatory (Mr = 31,000) proteins. The regulatory protein is identical to one of two heatstable inhibitors called inhibitor-2 (I2). Antiserum raised in sheep against I2 by repeated immunization potently blocked inhibitory activity, whereas preimmune serum did not. Immunoglobulins which blocked inhibitory activity were purified by affinity chromatography with I2 as the immobilized ligand. Using a "Western" immunoblotting procedure, as little as 1-5 ng of pure I2, obtained by electroelution of the Mr = 31,000 band of I2 from a polyacrylamide gel segment, were detected. Immunoblotting of the immunogen revealed only a band at Mr = 31,000, indicating the absence of contaminating antigenic proteins. When extracts of skeletal muscle and other rabbit tissues were denatured directly in dodecyl sulfate for immunoblotting the most intensely stained band was present at Mr = 60,000, rather than at Mr = 31,000 as expected. A small amount of I2 and other bands were detected, in particular at Mr = 36,000 and 25,000. Subsequent to heat treatment of the tissue extracts, there was an enrichment of I2 content relative to the Mr = 60,000 band. The results indicate the existence of a Mr = 60,000 protein related to I2. Activation of phosphorylase phosphatase in a muscle extract by treatment with Co2+ plus trypsin exactly coincided with digestion of the Mr = 60,000 immunoreactive protein. Available data indicate that this protein may function as a regulatory subunit of phosphorylase phosphatase.

Animals↗
PubMed1987

The interrelationship between cAMP-dependent alpha and beta subunit phosphorylation in the regulation of phosphorylase kinase activity. Studies using subunit specific phosphatases.

This study addresses the function of multisite phosphorylation of phosphorylase kinase catalyzed by the cAMP-dependent protein kinase. Using subunit specific protein phosphatases (the polycation-stimulated and ATP-, Mg2+-dependent enzymes), we show that the degree of phosphorylation of both the alpha and beta subunits modulates phosphorylase kinase activity. beta subunit phosphorylation is essential for activation and, independent of the degree of alpha subunit phosphorylation, enzyme fully dephosphorylated in the beta subunit is completely inactivated. alpha Subunit phosphorylation does, however, also regulate activity, and enzyme fully or partially phosphorylated in the beta subunit is inactivated as a consequence of alpha subunit dephosphorylation. The extent of inactivation caused by alpha subunit dephosphorylation is linearly dependent on the phosphorylation state of the beta subunit. Three peptide sites on the alpha subunit are phosphorylated by the cAMP-dependent protein kinase; the site primarily affecting activity is the one that is initially phosphorylated. These data provide evidence that subunit interrelationships play an important role in the regulation of phosphorylase kinase by multisite phosphorylation.

Adenosine Triphosphate↗
PubMed1987

Subunit phosphorylation and activation of phosphorylase kinase in perfused rat hearts.

The potential correlations between phosphorylase kinase subunit phosphorylation and activation have been examined using 32P-perfused rat hearts exposed to a variety of hormonal stimuli. Phosphate incorporation was measured after isolation of the enzyme by immunoprecipitation from heart extracts. Time courses of catecholamine or glucagon treatment produced a rapid rise in both the activity and the beta subunit phosphorylation of the enzyme, and a slightly slower increase in alpha' subunit phosphorylation. For short durations of catecholamine stimulation, the ratio of phosphate in the alpha' versus beta subunit was dependent upon hormone dose. After removal of hormone, both inactivation and alpha' subunit dephosphorylation were fairly slow, while the beta subunit was dephosphorylated more rapidly. For all of the above conditions, activation correlated with both alpha' and beta subunit phosphorylation. The maximum level of phosphate incorporation observed in response to hormonal stimulation is estimated to be approximately 1.3-1.7 mol of [32P]phosphate/mol of (alpha' beta gamma delta)4, divided about equally between the alpha' and beta subunits. When hearts were treated with hormone either in the absence of added calcium or in the presence of a calcium channel blocker, the time courses of subunit phosphorylation and activation were similar to those seen with standard perfusion conditions, suggesting that if any Ca2+-dependent autophosphorylation of phosphorylase kinase were occurring it does not make a major contribution to the observed hormonal responses. The complicated relationships observed here between phosphorylase kinase subunit phosphorylation and activation for the most part provide physiological affirmation of the patterns observed in vitro, but they also show some possible differences of potential interest.

Animals↗
PubMedDate not supplied

[Phosphorylase and gluco-6-phosphatase activity and glycogen level in tissues of vertebrates].

The activity of phosphorylase (EC 2.4.1.1), glucose-6-phosphatase (EC 3.1.3.9) and the content of glycogen have been determined in tissues of fish, amphibians, reptiles, mammalians. No differences in the activity of phosphorylase and glucose-6-phosphatase in the liver, myocardium, and brain of animals of the phylogenetic groups under study are found. The activity of glucose-6-phosphatase in the anaerobic muscles of poikilothermal animals is found to be rather high. The share of phosphorylase a in the skeletal muscles and brain as well as the glycogen content in the brain of these animals is essentially higher than that of adult mammalians.

Amphibians↗
PubMed1987

Autophosphorylation of the alpha subunit of phosphorylase kinase from rabbit skeletal muscle.

The autophosphorylation of the alpha subunit of phosphorylase kinase occurs simultaneously at multiple sites during incorporation of the first mol of phosphate. The predominant and initial autophosphorylation site on this subunit is different than the major site phosphorylated by cAMP-dependent protein kinase, which also phosphorylates multiple sites, as evidenced by two-dimensional phosphopeptide maps. All of the sites on the alpha subunit phosphorylated by cAMP-dependent protein kinase comigrate on peptide maps with autophosphorylation phosphopeptides; however, several phosphopeptides observed after autophosphorylation are not evident following phosphorylation by cAMP-dependent protein kinase. The phosphopeptide maps of the alpha subunit are the same whether autophosphorylation is carried out at pH 6.8 or 8.2 or whether MnATP is used instead of MgATP; there is only a slight difference in the maps brought about by EGTA-insensitive autophosphorylation. The autophosphorylation is shown to be an intrinsic activity of the phosphorylase kinase molecule; this conclusion is based on the observed copurification of the autophosphorylation activity with activities toward phosphorylase b and kappa-casein and the unaltered influence of various effectors on these activities throughout different sequential adsorption chromatography purification steps. Additional support to that already in the literature that the initial autophosphorylation events are predominantly intramolecular is gained by showing that previously autophosphorylated enzyme has little ability to catalyze the phosphorylation of nonphosphorylated enzyme.

Adenosine Triphosphate↗
PubMed1988

Changes of glycogen phosphorylase isozyme pattern, in rat tissues during pre- and postnatal development.

Quantitative estimates of M- and L-type phosphorylase subunits in adult and developing rat tissues have been obtained by immunotitration of tissue extracts with subunit-specific antibodies. It has been shown that M-type subunits occur in all studied tissues with exception of adult liver and placenta. L-type subunits account for most of the total phosphorylase activity in liver and bone marrow but are present also in a variety of other adult and in most of the fetal tissues examined. During maturation, selective increases in the expression of phosphorylase isozymes occur in certain tissues such as liver, brain, heart and skeletal muscle. The hitherto unknown hybrid LM has been produced by in vitro hybridization of M- and L-subunits.

Aging↗
PubMed1987

Glycogen phosphorylase isozyme pattern in rat liver and isolated non-parenchymal liver cells.

The isozyme distribution of glycogen phosphorylase (EC 2.4.1.1) was studied in adult rat liver and isolated non-parenchymal liver cells by means of immuno-titration. In adult rat liver, the L-type of glycogen phosphorylase was found to dominate, whereas the heterogeneous non-parenchymal liver cell population apparently contains all three glycogen phosphorylase isozymes.

Animals↗
PubMed1987

[Limited proteolysis and phosphorylation of phosphorylase kinase from chicken skeletal muscles].

The changes in the quaternary structure of chicken skeletal muscle phosphorylase kinase during limited proteolysis by trypsin and chymotrypsin were studied. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the products of phosphorylase kinase limited proteolysis revealed a similarity in the structure of the alpha'- and beta-subunits and some differences in the structure of the gamma-subunits of the chicken and rabbit enzymes. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol of 32P/mol of alpha' beta gamma' sigma monomer) and autophosphorylation (up to 8 mol of 32P/mol alpha' beta gamma' delta monomer) increased the activity of chicken phosphorylase kinase 1.5-fold and 2.0-fold, respectively. The incorporation of phosphate into the alpha' and beta-subunits in the course of the protein kinase-catalyzed reaction was demonstrated.

Animals↗
PubMed1987

Localization of phosphorylase kinase subunits at the sarcoplasmic reticulum of rabbit skeletal muscle by monoclonal and polyclonal antibodies.

Molecular structures related to phosphorylase kinase have been localized by light and electron microscopy in tissue sections of rabbit skeletal muscle employing polyclonal antibodies directed against the holoenzyme as well as monoclonal antibodies specific for its alpha-, beta- or gamma-subunits. In frozen sections of prefixed muscle fibres both known major regions of glycogen deposition, the intermyofibrillar space and the perinuclear area, are stained predominantly. In sections of unfixed muscle in which cytosolic phosphorylase kinase was removed by extensive washes prior to immunostaining the immunolabel is mainly associated with the sarcoplasmic reticulum (SR). This membrane location is further confirmed by immunoblot analysis of proteins solubilized from isolated SR with Triton X-114. Employing monoclonal antibodies two membrane proteins are identified as the alpha- and beta-subunits of phosphorylase kinase by Western blots. Immunoprecipitates reveal also the gamma-subunit; the delta-subunit, i.e., calmodulin, is enriched with the solubilized enzyme. It proves that a SR membrane associated form of holophosphorylase kinase exists in muscle. Functionally, this kinase might be involved in phosphorylation of phosphatidylinositol present on the SR Ca2+ transport ATPase and thereby might play a role in regulation of Ca2+ transport.

Animals↗