Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Neurospora crassa glycogen synthase (UDPglucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) was purified to electrophoretic homogeneity by a procedure involving ultracentrifugation, DEAE-cellulose column chromatography, (NH4)2SO4 fractionation and 3-aminopropyl-Sepharose column chromatography. The final purified enzyme preparation was almost entirely dependent on glucose-6-P and had a specific activity of 6.9 units per mg of protein. The subunit molecular weight of the glycogen synthase was determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel to be 88 000--90 000. The native enzyme was shown to have a molecular weight of 270 000 as determined by sucrose density gradient centrifugation. Thus, the glucose-6-P-dependent form of the N. crassa glycogen synthase can exist as trimer of the subunit. Limited proteolysis with trypsin or chymotrypsin converted the glucose-6-P-dependent form of the enzyme into an apparent glucose-6-P-independent form. The enzyme was shown to catalyze transfer of glucose from UDPglucose to glycogen as well as to its phosphorylase limit dextrin, but not to its beta-amylase limit dextrin. Moreover, glucose, maltose and maltotriose were not active as acceptors.
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PMID:Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the UDPglucose:glycogen 4-alpha-glucosyltransferase. 2 41

Phosphorylation of skeletal muscle glycogen synthase catalyzed by a protein kinase is stimulated up to 10-fold by the calcium-dependent regulator (CDR) protein. Half-maximal stimulation requires about 1 microgram of CDR/ml. Phosphorylation by the CDR-dependent synthase kinase is more rapid at pH 8.6 than at pH 6.8 and is blocked by ethylene glycol bis(beta-aminoethyl-ether)N,N'-tetraacetic acid and trifuloperazine. Approximately 60 to 70% of the phosphate is incorporated into the trypsin-insensitive region of glycogen synthase resulting in conversion of the a form to the b form of the enzyme. The CDR-dependent synthase kinase is not myosin light chain kinase, as this enzyme does not phosphorylate glycogen synthase. Furthermore, synthase phosphorylation by the cAMP-dependent protein kinase catalytic subunit is not affected by CDR. The possibility that CDR-dependent synthase kinase may be phosphorylase kinase is being investigated.
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PMID:Stimulation of glycogen synthase phosphorylation by calcium-dependent regulator protein. 10 93

The properties of purified mammalian adenosine 3':5'-cyclic monophosphate (cAMP)- and guanosine 3':5'-cyclic monophosphate (cGMP)-dependent protein kinases were compared. Several physical characteristics of the two enzymes were similar, including size, shape, affinity for cyclic nucleotide binding, and K(m) for ATP. In addition, the amino acid composition of the two proteins indicated a close composition homology (70-90%). Both cyclic nucleotide-dependent protein kinases catalyzed phosphorylation of rat liver pyruvate kinase (EC 2.7.1.40) and fructose 1,6-diphosphatase (EC 3.1.3.11), rabbit skeletal muscle glycogen synthase (EC 2.4.1.11) and phosphorylase b kinase (EC 2.7.1.38), and calf thymus histone H(2)b. The phosphorylation of several synthetic peptides and of trypsin-sensitive and trypsin-insensitive sites in glycogen synthase suggested similar recognition sites on the protein substrates for the two kinases. The cAMP-dependent protein kinase was the better catalyst with each protein or peptides substrate. The results suggest that the two enzymes evolved from a common ancestral protein.
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PMID:Adenosine 3':5'-cyclic monophosphate- and guanosine 3':5'-cyclic monophosphate-dependent protein kinases: possible homologous proteins. 19 77

Administration of adrenaline to an isolated rat hindlimb preparation rapidly decreased muscle phosphorylase phosphatase (EC 3.1.3.17) activity and increased heat-stable and trypsin-labile phosphatase inhibitor activity. This was associated with increased tissue cyclic AMP concentrations, phosphorylase (EC 2.4.1.1) activation and glycogen synthase (EC 2.4.1.11) inactivation.
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PMID:Control of rat skeletal-muscle phosphorylase phosphatase activity by adrenaline. 21 33

Skeletal muscle glycogen a4-synthase (EC 2.4.1.11) has been purified free of all synthase kinase and phosphatase activities by chromatography on a Glc-N-6-P-Sepharose affinity column and then on a phosphocellulose column. This preparation of glycogen synthase was tested as a substrate for purified skeletal muscle phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38). Phosphorylase kinase (1-10 microgram/ml or 0.03-0.3 microM) catalyzes rapid phosphorylation of glycogen synthase (4.5 microM) associated with conversion of the active a form to the less active b form. In the reaction, greater than 95% of the 32P incorporation from [gamma-32P]ATP goes into the synthase subunit almost exclusively in the trypsin-insensitive region which is responsible for synthase a-to-b conversion. Synthase phosphorylation or inactivations catalyzed by phosphorylase kinase is blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, is ATP dependent, is 10-fold more rapid at pH 8.6 than at pH 6.8, and is increased 10-fold by prior activation of the phosphorylase kinase with MgATP and cyclic AMP. With activated phosphorylase kinase at pH 8.2 the apparent Km and Vmax are approximately 70 microM and 4 mumol/min per mg with glycogen synthase and 70 microM and 9 mumol/min per mg with phosphorylase as substrate. It is concluded that glycogen synthase is a substrate in vitro for phosphorylase kinase, a Ca2+-dependent enzyme. The possible physiological significance of this reaction is discussed.
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PMID:Phosphorylation and inactivation of glycogen synthase by phosphorylase kinase. 22 47

Incubation of fat cells with insulin increased glycogen synthase I activity without changing total synthase activity. This effect of insulin was dependent upon the particular lot of albumin present in the medium and was abolished by incubating cells with trypsin. Half-maximal activation of glycogen synthase was obtained with 8 microunits/ml of insulin, a concentration very similar to that which half-maximally stimulated 3-O-methylglucose uptake. The basal percentage of phosphorylase a activity was not detectably altered by insulin, although it was decreased by incubating cells with 5 mM glucose. Insulin (50 microunits/ml) markedly opposed actions of epinephrine (0.05 to 10 muM) to increase phosphorylase a activity and decrease glycogen synthase I activity, effects which were observed without glucose. Partial activation of glycogen synthase by insulin was seen after 1 min and complete activation after 4 min. Glucose alone produced a transient increase in synthase I activity. When cells were incubated with insulin plus glucose for 4 min, the increase in the percent synthase I activity was much greater than the additive effects of insulin and glucose alone. This potentiation of the effect of insulin on glucogen synthase I activity depended on the time of incubation with glucose and on the concentration of the hexose. If cells were incubated with cytochalasin B before insulin plus glucose, the effect of glucose was abolished. These results suggest that there are at least two mechanisms by which insulin can increase fat cell glycogen synthase I activity. One requires glucose and activation occurs secondary to an increase in glucose transport; where another mechanism(s) is operative even in the absence of glucose.
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PMID:Activation of rat adipocyte glycogen synthase by insulins. 40 14

Protein phosphatases associated with the particulate fraction from rat liver were studied by chromatographing the fraction on a DEAE-cellulose column and assaying the eluate with phosphorylase alpha and glycogen synthase D as substrates. Phosphorylase phosphatase activity emerged as two peaks, termed P-1 and P-2 in order of elution, both of which were inhibited by Mn2+ and Mg2+. P-1 and P-2 were Mr = 50,000 and 32,000 proteins, respectively, and when treated with trypsin, P-1 converted to a form indistinguishable from P-2, to which protein phosphatase inhibitor-2 was a potent inhibitor. Thus P-2 appears to be the catalytic subunit of type-1 protein phosphatase even though it has been degrated proteolytically as evidenced by its relatively low Mr. The elution profile of glycogen synthase phosphatase activity was entirely different. The activity obtained with 5 mM Mn2+ resolved into three peaks, the second-migrating M-2 being the largest. M-2 is an Mr = 70,000 protein; but an attempt to purify it has been unsuccessful giving a product of Mr = 40,000 and closely similar to the type-1 catalytic subunit in properties including inhibition by inhibitor-2. These results suggest that phosphatases P-1 and M-2 have a common catalytic subunit (type-1), which is bound to different "regulatory" subunits. M-2 distributes in glycogen particles and microsomes evenly while P-1 is almost exclusively in microsomes.
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PMID:Characterization of protein phosphatases associated with the particulate fraction from rat liver. 216 61

A retro-inverso analogue of the pseudosubstrate sequence, Arg-Phe-Ala-Arg-Lys-Gly-Ala25-Leu-Arg-Gln-Lys-Asn-Val (1), found in the regulatory domain of all protein kinase C (PKC) subspecies was synthesized. It shows to be an inhibitor (IC50 = 31 microM) of the phosphorylation, by PKC, of [Ala9.10,Lys11.12] glycogen synthase (1-12). Its analogue in which D Ala25 is replaced by D Ser is not a PKC substrate, but a more potent inhibitor, competitive with the peptidic substrate (IC50 = 5 microM, Ki = 2 microM). Both retro-inverso peptides are highly specific for PKC versus adenosine cAMP-dependent protein kinase (PKA) and are totally stable towards proteolysis by trypsin or pronase.
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PMID:Inhibition of protein kinase C by retro-inverso pseudosubstrate analogues. 251 86

It has previously been demonstrated that the insulin-mimetic agent trypsin stimulates autophosphorylation of purified insulin receptors and activates the insulin receptor tyrosine kinase in vitro. We now report the effects of trypsin on whole cell tyrosine kinase activation and insulin receptor autophosphorylation. Trypsin treatment of intact adipocytes produces a time-dependent stimulation of tyrosine kinase activity as measured in lectin extracts containing the insulin receptor, or specifically immunoprecipitated insulin receptor samples. Trypsin treatment of adipocytes also results in a loss of insulin binding capacity, and a linear correlation exists between loss of binding and stimulation of tyrosine kinase activity. Exposure of adipocytes to trypsin is known to result in a time- and dose-dependent activation of intracellular glycogen synthase. Examination of the time courses of stimulation of tyrosine kinase and glycogen synthase activation in our system indicates that the stimulation of tyrosine kinase activity by trypsin occurs with sufficient rapidity and magnitude to be consistent with a role of phosphorylation in the activation of glycogen synthase. Trypsin has further been demonstrated to stimulate autophosphorylation of the beta-subunit of the insulin receptor in intact adipocytes. Cells prelabeled with [32P]PO4 for 2 h were exposed to trypsin, and receptors were partially purified over wheat germ agglutinin-agarose columns. Receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the beta-subunit was identified by autoradiography. The protein was extracted and hydrolyzed, and the phosphoamino acids were separated by electrophoresis and quantitated. Two- and five-fold increases in phosphotyrosine were observed with 3 and 10 min of trypsin treatment, respectively. We conclude that trypsin-induced cleavage of the insulin receptor alpha-subunit is relevant to the ability of trypsin to activate the insulin receptor tyrosine kinase in intact adipocytes. We further conclude that autophosphorylation of the insulin receptor and activation of its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic effects of trypsin.
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PMID:Insulin-mimetic effect of trypsin on the insulin receptor tyrosine kinase in intact adipocytes. 282

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.
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PMID:Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle. 282 16


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