Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Histone kinase activity was purified from human polymorphonuclear leukocytes by ammonium sulphate precipitation of a 180 000 x g supernatant, followed by DEAE-cellulose chromatography and gelfiltration. On DEAE-cellulose cAMP dependent kinase activity eluted in two peaks, I and III, at 1.2 mmho and 6.5 mmho, respectively. Catalytic subunit (C) from both peaks had Mr 33 000, 3.0S. Regulatory subunit (R) from peak I and III both had Mr 33 000 upon gelfiltration, but sedimented at 2.8--3.0S and 3.0--3.2S, respectively. R2 and R4 subunits were identified. The R-C dimer from peak I and III sedimented at 4.8S and (4.8)--5.1S, respectively. The holoenzyme from peak I had Mr 165 000, 6.7S, which suggest a R2C2 structure, while that of peak III sedimented at 6.7S, but eluted at Mr 330 000 (2R2C2) by gelfiltration. The Kmapp for peak I and III enzymes were, respectively: histone IIA 0.5 mg/ml (both forms), ATP 18 microM and 23 microM, and cAMP 5 X 10(-8) M and 6.3 x 10(-8) M. Both enzymes had pH optimum 6.7--6.9 and were equally sensitive to Ca2+, temperature and protein kinase inhibitor. The substrate specificity was histone VS greater than histone IIA = histone VIS greater than casein greater than phosvitin. Peak I enzyme, but not peak III enzyme, was dissociated by histone and high ionic strength and reassociation of R and C subunits were facilitated by ATP-Mg. It is concluded that peak I and III enzymes represent type I and II cAMP dependent protein kinases, respectively. Type I comprises 20--30% of cAMP dependent protein kinase activity and is absent from the 180 000 x g supernatant of gently disrupted cells. Purified catalytic subunit had Kmapp (ATP) 20 microM with rabbit muscle glycogen synthease I as substrates. Synthase I from rabbit muscle and human leukocytes were phosphorylated by catalytic subunit to synthase D (ratio of independence less than 0.07). cAMP independent histone kinase activity eluted in one peak (Peak II) at3 mmho. The enzymatic activity sedimented at 3.4S and eluted from gelfiltration with Mr 78 000. Kmapp for ATP was 78 microM and for histone IIA 0.5 mg/ml. The enzyme was sensitive to temperature, but less sensitive than cAMP dependent protein kinase to Ca2+, and insensitive to protein kinase inhibitor. The substrate specificity was histone IIA greater than histone VS = histone VIS, while casein and phosvitin were poor substrates. Glycogen synthase I was not phosphorylated. The cAMP independent histone kinase activity comprised 15% of the total histone kinase activity in a crude homogenate of leukocytes. Its physiological substrate is unknown.
Mol Cell Biochem 1979 Jul 15
PMID:Purification and properies of cAMP dependent and independent histone kinases from human leukocytes. 22 66

Glycogen synthase kinase-3 (GSK-3) was purified from rabbit liver to homogeneity by ultracentrifugation, ion-exchange chromatography on DEAE-cellulose, Cellulose phosphate, CM-Sephadex and Fast Protein Liquid Chromatography (FPLC) on Mono-S column. The enzyme was purified approximately 20,000 fold with an approximate 2% recovery. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. GSK-3 is a monomeric enzyme with a molecular weight of 50,000-52,000 as derived from SDS-polyacrylamide gel electrophoresis and gel filtration. The purified enzyme was indeed a GSK-3 since it phosphorylated three sites, i.e., 3a, 3b, and 3c on liver glycogen synthase. GSK-3 incorporated up to 2.6 mol Pi/mol glycogen synthase subunit with a concomitant inactivation of glycogen synthase activity.
Mol Cell Biochem 1990 Jun 25
PMID:Purification and partial characterization of glycogen synthase kinase-3 from rabbit liver. 216 42

Knowledge of the metabolic changes that occur in insulin-resistant type 2 diabetes is relatively lacking compared to insulin-deficient type 1 diabetes. This paper summarizes the importance of the C57BL/KsJ-db/db mouse as a model of type 2 diabetes, and illustrates the effects that insulin-deficient and insulin-resistant states have on hepatic glycogen metabolism. A longitudinal study of db/db mice of ages 2-15 weeks revealed that significant changes in certain parameters of hepatic glycogen metabolism occur during this period. The liver glycogen levels were similar between diabetic and control mice. However, glycogen particles from db/db mice were on average smaller in mass and had shorter exterior and interior chain lengths. Total phosphorylase and phosphorylase a activities were elevated in the genetically diabetic mice. This was primarily due to an increase in the amount of enzymic protein apparently the result of a decreased rate of degradation. It was not possible to find a consistent alteration in glycogen activity in the db/db mice. Glycogen synthase and phosphorylase from diabetic liver revealed some changes in kinetic properties in the form of a decrease in Vmax and altered sensitivity to inhibitors like ATP. The altered glycogen structure in db/db mice may have contributed to changes in the activities and properties of glycogen synthase and phosphorylase. The exact role played by hormones (insulin and glucagon) in these changes is not clear but further studies should reveal their contributions. The db/db mouse provides a good model for type 2 diabetes and for fluctuating insulin and glucagon ratios.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1990 Feb 09
PMID:Hepatic glycogen metabolism in the db/db mouse. 240 41

Glycogen synthase I from human polymorphonuclear leukocytes was phosphorylated with cAMP dependent protein kinase, synthase kinase or phosvitin kinase prepared from these cells. Limited tryptic hydrolysis released four phosphopeptides (t-A, t-B, t-C, t-D). Subsequent alpha-chymotryptic hydrolysis of the trypsin resistant core released three phosphopeptides (c-A, c-B, c-C). The kinetic changes of glycogen synthase were compared with the phosphorylation of the peptides. Equivalent kinetic changes (Kc equals 0.2-0.3 mM Glc-6-P) were obtained when 1 Pi/subunit was introduced by cAMP dependent protein kinase, 0.5 Pi/subunit by synthase kinase and 0.8 Pi/subunit by both kinases. Initially, cAMP dependent protein kinase phosphorylated peptides c-A and t-C in parallel and somewhat later also t-B, whereas synthase kinase initially phosphorylated only c-A. The ultimate effect of the two kinases on c-A was additive. It was concluded that the initial kinetic changes were dependent on phosphorylation of c-A, which contained two sites, one for each kinase. The same kinetic changes were induced by phosphorylation on each of the sites. In the subsequent phosphorylation the kinases, separately or together, phosphorylated peptide c-C indicating one non-specific phosphorylatable site in this peptide. The cAMP dependent protein kinase alone phosphorylated t-C maximally, whereas both kinases were required for an equal phosphorylation of t-A and t-B. It is suggested that the cAMP dependent protein kinase phosphorylated t-A and t-C, whereas the data did not allow a similar suggestion for t-B. The kinetic changes occurring during the later stages of phosphorylation were an increase in Kc for Glc. 6-P to 4-5 mM at 1.85 Pi/subunit and to 20 mM at 3.3 Pi/subunit, but the changes could not be assigned to phosphorylation of any specific peptide. Phosphorylation of the peptides t-D and c-B were insignificant, but c-B may be phosphorylated under other experimental conditions (25). The phosvitin kinase phosphorylated glycogen synthase extremely slowly to an extent of 0.8 Pi/subunit, mainly in peptide c-C. Glycogen synthase would appear without physiological importance as substrate for this kinase. Phosphorylase kinase from rabbit skeletal muscle incorporated 0.7 Pi/subunit, mainly in peptide c-A causing a decrease in RI to 0.3, which upon further incubation remained constant. The rate of decrease in RI in 0.5 was unaffected by several synthase modifiers, including Glc-6-P, but was inhibited by ADP and Pi. The rate of phosphorylation by cAMP dependent protein kinase and synthase kinase was diversely affected in different buffers, however, without affecting the ultimate phosphorylation pattern.
Mol Cell Biochem 1981 Mar 13
PMID:Phosphorylation of glycogen synthase I from human polymorphonuclear leukocytes. 626 29

Lithium ion, like insulin, activated adipocyte glycogen synthase with or without glucose in the medium. However, the effect of lithium ion was much greater than that of insulin under both conditions. The lithium-activated glycogen synthase was stable to both Sephadex chromatography and ethanol precipitation of the enzyme, indicating that the effect of lithium ion on glycogen synthase was through covalent modification of the enzyme. Glycogen synthase was significantly activated by lithium ion under conditions where concentrations of cellular ATP were unaffected. The effect of lithium ion on glycogen synthase was rapid and observed at concentrations as low as 1 to 3 mM, reaching a maximum at the concentration of 40 mM. It was thus the most sensitive of all the effects studied (see previous paper). Insulin further stimulated glycogen synthase at low concentrations but not at maximal concentration of lithium ion. Lithium-activated glycogen synthase was inhibited by both epinephrine and dibutyryl cyclic AMP, but was not affected by the removal of extracellular Ca++. Interestingly, lithium ion had no detectable effect on basal pyruvate dehydrogenase as well as on epinephrine-stimulated phosphorylase. The failure of lithium ion to thus mimic insulin actions on pyruvate dehydrogenase and on phosphorylase suggests that the action of lithium ion on glycogen synthase is quite specific and may be mediated by stimulating a phosphatase or by inhibiting a protein kinase acting specifically on glycogen synthase.
Mol Cell Biochem 1983
PMID:'Insulin-like' effects of lithium ion on isolated rat adipocytes. II. Specific activation of glycogen synthase. 641 71

Glycogen synthase I in a homogenate of human polymorphonuclear leukocytes was phosphorylated under imitated physiological conditions utilizing the endogenous protein kinases. At subsequent steps of phosphorylation the 32P-labelled synthase was purified and characterized. Limited tryptic hydrolysis of the 32P-labelled synthase released four phosphopeptides (t-A, t-B, t-C, t-D) and subsequent chymotrypsinization of the trypsin resistant core released three phosphopeptides (c-A, c-B, c-C). One Pi/subunit was incorporated within 8-10 min and 2.2 Pi/subunit within 60 min increasing the Kc for Glc-6-P to 4-6 mM. The initial phosphorylation up to 0.8 Pi/subunit occurred mainly in peptide c-A and a linear relation between ratio of independence (RI) of glycogen synthase in the interval RI 0.85 to RI 0.05 and phosphorylation of this peptide of 0.5 Pi was observed. Phosphorylation of this peptide is responsible for the decrease in ratio of independence. From experiments with inhibitors and activators, the initial phosphorylation was found predominantly catalysed by the endogenous cAMP independent synthase kinase, however, the endogenous cAMP dependent protein kinase and phosphorylase kinase also phosphorylate endogenous glycogen synthase I to a minor degree. Circumstantial evidence for a Ca-dependent synthase kinase different from phosphorylase kinase is presented. The endogenous Glc-6-P dependent glycogen synthase occurring in a homogenate of leukocytes disrupted in the presence of NaF incorporated 1.07 Pi/subunit and Kc for Glc-6 was increased from 6-8 mM to 20 mM. From the present and previous experiments [7] a total of 8 major phosphorylatable sites have been defined, one on each of the peptides t-A, t-B, c-B, c-C and two on peptide c-A, which in addition may contain a third site for phosphorylase kinase. Assuming identical subunits, only 13 out of 32 sites are thus covalently modified at maximum phosphorylation. The operational defined synthase R (Kc for Glc-6-P 0.5 mM) and D (Kc for Glc-6-P 2-8 mM) activities correspond to synthase with about 0.8 Pi and 1.8-2.3 Pi/subunit, respectively.
Mol Cell Biochem 1981 Mar 13
PMID:Phosphorylation of glycogen synthase in a homogenate of human polymorphonuclear leukocytes. 678 73

Regulation of the dephosphorylation of glycogen synthase in extracts from rat heart has been studied by adding exogenous phosphatase to the extract. These experiments were possible only because the endogenous protein phosphatase activity of the extract could be inhibited by KF under conditions where alkaline phosphatase activity was not. The concentration of substrate (glycogen synthase from the heart extract) and catalyst (purified E. coli alkaline phosphatase) could be varied independently, by adding known amounts of alkaline phosphatase to the KF-containing heart extracts. Alkaline phosphatase could completely dephosphorylate glycogen synthase while phosphorylase was unchanged. The rate of dephosphorylation was proportional to both the concentration of alkaline phosphatase added to the tissue extract and the amount of glycogen synthase in the extract. The Km for glycogen synthase was close to the concentration found in heart tissue. The Km and the maximum rate of dephosphorylation were both dependent on the phosphorylation state of the glycogen synthase. Less phosphorylated enzyme forms were dephosphorylated faster. These results indicate the necessity for precise control of many variables in studying the rate of glycogen synthase dephosphorylation. Alkaline phosphatase-catalyzed dephosphorylation could be inhibited by physiological concentrations of glycogen. Glycogen synthase dephosphorylation in extracts from fasted-refed rats was less sensitive to glycogen inhibition than in extracts from normal animals. The phosphorylation state of the glycogen synthase in these animals was assessed by kinetic studies to show that differences in phosphorylation state probably could not account for the observations. Fasting led to a decreased rate of dephosphorylation of glycogen synthase due to both an apparent change in kinetic properties of glycogen synthase as a substrate for alkaline phosphatase, and an increased inhibitory effect of glycogen. Stable modifications of glycogen synthase caused by altered nutritional states in the animals are thought to produce these effects.
Mol Cell Biochem 1982 May 14
PMID:Dephosphorylation of glycogen synthase in rat heart extracts by E. coli alkaline phosphatase. Use of an exogenous phosphatase to study substrate-mediated regulation of dephosphorylation. 681 91

Glycogen synthase in skeletal muscle of 3-day alloxan-diabetic rats was found to be in a less active state than in normal muscle. Both the activity ratio (activity without G6P divided by activity with 7.2 mM G6P at 4.4 mM UDPG, pH 7.8) and fractional velocity (activity with 0.25 mM G6P divided by activity with 10 mM G6P at 0.03 mM UDPG, pH 6.9) were significantly lower in the diabetic tissue. Correspondingly, the S0.5 for UDPG and A0.5 for G6P were significantly higher in diabetic tissue, suggesting decreased affinity for substrate and activator, respectively. The kinetic changes in the diabetic synthase were identical whether the alloxan-treated animals were maintained on insulin for 7 days prior to withdrawal for 3 days, or studied 3 days immediately after alloxan treatment. The diabetes-induced changes in synthase could be reversed by injecting the diabetic rat with insulin 10 min prior to sacrifice. After insulin treatment, the S0.5 for UDPG and A0.5 for G6P decreased to control levels or lower and the activity ratios and fractional velocities increased to control levels or higher. The activity of glycogen synthase phosphatase was not decreased in diabetic skeletal muscle. This observation, coupled with the rapid response of the diabetic synthase to in vivo insulin treatment, suggests that, unlike the phosphatase in cardiac muscle and liver, the glycogen synthase phosphatase in skeletal muscle is not altered by the diabetic state.
Mol Cell Biochem 1982 Oct 29
PMID:Glycogen synthase in diabetic rat skeletal muscle: activation by insulin. 681 78

High glycogen content and abnormal mitochondria have been seen in muscles from RN- carrier pigs in a previous work. Glycogen synthase, branching enzyme, phosphorylase and debranching enzyme activities, and mitochondrial characteristics were studied in normal and RN- carrier pigs. Branching enzyme activity was higher (P < 0.01) and glycogen synthase activity tended to be higher in longissimus dorsi muscle from RN- carrier pigs compared to normal pigs. There were no differences in the activities of either phosphorylase and debranching enzyme between both types of pigs. Citrate synthase activity and mitochondrial respiration were slightly higher in muscle from RN- pigs compared to normal pigs. Glycogen content in muscle from RN- pigs could result from the imbalance between anabolic and catabolic enzyme activities of glycogen metabolism. The higher specific activity in mitochondria of RN- pigs muscle might be the compensatory effect of an abnormal glycolytic metabolism.
Comp Biochem Physiol Biochem Mol Biol 1994 Jul
PMID:Enzyme activities of glycogen metabolism and mitochondrial characteristics in muscles of RN- carrier pigs (Sus scrofa domesticus). 808 56

Glycogen synthase, the regulatory enzyme of glycogen synthesis undergoes multisite phosphorylation leading to its inactivation. The kinases responsible for this covalent modification (ex. cAMP-dependent protein kinase, protein kinase C and glycogen synthase kinase-3) are controlled by the second messengers generated by different hormones. The isolated hepatocytes has been used as one of the experimental models for studying this complex regulatory process. Inactivation of glycogen synthase by glucagon and vasopressin has been shown to be accompanied with incorporation of phosphate into the enzyme protein. Insulin has been shown to activate glycogen synthase by inhibition of kinases and activation of synthase phosphatase. Glycogen synthase is activated by several gluconeogenic substrates, in addition to glucose. Studies in hepatocytes with activators and inhibitors of protein kinase C show that this enzyme negatively controls glycogen synthase. The differential effects of the phosphatase inhibitors, calyculin A and okadaic acid in liver cells provide supporting evidence that protein phosphatase type-1 plays a major role in the regulation of glycogen synthase. Hepatocytes isolated from diabetic rats of both types (insulin-dependent and non-insulin-dependent) mimic the defective glycogen synthase activation seen in vivo.
Mol Cell Biochem
PMID:Regulation of glycogen synthase activation in isolated hepatocytes. 856 54


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