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At least 703 records · Page 39Linked to original sources
PubMed1992

Characterization of neuronal protein phosphatases in Aplysia californica.

Biochemical properties of neuronal protein phosphatases from Aplysia californica were characterized. Dephosphorylation of phosphorylase alpha by extracts of abdominal ganglia and clusters of sensory neurons from pleural ganglia was demonstrated. Type-1 protein phosphatase (PrP-1) was identified in these extracts by the dephosphorylation of the beta-subunit of phosphorylase kinase and its inhibition by the protein, inhibitor-2. Type-2A protein phosphatase (PrP-2A) was demonstrated by the dephosphorylation of the alpha-subunit of phosphorylase kinase, which was insensitive to inhibitor-2. As in vertebrate tissues, only four enzymes, PrP-1 (47%), PrP-2A (42%), PrP-2B (11%), and PrP-2C (less than 1%), accounted for all the cellular protein phosphatase activity dephosphorylating phosphorylase kinase. Aplysia PrP-1 and PrP-2A were potently inhibited by okadaic acid, with PrP-1 being approximately 20-fold more sensitive than PrP-2A. By comparison, purified PrP-2A from rabbit skeletal muscle was 15- to 20-fold more sensitive to okadaic acid than PrP-1 from the same source. Only PrP-1 was associated with the particulate fractions from Aplysia neurons, whereas PrP-1 and PrP-2A, -2B, and -2C were all present in the cytosol. Extraction of the particulate PrP-1 decreased its sensitivity to okadaic acid by sixfold, suggesting that cellular factor(s) affect its sensitivity to this inhibitor. In most respects, protein phosphatases from Aplysia neurons resemble their mammalian counterparts, and their biochemical characterization sets the stage for examining the role of these enzymes in neuronal plasticity, and in learning and memory.

Abdomen↗
PubMed1985

Quantitation and significance of 125I-calmodulin binding to myosin light chain kinase and phosphorylase distributed on polyacrylamide gels.

Glycogen phosphorylase (a or b) binds 125I-calmodulin in a Ca2+-dependent manner, in the 125I-calmodulin overlay technique. This binding is quantitatively identical to 125I-calmodulin binding to myosin light chain kinase. In an in vitro assay, calmodulin stimulates phosphorylase activity at limiting concentrations of either glucose-1-phosphate or glycogen, but the Ka is 1000 fold higher than for the kinase, and is not Ca2+-dependent. Activation of phosphorylase, but not myosin light chain kinase, by calmodulin can be mimicked by troponin C or bovine serum albumin. These results demonstrate that the properties of calmodulin interaction with proteins can vary between the 125I-calmodulin technique and a functional assay of calmodulin effect on the same protein.

Animals↗
PubMed2000

The effects of glucose and the hexosamine biosynthesis pathway on glycogen synthase kinase-3 and other protein kinases that regulate glycogen synthase activity.

BACKGROUND: Glycogen synthase (GS) activity is determined by its phosphorylation state. We have previously demonstrated that high glucose (HG) downregulates both basal and insulin-stimulated GS activity in rat-1 fibroblasts and that the hexosamine biosynthesis pathway (HBP) may be involved in mediating some of the effects of glucose. In this study we investigate the influence of high glucose and glucosamine (GlcN) on the activity of several kinases that phosphorylate and inactivate GS. METHODS: Glycogen synthase kinase (GSK) 3, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), casein kinase (CK) 1, and phosphorylase kinase (PhK) activities were assayed in cellular extracts from control rat-1 fibroblasts and those that overexpress human cDNA for glutamine:fructose 6-phosphate amidotransferase (GFA), the rate-limiting enzyme in the HBP. RESULTS: Culturing rat-1 fibroblasts in HG (20 mmol/L) or GlcN (3-5 mmol/L) for 16-20 hours increases GSK-3 activity by 23.9 and 50%, respectively, when compared to activity at low glucose (LG, 1 mmol/L). The effects of HG on GSK-3 activity are greater in cells overexpressing GFA (38.8% increase). Insulin (1.7 nmol/L) treatment leads to a 20-25% decrease in GSK-3 activity that is not affected by HG, GlcN, or GFA overexpression. Culturing control cells in HG increases PKA and CK-1 activities by 56 and 95%, respectively, and HG diminishes insulin action on CK-1 activity. GlcN inhibits insulin action on both PKA and CK-1 activities. HG, GlcN, and GFA overexpression blunted insulin's ability to downregulate PhK activity in LG conditions. PKC activity is not significantly altered in either cell line in the above conditions. CONCLUSIONS: These results suggest that HG alters both basal and insulin-regulated activity of several kinases that phosphorylate GS, and some of the effects of glucose may be mediated by its metabolism via the HBP.

Amino Acid Sequence↗
PubMed1983

Effects of the calmodulin antagonist, fluphenazine, on phosphorylation of myosin and phosphorylase in intact smooth muscle.

The activities of phosphorylase kinase and myosin light-chain kinase are regulated by Ca2+ binding to calmodulin. However, differences in the activation properties of the purified enzymes are apparent, since calmodulin binds to phosphorylase kinase in the absence of Ca2+ whereas prior formation of a Ca2+ . calmodulin complex is necessary for calmodulin to bind to and activate myosin light chain kinase. Since the phenothiazines have been implicated as anticalmodulin drugs and inhibit contractile activity in smooth muscle, we examined the effects of the phenothiazine, fluphenazine, on isometric tension development and phosphorylation of phosphorylase and the phosphorylatable light chain (P-light chain) of myosin in intact bovine tracheal smooth muscle. Preincubation with 50 microM fluphenazine for 5 min inhibited the maximal rate and extent of isometric tension development and P-light chain phosphorylation in the presence of 60 mM KCl. Application of fluphenazine after tension and the phosphate content of the P-light chain had reached steady-state levels in response to 60 mM KCl produced little relaxation or dephosphorylation of the P-light chain. KCl-mediated phosphorylase a formation was not inhibited by preincubation with fluphenazine for 5 min. However, long periods of preincubation (30-60 min) produced significant inhibition of phosphorylase a formation and proportionally greater inhibition of tension and P-light chain phosphorylation. Since phosphorylase a formation was not inhibited during short-term preincubation with fluphenazine, KCl-dependent increases in the concentration of free intracellular Ca2+ may not have been affected. Moreover, since both isometric tension development and P-light chain phosphorylation were attenuated in a parallel manner, inhibition of contractile activity in intact smooth muscle by anticalmodulin agents may be directly related to inhibition of P-light chain phosphorylation.

Animals↗
PubMed1985

Cytological localization of adenosine kinase, nucleoside phosphorylase-1, and esterase-10 genes on mouse chromosome 14.

We have determined the regional locations on mouse chromosome 14 of the genes for mouse adenosine kinase (ADK), nucleoside phosphorylase-1 (NP-1), and esterase-10 (ES-10) by analysis of rearranged mouse chromosomes in gamma-irradiated Chinese hamster X mouse hybrid cell lines. Irradiated clones were screened for expression of the murine forms of these enzymes; segregant clones that expressed only one or two of the three markers were karyotyped. The patterns of enzyme expression in these segregants were correlated with the presence of rearranged chromosomes. The Adk gene was localized to bands A2 to B, Np-1 to bands B to C1, and Es-10 to bands D2 to E2.

Adenosine Kinase↗
PubMed1990

Metabolic causes of myoglobinuria.

To evaluate the proportion of cases of myoglobinuria that can be ascribed to specific metabolic defects, we have studied eight enzymes--phosphorylase, phosphorylase kinase, phosphofructokinase (PFK), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGAM), lactate dehydrogenase (LDH), carnitine palmitoyltransferase (CPT), and myoadenylate deaminase (MAD)--in muscle biopsy specimens from 77 consecutive patients with myoglobinuria (documented in 44, suspected in 33). Enzyme defects were found in 36 patients: CPT deficiency in 17, phosphorylase deficiency in 10, phosphorylase kinase deficiency in 4, MAD deficiency in 3, PGK deficiency in 1, and a combined defect of CPT and MAD in 1. Exercise was the main precipitating factor, both in patients with and in those without detectable enzymopathies. Thirty patients had specific enzymopathies without myoglobinuria: 14 had phosphorylase deficiency, 9 had MAD deficiency, 3 had phosphorylase kinase deficiency, 3 had PFK deficiency, and 1 had PGAM deficiency. Systematic biochemical evaluation of muscle biopsy specimens revealed specific enzymopathies in about half of the patients with idiopathic myoglobinuria. The rest may have blocks of metabolic pathways not yet studied routinely, such as beta oxidation, or genetic defects of the sarcolemma, such as Becker's muscular dystrophy.

AMP Deaminase↗
PubMed1985

Characterization of Ca2+-activated protein phosphatase activity in exocrine pancreas.

Ca2+-activated protein phosphatase activity was demonstrated in mouse pancreatic acinar cytosol with alpha-casein and skeletal-muscle phosphorylase kinase as substrates. This phosphatase activity preferentially dephosphorylated the alpha subunit of phosphorylase kinase. After DEAE-cellulose chromatography, the Ca2+-activated phosphatase activity became dependent on exogenous calmodulin for maximal activity. Half-maximal activation was achieved at 0.5 +/- 0.1 microM-Ca2+. Trifluoperazine completely inhibited Ca2+-activated phosphatase activity, with half-maximal inhibition occurring at 8.5 +/- 0.6 microM. Mn2+, but not Mg2+, at 1 mM concentration could substitute for Ca2+ in eliciting full enzyme activation. The apparent Mr of the phosphatase as determined by Sephadex G-150 chromatography was 93000 +/- 1000. Submitting active fractions obtained after Sephadex chromatography to calmodulin affinity chromatography resulted in the resolution of a major protein of Mr 55500 +/- 300. In conclusion, Ca2+-activated protein phosphatase activity has been identified in exocrine pancreas and has several features in common with Ca2+-activated calmodulin-dependent protein phosphatases previously isolated from brain and skeletal muscle. It is possible that this Ca2+-activated phosphatase may utilize as substrates certain acinar-cell phosphoproteins previously shown to undergo dephosphorylation in response to Ca2+-mediated secretagogues.

Animals↗
PubMed1989

Transient changes in cyclic AMP and in the enzymic activity of protein kinase and phosphorylase during the cardiac cycle in the canine myocardium and the effect of propranolol.

Oscillation of cyclic AMP and in the activity ratio of cyclic AMP-dependent protein kinase and of glycogen phosphorylase with the cardiac cycle were demonstrated in the canine heart in situ. For tissue sampling an ECG (R-wave)-triggered, automatically working push-freeze-drill apparatus was developed which allows intraventricular cryobiopsies from the left ventricular muscle of anaesthetized open-chest dogs. The nucleotide cyclic AMP oscillated with the cardiac cycle during normal working condition, the higher cyclic AMP level occuring during systole. Cyclic GMP was assayed to be without oscillatory changes during the contraction-relaxation cycle. 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 released from the sympathetic nerve endings within the heart. Contrary the activity ratio of phosphorylase retains its transient changes during the cardiac cycle in the presence of propranolol, indicating a Ca-mediated activation of phosphorlase kinase during the contraction process.

1-Propanol↗
PubMed1990

Mechanisms underlying enhanced glycogenolysis in livers of 3,5,3'-triiodothyronine-treated rats.

Rats trained on a diurnal controlled meal-feeding schedule and injected with a single dose of 3,5,3'-triiodothyronine (T3) failed to accumulate liver glycogen and incorporated less D-[6-3H]glucose into glycogen than normally observed during the feeding period. In the experimental group, the concentration of liver adenosine 3',5'-cyclic monophosphate (cAMP) did not fall during feeding and the pattern of activities of glycogen phosphorylase, glycogen synthase, and phosphorylase kinase remained conductive to glycogenolysis. Liver lysosomal alpha-glucosidase activity normally fell during feeding periods. After T3 treatment the activities of alpha-glucosidase and two lysosomal cathepsins (B1 and D) were elevated. The evidence suggests that T3 may induce both liver phosphorylase kinase and lysosomal alpha-glucosidase. This outcome of T3 excess, in concert with previously described T3-inducible systems, provides a plausible explanation for the failure of glycogen accumulation in this experimental model.

Adenine Nucleotides↗
PubMed1992

Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets.

The catalytic subunits of bovine platelet protein phosphatases were separated into three distinct forms by chromatography on heparin-Sepharose. Each phosphatase was further purified to apparent homogeneity as judged in sodium dodecyl sulfate-polyacrylamide gel yielding single protein bands of 37, 41, and 36 kDa. The 37-kDa phosphatase was excluded from heparin-Sepharose and preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. It was stimulated by polycations (polybrene or histone H1) and was inhibited by okadaic acid (IC50 = 0.3 nM), but its activity was not influenced by inhibitor-2 or heparin. The 41-kDa phosphatase was eluted from heparin-Sepharose by 0.20-0.25 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was stimulated by polycations and inhibited by okadaic acid (IC50 = 2 nM), but its activity was not affected by inhibitor-2 or heparin. The 36-kDa phosphatase was eluted from heparin-Sepharose by 0.45-0.50 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was inhibited by inhibitor-2, heparin, histone H1, and okadaic acid (IC50 = 70 nM). The 37- and 36-kDa phosphatases can be classified as type-2A and type-1 enzymes, respectively. The 41-kDa phosphatase does not precisely fit the criteria of either type, showing only partial similarities to both type-1 and type-2A enzymes and it may represent a novel type of protein phosphatase in bovine platelets.

Animals↗