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)

Ram spermatozoa incubated in the presence of Ca2+ and the Ca2+-ionophore A23187 undergo a process which is known as the acrosome reaction. This reaction is characterized by fusion of the outer acrosomal membrane and the overlying plasma membrane to form mixed vesicles which can be seen in the electron microscope. As a result, the trypsin-like acrosin is released from the cells to the medium. The occurrence of the acrosome reaction was determined by following acrosin activity in the medium. After 2 h of incubation of the cells in the presence of ionophore and Ca2+, the released acrosin activity is related to the ionophores according to the sequence: A23187 greater than monensin greater than valinomycin greater than FCCP = without ionophore. The study of Ca2+ uptake by the cells revealed that Ca2+ enters the cell prior to the release of acrosin. Monensin can induce Ca2+ uptake and acrosin release only when Na+ is present in the incubation medium. There is no increase in Ca2+ uptake with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). We suggest that the Na+/H+ exchange induced by monensin causes an increase in intracellular Na which is the driving force for the Ca2+ entry via a Ca2+/Na+ antiporter. Since monensin can induce an increase in Ca2+ uptake only in the presence of Na+, FCCP enhances Ca2+ uptake in the presence of valinomycin, and A23187 is a Ca2+/2H+ exchanger, we suggest that alkalization of the intracellular space is involved in the acrosome reaction. Calcium uptake in the presence of monensin is not affected by the uncoupler FCCP, a result which indicates that Ca2+ is not accumulated in the mitochondria. Incubation of cells for 3 h in the absence of Ca2+ or ionophore caused a 3-fold increase in the rate of acrosin release when monensin and Ca2+ were added together. There was no change in this rate when A23187 was used. We suggest that during the preincubation time (known as capacitation) the permeability of the plasma membrane to Ca2+ is enhanced. This study shows that acrosin release and Ca2+ uptake can be used as a quantitative asay for the determination of the acrosome reaction.
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PMID:Induction of acrosomal reaction and calcium uptake in ram spermatozoa by ionophores. 312 23

A reaction of the basic amino acids, lysine and arginine, with components of cigarette smoke has been observed. The adducts produced have been identified as cyanomethyl derivatives. Both formaldehyde and cyanide, which are known to be present in cigarette smoke, are involved in the reaction with the primary amino group. The reaction is time-dependent and can be enhanced by an increase of temperature or by incubation under alkaline conditions. Cyanomethyl adduct formation was found to be increased when smoke from cigarettes with higher tar and nicotine content was used. When proteins, such as bovine serum albumin, trypsin inhibitors or crude rat lung proteins were incubated with the cigarette smoke solution, new protein adducts with increased pI values were produced which are separable from the original proteins by gel isoelectric focussing. Radioisotopically labelled cyanide can be irreversibly linked to protein and the linkage is enhanced in the presence of formaldehyde.
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PMID:Formation of cyanomethyl derivatives of basic amino acids and proteins with components in cigarette smoke. 314 35

A 1.6-kb cDNA for human liver branched-chain acyltransferase [E2b] was placed in a transcription vector under the control of the SP6 promoter. In vitro translation of transcripts from this vector produced a pre-E2b fragment of Mr 39,000. Following import into mitochondria, this protein was processed to a protein with an Mr of 36,000. The processed protein was fully protected from trypsin digestion. Import and processing did not occur in the presence of rhodamine 123 or carbonyl cyanide m-chlorophenyl hydrazone, suggesting that membrane potential and coupled respiration were required. Uptake and processing were species and tissue independent, since both mouse-liver and human-lymphoblast mitochondria converted the human pre-E2b protein fragment. Mitochondria from patient cells that lack E2b through an inherited defect were able to import and process the in vitro-made protein, suggesting that the inherited defect was in the gene for E2b and not in the organelle-structure function. This system now provides additional methods for investigation of mechanisms responsible for the human inherited disorders affecting the branched-chain alpha-ketoacid dehydrogenase complex.
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PMID:Mitochondrial import and processing of an in vitro synthesized human prebranched chain acyltransferase fragment. 318 39

Physicochemical and chemical properties of small proteoglycans containing galactosaminoglycan chains from cultured human skin fibroblasts and human smooth-muscle cells were compared to determine the extent of structural similarity. The proteoglycan secreted by smooth-muscle cells was of larger molecular size and of higher buoyant density, due to longer glycosaminoglycan chains, than the secretion product of skin fibroblasts. Additionally, both proteoglycans differed in the ratio of iduronic acid and glucuronic acid residues. On the other hand, degradation of secreted [3H]leucine-labelled proteoglycans with chondroitin ABC lyase followed by SDS/polyacrylamide-gel electrophoresis resulted in the appearance of core protein bands of identical size (Mr 48,000 and 45,000, depending on the number of asparagine-bound oligosaccharides). An Mr value of 40,000 was determined for the core protein of cells pretreated with tunicamycin. An antibody against the core protein from fibroblast secretions was cross-reactive with the core protein from smooth-muscle cells. Core protein accumulating intracellularly after treatment with carbonyl cyanide m-chlorophenylhydrazone exhibited, on reduction and alkylation, an isoelectric point of 7.8 in both cell types. Limited proteolysis by staphylococcal V8 serine proteinase or endoproteinase Lys-C led in both instances to the formation of peptides of identical size. Peptides bearing asparagine-bound oligosaccharides were free of glycosaminoglycan chains. Similar peptide patterns were obtained when 125I-labelled core proteins were digested with either trypsin or chymotrypsin. Thus small proteoglycans from fibroblasts and smooth-muscle cells can be differentiated by their glycosaminoglycan moieties but not by the nature of their core proteins.
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PMID:Comparison of small proteoglycans from skin fibroblasts and vascular smooth-muscle cells. 380 Sep 48

Conditions are described for the production of large amounts of an extracellular elastolytic protease by Vibrio vulnificus. The yield of enzyme was maximal during the late exponential growth phase and was stable during the stationary growth phase in a medium composed of 2% Proteose Peptone and 1.5% NaCl. The protease has a molecular weight of ca. 50,500 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), an isoelectric point of ca. 5.8, and a pH optimum range against azocasein and elastin of pH 7 to 8. The caseinolytic and elastase activities in protease preparations partially purified by ammonium sulfate precipitation were inseparable by gel filtration, hydrophobic interaction chromatography, and isoelectric focusing. Both activities were deleteriously affected by heat, low pH, heavy-metal ions, chelating agents, reducing agents, sodium cyanide, N-bromosuccinimide, alpha-2-macroglobulin, and phosphoramidon, but were unaffected by various trypsin inhibitors, chymostatin, aprotinin, leupeptin, pepstatin A, phenylmethylsulfonyl fluoride, and N-ethylmaleimide.
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PMID:Production and partial characterization of an elastolytic protease of Vibrio vulnificus. 390 48

Alkylation of the ADP/ATP carrier protein in beef heart mitochondria by N-ethylmaleimide (NEM) results in inactivation of transport. One out of the four cysteinyl residues contained in 1 mol of carrier subunit of Mr 32 000 is alkylated by NEM. The identification of the alkylated residue to Cys-56 has been achieved by chemical and enzymatic cleavages. The chemical cleavages included cleavages at the nonalkylated cysteinyl residues by cyanide at alkaline pH and at methionyl residues by cyanogen bromide. Enzymatic cleavage involved the use of trypsin and chymotrypsin; the resulting peptides were resolved by high-performance liquid chromatography. Analysis of a small size [14C]NEM-labeled peptide obtained by tryptic and chymotryptic digestion of the [14C]NEM-labeled carrier protein yielded the following amino acid sequence: (Formula: see text) where X is probably a substituted lysine.
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PMID:Localization of the N-ethylmaleimide reactive cysteine in the beef heart mitochondrial ADP/ATP carrier protein. 609 66

Electron transfer to rat liver microsomal cytochrome P-450 of 14 alpha-methyl group demethylation of 24,25-dihydrolanosterol (C30-sterol) has been studied with a new radio-high-performance liquid chromatography assay. The monooxygenase is dependent upon NADPH plus oxygen, insensitive to CN-, and sensitive to CO. Microsomal oxidation is also sensitive to trypsin digestion, and reactivation is dependent upon the addition of purified, detergent-solubilized cytochrome P-450 reductase. Electron transport of C-32 sterol demethylation can be fully supported by very low concentrations of NADPH (approximately 10 microM) only in the presence of saturating concentrations of NADH (approximately 200 microM) suggesting involvement of cytochrome b5-dependent electron transfer in addition to the NADPH-supported pathway. The cytochrome P-450 of 14 alpha-demethylation has been solubilized with detergents, resolved chromatographically from cytochrome P-450 reductase and cytochrome b5, and fully active C-32 demethylase reconstituted. Incubation of intact microsomes with NADH and very low concentrations of NADPH described above leads to interruption of demethylation without 14 alpha-methyl group elimination. Under these conditions, C-32 oxidation products of the C30-sterol substrate accumulate at the expense of formation of demethylated, C29-sterol products. This enzymic interruption of C-32 demethylation, accumulation of oxygenated C30-sterols, along with subsequent demethylation of the isolated C30-oxysterols under similar oxidative conditions supports the suggestion that 14 alpha-hydroxymethyl and aldehydic sterols are metabolic intermediates of sterol 14 alpha-demethylation. Only very modest inductions of the constitutive cytochrome P-450 isozyme of 14 alpha-methyl sterol oxidase can be obtained with just 2 out of 12 known, potent inducers of mammalian hepatic cytochrome P-450s. Alternatively, administration of complete adjuvant in mineral oil drastically reduces amounts of total microsomal cytochrome P-450 while activity of 14 alpha-methyl sterol oxidase is not affected dramatically. Thus, as much as 2.5-fold enhancement of C-32 oxidase specific activity is obtained when expressed per unit of cytochrome P-450.
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PMID:Cytochrome P-450-dependent oxidation of lanosterol in cholesterol biosynthesis. Microsomal electron transport and C-32 demethylation. 620 95

The Ca2+-dependent ATPase was solubilized from rat heart sarcolemmal membranes upon digestion with trypsin and was found to be different from Ca2+-stimulated Mg2+-dependent ATPase (Dhalla, N. S., Anand-Srivastava, M. B., Tuana, B. S., and Khandelwal, R. L. (1981) J. Mol. Cell. Cardiol. 13, 413-423). The enzyme was purified by high speed centrifugation, ammonium sulfate fractionation, and column chromatography and was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis. In sodium dodecyl sulfate-acrylamide gels, the enzyme dissociated into two subunits or fragments with molecular weights of about 55,000 and 12,000. The molecular weight of the enzyme, estimated by gel filtration on a Sephadex G-100 column, was found to be about 67,000. The enzyme utilized ATP with a Km of 0.20-0.26 mM but was also able to utilize ITP, CTP, GTP, and ADP as substrates at much lower rates. It was activated by Ca2+ with a Ka of 0.13-0.21 mM; it was also activated by other cations in the order Ca2+ greater than Mn2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. Divalent cations like Cu2+, Ni2+, and Mg2+ were potent inhibitors. The enzyme was insensitive to ouabain, verapamil, oligomycin, cyanide, and vanadate but was markedly inhibited by N-ethylmaleimide. Calmodulin failed to stimulate Ca2+-dependent ATPase and instead inhibited slightly. Unlike K+, Na+ produced a marked inhibition of the Ca2+-dependent ATPase activity, and this inhibition was associated with an 8- 10-fold decrease in the affinity of the enzyme for Ca2+. The competitive action of Na+ indicates that the Ca2+-dependent ATPase may be a site of Na+-Ca2+ antagonism in the cell membrane.
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PMID:Purification and characterization of a Ca2+-dependent ATPase from rat heart sarcolemma. 621 55

Phagocytic vesicles from rabbit lung macrophages produced superoxide in the presence of NADH or NADPH. At 37 degrees C, these vesicles generated 51+/-7.8 nmol O(2) (-)/min per mg protein in the presence of 0.5 mM NADPH. The apparent K(m) for NADPH and NADH (66 and 266 muM, respectively), the pH optimum for the reaction (6.9), and the cyanide insensitivity were similar to properties of plasma membrane-rich fractions of stimulated polymorphonuclear leukocytes studied by others. The activity of the phagocytic vesicles was trypsin sensitive. The specific superoxide-generating activity of macrophage phagocytic vesicles isolated from cells incubated up to 90 min with phagocytic particles remained constant. Calcium in micromolar concentrations inhibited the NADPH-dependent O(2) (-)-generating activity of phagocytic vesicles. In a physiological ionic medium (100 mM KCl, 2.5 mM MgCl(2), 30 mM imidazole-HCl, pH 6.9), a maximal inhibition of O(2) (-) generation by phagocytic vesicles of 80% was observed at 40 muM free Ca(2+). The half maximum inhibitory effect was at 0.7 muM Ca(2+). Variations of the calcium concentration resulted in rapid and reversible alterations in O(2) (-)-forming activity. Preincubation of phagocytic vesicles in the presence of EGTA rendered their O(2) (-) generation rate in the presence of NADPH insensitive to alterations in the free calcium concentration. This desensitization by low EGTA concentrations (</=100 muM) was reversible by the addition of excess calcium, but desensitization by high EGTA concentrations (>1 mM) was not reversible by the addition of calcium either in the presence or absence of purified rabbit lung macrophage or bovine brain calmodulins. Furthermore, trifluoperazine, a drug that inhibits calmodulin-stimulated reactions, did not alter the activity or the calcium sensitivity of the superoxide-generating system of sensitive phagocytic vesicles. Peripheral plasma membrane vesicles (podosomes) prepared by gentle sonication of macrophages possessed on O(2) (-)-generating system with similar properties to those of phagocytic vesicles. We conclude that the activated O(2) (-)-generating system of rabbit lung macrophages has its initial localization in the plasmalemma and undergoes subsequent internalization into phagocytic vesicles, where it can function for prolonged periods of time. Calcium at concentrations likely to exist in macrophage cytoplasm exerts a regulatory effect on the activated system.
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PMID:Effect of calcium on superoxide production by phagocytic vesicles from rabbit alveolar macrophages. 625 9

We have isolated an outer mitochondrial membrane (OMM) fraction from baker's yeast. Saccharomyces cerevisiae, that possesses porin activity and contains a major polypeptide of 29,000 daltons. By analogy to similar data for an OMM fraction from rat liver and mung bean [Zalman, L. S., Nikaido, N. & Kagawa, Y. (1980) J. Biol. Chem. 255, 1771-1774], the 29,000-dalton polypeptide of the isolated yeast OMM fraction has been tentatively identified as porin. Evidence to substantiate this identification was provided by the finding that both the porin activity and the 29,000-dalton polypeptide were entirely resistant when the OMM fraction was exposed to trypsin digestion, with the 29,000-dalton polypeptide being virtually the only polypeptide in the OMM fraction to be unaffected by trypsin digestion. There was no protection when trypsin digestion was carried out in the presence of detergent. Using monospecific antibodies, we have shown that yeast porin is apparently not synthesized as a larger precursor in a cell-free translation system. In vitro-synthesized porin could not be integrated into dog pancreas microsomal vesicles or into an isolated OMM fraction from yeast, either co- or posttranslationally. In vitro-synthesized porin, however, could be integrated posttranslationally into whole isolated mitochondria. This membrane specificity suggests that integration does not proceed by unassisted partitioning. The integration of porin into whole mitochondria occurred with fidelity by the criterion of its resistance to trypsin. Moreover, integration was not inhibited in the presence of the protonophore carbonyl cyanide m-chlorophenyl-hydrazone whereas translocation into the mitochondrial matrix of the in vitro-synthesized gamma subunit of F1-ATPase was inhibited.
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PMID:In vitro synthesis and integration into mitochondria of porin, a major protein of the outer mitochondrial membrane of Saccharomyces cerevisiae. 629 16


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