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 purpose of this work was to solubilize vasoactive intestinal peptide (VIP) receptors from rat small intestinal plasma membranes and to analyze the nature and function of its molecular form(s) in a nondenaturing environment. Membranes were incubated with 3 nM 125I-VIP, washed, and treated with 1% Triton X-100. Chromatography on Sephadex G-50 showed that 60% of the extractable radioactivity was eluted with macromolecular components in the void volume. This radioactive material was dramatically reduced when 1 microM unlabeled VIP was present in the incubation medium or when membranes were pretreated with trypsin or dithiothreitol. Macromolecular components that had bound 125I-VIP were further chromatographed on Sephacryl S-300. Two peaks were observed: a major one (80%) and a minor one (20%) with Stokes radii of 5.2 and 3.1 nm, respectively. The labeling of both components was inhibited by unlabeled VIP or peptide with NH2-terminal histidine and COOH-terminal isoleucine amide (a VIP agonist). The presence of GTP (0.1 mM) in the incubation medium of membranes completely abolished the labeling of the 5.2-nm component but did not affect that of the 3.1-nm one. Moreover, GTP induced dissociation of 125I-VIP from the 5.2-nm component isolated by Sephacryl S-300 chromatography. This effect was time dependent and nucleotide specific. In contrast, GTP did not affect the stability of the 3.1-nm component. After cholera toxin catalyzed [32P]ADP-ribosylation of membranes, chromatography of solubilized material on Sephacryl S-300 showed that a peak of 32P radioactivity was coeluted with the 5.2-nm component.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Solubilization and hydrodynamic characterization of guanine nucleotide sensitive vasoactive intestinal peptide-receptor complexes from rat intestine. 254 61

Recently, we reported that pp60c-src kinase activity was present in adult bovine coronary arterial smooth muscle and showed that the activity of the enzyme in in vitro immunoprecipitation assays was stimulated 20-60-fold by ATP (Di Salvo, J., Gifford, D., and Kokkinakis, A. (1988) Biochem. Biophys. Res. Commun. 153, 388-394). In the present study, ATP-mediated stimulation of activity was also demonstrated in extracts from aortic vascular smooth muscle. In contrast, no stimulation was apparent in extracts from brain. Stimulation of activity in vascular preparations was also induced with beta,gamma-imidoadenosine 5'-triphosphate (AMP.PNP), a nonmetabolizable analog of ATP, and with several other polyphosphates including ADP and sodium pyrophosphate. No stimulation occurred in response to monophosphates such as AMP or KH2PO4. As expected, the specific activity of pp60c-src in brain extracts did not change when the amount of extracted protein included in immunoprecipitation mixtures was increased. Unexpectedly, however, the specific activity of the vascular enzyme decreased markedly as the amount of extracted protein subjected to immunoprecipitation was increased. Following stimulation of pp60c-src in vascular extracts with ATP, the enzyme behaved in a fashion similar to pp60c-src extracted from brain. That is, the enhanced specific activity of the stimulated vascular enzyme did not decrease with increasing amounts of extracted protein. Moreover, mixing experiments in which vascular smooth muscle extracts were added to brain extracts showed that the muscle extracts contained a factor which inhibited pp60c-src kinase activity. This inhibition was blocked when the mixed extracts were immunoprecipitated in the presence of ATP, or when inhibitory extract was treated with trypsin. Taken together, these data suggest that pp60c-src kinase activity in vascular tissue may be subject to a novel regulatory mechanism involving an inhibitory protein factor which can be nullified by polyphosphates.
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PMID:ATP- and polyphosphate-mediated stimulation of pp60c-src kinase activity in extracts from vascular smooth muscle. 254 81

The possibility that thrombin-induced platelet reactivity could occur via both a receptor-related and a proteolytic process was examined. Thrombin elicited the formation of considerably more [32P]phosphatidic acid (an index of phospholipase C catalysed phosphoinositide metabolism) than did platelet activating factor, 5-hydroxytryptamine, ADP, and the thromboxane A2 analogue EP171, when these agents were added either alone or in combination. Co-addition of thrombin and EP171 did not evoke significantly more [32P]phosphatide acid than did thrombin alone. The protease inhibitor leupeptin, decreased but did not abolish [32P]phosphatidic acid formation elicited by either thrombin alone or thrombin in combination with EP171. The serine protease, trypsin, stimulated an increase in [32P]phosphatidic acid and this effect was additive with that of EP171. This augmentation by trypsin of EP171-induced [32P]phosphatidic acid formation was inhibited by leupeptin. These results are consistent with the concept that thrombin-induced activation of phospholipase C occurs by two distinct mechanisms: one via proteolysis, which is sensitive to leupeptin, and the other via receptor activation, a process shared by EP171. The individual components of this dual mechanism can be mimicked by the co-addition of a receptor-directed agonist (EP171) and a proteolytic agent (trypsin).
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PMID:Evidence for two mechanisms of thrombin-induced platelet activation: one proteolytic, one receptor mediated. 255 47

Limited tryptic digestion of Escherichia coli transcription termination factor rho [an RNA-dependent nucleoside triphosphatase (NTPase)] yields predominantly two fragments (f1 and f2) when the protein is bound to both poly(C) and ATP. The apparent molecular masses of the two fragments are 31 kDa for f1 and 15 kDa for f2, adding up to the molecular mass of the intact rho polypeptide chain (46 kDa). Sequence analysis of the amino termini demonstrates that f1 is derived from the amino-terminal portion of rho and that the trypsin cleavage that defines f2 occurs at lysine-283. These results suggest that, in the liganded (activated) form, the native rho protein monomer is organized into two distinct structural domains that are separable by a single proteolytic cleavage. The f1 fragment, purified from NaDodSO4/polyacrylamide gels and renatured, binds poly(C) but the f2 fragment does not; neither regains any ATPase activity. ATP- and polynucleotide-dependent changes in the rate of proteolysis and in the character of the fragments produced suggest that rho undergoes a series of conformational transitions as a consequence of RNA binding, NTP binding and NTP hydrolysis. The rate of loss of rho ATPase activity and of intact rho monomers is slower in the presence of adenosine 5'-[gamma-thio]triphosphate than in the presence of either ATP or ADP, indicating that the hydrolysis of ATP may result in different conformational effects than does the binding of this ligand. These findings are discussed within the context of recent models of rho-dependent transcription termination.
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PMID:Escherichia coli transcription termination factor rho has a two-domain structure in its activated form. 258 Mar 3

Aggregation and autophosphorylation of the insulin receptor-protein kinase, from cultured 3T3-L1 adipocytes, were studied in the presence of cationic polyamino acids. Poly-L-lysine and poly-L-arginine produced the following effects with the purified receptor: first, the autophosphorylation rate was increased by polycations. Half-maximal stimulation was proportional to polymer length. The rate enhancement was greater at lower ATP concentrations. Second, near-endpoint (equilibrium) autophosphorylation was greater in the presence of the polycations. Polycations inhibited the reverse reaction: ADP + phosphoreceptor yielding ATP + aporeceptor. Third, the [32P]phosphopeptides generated by trypsin digestion of the 32P-beta-subunit, showed that no new autophosphorylation sites resulted from the presence of polycations. Fourth, the polycations, but not insulin, promoted receptor aggregation, and phosphoreceptor aggregated more readily than aporeceptor. Insulin receptor enriched through the wheat germ agglutinin eluate step was compared with purified receptor. Higher concentrations of poly-L-arginine were required to stimulate autophosphorylation and to promote aggregation. Finally, several polycation-dependent substrates present in the wheat germ agglutinin eluate co-aggregated with the insulin receptor. Polycation-stimulated receptor autophosphorylation is linked to a lower KM,app for ATP, but substrate phosphorylation may require the aggregation.
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PMID:Insulin receptor aggregation and autophosphorylation in the presence of cationic polyamino acids. 259 62

Myosin (opaque myosin) isolated from the opaque portion of scallop smooth muscle, a catch muscle, was subjected to limited digestion by trypsin during the steady-state ATPase reaction. The 200-kDa heavy chain of opaque myosin was cleaved into 125- and 74-kDa fragments. The proteolytic rate in the absence of Ca2+ was lower than that in the presence of Ca2+, and was similar to that in the presence of ADP and absence of Ca2+. The results suggest that the steady-state intermediate of opaque myosin ATPase in the absence of Ca2+ is EADP, which is consistent with the previous results based on the difference UV-absorption spectrum (Takahashi, M., Sohma, H., & Morita, F. (1988) J. Biochem. 104, 102-107). In the presence of F-actin, the proteolytic rates were decreased, but the digestive patterns by trypsin were similar to those of myosin alone. Even in the presence of F-actin, the proteolytic rate during the ATPase reaction in the absence of Ca2+ was lower than that in the presence of Ca2+, and was similar to that in the presence of ADP and absence of Ca2+. In addition, there was another trypsin-susceptible site which is probably located at 18 kDa from the N-terminal of the heavy chain. The site in the absence of Ca2+ was hardly cleaved when ATP or ADP was present. Similar tendencies were observed even in the presence of F-actin. These findings suggest that the intermediate of opaque myosin ATPase at the steady state in the absence of Ca2+ is EADP even in the presence of F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Myosin may stay in EADP species during the catch contraction in scallop smooth muscle. 261 94

The nucleotide affinity label 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate (2-BDB-TADP) reacts covalently with pig heart NAD+-dependent isocitrate dehydrogenase with a limiting value of 75% inactivation and loss of ADP activation concomitant with incorporation of about 1 mol of reagent/mol of average enzyme subunit (Huang, Y.-C., Bailey, J. M., and Colman, R. F. (1986) J. Biol. Chem. 251, 14100-14107). Complete protection against the functional changes is provided by ADP + Mn2+, and reagent incorporation is decreased to about 0.5 mol/mol of average enzyme subunit. We have now identified the critical modified peptide by comparison of the peptides labeled by 2-BDB-TADP at pH 6.8 in the absence and presence of ADP + Mn2+. After removal of excess reagent, modified enzyme was treated with [3H]NaBH4 to reduce the keto groups of the reagent and introduce a radioactive tracer into the reagent which is covalently linked to the protein. Following carboxymethylation and digestion with trypsin, the specific modified peptide was isolated using two successive high performance liquid chromatography steps: 1) 0.1% trifluoroacetic acid with an acetonitrile gradient; and 2) 20 mM ammonium acetate, pH 5.8, with an acetonitrile gradient. Gas phase sequencing gave the modified peptide Leu-Gly-Asp-Gly-Leu-Phe-Leu-Gln in which aspartic acid is the target of 2-BDB-TADP. Isolation of the corresponding tryptic peptide from unmodified enzyme yielded the sequence Leu-Gly-Asp-Gly-Leu-Phe-Leu-Gln-CmCys-CmCys-Lys. Isocitrate dehydrogenase is composed of three distinct subunits (alpha, beta, and gamma), separable by chromatofocusing in urea and identified by analytical gel isoelectric focusing. The evidence indicates that the specific peptide labeled by 2-BDB-TADP, which is at or near the ADP site, can be derived from the gamma subunit.
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PMID:Aspartyl peptide labeled by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate in the allosteric ADP site of pig heart NAD+-dependent isocitrate dehydrogenase. 274 37

Pig gastric membrane vesicles enriched in (H+ + K+)-ATPase were covalently modified with pyridoxal 5'-phosphate (PLP). The modification resulted in inhibition of K+-dependent ATP hydrolysis, formation of phosphoenzyme and ATP-driven H+-uptake catalyzed by (H+ + K+)-ATPase. ATP, ADP, and adenyl-5'-yl imidodiphosphate were protective ligands, whereas Mg2+ and K+ were not. Specific PLP-binding of about 4.5 nmol/mg membrane protein was necessary for complete inhibition of the enzyme activity, indicating that the stoichiometry of PLP-binding to the enzyme was about 1:1. Limited proteolysis of the enzyme modified with [3H]PLP by trypsin suggests that PLP specifically modifies the lysine residue located in the 16-kDa fragment of the enzyme cleaved by trypsin. These results suggested that PLP binds to a specific lysine residue in the nucleotide-binding site or a region in its vicinity and inhibits the substrate binding or phosphorylation step of (H+ + K+)-ATPase.
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PMID:Modification of gastric (H+ + K+)-ATPase with pyridoxal 5'-phosphate. 283 Nov 98

The regulatory site peptide sequence of phosphorylated inactive pyruvate, orthophosphate dikinase from maize leaf tissue was determined by automated Edman degradation analysis of 32P-labeled peptides purified by reversed-phase high performance liquid chromatography. The overlapping phosphopeptides were products of a digestion of the [beta-32P]ADP-inactivated dikinase with either trypsin or Pronase E. The sequence is Thr-Glu-Arg-Gly-Gly-Met-Thr(P)-Ser-His-Ala-Ala-Val-Val-Ala-Arg. The phosphothreonine residue, which appeared as either an anomalous proline or an unidentifiable phenylthiohydantoin derivative during sequencing, was verified by two-dimensional phosphoamino acid analysis of the phosphopeptides and by resequencing the tryptic peptide after dephosphorylation with exogenous alkaline phosphatase. This sequence, starting at position 4, is completely homologous to the previously published sequence of the tryptic dodecapeptide harboring the catalytically essential (phospho)histidyl residue in the active-site domain of the dikinase from the nonphotosynthetic bacterium, Bacteroides symbiosus (Goss, N.H., Evans, C.T., and Wood, H.G. (1980) Biochemistry 19, 5805-5809). These comparative results indicate that the regulatory phosphothreonine causing complete inactivation of maize leaf dikinase is separated from the critical active-site (phospho)histidine by just one intervening residue in the primary sequence.
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PMID:Sequence of the phosphothreonyl regulatory site peptide from inactive maize leaf pyruvate, orthophosphate dikinase. 283 85

Three to six mg of the millimolar Ca2+-requiring proteinase (m-calpain) were obtained from 1 kg bovine cardiac muscle (fresh wt) and some enzymatic properties of this proteinase were determined. Activity of bovine cardiac m-calpain decreases as ionic strength increases from 75 to 1000 mM. Maximal activation of m-calpain by Ca2+, La3+, Ba2+, and Mn2+ occurs at 2 to 3 mM concentrations of each of these divalent cations, but La3+ activation is only 20 to 25% and Ba2+ and Mn2+ activation only 6 to 10% as great as Ca2+ activation. Maximum Sr2+ activation occurs at 20 mM Sr2+ and is 90 to 95% of maximum Ca2+ activation. Mg2+, Zn2+, Cr2+, and Cd2+ do not activate m-calpain when added alone; Mg2+ does not affect, but Zn2+ inhibits, Ca2+-stimulated activity. The nonionic detergents, Triton X-100 and Brij 35, activate m-calpain 1.6- to 2.0-fold but do not change its Ca2+ requirement. Sodium dodecyl sulfate and urea inhibit m-calpain completely at 0.045% and 2.0 M, respectively. Because they bind Ca2+ needed for activation, ATP, ADP, and ITP inhibit m-calpain. The trypsin inhibitors, phenylmethylsulfonyl fluoride, ovomucoid trypsin inhibitor, ovoinhibitor, aprotinin, alpha 1-antiproteinase inhibitor, soybean trypsin inhibitor, and lima bean trypsin inhibitor do not affect m-calpain activity; m-calpain does not release measureable quantities of acid-soluble peptides from a rabbit skeletal sarcoplasmic protein fraction but does degrade rabbit skeletal myofibrils and casein.
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PMID:Some properties of the millimolar Ca2+-dependent proteinase from bovine cardiac muscle. 285 32


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