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 influence of various proteinases on GTP hydrolysis was studied in membranes of human platelets. Of the proteinases examined, trypsin, acrosin and a recently described trypsin-like proteinase from bovine sperm, but not chymotrypsin, increased GTP hydrolysis. Similar to what was described previously for hormone-like agents, the stimulation of GTP hydrolysis by the proteinases was only observed at low GTP concentrations, with apparent Km values of 0.2-0.3 microM-GTP. Stimulation of the high-affinity GTPase by the proteinases occurred without apparent lag phase and was constant over a long period of incubation. The proteinase inhibitors leupeptin and soya-bean trypsin inhibitor blocked the stimulation of GTP hydrolysis, but did not reverse the effect of the proteinases. Treatment of platelet membranes with N-ethylmaleimide, which eliminates Gi-protein (inhibitory guanine-nucleotide-binding protein)-related GTPase stimulation by adrenaline, decreased stimulation of GTP hydrolysis by the proteinases only partially. Activation of GTP hydrolysis by the proteinases was partially additive with that caused by adrenaline, whereas thrombin stimulation was not increased further. The data indicate that, similarly to the proteinase thrombin, trypsin and trypsin-like proteinases can activate GTP-hydrolysing protein(s) that exhibit high affinity for GTP in platelet membranes. It is suggested that the proteinases interact in platelet membranes with a receptor site similar to that used by thrombin and that the observed GTPase stimulation is a reflection of a proteinase-receptor interaction with a guanine-nucleotide-binding regulatory protein.
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PMID:Stimulation of high-affinity GTPase by trypsin and trypsin-like proteinases in membranes of human platelets. 283 22

The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.
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PMID:Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin. 283 61

The synthesis of lauroyl sucrose capable of solubilizing 100% of beta-adrenergic receptors from bovine cerebellum membranes has been carried out. The preparative procedure for isolation of homogeneous beta-adrenergic receptors including affinity chromatography on the novel support, oxprenolol-Sepharose, is described. According to SDS-PAAG electrophoresis data, the Mr value for the beta-adrenergic receptor is 61 kD. The purified beta-adrenergic receptor can interact with the purified GTP-binding regulatory protein of adenylate cyclase (Gs) after their reconstitution into liposomes. Trypsin treatment of the purified receptor does not interfere with its functional properties, nor does it change the hydrodynamic parameters under non-denaturing conditions despite the fact that the polypeptide chain of the receptor is cleaved by trypsin.
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PMID:[Isolation of a homogeneous functionally active beta-adrenergic receptor from bovine cerebellum using lauroyl sucrose. Effect of trypsin on receptor activity]. 285 8

gamma-Glutamyl transpeptidase (gamma GTP) is an enzyme found in cerebral capillary endothelial cells, the presumed site of the blood-brain barrier, but not in endothelial cells lining blood vessels in other parts of the body. Using a line of mouse cerebral microvessel endothelial cells (ME-ly cells) and a sensitive colorimetric assay to measure gamma GTP levels we demonstrated that primary cultures of mouse astrocytes and a line of rat C6 glioma cells released a soluble product(s) that induced the production of gamma GTP in cultured endothelial cells by 34% and 39%, respectively, over control levels. Cerebrovascular smooth muscle cells had no significant effect on gamma GTP levels in ME-ly cells, and the astrocyte product(s) had no effect on rabbit aortic endothelial cells. The induction of gamma GTP levels in ME-ly cells was apparent after one day of exposure to the astrocyte product(s) and increased in magnitude with increasing time of exposure of the ME-ly cells to the product(s). Removal of the product(s) from the ME-ly cells resulted in a return to control levels of gamma GTP in the ME-ly cells within 2 days. The presence of a protein synthesis inhibitor during incubation with the product(s) blocked the induction of gamma GTP in ME-ly cells, and treatment of the product(s) with 200 U/ml TPCK-trypsin destroyed its inductive properties.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Induction of gamma-glutamyl transpeptidase in cultured cerebral endothelial cells by a product released by astrocytes. 288 71

The affinity of many types of membrane-bound receptors coupled negatively to adenylate cyclase is regulated by divalent and monovalent cations and by guanine nucleotides (GTP). We used alpha 2-adrenoreceptors of human platelets as a model system to find out the effect of limited proteolysis with trypsin on the regulation of the alpha 2-adrenoreceptor-agonist interactions by GTP and Na+. We found that partial proteolysis of the membranes with trypsin for 3 min at 35 degrees C reduced specific [3H]yohimbine binding to platelet membranes to 40-50% of control. The following characteristics of the receptors remaining after proteolysis were similar to those of untreated membranes: affinity for the agonist and antagonists, stereospecificity, and kinetic properties. Trypsin also did not modify the ability of the receptor's change from a high to low affinity state in the presence of Na+. These findings suggested that the capability of the receptors to recognize the ligand and their ability to undergo a conformational change in the presence of the agonist were retained despite a reduction in the total number of receptors by trypsin. However, the modulation of the receptor--agonist interactions by GTP or Mg2+ was lost in the trypsin-pretreated membranes, while the modulation by Na+ remained intact. It is suggested that the loss of GTP or Mg2+ effects on receptor--ligand interactions produced by trypsin may be due to trypsin-induced disruption of subunits (alpha i, beta gamma) interactions of Gi protein.
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PMID:Pretreatment of human platelet membranes with trypsin abolishes GTP but not Na+ effects on alpha 2-adrenoreceptor-agonist interactions. 288 68

The function of structural domains of the beta-adrenergic receptor were probed by studying the ability of tryptic fragments of the receptor to catalyze the binding of guanosine-5'-O-(3-thiotriphosphate (GTP gamma S) to the GTP-binding regulatory protein, Gs. beta-Adrenergic receptor purified from turkey erythrocytes was treated with trypsin under nondenaturing conditions. Such treatment decreased beta-adrenergic ligand binding activity by only 15-25%. Active components of the limit digest were repurified by affinity chromatography on alprenolol-agarose and then reconstituted with purified Gs into unilamellar phospholipid vesicles. After reconstitution, the proteolyzed receptor was able to catalyze agonist-stimulated binding of GTP gamma S to Gs at a rate and extent equivalent to that of the nonproteolyzed receptor. The proteolyzed receptor was also partially activated upon reduction by dithiothreitol, as previously reported for the intact receptor (Pedersen, S.E., and Ross, E.M. (1985) J. Biol. Chem. 260, 14150-14157). The repurified, active tryptic digest contained two detectable peptides. One, of approximately 2 X 10(4) Da, contained either four or five of the amino-terminal membrane-spanning domains plus the intervening hydrophilic loops but not the amino-terminal extracellular, glycosylated peptide. The second, of 9,000-10,000 Da, was composed essentially of the two carboxyl-terminal membrane-spanning domains and the intervening extracellular, hydrophilic loop. These data indicate that most of the large intracellular hydrophilic loop and the hydrophilic, carboxyl-terminal region of the receptor are not necessary for the agonist-stimulated regulation of Gs.
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PMID:The hydrophobic tryptic core of the beta-adrenergic receptor retains Gs regulatory activity in response to agonists and thiols. 289 Jun 39

By treatment with detergent and NaCl, particulate (1----3)-beta-D-glucan synthase (EC 2.4.1.34) from Hansenula anomala or Neurospora crassa was dissociated into a "soluble fraction" and a "membrane fraction." Each fraction alone was almost inactive, but enzymatic activity could be reconstituted by mixing the two fractions and adding GTP or one of its analogs. Based on their lability to heat and to incubation with trypsin, the activity in both fractions is proteinaceous. The active component in the soluble fraction appears to bind guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), since it was specifically protected by this nucleotide against heat inactivation and against inactivation in the presence of EDTA. Furthermore, precipitation of the soluble component with ammonium sulfate in the presence of GTP[gamma S] gave rise to a fraction that was highly active in the absence of added nucleotide, indicating either tight binding or covalent interaction between GTP[gamma S] and the soluble component. The membrane fraction probably contains the catalytic moiety, because it was partially protected against heat inactivation by the substrate, UDP-glucose. Soluble fractions that stimulated membrane fractions from H. anomala and N. crassa were obtained from several other fungi, including Saccharomyces cerevisiae. We propose that the soluble fraction contains a GTP-binding protein that modulates the biosynthesis of (1----3)-beta-D-glucan of fungal cell walls and probably has a major role in the regulation of cell wall morphogenesis.
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PMID:Regulation of fungal cell wall growth: a guanine nucleotide-binding, proteinaceous component required for activity of (1----3)-beta-D-glucan synthase. 294 41

Whereas the ribosome-dependent ATPase activity of EF-3 required highly active ribosomes for its full activity, a catalytic site for ATP hydrolysis may reside in the EF-3 as being supported by the activity-EF-3/ribosome amount profiles. The direct interaction of EF-3 with various nucleotides such as GTP, UTP, CTP, dATP, ADP and AMPPNP as well as ATP was analyzed by protection experiments against trypsin digestion of the factor according to SDS-gel electrophoresis. The protection effect varied with the used nucleotides roughly in accordance with the inhibitory effect of those on the ribosome-dependent ATPase. The ATPase activity of EF-3 alone in the absence of ribosome was observed by using large amounts of the factor and the rate was two orders of magnitude lower than that of the ribosome-dependent.
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PMID:Intrinsic ATPase activity of yeast peptide chain elongation factor 3(EF-3) and its direct interaction with various nucleotides. 295 56

The properties of protein kinase activity associated with Fc receptor specific for IgG2a (Fc gamma 2aR) of a murine macrophage like cell line, P388D1, were investigated. IgG2a-binding protein isolated from the detergent lysate of P388D1 cells by affinity chromatography on IgG-Sepharose was found to contain four distinct proteins of Mr 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with [gamma-32P]ATP. The autophosphorylation of Fc gamma 2a receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Casein was found to be a much better phosphate acceptor than histone in this system, as casein incorporated about 32-fold more 32P than histone did. Phosphorylation of casein catalyzed by Fc gamma 2a receptor complex was dependent on casein concentration (maximum phosphate incorporation being at 0.5 mg/mL), increased with time or temperature, was dependent on the concentration of ATP and Mg2+, and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn2+ (greater than 25 mM) or KCl (greater than 100 mM) or by a small amount of heparin (greater than 10 units/mL) and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fc gamma 2a receptor used ATP as substrate with an apparent Km of 2 microM as well as GTP with an apparent Km of 10 microM. Prior heating (60 degrees C for 15 min) or treatment with protease (trypsin or Pronase) of Fc gamma 2a receptor complex almost totally abolished casein kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein kinase activity associated with Fc gamma 2a receptor of a murine macrophage like cell line, P388D1. 296 64

The Ca2+/Mg2+ ATPase of the rat heart sarcolemmal particles was solubilized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in non-denaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240,000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12-0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca2+ (Ka = 0.4 mM) and Mg2+ (Ka = 0.2 mM) as well as by other cations in the order Ca2+ greater than Mg2+ greater than Mn2+ greater than Sr2+ greater than Ba2+ greater than Ni2+ greater than Cu2+. The ATPase activity in the presence of Ca2+ was markedly inhibited by Mg2+, Mn2+, Ni2+ and Cu2+ whereas the monovalent cations such as Na+ and K+ were without effect. The enzyme did not exhibit Ca2+ stimulated Mg2+ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca2+ or Mg2+ ATPase activity was 8.5-9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca2+ metabolism in the myocardium is discussed.
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PMID:Purification and characterization of a Ca2+/Mg2+ ecto-ATPase from rat heart sarcolemma. 297 73


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