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)

Quiescent cultures of chick embryo fibroblasts incubated with human alpha-thrombin (14-219 pM) incorporated [methyl-3H]thymidine proportional to concentration. Inactivated forms of this protease (e.g. active-site-conjugated alpha-thrombin or its hirudin complex) had no mitogenic activity and did not compete with 124I-alpha-thrombin for binding to specific plasma membrane receptors. The noncoagulant but esterolytic active forms, gamma- and nitro-alpha-thrombins, were weakly mitogenic and correspondingly competed weakly for binding. Trypsin competed equally as well as native thrombin for binding, whereas chymotrypsin, elastase, and human urokinase competed with 80-fold less affinity. Plasma, arginine-specific proteases associated with nerve or epidermal growth factors, insulin, and insulin-like growth factors did not compete for binding. These data demonstrate that (a) functional catalytic residues of the thrombin active site are necessary for mitogenic activity and for specific binding; (b) regions adjacent to the active site, i.e. the high affinity protein recognition site, appear to enhance binding; and (c) the receptor can discriminate between other proteases and binds those which are also mitogens for the avian cells. The characteristics of 125I-alpha-thrombin binding were determined, and it was found to be (i) proportional to cell number; (ii) optimal at pH 6.8; (iii) 70-90% specific; (iv) at equilibrium after 60 min of incubation at 22-24 degrees C or 180 min at 0-4 degrees C (the rate constants for association, i.e. ka, at 22 and 4 degrees C were 18 and 1.1 x 10(7) M-1 min-1, respectively); and (v) essentially nondissociable. Nondissociable thrombin that bound during incubation at 0-4 degrees C was distributed equally between trypsin-sensitive and insensitive compartments. Thrombin associated with the former was released into the media when the cells were incubated at 0-4 degrees C with hirudin or hydroxylamine, or transferred to the insensitive compartment when incubated at 22 degrees C. Finally, confluent cultures of fibroblasts bind 2-3 x 10(4) 125I-alpha-thrombin molecules/cell with an apparent binding constant, i.e. Kd, of 0.7 nM (a true Kd could not be determined because of the irreversible nature of thrombin binding). The binding capacity per cell and the apparent Kd value increased proportionally to an increase in culture density.
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PMID:Protease mitogenic response of chick embryo fibroblasts and receptor binding/processing of human alpha-thrombin. 625 43

The human transferrin receptor could be fluorographically detected after immunoprecipitation from a leukemic T-cell line labeled with [3H]palmitic acid. The label was found ony in association with the human transferrin receptor and not in association with two other major plasma membrane glycoproteins, demonstrating that the incorporation of radioactivity was not due to metabolism of the palmitate. Treatment of sodium dodecyl sulfate-polyacrylamide gels containing the [3H]palmitate-labeled transferrin receptor with hydroxylamine, prior to fluorography, resulted in release of a substantial fraction of the label from the molecule. In addition, at least part of the label released from immunoprecipitates of the transferrin receptor by treatment with hydroxylamine was identified as palmitohydroxamate, providing further evidence that the labeled fatty acid is covalently bound to the receptor. A proteolytic fragment (Mr = 70,000) derived from the portion of the transferrin receptor exposed on the cell surface can be obtained by trypsin digestion of intact or Nonidet P-40-solubilized cells. When cells were labeled with [3H]palmitic acid, none of the radioactivity could be detected in the tryptic fragment. Thus, the bound palmitate appears to be associated with the region of the molecule that is in close proximity to the plasma membrane.
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PMID:Covalent binding of fatty acid to the transferrin receptor in cultured human cells. 626

Incubation of HeLa cell mRNA guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) with [alpha-32P]GTP and a divalent cation in the absence of an RNA acceptor results in the formation of a covalent enzyme-guanylate complex. The complex, after purification by phosphocellulose chromatography, can transfer its bound GMP moiety to pyrophosphate, regenerating GTP, or to the 5'-diphosphate end of poly(A), forming a cap structure G(5')pppA(pA)n. The GMP-polypeptide has a molecular weight of 65,000 and is stable to heating in the presence of sodium dodecyl sulfate. On the basis of the alkali-stable and acid-labile nature of the bond and its susceptibility to nucleophilic attack by hydroxylamine at low pH, the GMP-polypeptide linkage appears to be a phosphoamine bond. After digestion with trypsin, a single GMP-peptide was resolved by two dimensional electrophoresis and chromatography.
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PMID:Eukaryotic mRNA capping enzyme-guanylate covalent intermediate. 628 66

Protein C inhibitor isolated from human plasma inhibited thrombin, factor Xa, trypsin and chymotrypsin as well as activated protein C, but had very little effect on urokinase and plasmin. The inhibition constants (K1) of protein C inhibitor for activated protein C, thrombin and factor Xa were 5.6 X 10(-8) M, 6.7 X 10(-8) M and 3.1 X 10(-7) M, respectively. The second-order rate constant for inhibition of activated protein C by the inhibitor increased about 30-fold in the presence of an optimal heparin concentration (5-10 units/ml). The inhibition of activated protein C by plasma protein C inhibitor was also accelerated by heparin. When activated protein C (Mr = 62,000) was incubated with protein C inhibitor (Mr = 57,000), enzyme-inhibitor complexes with apparent Mr = 102,000 and 88,000 were observed in the nonreduced and the reduced samples, respectively, on SDS-polyacrylamide gel electrophoresis. In addition to these complexes, a band of unbound enzyme and a band with Mr = 54,000 were detected. When 125I-labeled protein C inhibitor was exposed to activated protein C, the inhibitor band was converted to bands with apparent Mr = 102,000 and 54,000 in the nonreduced samples, as determined by autoradiography after gel electrophoresis in SDS. The band with Mr = 54,000 also appeared when the inhibitor reacted with other serine proteases. The activated protein C was released from the inactive complex by treatment with 1 M ammonia or hydroxylamine. This phenomenon was found by SDS-polyacrylamide gel electrophoresis to represent the dissociation of the enzyme-inhibitor complex by ammonia or hydroxylamine into the free enzyme and the proteolytically modified inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of inhibition of activated protein C by protein C inhibitor. 632 92

Based on the partial sequence of the cyanogen bromide fragments [Tratschin, J.D., Wirz, B., Frank, G. and Zuber, H. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 879-892], the amino-acid sequence of thermophilic lactate dehydrogenase from B. stearothermophilus was completed by the preparation and sequencing (sequenator, carboxypeptidase A and Y) of further overlapping fragments. Suitable peptide fragments were obtained by lactate dehydrogenase cleavage with hydroxylamine, o-iodosobenzoic acid and trypsin. The polypeptide chain of thermophilic lactate dehydrogenase from B. stearothermophilus consists of 317 amino-acid residues. While sequence homology with mesophilic lactate dehydrogenase of higher organisms reaches 35%, it is substantially higher with this mesophilic enzyme of bacillae (greater than 60%, B. megaterium, B. subtilis). The secondary structure elements and amino-acid residues of the active site of thermophilic lactate dehydrogenase deducted from primary structure data were compared with those from the mesophilic enzyme, the same was done for the internal sequence homology at the nucleotide-binding units. A comparative structure analysis (matrix system) based on the primary structure data of thermophilic enzyme should provide insight into the characteristic structure differences between thermophilic and mesophilic lactate dehydrogenase.
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PMID:Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. III) The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus. Hydroxylamine-, o-iodosobenzoic acid- and tryptic-fragments. The complete amino-acid sequence. 635 52

The complete amino acid sequence of the 279-residue CNBr peptide CB8 from the alpha 1 chain of type I calf skin collagen is presented. It was determined by sequencing overlapping fragments of CB8 produced by Staphylococcus aureus V8 proteinase, trypsin, Endoproteinase Arg-C and hydroxylamine. Tryptic cleavages were also made specific for lysine by blocking arginine residues with cyclohexane-1,2-dione. This completes the amino acid sequence analysis of the 1054-residues-long alpha (I) chain of calf skin collagen.
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PMID:Completion of the amino acid sequence of the alpha 1 chain from type I calf skin collagen. Amino acid sequence of alpha 1(I)B8. 635 80

Chicken pepsinogen is a glycoprotein consisting of a single polypeptide chain and containing the following 367 amino acid residues: Asp23, Asn16, Thr26, Ser41, Glu14, Gln11, Pro18, Gly31, Ala17, Cys7, Val25, Met9, Ile23, Leu28, Tyr22, Phe20, His8, Lys17, Arg7, Trp4. The Mr-value of the protein is 42 074. This value includes the carbohydrate moiety of the protein, i.e. Man3, (GlcNAc)7, (-SO3H)5. The primary fragmentation of the molecule was effected by limited trypsinolysis at arginine residues after preceding modification of the lysines with citraconic anhydride. All eight peptides expected in theory were obtained and their size, amino acid composition, and N-terminal amino acid sequence were characterized. To elucidate the amino acid sequence of these large fragments the latter were subjected to secondary cleavage by CNBr, trypsin (after removal of the protecting groups from the lysines), the proteinase from Staphylococcus aureus V8 strain, alpha-chymotrypsin, hydroxylamine, or dilute acid; the resulting peptides were isolated by gel permeation and ion-exchange chromatography and by the fingerprint techniques. Overlaps at sites of the arginine residues were obtained in an earlier study [Baudys, M. & Kostka, V. (1982) Collect. Czech. Chem. Commun. 47, 2814-2832]. Chicken pepsinogen shows the highest degree of homology with the primary structures of pepsinogens A. The internal homologies are apparent in the neighborhood of the two active aspartic acid residues. We have assigned tentatively chicken pepsinogen to the group of pepsinogens A (EC 3.4.23.1); this assignment is a result both of our sequence studies and of an investigation of the kinetic characteristics of the enzyme.
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PMID:Covalent structure of chicken pepsinogen. 661 63

Bovine nasal cartilage was repeatedly extracted with the dissociative solvent, 4 M guanidinium chloride. About 25% of the total tissue proteoglycans could not be solubilized as judged by galactosamine analysis. The inextractable proteoglycan could be at least partially solubilized by treating the guanidine-extracted tissue with reagents which completely or partially degraded the proteoglycan protein core. Digestion with papain or cyanogen bromide completely solubilized the tissue and released all of the galactosamine-containing material while trypsin or hydroxylamine treatment left the cartilage macroscopically unchanged and extracted about 50% of the residual galactosamine. The degradatively solubilized material was compared to that extracted with guanidinium chloride. The papain-released glycosaminoglycan chains from the two proteoglycan preparations were similar with respect to size, degree of sulfation, position of sulfation, and hexosamine content. Furthermore, the fragments released from the cartilage residue by either cyanogen bromide, trypsin, or hydroxylamine treatment were of the same size, as judged by gel chromatography, as those derived from similarly digested guanidine-extracted proteoglycan. Trypsin digestion also released the keratan sulfate-enriched peptide as well as peptides from the hyaluronic acid-binding region. By the methods used, the inextractable proteoglycan appears to be similar to the fraction which is readily soluble under dissociative conditions and thus may be held tightly within the tissue by a nonspecific mechanism(s) such as entanglement in the collagen fibril network.
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PMID:Studies on the properties of the inextractable proteoglycans from bovine nasal cartilage. 664 84

The amino acid sequence of the NADP+-dependent enzyme ovine 6-phosphogluconate dehydrogenase has been determined by conventional direct protein sequence analysis of peptides resulting from digestion of the protein with trypsin and chemical cleavages with cyanogen bromide, hydroxylamine, and iodosobenzoic acid. The polypeptide contains 466 amino acids and its NH2 terminus is acetylated. The Candida utilis enzyme is inactivated by reaction of pyridoxal phosphate with two lysine residues (Minchiotti, L., Ronchi, S., and Rippa, M. (1981) Biochim. Biophys. Acta 657, 232-242). These residues are conserved in the ovine enzyme. In contrast to NAD+ dehydrogenases which have weakly related sequences and spatially related folds in their nucleotide-binding sites, no significant sequence homologies were detected between 6-phosphogluconate dehydrogenase and any of three other NADP+-requiring enzymes, glutamate dehydrogenase, p-hydroxybenzoate hydroxylase, and dihydrofolate reductase. This is in accord with structural data that show no spatial relationship between NADP+-binding sites in these enzymes.
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PMID:Amino acid sequence of ovine 6-phosphogluconate dehydrogenase. 668 25

Branched-chain alpha-keto acid dehydrogenase is a multienzyme complex consisting of three catalytic components, i.e. branched-chain alpha-keto acid decarboxylase (E1), dihydrolipoyl transacylase (E2), and dihydrolipoyl dehydrogenase (E3). In this report the E2 component of highly purified branched-chain alpha-keto acid dehydrogenase from bovine kidney and liver was characterized with an independent radiochemical assay for this component. The assay uses the model reaction: R-14CO-S-CoA + Lip-(SH)2 in equilibrium R-14CO-S-Lip-SH + CoA-SH, which is similar to that catalyzed by the transacetylase component of the pyruvate dehydrogenase complex. In this reaction, exogenous dihydrolipoamide substitutes for the protein (E2)-bound dihydrolipoyl moiety, and [1-14C]acyl-CoA synthesized enzymatically is the acyl-CoA substrate. The thioester structure of the reaction product, S-acyldihydrolipoamide, was identified by mass spectrometry, its characteristic absorption at 232-245 nm and by formation of hydroxamate with hydroxylamine. Rates of the E2-catalyzed transacylation reaction with various [1-14C]acyl-CoAs are in the order of [1-14C]isobutyryl-CoA greater than [1-14C] isovaleryl-CoA greater than [1-14C]acetyl-CoA. The activity with acetyl-CoA is 15% of that with isobutyryl-CoA. The E2 activity is strongly inhibited by arsenite. Modification of the covalently bound lipoyl moiety through reductive acylation in the presence of N-ethylmaleimide is without effect on the transacylation reaction. These data, along with results of initial velocity and product inhibition suggest that the model reaction proceeds via a random Bi Bi mechanism. Limited proteolysis of purified bovine liver branched-chain alpha-keto acid dehydrogenase with trypsin results in complete loss of the overall activity catalyzed by the complex. Nonetheless the activity of the E2 component is not affected. The tryptic digestion cleaves E2 subunits (Mr = 52,600) into a major fragment of Mr = 25,700. By contrast, E1 alpha and E1 beta subunits of the complex are relatively resistant to proteolysis with trypsin. The results indicate that structural properties of the E2 component of branched-chain alpha-keto acid dehydrogenase are similar but not identical to those of the transacetylase component of the pyruvate dehydrogenase complex.
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PMID:Catalytic and structural properties of the dihydrolipoyl transacylase component of bovine branched-chain alpha-keto acid dehydrogenase. 674 48


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