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)

Protein kinase associated with rat liver microsomes was only partly extracted by treatment with 1.5 M KCl. The enzyme was solubilised by Triton X-100 or sodium deoxycholate at the same or slightly higher detergent concentrations than microsomal marker components. The enzyme activity increased 2-3 fold upon solubilisation. Three peaks with protein kinase activity (fractions MI, MII and MIII) were resolved on DEAE-cellulose chromatography. Fraction MIII but not fractions MI or MII was activated by adenosine 3':5'-monophosphate (cyclic AMP). All fractions catalysed the phosphorylation of protamine and histones but not that of casein or phosvitin. Fractions MI and MIII had a similar substrate specificity and phosphorylated histones at a relatively much higher rate than did fraction MII. The isoelectric points were 8.1 for fraction MI, 5.5 for fraction MII and 4.9 for fraction MIII. On incubation of fraction MIII with cyclic AMP it was split into two catalytically active components with pI 8.1 and 7.35. The component with pI 8.1 was predominant and corresponded to fraction MI. Five protein kinase peaks were resolved from rat liver cytosol by DEAE-cellulose chromatography. Three of them (fractions CIa, CIIb and CIII) had the same properties as each of the microsomal kinase fractions. A forth fraction (CIIa) was cyclic-AMP-dependent and had the same substrate specificity as fractions MI and MIII. Its pI was 5.1, and it was split into two components by cyclic AMP (pI 8.1 and 7.35). In binding studies fraction CIIb bound more efficiently to microsomes than fraction CIII, while fractions CIa, CIIa and the microsomal protein kinase fractions did not bind appreciably. When microsomes were treated with trypsin exposed protein kinase was inactivated and the latency of the remaining enzyme increased substantially. Most of fraction MII was inactivated by trypsin while fraction MIII was resistant. The possible orientation of protein kinase fractions MII and MIII in the microsomal membrane is discussed.
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PMID:Protein kinases of rat liver endoplasmic reticulum. Solubilisation, partial characterisation and comparison with protein kinases of rat liver cytosol. 20 48

Two cyclic AMP-binding proteins, not identical with regulatory subunits of protein kinases, have been isolated from Trypanosoma gambiense. The cyclic AMP receptors were separated by gel chromatography on the basis of their molecular weights. The binding constants of the high and the low molecular weight receptors for cyclic AMP were determined to be 0.4 muM and 0.6 muM, respectively. Cyclic IMP and cyclic GMP compete with cyclic AMP for the binding sites of both receptors. The cyclic AMP binding of the low molecular weight receptor was competitively inhibitied by adenine derivatives. The binding capacity of the high molecular weight receptor was enhanced about two-fold by proteolytic modification with trypsin.
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PMID:Adenosine 3', 5'-cyclic monophosphate-binding proteins from Trypanosoma gambiense. 20 48

The relative potencies of various prostaglandins were investigated in trypsin-dispersed cat adrenocortical cells. Prostacyclin proved to be the most potent steroidogenic prostaglandin, being 100-1000 times more potent than PGE2. This stimulant effect of prostacyclin was only partially dependent upon the presence of extracellular calcium and was associated with increased levels of cyclic AMP. These data suggest a possible role for prostacyclin in corticosteroidogenesis.
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PMID:Prostacyclin: a potent stimulator of adrenal steroidogenesis. 21 24

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

BHK fibroblasts contain two forms of cyclic AMP phosphodiesterase 3':5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) as analyzed by linear sucrose gradient fractionation; a 3.6-S form (peak I) and a 6.7-S form (peak II). Peak I is specific for cyclic AMP as substrate and displays Michaelis-Menten kinetics with an apparent Km of 2--3 micrometer. Peak II hydrolyzes cyclic GMP and displays anomalous kinetics for cyclic AMP hydrolysis. The activity of isolated peak II for cyclic AMP is increased by storage at 4 degrees C, treatment with trypsin, or treatment with rat brain and BHK fibroblast activator proteins. The activity of isolated peak I is unaffected by these conditions. Linear sucrose gradient fractionation demonstrates that activation of peak II by trypsin leads to the formation of a 3.6-S cyclic AMP-specific enzyme form, possibly peak I. In contrast to BHK fibroblasts (and most other mammalian tissues), rat uterus contains only one form of cyclic nucleotide phosphodiesterase on linear sucrose gradients, a 7-S form capable of hydrolyzing both cyclic AMP and cyclic GMP. Treatment of rat uterine supernatant with trypsin leads to the appearance of a 4-S, cyclic AMP-specific form with properties similar to that of BHK peak I. These data suggest that the kinetically complex, higher molecular weight cyclic nucleotide phosphodiesterases may consist of more than one catalytically active site and that multiple forms of the enzyme arise through dissociative mechanisms, possibly as a means of in vivo regulation.
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PMID:Activation of mammalian cyclic AMP phosphodiesterases by trypsin. 21 13

Purified rat Schwann cells were found to proliferate very slowly in normal growth medium containing 10% fetal calf serum (FCS). Crude extracts of bovine pituitary or brain markedly enhanced Schwann cell growth, while similar extracts of nerve roots, liver and kidney did not. Pituitary extracts were more potent than brain extracts, and extracts from both anterior and posterior pituitary were active. The mitogenic activity of pituitary extracts was reduced by treatment with trypsin, and abolished by pronase and by boiling. A variety of known anterior and posterior pituitary hormones, as well as fibroblast, epidermal and nerve growth factors, were not mitogenic. FCS (greater than 1%) was required for Schwann cell proliferation, but even high concentrations of FCS did not substitute for pituitary or brain extracts, and serum from various other species did not support Schwann cell growth. Although various agents that increase cyclic AMP levels (such as cholera toxin) had been shown to be Schwann cell mitogens, extracts of pituitary or brain did not increase cyclic AMP levels. Extracts of various bovine tissues, including pituitary, brain, liver and kidney, acted synergistically with cholera toxin in stimulating Schwann cell proliferation, although the increase in cyclic AMP induced by the mixture was not greater than that seen with cholera toxin alone. We conclude that there are at least two separate pathways for stimulating Schwann cell division, only one of which involves an increase in intracellular cyclic AMP.
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PMID:Schwann cell growth factors. 21 15

Cyclic AMP-dependent protein kinases prepared from crude cytosols of chick kidney, rat kidney and rat liver were found on dilution to exhibit complex kinetics. Dilution of the cytosols appears to increase the state of activation of the enzymes. This effect was due to the presence of inhibitory agents in the cytosol which had a greater inhibitory effect on the cyclic AMP-dependent than on the cyclic AMP-independent enzyme. Two types of inhibitory activity were found by column chromatography, one resistant to trichloroacetic acid precipitation and boiling but affected by trypsin digestion and the other resistant to boiling and trypsin digestion but precipitated by trichloroacetic acid. Inhibitory activity corresponding to the former characteristics has been described previously but the presence of additional soluble inhibitory agents in the cytosol has not been documented. The complete characterisation of this previously undescribed inhibitory activity requires further investigation. The relevance of such cytosolic inhibitory activity to the interpretation of states of activation of protein kinase enzymes is discussed.
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PMID:Soluble cyclic AMP-dependent protein kinases from chick kidney. Effects of dilution and non-protein inhibitors. 21 42

Trypsin, thrombin, and ionophore A23187 activate phospholipid breakdown of platelets that have been labeled with [(14)C]arachidonate, releasing their cyclooxygenase and lipoxygenase products. Intact platelets can also very effectively directly degrade low concentrations of exogenous, free [(14)C]arachidonate. Pretreatment of platelets with trypsin, thrombin, or ionophore A23187 for a minimum time of 30 sec leads to complete inactivation of cyclooxygenase activity, as demonstrated by subsequent exposure to [(14)C]arachidonate. Lipoxygenase activity is lost after 5 min. The thrombin-induced inactivation of cyclooxygenase and lipoxygenase is prevented by cyclic AMP (which inhibits the stimulated activity of phospholipase A(2)), although cyclic AMP does not affect the degradation of exogenous [(14)C]arachidonate. Exposure of platelets labeled with [(14)C]arachidonate to unlabeled arachidonate under conditions that lead to use of the latter also results in a similarly rapid inhibition of cyclooxygenase activity, as determined by subsequent challenge with thrombin. Under these conditions lipoxygenase activity is much less markedly inactivated. The arachidonate-induced inhibition of cyclooxygenase activity is not prevented by cyclic AMP. Trypsin does not induce platelet aggregation, and platelets whose cyclooxygenase activity has been inactivated are intact insofar as they are still able to undergo aggregation. These studies demonstrate that operation in intact platelets of the cyclooxygenase pathway, through use of endogenous or exogenous substrate, leads to a very rapid, irreversible inactivation of this enzyme. The lipoxygenase pathway is also progressively impaired, but much less rapidly than the cyclooxygenase enzyme and much less markedly on use of exogenous compared to endogenous substrate. The possible consequences of these physiological processes of spontaneous inactivation are considered.
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PMID:Rapid inactivation of cyclooxygenase activity after stimulation of intact platelets. 21 91

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

Cardiac microsomes were incubated with [gamma-32P]ATP and a cardiac adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase in the presence of ethylene glycol bis(bets-aminoethyl ether)-N,N'-tetraacetic acid. After solubilization in sodium dodecyl sulfate and fractionation by polyacrylamide gel electrophoresis, a single microsomal protein component of approximately 22,000 daltons was found to bind most of the 32P label. The 32P labeling of this component increased several fold when NaF was included in the incubation medium. No other component of cardiac microsomes, including sarcoplasmic reticulum ATPase protein, contained significant amounts of 32P label. This 22,000-dalton phosphoprotein formed by cyclic AMP-dependent protein kinase had stability characteristics of a phosphoester rather than an acyl phosphate. Washing of microsomes with buffered KCl did not decrease the amount of 32P labeling to the 22,000-dalton protein, suggesting that this protein is associated with the membranes of sarcoplasmic reticulum rather than being a contaminant from other soluble proteins. The 22,000-dalton protein was susceptible to trypsin. Brief digestion with trypsin in the presence of 1 M sucrose did not significantly affect microsomal calcium transport activity, but prevented both subsequent phosphorylation of the 22,000-dalton protein and stimulation of calcium uptake by cyclic AMP-dependent protein kinase, suggesting that this protein is a modulator of the calcium pump. These results are consistent with previous findings (Kirchberger, M.A., Tada, M., and Katz, A.M. (1974) J. Biol. Chem. 249, 6166-6173; Tada, M., Kirchberger, M.A., Repke, D.I., and Katz, A.M. (1974) J. Biol. Chem. 249, 6174-6180) that cyclic AMP-dependent protein kinase-catalyzed phosphorylation is associated with stimulation of calcium transport in the cardiac sarcoplasmic reticulum, and further indicate that this phosphorylation occurs at a component of low mass (22,000 daltons) of the cardiac sarcoplasmic reticulum which, while separable from the calcium transport ATPase protein (100,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has the ability to regulate calcium transport by the cardiac sarcoplasmic reticulum.
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PMID:Phosphorylation of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase. 23 23


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