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)

The histidine residues in human chorionic gonadotropin (hCG) were chemically modified using diethyl pyrocarbonate. Derivatives of hCG with an average of 0.5-3.5 histidines modified (maximum of 4 per hCG) had reduced receptor-binding and cell-stimulating activities. Acylation of hCG at progressively lower pH values (conditions in which 1 of the 2 absolutely conserved histidines alpha His-83 is not titratable, whereas alpha His-94 becomes increasingly protonated and resistant to modification) produced hCG derivatives with a greater retention of receptor-binding activity than cell-stimulating activity. The involvement of alpha His-94 as part of the receptor-binding region of the hormone and of alpha His-83 as a putative active site residue was inferred. Proteinaceous protease inhibitors were shown to neutralize the agonist activity of hCG and to reduce the binding of hCG to its receptor and also to specific antisera. It was presumed that an inhibitor-hormone complex was formed which was analogous to the complexing of inhibitor with the "substrate pocket" of a serine protease. The discovery of primary sequence analogies between hCG and the serine protease chymotrypsin enabled the prediction of hCG structure using the enzyme as a folding template. Solvent-exposed and buried core regions of the peptide chain were delineated using smoothed hydrophobicity profiles in combination with Chou-Fasman secondary structure predictions. Hypervariable hydrophobicity indices between residues 38 and 80 of the human beta subunits reflected different folding arrangements which presumably conferred the individual receptor specificities. When mapped to the putative structure these receptor-determinant loops were adjacent to an area of the alpha subunit analogous to the substrate pocket of serine proteases. Disulfide bond assignments and intersubunit contact regions were identifiable. The proposed tertiary structure for hCG manifests the topographical epitopes defined using monoclonal antibodies and satisfies the currently available data on specific modification and its effects upon hormonal structure and function. This paper is considered to be the first report of a differential effect upon the agonist and receptor binding abilities of a glycoprotein hormone after modification of the proteinaceous, as opposed to the glycosylated, moiety of the molecule.
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PMID:Functionally distinct agonist and receptor-binding regions in human chorionic gonadotropin. Development of a tertiary structure model. 258 90

We investigated the location of platelet-specific alloantigen Baka on platelet membrane glycoproteins. In indirect immunoprecipitation experiments, the anti-Baka antibody precipitated glycoprotein (GP) II b and a small amount of GP III a. The immunoblots using partially purified GP II b/III a complex as the target antigen indicated that GP II b alpha carried the Baka alloantigen. When the partially purified GP II b/III a complex digested with chymotrypsin was examined, the Baka alloantigen was found on a 65 kD fragment derived from GP II b alpha under reducing conditions. In addition, the immunoblots after two-dimensional nonreduced-reduced SDS-PAGE directly indicated that the 65 kD fragment had a mol. wt. of 80 kD under nonreducing conditions. The immunoblots using platelets digested in situ with chymotrypsin indicated that the 65 kD fragment of GP II b alpha was retained by the platelet membrane. We conclude, therefore, that the Baka alloantigen is located on a 65 kD fragment that represents the membrane side of the cleavage site of chymotrypsin on GP II b alpha.
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PMID:[Immunochemical characterization of platelet-specific alloantigen Baka]. 258 47

Thrombin interacts with a platelet protein which is immunologically related to fibroblast protease nexin and has been termed platelet protease nexin I (PNI). Conflicting hypotheses about the relationship of the thrombin-PNI complex formation to platelet activation have been proposed. The studies presented here demonstrate that the platelet-associated and supernatant complexes with added 125I-thrombin are formed only under conditions which produce platelet activation in normal and chymotrypsin-modified platelets. The platelet-associated complex is formed prior to the appearance of complexes in supernatants. Appearance of the supernatant complex coincides with the appearance of thrombospondin in the reaction supernatants. Excess native thrombin, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide or hirudin can prevent radiolabeled platelet-associated complex formation if added before 125I-thrombin. DAPA or hirudin can prevent or dissociate complex formation if added up to one minute after thrombin but not at later time points. The surface associated complex is accessible to trypsin although a portion remains with the cytoskeletal proteins when thrombin-activated platelets are solubilized with Triton X 100. The surface-associated complex formation parallels many aspects of the specific measurable thrombin binding, yet it does not appear to involve other identified surface glycoprotein thrombin receptors or substrates. Although the time course of appearance of the complexes in supernatants is consistent with other data which suggest that PNI may be released from platelet granules during platelet activation, other explanations for the appearance of PNI on the platelet surface and in supernatants during platelet activation are possible.
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PMID:The interaction of thrombin with platelet protease nexin. 259 74

Optimum conditions were determined for solubilizing cell membrane receptors for binding measles virus (MV). Evidence for specific receptors was shown from the saturability of MV binding to intact Vero cells or African Green Monkey erythrocytes (MRBCs). Receptors, solubilized from Vero cells and MRBC with 1.5% octyl glucoside, inhibited MV attachment, infectivity, and hemagglutination activities. Extracts from chicken erythrocytes, which did not bind MV, were inactive in all assays. MV binding activity in Vero or MRBC extracts was stable to heating (100 degrees, 10 min) or neuraminidase treatment, but was inactivated by a range of proteases, including chymotrypsin, and bound to lentil and pea lectin agaroses, to indicate a glycoprotein component.
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PMID:Characterization of octyl glucoside-solubilized cell membrane receptors for binding measles virus. 267 66

The Minnesota strain of turkey enteric coronavirus (TCV) was grown on a human rectal tumor (HRT-18) cell line in the presence of radiolabeled amino acids and glucosamine to analyse virion structural proteins. In addition to the 52,000 unglycosylated nucleocapsid protein, three major glycoprotein species were found to be associated with the viral envelope. A predominant glycosylated protein with a molecular weight of 22-24,000 represented the transmembrane matrix protein. Larger glycoproteins with apparent molecular weights of 180-200,000 (gp 200), 120-125,000 (gp 120) and 95-100,000 (gp 100) were associated to the characteristic large bulbous projections (peplomers) located at the surface of the virion. The gp 100 and gp 120 species apparently arose from a proteolytic cleavage of gp 200, as suggested by digestion studies with trypsin and chymotrypsin. An additional large glycoprotein with mol. wt. of 140,000 (gp 140), that behaved as a disulfide-linked dimer of a 66,000 molecule, was found to be associated to granular projections located near the base of the large peplomers. Digestion studies with trypsin, bromelain and pronase demonstrated that gp 140 was related to the hemagglutinating activity of the virus. An inner membranous sac or tongue-shaped structure could be visualized in the interior of the viral particles following treatment with pronase. In contrast, trypsin or chymotrypsin treatments resulted in evaginations ("budding") on the virus surface. Progeny viral particles produced in TCV-infected cell cultures in the presence of tunicamycin lacked both types of surface projections, as demonstrated by electron microscopy and electrophoresis. The matrix protein also appeared to be reduced to its unglycosylated form, concomitant with a considerable loss of its antigenicity. Thus, with respect to its morphological and biochemical characteristics, TCV resembles viruses belonging to the group of mammalian hemagglutinating coronaviruses, but differs in that both types of envelope glycoproteins are N-glycosylated as in case of the avian infectious bronchitis virus.
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PMID:Identification and location of the structural glycoproteins of a tissue culture-adapted turkey enteric coronavirus. 267 55

The levels of proteolytic activity in cell washes, lysates and pellets of C. pylori and gastric Campylobacter-like organisms isolated from humans and ferrets, respectively, have been studied using porcine mucus glycoprotein and bovine haemoglobin substrates. The total haemoglobin degrading activity, expressed by 10(12)-10(13) cfu of either organism, was no greater than 3 micrograms chymotrypsin equivalents. The mucolytic specific activity (rate of mucus peptide bond hydrolysis by bacterial protein) of the fractions tested from both organisms did not exceed 2nmol/min/mg protein. This value is 1000-fold lower than expected from published data. Electrophoretic profiles suggested that the mucolytic activity assessed by fluorimetry was insufficient to alter the quaternary structure of mucus and hence may not significantly contribute to the undermining of gastric mucus integrity.
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PMID:Proteolytic activities of human Campylobacter pylori and ferret gastric Campylobacter-like organism. 267 33

The authors isolated a product of proteolytic degradation of glycoprotein IIIa (GPIIIa) which is formed on the surface of human platelets during incubation with chymotrypsin and which was previously described as the 66 kDa platelet membrane component. This component migrated with an apparent Mr 62,400 in a non-reduced system of sodium dodecyl sulfate polyacrylamide gel electrophoresis. In a reduced system it yielded two major subunits migrating with apparent Mr 14,000-17,000 and 65,000. The low-molecular weight component began with the NH2-terminal sequence of GPIIIa (GPNICTTR...) and the larger component with residue 348 of GPIIIa (GKIRSKKA...) as deduced from a cDNA clone of this glycoprotein. The two subunits appeared to be linked by one or more S-S bridges supporting the contention that GPIIIa is a highly folded molecule on the platelet membrane. In contrast to GPIIIa, the '66 kDa component' did not bind to GRGDSPK-agarose, to fibrinogen-agarose nor to insolubilized monoclonal antibody recognizing the GPIIb/IIIa complex. The exposure of fibrinogen receptors during the course of incubation of platelets with chymotrypsin preceded the formation of the '66 kDa component' characterized in this study. An intermediate product of GPIIIa proteolysis migrating with an apparent Mr 120,000 in a non-reduced system and Mr 80,000 in a reduced system was identified as a precursor of the '66 kDa component'. The '120 kDa component' was not retained on GRGDSPK-agarose or on fibrinogen-agarose but it was retained on insolubilized antibody recognizing the GPIIb/IIIa complex. Incubation of platelets with porcine pancreatic elastase or human granulocytic elastase resulted in the formation of similar proteolytic degradation fragments.
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PMID:Structural and functional characterization of major platelet membrane components derived by limited proteolysis of glycoprotein IIIa. 275 53

Allantoic fluids (n = 65) of Day 24-37 bovine conceptuses were collected and assayed for luteotrophic activity in vitro with dispersed bovine luteal cells. Significant luteotrophic activity was found in 41% of the samples, with the highest percentage occurring between Days 25 and 28. The activity is ammonium sulphate-precipitable, heat-labile and inactivated by trypsin and chymotrypsin. Gel filtration column chromatography identified one peak of luteotrophic activity with a molecular weight of 68,000. Concanavalin A bound the luteotrophic activity, thus allowing rapid and substantial purification from a major protein of Mr 68,000 which was concanavalin A non-reactive. The results of one- and two-dimensional SDS-PAGE of concanavalin A-reactive fractions containing activity suggest that the luteotrophic activity present in allantoic fluid is associated with a glycoprotein of Mr 68,000 present in very low concentrations. The active factor does not appear to be alpha-fetoprotein. This protein may be an important conceptus-derived luteotrophin that stimulates progesterone production by the corpus luteum of cows during pregnancy.
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PMID:Identification of a luteotrophic protein in bovine allantoic fluid. 281 Feb 32

The glycoprotein thrombospondin is distributed between the extracellular matrix and the platelet-sequestered pool in the resting state and it undergoes redistribution upon platelet stimulation. It is believed to play a role in matrix structure and in coagulation. We have studied the structural domains of endothelial cell (EC) thrombospondin by use of the serine proteases thrombin, trypsin and chymotrypsin and have characterized the heparin-binding domains of this molecule. For this purpose we used purified thrombospondin synthesized and secreted by bovine aortic endothelial cells grown in the presence of radiolabeled methionine. We find that the susceptibility of EC thrombospondin to proteolysis is five-fold smaller than that of platelet thrombospondin. In the presence of 2 mM Ca ions the molecule is cleaved by 20 U/ml thrombin at a single locus, to yield fragments of 160 kDa and 35 kDa. Trypsin digestion for 5 min at room temperature at an enzyme-to-substrate ratio of 1:20 produces a stable fragment of 140 kDa but not the 30-kDa fragment observed in platelet thrombospondin. Chymotrypsin, under identical conditions to those used for trypsin, cleaves EC thrombospondin into four stable fragments of 160 kDa, 140 kDa, 27 kDa and 18 kDa. Chelation of Ca by EDTA increases susceptibility of the molecule to proteolysis. Under the conditions used a cryptic thrombin-cleavage site, not hitherto observed in platelet thrombospondin, was observed in EC thrombospondin. The location of this site is near a chymotrypsin-susceptible site, which has been observed in the long connecting arm, which is particularly Ca-stabilized. Heparin-binding capacity of EC thrombospondin was observed in at least two separate loci. Both thrombin and chymotrypsin produced small fragments (35 kDa and 27 kDa respectively) which bound to heparin with high affinity, and large fragments (160 kDa for thrombin and 140 kDa for chymotrypsin) which had low affinity. Chelation of Ca substantially decreased the low-affinity binding of the large fragments but not the high-affinity binding of the small fragments. Two-dimensional gel electrophoresis of the chymotryptic heparin-binding fragments shows that each molecule gave rise to a heterogeneous array of fragments of high molecular mass bound by disulfide bonds, indicating that there is a difference in the rate of cleavage between the three subunits of EC thrombospondin. Trypsin, despite its limited degradation, completely eliminated the heparin-binding capacity of both high and low-affinity loci, in contrast to platelet thrombospondin where the high affinity remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The structure of endothelial cell thrombospondin. Characterization of the heparin-binding domains. 282 10

We have defined three categories of cultured cell lines on the basis of their permissiveness (susceptibility to initial infection) to mouse hepatitis virus (MHV). Fully permissive L-2 cells gave rise to 100-1000-fold higher numbers of infectious centers than did semi-permissive LM, LM-K or C-1300 cells, whereas non-permissive Vero or C-6 cells were refractory to MHV infection. On an infected cell basis, there was no deficiency on the part of semi-permissive cell lines to replicate total viral RNA, viral polypeptides or progeny virions. Two of the semi-permissive cell lines (LM and LM-K) supported persistent MHV infection, while a third (C-1300) succumbed to lytic infection. LM and LM-K cells, but not C-1300 cells showed resistance to MHV-induced membrane fusion, even when placed in contact with fusion-active MHV-infected L-2 cells. The ability of a given cell to undergo fusion did not correlate with membrane lipid characteristics (unsaturated fatty acid and sterol content) which contribute to membrane "fluidity". In order to more closely study the parameters of MHV-induced cell fusion, membranes were prepared from MHV-infected L-2 cells and monitored for their fusogenic potential with permissive L-2 cells, semi-permissive LM cells and non-permissive vero cells. Fusion was only observed with the permissive L-2 cells, and only when exogenous protease (trypsin or chymotrypsin) was added. When the membranes were prepared from 35S-methionine-labeled MHV-infected L-2 cells and subjected to protease treatment, the radiolabeled 180,000 dalton form of the E2-glycoprotein underwent proteolytic cleavage to yield a major product of approximately 90,000 daltons. Both trypsin and chymotrypsin were effective in this proteolytic cleavage and in activating membrane fusion. In a normally permissive, fusogenic infection of MHV in L-2 cells, the protease inhibitors TPCK and ZPCK, but not TLCK, were found to inhibit cell fusion. In MHV-infected L-2 cells, E2 was found almost exclusively as the 180,000 dalton form but turned over rapidly as shown by pulse-chase studies. TPCK and ZPCK but not TLCK inhibited turnover. The results suggest that L-2 cells contain a protease which cleaves at aromatic amino acids such as phenylalanine, and that this protease cleaves the 180,000 dalton form of the E2 to peptide fragments, one or more of which may activate cell fusion.
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PMID:The role of protease-dependent cell membrane fusion in persistent and lytic infections of murine hepatitis virus. 282 27


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