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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A plasmalemmal Na(+)-Ca2+ exchange mechanism is an important electrogenic determinant of contractility in cardiac cells. As in other cell types, calcium influx by Na(+)-Ca2+ exchange is secondarily activated by cytoplasmic calcium and probably ATP, but these modulatory mechanisms are either absent or altered in isolated cardiac sarcolemmal vesicles. Involvement of a calcium-dependent protein kinase in exchange regulation has been suggested but not verified. Here I describe measurements of outward Na(+)-Ca2+ exchange current, corresponding to calcium influx, in giant excised sarcolemmal patches from guinea pig myocytes. The exchange current is stimulated by both calcium and Mg-ATP from the cytoplasmic face, evidently through separate mechanisms. Activation by cytoplasmic calcium takes place within seconds, is reversible, and does not require ATP. Stimulation by Mg-ATP reverses only slowly over greater than 10 min, or not at all. Unexpectedly, a substantial decrease in exchange current occurs during activation by cytoplasmic sodium, which seems to reflect an inactivation process rather than ion concentration changes or a 'first pass' exchange cycle. This apparent inactivation, and the modulations by cytoplasmic calcium and Mg-ATP, are all abolished by brief treatment of the cytoplasmic surface with chymotrypsin, leaving the exchanger in a maintained state of high activity. Therefore, limited proteolysis deregulates Na(+)-Ca2+ exchange and could contribute to the loss of secondary regulation of the exchange in isolated sarcolemmal vesicles.
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PMID:Regulation and deregulation of cardiac Na(+)-Ca2+ exchange in giant excised sarcolemmal membrane patches. 231 60

p36 is a major substrate of both viral and growth factor-receptor-associated tyrosine protein kinases. p36 can be isolated as a complex consisting of a subunit of Mr 36,000 (p36) and a subunit of Mr 10,000 (p10), and it represents an abundant cellular protein. We have isolated the p36-p10 complex from bovine intestinal epithelium and analyzed the amino terminus of both subunits. Sequence analysis of the first 56 amino acids of p10 demonstrates a striking sequence homology (48% identically placed residues) with the Mr 10,000 calcium-binding proteins from bovine brain, termed S-100. Intestinal p36 could be effectively labeled on a single tyrosine in vitro with immunoprecipitated pp60v-src and [gamma-32P]ATP. Mild proteolysis of p36 with chymotrypsin resulted in the cleavage into large (Mr, 33,000) and small domains (Mr, 3000), with the latter representing the phosphorylated amino terminus. Although the amino terminus is apparently blocked, sequence analysis of a secondary tryptic peptide of the Mr 3000 fragment as well as the amino-terminal sequence of the Mr 33,000 domain and overlapping peptides clearly established the site of tyrosine phosphorylation.
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PMID:Amino-terminal sequence of p36 and associated p10: identification of the site of tyrosine phosphorylation and homology with S-100. 241 74

Two intrinsic proteins of bovine lens membranes with apparent relative molecular masses (Mr, app) of 26,000 and 18,000 were phosphorylated in intact membranes by protein kinase C prepared from either bovine brain or lens. The kinase preparations exhibited histone H1 phosphorylation dependent on calcium and phospholipid but not on cAMP. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the lens membranes showed a major band at Mr, app = 26,000 (identified as MP26, the main intrinsic protein of lens fiber cells), an intermediate band at Mr, app = 18,000 and several minor bands. Autoradiography of complete assay mixture containing protein kinase C, calcium, magnesium and [gamma-32P]ATP showed major bands at Mr, app = 18,000 and 26,000. Several lines of evidence indicated that the label at Mr, app = 26,000 was associated with MP26, a protein which has been found in lens junctions and which may form cell-cell channels. Treatment of the phosphorylated membranes with chymotrypsin and V8 protease cleaved the major band at Mr, app = 26,000 to fragments of Mr, app .= 22,000 and 24,000. Label was not detected in the resulting Mr, app = 22,000 peptide, but the Mr, app = 24,000 peptide was found to be labeled. Phosphoamino acid analysis of MP26 indicated that approximately 75% of the label was on phosphoserine and 25% was on phosphothreonine. No label was found on phosphotyrosine. These results differ from those reported for cAMP-dependent phosphorylation of lens proteins. Phosphorylation by protein kinase C may account for some of the labeling of MP26 detected in vivo.
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PMID:Phosphorylation of lens intrinsic membrane proteins by protein kinase C. 242 29

In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined. In order to visualize the proteolyzed EP forms we have induced the formation of a maximum level EP from [gamma-32P]ATP in the presence of Ca2+ + La3+ and applied a good-resolution acidic discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteolysis of inside-out vesicle membranes by trypsin, Pronase, papain, or chymotrypsin produces a calmodulin-like activation of the calcium pump, abolishes its calmodulin sensitivity, and decreases the original 140-kDa EP complex to a limit polypeptide of 80 kDa. Trypsin digestion produces another major intermediary fragment of 90 kDa, which is still a low-activity calmodulin-sensitive form of the pump. The red cell calcium pump is activated by trypsin both in the absence and presence of Ca2+ during digestion although the rate of activation and the appearance of the 80-kDa polypeptide are enhanced by Ca2+. If proteolytic digestion is carried out by chymotrypsin, a calmodulin-insensitive maximum activation of the calcium pump coincides with the formation of a 125-130-kDa EP-forming polypeptide. Chymotrypsin and carboxypeptidase A have synergistic effects on the formation of this latter high-activity species. Based on these data we suggest a probable molecular arrangement for the functional parts of the red cell membrane calcium pump.
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PMID:Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis. 242 14

A bland procedure, conducted in ice, is described for the extraction with HCl of smooth-muscle-contracting substances from plexus-containing ileal longitudinal muscle (l.m.) sheets obtained mainly from rabbits and some guinea-pigs. The spasmogenic activity in rabbit extracts was distinguished from acetylcholine, histamine and 5-hydroxytryptamine by antagonists; and from prostaglandins, by its insolubility in ether at acid pH and by pretreatment of the animals with indomethacin. The fact that it contracts the separated l.m. of the guinea-pig ileum, whether plexus-containing or plexus-free, and in atropine distinguishes it also from methionine-enkephalin, somatostatin, 13-norleucine motilin, bombesin, and cholecystokinin octapeptide (CCK8). This activity was partially purified, first by several partitions with ether at pH 1.4-2.2 and then by treatment at pH 4.5-5 with lead acetate. The virtual absence of ATP was confirmed by the firefly bioluminescence technique. The guinea-pig-ileum-contracting component in the partially purified extracts was destroyed by pepsin, chymotrypsin and DPCC-treated trypsin, indicating its peptide nature and distinguishing it from oxytocin, vasopressin, bradykinin, etc. In parallel assays the partially purified rabbit extracts were considerably more active than Substance P on jird or rat ascending colons than on the guinea-pig l.m., suggesting the presence of a second spasmogenic component in the extracts. In guinea-pig extracts the partially purified activity was 8-16 times greater when plexus-containing than when plexus-free, pointing to Auerbach's plexus as the source of the activity.
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PMID:Extraction and partial purification of spasmogenic substances in Auerbach's plexus. 242 21

Incubation of human plasma with 27 nM [gamma-32P]ATP in the presence of 20 mM MnCl2 results in the phosphorylation of several proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. About 60% of the incorporated radioactivity is found in a 75-kDa protein containing [32P] phosphoserine. The amino-terminal amino acid sequence of the purified 75-kDa [32P]phosphoprotein is identical to that of vitronectin (also termed serum spreading factor or complement S protein). Rabbit antiserum against vitronectin precipitates greater than 90% of the 75-kDa [32P]phosphoprotein from plasma. Reverse phase chromatography of [32P]vitronectin degraded sequentially with CNBr and chymotrypsin yields one major labeled peptide. The sequence of the peptide, Ser-Arg-Arg-Pro-[32PO4]Ser-Arg-Ala-Thr, corresponds to residues 374-381 which are located in the heparin-binding fragment of vitronectin identified by Suzuki et al. [1984) J. Biol. Chem. 259, 15307-15314). Vitronectin could potentially be phosphorylated in vivo with ATP released from injured cells or secreted by platelets activated during hemostasis.
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PMID:Phosphorylation of vitronectin by a protein kinase in human plasma. Identification of a unique phosphorylation site in the heparin-binding domain. 244

1. The effect of an acid extract of the carp intestinal bulb (ECI) on guinea-pig ileum longitudinal smooth muscle (GPLM) and carp intestinal bulb longitudinal smooth muscle (CIBLM) was examined. 2. ECI caused a concentration-dependent contraction of GPLM and CIBLM. This ECI-induced response was reduced by atropine to 30-40% of the control, indicating that part of the contracting activity of ECI is attributable to acetylcholine. The atropine-resistant contracting activity of ECI was not mediated by histamine, 5-hydroxytryptamine, ATP, ADP, angiotensin II, neurotensin, vasoactive intestinal peptide or an opioid peptide. 3. The active material mediating the atropine-resistant contracting activity is probably a peptide, because the contraction in response to ECI was abolished on incubation with pepsin or alpha-chymotrypsin. 4. [D-Pro2, D-Trp7,9]-substance P, [D-Pro4, D-Trp7,9]-substance P (4-11) decreased the atropine-resistant contracting activity of ECI as did desensitization induced by substance P. 5. On a Sephadex G 25 column, the active material was eluted as one peak. The active fractions were pooled and then applied to another Sephadex G25 column to compare the Ve/Vo value for the active material with those for peptides of known molecular weights. The molecular weight of the active material was estimated to be 1200-1700 (1410 +/- 70, n = 6). 6. The results indicate the presence of a substance P-like peptide in the carp intestinal bulb.
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PMID:Presence of a substance P-like peptide in an acid extract of the intestinal bulb of the carp (Cyprinus carpio). 246 88

Monoclonal antibodies raised against chicken gizzard smooth muscle myosin light chain kinase were used for immunological and structural studies of this enzyme. Epitope mapping of trypsin-digested chicken gizzard enzyme showed that MM-1, 2, 3, 4, 5, 6, and 7 bind to 65 kDa (trypsin-digested) and 60 kDa (chymotrypsin-digested) fragments which contain the catalytic domain of the kinase. Kinetic analysis demonstrated that MM-7 inhibited kinase activity competitively with respect to ATP and noncompetitively with respect to myosin light chain, thereby indicating that MM-7 binds at or near the ATP binding site of the enzyme. Immunoblot analysis revealed that all these antibodies (MM-1 to 12) reacted with the enzyme (130 kDa) from intestinal and vascular smooth muscles, whereas 5 (MM-1, 3, 4, 6, and 9) or 3 (MM-1, 3, and 4) of 12 antibodies did not cross-react with chicken cardiac muscle or with blood platelet myosin light chain kinase (130 kDa), respectively. None of these antibodies showed cross-reactivity against skeletal muscle myosin light chain kinase. As for mammalian species, MM-11 and 12 reacted with myosin light chain kinase of vascular smooth muscle (140 kDa) and MM-11 cross-reacted with the enzyme (140 kDa) from cardiac muscle of rat and rabbit. These data suggest the existence of at least 4 subspecies of myosin light chain kinase in chicken tissues and the heterogeneity of tissue- and species-specific isozyme forms.
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PMID:Monoclonal antibody assessment of tissue- and species-specific myosin light chain kinase isozymes. 247 31

We recently identified a novel protein tyrosine kinase that specifically phosphorylates truncated pp60c-src (Mr = 53,000) at a tyrosine residue(s) distinct from its autophosphorylation site. In this study, we examined whether this enzyme phosphorylates intact pp60c-src (Mr = 60,000) and determined its phosphorylation site. Non-neuronal and neuronal forms of intact pp60c-src were separately purified from the membrane fraction of neonatal rat brain by sequential column chromatographies. The novel kinase phosphorylated tyrosine residues of both forms of intact pp60c-src. The phosphorylation occurred in parallel with autophosphorylation of pp60c-src, and in both forms the final stoichiometry estimated was quite similar to that of autophosphorylation (about 5%). The enzyme also phosphorylated pp60c-src in which the kinase activity had been destroyed by an ATP analogue, p-fluorosulfonylbenzoyl 5'-adenosine. The phosphorylation site of the non-neuronal form was analyzed by sequential peptide mapping with tosylphenylalanyl chloromethyl ketone-treated trypsin and alpha-chymotrypsin. Tryptic digestion of the phosphorylated pp60c-src yielded a unique phosphopeptide that cross-reacted with an antibody specific for the carboxyl-terminal sequence of chicken pp60c-src. Digestion of the phosphopeptide with chymotrypsin yielded a product that comigrated with a synthetic phosphopeptide corresponding to the carboxyl-terminal 15 residues of chicken pp60c-src. These results clearly indicate that the carboxyl-terminal sequence of rat pp60c-src is identical to that of chicken pp60c-src, and a tyrosine residue corresponding to chicken Tyr527 is the phosphorylation site. This phosphorylation resulted in a decrease in the enolase phosphorylating activity of pp60c-src. Kinetic experiments indicated that this decrease in activity was due to a decrease in the Vmax value of pp60c-src. These findings support our previous proposal that the novel tyrosine kinase acts as a specific regulator of pp60c-src in cells.
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PMID:A protein tyrosine kinase involved in regulation of pp60c-src function. 248 Mar 46

Four proteases have been used to assess the topology of the H+-ATPase from Saccharomyces cerevisiae reconstituted into phosphatidylserine vesicles. Limited proteolysis by trypsin and alpha-chymotrypsin inactivates the enzyme and produces stable, membrane-bound fragments. Sequence analyses of these peptides have located the peptide bonds hydrolyzed. The labile bonds are on opposite sides of a central hydrophilic domain containing consensus sequences for the site of phosphorylation and fluorescein isothiocyanate binding of several related ATPases. Limited proteolysis of the ATPase by elastase cuts approximately 50 amino acids from the C terminus, leaving the remaining membrane-bound fragments active. Proteolysis by carboxypeptidase Y in the presence and absence of detergent suggests that the C terminus is on the inside of the vesicle in this reconstitution. A model for the transmembrane arrangement of the polypeptide is proposed. In this model, the C terminus is on the inside of the vesicle, the N terminus is on the outside, the ATP binding region is on the outside, and the polypeptide passes through the membrane a minimum of five times.
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PMID:Topology of the yeast plasma membrane proton-translocating ATPase. 252 Dec 19


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