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)

Crystals of a complex of chicken gizzard G-actin and DNase I were soaked in a solution of radioactive 4-hydroxymercuribenzoate (MB). The soaked crystals, which contained 0.93 mol of MB per mol of G-actin, were dissolved in "G-buffer" and digested with trypsin, and the resulting peptides were fractionated by thin-layer chromatography. The MB is exchangeable between peptides that contain cysteine residues, but the data obtained here suggested that MB attached to the cysteine residue at the 373rd position of the G-actin molecule.
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PMID:Site of attachment of mercuribenzoate in crystals of an actin:DNase I complex. 129 88

Thiol protease inhibitor (TPI) proteins in embryos of the brine shrimp Artemia were purified to apparent homogeneity and several of their properties were studied. Four protein fractions containing thiol protease inhibitor activity were obtained by high performance liquid chromatography of Artemia embryo proteins on a C-18 reverse-phase column and these were designated as TPI-1a, -1b, -2, and -3. Acrylamide gel electrophoresis showed that TPI-1a and TPI-1b each consisted of two bands of 11.8 and 13.6 kilodaltons (kDa), while TPI-2 and TPI-3 consisted of only one band of 12.5 kDa. Isoelectric focusing experiments demonstrated that TPI-3 contained one band at pH 5.3, while both TPI-1b and TPI-2 yielded bands at pH 5.2 and 5.3. TPI-1a did not yield any major bands. Amino acid composition analyses of the Artemia TPI proteins showed them to be remarkably similar to one another. All were rich in valine and aspartic and glutamic acids, and devoid of cysteine. Partial trypsin digestion of TPI-1b, TPI-2, and TPI-3 yielded several peptides with identical mobilities on a reverse-phase column and several other peptides with different mobilities, suggesting that the multiple forms of Artemia TPIs may have originated from the same parental protein. N-terminal amino acid sequence analyses of TPI-3 suggest that Artemia TPI proteins are members of the type I cystatin family of protease inhibitors.
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PMID:Purification and partial characterization of thiol protease inhibitors from embryos of the brine shrimp Artemia. 129 29

The primary processing of the poliovirus polyprotein is catalyzed by 2A protease (2Apro) which cleaves at the 1D/2A junction in a very rapid cotranslational reaction. In addition, 2Apro also indirectly induces cleavage of the p220 component of eIF-4F, which results in selective inhibition of host protein synthesis. Earlier studies have indicated that 2Apro is related to 3C protease (3Cpro) and is structurally similar to trypsin-like serine proteases with the substitution of Cys109 as the nucleophile. We noticed that 2Apro of enteroviruses and rhinoviruses contains a specific motif of Cys55-Xaa-Cys57-Xaan-Cys115-Xaa-His117 which is absolutely conserved, but which is not found in viral 3Cpro or known cellular serine proteases. To better understand the specific roles these conserved cysteine and histidine residues played in the structure/function of 2Apro, we constructed a series of 2Apro mutants by site-specific mutagenesis and analyzed the mutant enzymes with respect to their biochemical properties. Conservative amino acid replacements at Cys55, Cys57, Cys115, or His117 resulted, in each case, in a complete loss of both in cis and in trans activities of 2Apro. To determine the function of these residues, we examined the biochemical/structural features of 2Apro expressed in a cell-free rabbit reticulocyte lysate system. Gel mobility shift and chemical modification data suggest that these cysteine residues do not form intra-molecular disulfide linkages as a structural feature of 2Apro. However, studies with metal chelators did not eliminate the possibility that 2Apro contains a metal-binding ligand. Finally, our results suggest that these conserved cysteine and histidine residues, including Cys55, Cys57, Cys115, and His117, are critical in maintaining the active conformation of 2Apro structure and essential in supporting the catalytic activity of 2Apro.
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PMID:Characterization of the roles of conserved cysteine and histidine residues in poliovirus 2A protease. 131 Jan 93

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with various forms of periodontal disease. Several characteristics of P. gingivalis are thought to contribute to its pathogenicity; these include haemagglutination and trypsin-like protease activity. Previous studies suggest an association between haemagglutination and trypsin-like protease activity of P. gingivalis. To investigate this, two complementary quantitative experimental approaches were taken. Five independent mutants of P. gingivalis deficient in trypsin-like protease activity were shown to exhibit reduced haemagglutination activity. In addition, enhancers (cysteine and dithiothreitol) and inhibitors (N-ethylmaleimide, N-p-tosyl-L-lysine-chloromethyl ketone, and phenylmethylsulphonyl fluoride) of trypsin-like protease activity were shown, respectively, to significantly enhance and inhibit haemagglutination activity of washed, wild-type P. gingivalis cells (p less than 0.05, paired t-test). Statistical analysis indicated a strong correlation between haemagglutination and trypsin-like protease activity (r = 0.85, p less than 0.001, Spearman rank correlation). The effect of the protease enhancers and inhibitors on haemagglutination activity was specific for P. gingivalis, as they did not significantly change the haemagglutination activity of Fusobacterium nucleatum. These results suggest that the proteolytic site of the trypsin-like protease participates in haemagglutination activity of P. gingivalis.
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PMID:Correlation of haemagglutination activity with trypsin-like protease activity of Porphyromonas gingivalis. 133 63

The regulation of acid secretion was clarified by the development of H2-receptor antagonists in the 1970s. It appears that gastrin and acetylcholine exert their effects on acid secretion mainly by stimulation of histamine release from the enterochromaffin-like (ECL) cell of the fundic gastric mucosa. The isolated ECL cell of rat gastric mucosa responds to gastrin/cholecystokinin (CCK), acetylcholine, and epinephrine with histamine release and to somatostatin and R-alpha-methyl histamine by inhibition of histamine release. Histamine and acetylcholine stimulate the parietal cell by elevation of cAMP or [Ca]i by activation of H2 or M3 receptors, respectively. These independent pathways converge to activate the gastric acid pump, the H+,K+ ATPase. Activation is a function of the association of the ATPase with a potassium chloride transport pathway that occurs in the membrane of the secretory canaliculus of the parietal cell. Hence the secretory canaliculus is the site of acid secretion, the acid being pumped into the lumen of the canaliculus. The pump is composed of two subunits, a large catalytic and a smaller glycosylated protein. This final step of acid secretion has become the target of drugs also designed to inhibit acid secretion. The target domain of the benzimidazole class of acid pump inhibitors is the extracytoplasmic domain of the pump that is secreting acid, and the target amino acids are the cysteines present in this domain. The secondary structure of the pump can be analyzed by determining trypsin-sensitive bonds in intact, cytoplasmic-side-out vesicles of the ATPase, and it has been shown that the alpha subunit has at least eight membrane-spanning segments. Omeprazole, the first acid pump inhibitor, forms a disulfide bond with cysteines in the extracytoplasmic loop between the fifth and sixth membrane-spanning segment and to a cysteine in the extracytoplasmic loop between the seventh and eight segments, preventing phosphorylation of the pump by ATP. As a result of the effective and long-lasting inhibition of acid secretion by the acid pump inhibitor, superior clinical results have been found in all forms of acid-related disease.
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PMID:Acid secretion and the H,K ATPase of stomach. 134 Oct 65

S-Alkylcysteine alpha, beta-lyase was found in a thermophile, Bacillus sp. 41A, which was newly isolated from soil, and purified to homogeneity from the cell extract. The enzyme has a molecular weight of about 76,000, and is composed of two subunits identical in molecular weight (39,000). The enzyme requires pyridoxal 5'-phosphate as a coenzyme, and catalyzes alpha, beta-elimination of S-methyl-L-cysteine and its analogs such as S-ethyl-L-cysteine, L-djenkolate, L-cystine, Se-methyl-L-selenocysteine, and O-methyl-DL-serine. However, S-methyl-D-cysteine, D-cystine, L-methionine, and L-norleucine were inert. The enzyme also catalyzes the beta-replacement reaction of S-methyl-L-cysteine with various thiols to yield the corresponding S-substituted cysteines. In addition to S-methyl-L-cysteine, Se-methyl-L-selenocysteine and O-methyl-DL-serine also serve as beta-substituent acceptors in the beta-replacement reaction. The enzyme is most active at 70 degrees C and stable at high temperatures. Automated Edman degradation provided the N-terminal sequence of the first 44 amino acids. The amino acid sequence in the vicinity of the lysyl residue to which pyridoxal 5'-phosphate is bound, was -Lys-His-Gln-Arg- by Edman degradation of the pyridoxyl peptide obtained by digestion with trypsin after reduction with sodium borohydride.
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PMID:Thermostable S-alkylcysteine alpha, beta-lyase from a thermophile: purification and properties. 136 9

Normal rabbit serum contained two kinds of growth-inhibitory protein, GI-I and GI-II, in latent forms. These latent inhibitors were activated by incubation at 37 degrees C for 12 h, and their activation was lowered by inhibitors for serine, cysteine and metalloproteinases. Both growth inhibitors were highly purified in active forms by successive column chromatographies. GI-I showed a major protein band with an Mr of 18,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while GI-II showed a major protein band with an Mr of 36,000. GI-I and GI-II half-inhibited the growth of rat tumorigenic cell line (RSV-BRL) at concentrations of 0.5 ng/ml and 10 ng/ml, excess concentrations. Of the 15 cell lines tested, GI-I specifically inhibited the growth of rodent and lagomorph cells, whereas GI-II nonspecifically inhibited the growth of all cell lines tested. Specificities for cell type and malignancy were not observed with either inhibitor. These growth inhibitors were stable to a reducing reagent and proteinase inhibitors, but labile to urea, acid, organic solvents, trypsin, plasmin and heating at 95 degrees C for 5 min. These properties suggested that both growth inhibitors might be distinct from known growth-inhibitory factors.
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PMID:Purification and partial characterization of two types of growth-inhibitory protein latently present in rabbit serum. 137 Oct 74

In the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) at neutral pH, only two one-disulfide intermediates accumulate to a significant extent, namely [5-55] and [30-51]. In this paper we describe a recombinant model of [5-55], designated [5-55]Ala, which was made by replacing the cysteine residues not involved in the disulfide bond with alanine. As judged by two-dimensional NMR, [5-55]Ala folds into essentially the same conformation as native BPTI. Moreover, like native BPTI, [5-55]Ala inhibits trypsin stoichiometrically. Thus, the disulfide-bonded intermediate [5-55] corresponds not to a partially folded protein folding intermediate but rather to an essentially completely folded protein. This conclusion provides an explanation for many of the thermodynamic and kinetic properties of [5-55] in the folding pathway of BPTI.
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PMID:Complete folding of bovine pancreatic trypsin inhibitor with only a single disulfide bond. 137 75

The monoclonal antibody, 3/4/2, which was raised against purified rat cytochrome P450 isoenzyme 1A1 (CYP1A1) binds to cytochromes P4501A in many species. It was shown by immunoblotting that the antibody binds to CYP1A1 in microsomal fractions prepared from rat, mouse, rabbit, hamster and human. The antibody also binds to cytochrome P450 isoenzyme 1A2 in microsomal fractions prepared from rabbit and human, but not rat or mouse. Using purified isoenzymes in an enzyme-linked immunosorbent assay it was found that the affinity of binding to the two rabbit hydrocarbon-inducible isoenzymes is reduced compared with that for rat CYP1A1. Binding is not affected by denaturation of the antigens. The effects of chemical and enzymatic treatments on rat CYP1A1 showed that the epitope contains a trypsin-sensitive site that includes arginine, but lacks lysine. The epitope does not contain methionine, cysteine, aspartic acid or glutamic acid residues. In addition, digestion of the protein with cyanogen bromide produces a fragment of Mr 20,000 which contains the antibody binding site. By comparing the cross-reactivity of the antibody with the primary structures of CYP1A1 and 1A2 from the rat, mouse, rabbit and human, and by considering the results of the chemical and enzymatic treatments, it was possible to deduce the likely location and structure of the binding site of 3/4/2 on members of the CYP1A subfamily. It is concluded that the epitope for this antibody is Phe-Arg-His-Ser-Ser-Phe, which lies at positions 380-385 in rat CYP1A1. Further, it is predicted from a model of the tertiary structure of eukaryotic cytochrome P450 that a part of this binding site lies within a helix in the native protein.
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PMID:Identification of the epitope of a monoclonal antibody which binds to several cytochromes P450 in the CYP1A subfamily. 137 49

Decay accelerating factor (DAF) has 4 SCR (short consensus repeat) units. Each SCR unit consists of approx. 60 amino acids characterized by having four conserved cysteine residues and several other highly conserved residues which include proline, tryptophan, tyrosine/phenylalanine and glycine. To determine the disulfide-bonding pattern, we used the urine form of DAF. After thermolysin and trypsin digestion, we isolated seven disulfide-linked peptides by HPLC purification. Because all of the cysteine residues are disulfide-bonded, DAF should contain eight disulfide bonds. After subtilisin and trypsin digestion, we isolated the eighth disulfide-bonded peptides by HPLC purification. From sequence analyses of these peptides, we could identify all disulfide bonds in the 4 SCR units of DAF as being between the first and the third and between the second and the fourth half-cystines within each SCR unit.
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PMID:Complete determination of disulfide bonds localized within the short consensus repeat units of decay accelerating factor (CD55 antigen). 137 29


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