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)

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 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

1. The time-course of tryptic hydrolysis of aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) was followed by activity measurements in the presence and absence of allosteric effectors, and by polyacrylamide-gel electrophoresis. 2. Two proteins with enzyme activity are formed in this way from native enzyme, and the isolation and some properties of these species are reported. The larger protein (10.6S) resembles native enzyme in that it contains regulatory subunits and is sensitive to allosteric effectors, as well as in a more detailed kinetic investigation. It appears from the time-course of tryptic digestion to be an intermediate in the formation of a catalytic subunit (5.5S) which is similar to, but not identical with, the catalytic subunit produced by mercurial treatment of the native enzyme. 3. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis of the different enzyme forms demonstrates that trypsin can hydrolyse bonds in the catalytic polypeptide chains as well as completely remove the regulatory polypeptide chains. 4. Both preparations of catalytic subunit can recombine with regulatory subunit to form enzymes which resemble the native enzyme in being activated by ATP, although they do not appear to be inhibited by CTP. 5. This study is consistent with the models of the enzyme that propose that the catalytic subunits are held together in the native enzyme by three pairs of regulatory polypeptide chains.
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PMID:Enzyme forms produced from aspartate transcarbamoylase by digestion with trypsin. 477 64

A protein kinase was solubilized from whole vaccinia virions by using a solution containing deoxycholate, dithiothreitol, and sodium or potassium chloride. The released enzyme was completely dependent on Mg(2+) and was greatly stimulated by added basic proteins such as protamine or histones. Dithiothreitol was also stimulatory, whereas GTP, CTP, UTP, and P(i) at concentrations equimolar with ATP had little or no effect. Attempts to purify the protein kinase were initially unsuccessful, leading us to consider that either the enzyme was extremely labile or that two readily separable components were required for activity. The observation that the material extracted with NP-40 detergent during the preparation of viral cores stimulated the protein kinase activity of the intact cores supported the second possibility. As the protein kinase, now solubilized from viral cores, was passed through successive DEAE-cellulose columns, it became increasingly dependent for activity on addition of the NP-40 extract. A 30- to 40-fold stimulation of protein kinase activity, which afforded recovery of essentially all starting activity, could be effected by addition of the NP-40 extract to the partially purified enzyme. The NP-40 extract was shown to contain a heat stable, trypsin-sensitive protein, whose action could not be duplicated by cyclic nucleotides.
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PMID:Protein kinase activity from vaccinia virions: solubilization and separation into heat-labile and heat-stable components. 477 67

A Mg-dependent adenosine triphosphatase (ATPase) activated by submicromolar free Ca2+ was identified in detergent-dispersed rat liver plasma membranes after fractionation by concanavalin A-Ultrogel chromatography. Further resolution by DE-52 chromatography resulted in the separation of an activator from the enzyme. The activator, although sensitive to trypsin hydrolysis, was distinct from calmodulin for it was degraded by boiling for 2 min, and its action was not sensitive to trifluoperazine; in addition, calmodulin at concentrations ranging from 0.25 ng-25 micrograms/assay had no effect on enzyme activity. Ca2+ activation followed a cooperative mechanism (nH = 1.4), half-maximal activation occurring at 13 +/- 5 nM free Ca2+. ATP, ITP, GTP, CTP, UPT, and ADP displayed similar affinities for the enzyme; K0.5 for ATP was 21+/- 9 microM. However, the highest hydrolysis rate (20 mumol of Pi/mg of protein/10 min) was observed at 0.25 mM ATP. For all the substrates tested kinetic studies indicated that two interacting catalytic sites were involved. Half-maximal activity of the enzyme required less than 12 microM total Mg2+. This low requirement for Mg2+ of the high affinity (Ca2+-Mg2+)ATPase was probably the major kinetic difference between this activity and the nonspecific (Ca2+ or Mg2+)ATPase. In fact, definition of new assay conditions, i.e. a low ATP concentration (0.25 mM) and the absence of added Mg2+, allowed us to reveal the (Ca2+-Mg2+)ATPase activity in native rat liver plasma membranes. This enzyme belongs to the class of plasma membrane (Ca2+-Mg2+)ATPases dependent on submicromolar free Ca2+ probably responsible for extrusion of intracellular Ca2+.
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PMID:A high affinity calcium-stimulated magnesium-dependent ATPase in rat liver plasma membranes. Dependence of an endogenous protein activator distinct from calmodulin. 611 12

The topography of the dolichyl phosphate biosynthetic enzymes within the plane of rat liver microsomes was investigated by the use of two impermeant inhibitors of enzyme activity: trypsin and mercury-dextran. Mercury-dextran was found to inactivate over 50% of the activities of the CTP-dependent dolichol kinase and the long-chain prenyltransferase. Trypsin caused over 90% inactivation of the long-chain prenyltransferase and 60% inactivation of the dolichol kinase. In addition, the CTP-dependent dolichol kinase was inhibited over 90% by CDP applied externally to sealed microsomes. Inactivation of the dolichyl phosphate biosynthetic enzymes by the impermeant probes occurred under conditions where the mannose-6-phosphatase activity was highly latent. It was concluded that the active sites of these two enzymes are located on the external surface of the microsomal membranes and that dolichyl phosphate biosynthesis occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.
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PMID:Topography of dolichyl phosphate synthesis in rat liver microsomes. Transbilayer arrangement of dolichol kinase and long-chain prenyltransferase. 618 71

The Ca2+-dependent ATPase was solubilized from rat heart sarcolemmal membranes upon digestion with trypsin and was found to be different from Ca2+-stimulated Mg2+-dependent ATPase (Dhalla, N. S., Anand-Srivastava, M. B., Tuana, B. S., and Khandelwal, R. L. (1981) J. Mol. Cell. Cardiol. 13, 413-423). The enzyme was purified by high speed centrifugation, ammonium sulfate fractionation, and column chromatography and was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis. In sodium dodecyl sulfate-acrylamide gels, the enzyme dissociated into two subunits or fragments with molecular weights of about 55,000 and 12,000. The molecular weight of the enzyme, estimated by gel filtration on a Sephadex G-100 column, was found to be about 67,000. The enzyme utilized ATP with a Km of 0.20-0.26 mM but was also able to utilize ITP, CTP, GTP, and ADP as substrates at much lower rates. It was activated by Ca2+ with a Ka of 0.13-0.21 mM; it was also activated by other cations in the order Ca2+ greater than Mn2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. Divalent cations like Cu2+, Ni2+, and Mg2+ were potent inhibitors. The enzyme was insensitive to ouabain, verapamil, oligomycin, cyanide, and vanadate but was markedly inhibited by N-ethylmaleimide. Calmodulin failed to stimulate Ca2+-dependent ATPase and instead inhibited slightly. Unlike K+, Na+ produced a marked inhibition of the Ca2+-dependent ATPase activity, and this inhibition was associated with an 8- 10-fold decrease in the affinity of the enzyme for Ca2+. The competitive action of Na+ indicates that the Ca2+-dependent ATPase may be a site of Na+-Ca2+ antagonism in the cell membrane.
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PMID:Purification and characterization of a Ca2+-dependent ATPase from rat heart sarcolemma. 621 55

Micromolar concentrations of GDP or GTP stimulate protein synthesis by isolated yeast mitochondria 3- to 10-fold even if alpha-ketoglutarate and an ATP-regenerating system are present. No stimulation is observed with GMP, UTP, CTP, TTP, and the nonhydrolyzable GTP analogues guanyl(beta, gamma-methylene) diphosphate and guanyl imidodiphosphate. This stimulatory effect of exogenously added guanyl nucleotides may answer the long standing question why protein synthesis by isolated mitochondria is so slow. It can also explain previous reports by two other laboratories that a high speed supernatant from yeast cells stimulates protein synthesis by isolated mitochondria. The supernatant contains nondialyzable GMP which is converted to GDP under the conditions used for assaying mitochondrial protein synthesis. The stimulatory effect of high speed supernatants is abolished by 5'-nucleotidase (which degrades GMP) or by trypsin (which destroys supernatant protein(s) necessary for converting GMP to GDP). No evidence was obtained that the stimulatory effect of high speed supernatants was caused by precursors to cytoplasmically made cytochrome c oxidase subunits.
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PMID:Stimulation of in vitro mitochondrial protein synthesis by yeast cytoplasmic extracts is caused by guanyl nucleotides. 624 10

We have identified a proteolytic system that selectively degrades histone H1 in normal human lymphocytes. Treatment of permeabilized human lymphocytes with a series of nucleotides produced a marked decrease in their histone H1 content compared to untreated cells. The nucleotide-stimulated process was selective for histone H1 because gel electrophoresis showed that almost all other lymphocyte protein bands remained constant while histone H1 disappeared. The elimination of histone H1 appears to be the result of proteolysis by a trypsin-like enzyme because it was inhibited by phenylmethylsulfonyl fluoride, antipain, soybean trypsin inhibitor, and diisopropyl fluorophosphate. Proteolysis was stimulated by P1,P4-di(adenosine-5') tetraphosphate, P1,P3-di(adenosine-5') triphosphate, P1,P5-di(adenosine-5') pentaphosphate, adenosine 5'-tetraphosphate, ATP, adenosine 5'-[alpha, beta-methylene]triphosphate, adenosine 5'-[beta, gamma-methylene]triphosphate, ADP, CTP, GTP, UTP, dATP, or pyrophosphate, whereas AMP, adenosine, adenosine diphosphoribose, NAD+, cAMP, or sodium phosphate did not show this stimulation of proteolysis. ATP, [alpha, beta-methylene]ATP, [beta, gamma-methylene]ATP, and pyrophosphate all stimulated proteolysis, suggesting that a pyrophosphate linkage was necessary for this process. Thus, resting human lymphocytes contain a trypsin-like protease that is stimulated by nucleotides or pyrophosphate to selectively degrade histone H1.
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PMID:Nucleotide-stimulated proteolysis of histone H1. 631 May 79

The presence of a soluble, Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity in Ehrlich ascites tumor cell homogenates is reported. The crude homogenate was fractionated over Sephadex G-150 gel-filtration and DEAE-Sephacel anion-exchange columns, and two p-nitrophenylphosphatase activities were resolved. The most active fraction, Peak I, was characterized and found to be similar to phosphotyrosyl-protein phosphatases characterized elsewhere in that it has optimal activity at neutral pH; it is inhibited by phosphate, Zn2+, and vanadate; and it is not inhibited by levamisole. However, Peak I differs from phosphotyrosyl-protein phosphatases in that Mg2+ or Mn2+ is required for activity, fluoride is an inhibitor, and pyrophosphate is not inhibitory. Inhibition by the phosphorylated compounds phosphotyrosine, phosphoserine, phosphothreonine, ATP, CTP, GTP, ITP, NADP, fructose 6-phosphate, glucose 1-phosphate, galactose 1-phosphate, 2-phosphogluconic acid, and 6-phosphogluconic acid was also observed. Ehrlich ascites tumor cell p-nitrophenylphosphatase is shown to be sensitive to inactivation by trypsin, N-ethylmaleimide, or heat treatments.
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PMID:Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity is present in Ehrlich ascites tumor cells. 633 18


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