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)

Human embryonic lung (HuEL) cells in culture produce large amounts of the enzyme, plasminogen activator, and thus generate substantial amounts of active plasmin. Despite the presence of plasmin, however, HuEL cells grow in ordered, flattened, adherent sheets. It seemed of interest to characterize protease inhibitors that might be present in HuEL cultures and which might account for this apparent contradiction. This paper reports the isolation and purification of the major serine protease inhibitor in 5-day serum-free conditioned medium (CM) from HuEL cells, and the purification of an identical molecule from fetal bovine serum (FBS). Both the CM-derived inhibitor and the FBS-derived inhibitor are identical with fetuin, the major glycoprotein of FBS. The CM-derived inhibitor is apparently derived from the FBS used to supplement the growth medium of HuEL cells between serum-free CM collection periods. It is not labeled metabolically with 3H-leucine. Its electrophoretic behavior is indistinguishable from that of standard fetuin in SDS-PAGE, non-SDS basic pH,PAGE, and isoelectric focusing. The CM-derived inhibitor and standard fetuin inhibit trypsin and plasmin with similar efficiencies, but neither inhibits chymotrypsin, pancreatic elastase, or plasminogen activator. They are immunologically indistinguishable. The suggestion is made that fetuin, and possibly other protease inhibitors present in HuEL cell cultures, may be concentrated locally by HuEL cells and gradually released back into the medium in the absence of serum. These molecules may serve to protect HuEL cells against proteases they generate.
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PMID:Isolation of the major serine protease inhibitor from the 5-day serum-free conditioned medium of human embryonic lung cells and demonstration that it is fetuin. 617 91

Camostate, a synthetic serine protease inhibitor, specifically inhibits the trypsin activity in vitro. Immediately after intravenous administration of camostate (5, 2.5, 0.5, and 0.1 mg/kg body weight/h) the trypsin activity in the biliary-pancreatic juice was diminished dose-dependently in anaesthetized rats. The amylase release was not influenced. Camostate and its metabolites were detected by high-pressure liquid chromatography in the pancreatic juice and tissue; in plasma, only the metabolites were found. Therefore, the inhibitory action of camostate on the trypsin activity in the biliary-pancreatic juice may be due to penetration of camostate into the juice.
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PMID:Effect of a specific serine protease inhibitor on the rat pancreas: systemic administration of camostate and exocrine pancreatic secretion. 620 86

Trypsin(ogen) was isolated from the pyloric ceca of Greenland cod. Greenland cod trypsin catalyzed hydrolysis of N alpha-benzoyl-DL-arginine p-nitroanilide, tosyl arginine methyl ester and protein and was inhibited by the serine protease inhibitor PMSF and other well-known trypsin inhibitors. Greenland cod trypsin was more stable at alkaline pH than at acid pH; and was inactivated by relatively low thermal treatment. Like other trypsins, the enzyme was rich in potential acidic amino acid residues but poor in basic amino acid residues and had a molecular weight of 23,500; but it had less potential disulfide pairs, less alpha-helix and a lower H phi ave than other trypsins previously characterized. Reactions catalyzed by Greenland cod trypsin were not very responsive to temperature change, such that specific activity was relatively high at low reaction temperature.
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PMID:Trypsin from Greenland cod, Gadus ogac. Isolation and comparative properties. 651 65

The interaction of alpha 1-Antitrypsin, the major serine protease inhibitor in plasma, with pathogenic Trichomonas vaginalis and the acquisition by trichomonads of this host protein from normal human plasma were investigated. alpha 1-Antitrypsin acquired by intact parasites could not be removed by repeated washings in phosphate-buffered saline. Saturation kinetics were observed after incubation of glutaraldehyde-fixed organisms with 125I-labeled alpha 1-antitrypsin. Evidence suggesting that specific parasite membrane sites were responsible for trichomonal acquisition of alpha 1-antitrypsin was obtained through competitive binding experiments using purified preparations of homologous versus heterologous plasma proteins. No evidence of degradation of bound antitrypsin by live parasites was observed. The avid binding of alpha 1-antitrypsin by pathogenic T. vaginalis after incubation in normal human plasma was demonstrated by using sensitive electrophoretic and immunodetection techniques. Radioimmunoprecipitation of intrinsically labeled, detergent-solubilized extracts of T. vaginalis incubated with monospecific antisera against alpha 1-antitrypsin and other human plasma proteins revealed the inability of parasites to biosynthesize any substance cross-reactive with host plasma proteins. Finally, T. vaginalis organisms pretreated with alpha 1-antitrypsin inhibited trypsin caseinase activity in an in vitro assay. The implications of these observations are discussed.
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PMID:Acquisition of alpha 1-Antitrypsin by a pathogenic strain of Trichomonas vaginalis. 660 23

Carboxamidopeptidase, an enzyme which inactivates neurohypophyseal hormones, has been purified 3800-fold in an overall yield of 22% from toad skin, a neurohypophyseal hormone target organ, by (NH4)2SO4 fractionation, DEAE-Sephadex chromatography, and affinity chromatography on immobilized p-aminobenzamidine and concanavalin A-agrose. The purified enzyme is capable of inactivating both [8-arginine]vasopressin (AVP) and oxytocin by hydrolyzing the Arg8-Gly9-NH2 and the Leu8-Gly9-NH2 bonds, respectively, and can hydrolyze the ester substrates, benzoyl-L-arginine ethyl ester (BzArgOEt) and acetyl-L-trypsine ethyl ester, suggesting that the enzyme has both trypsin-like and chymotrypsin-like activities. Carboxamidopeptidase is maximally active at pH 7.5-8.5 for AVP and BzArgOEt and pH 7.0 for oxytocin. Carboxamidopeptidase is inhibited by ovoinhibitor, ovomucoid, Trasylol. lima bean trypsin inhibitor, concanavalin A, antipain, leupeptin, chymostatin, elastatinal, p-nitrophenyl p-guanidinobenzoate, and 4-methylumbelliferyl p-guanidinobenzoate but not by soybean trypsin inhibitor, alpha 1-antitrypsin, hirudin, pepstatin, bestatin, phosphoramidon, or cysteine. The enzyme is also inhibited by the serine protease inhibitor, diisopropyl phosphofluoridate (i-Pr2PF), and by the chloromethyl ketone derivatives of tosyllysine, tosylphenylalanine, and (benzyloxycarbonyl)phenylalanine, as well as by the sulfhydryl group reagent, p-(chloromercuri)benzoate (PCMB). Inhibition by PCMB is reversed by cysteine. The molecular weight determined by gel filtration in the presence of 1 MNaCl is approximately 100 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of two identical subunits of 48 000 daltons. Each subunit consists of a heavy chain (28 000 daltons) and a light chain (19 000 daltons) joined by a disulfide bond(s). Labeling experiments using [3H]-i-Pr2PF showed that the enzyme active site is located in the heavy chain.
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PMID:Carboxamidopeptidase: purification and characterization of a neurohypophyseal hormone inactivating peptidase from toad skin. 676 14

Mutants of Bacillus subtilis capable of secreting high amounts of protease were highly tolerant to the lethal and lytic effects of nafcillin. Protease-deficient mutants were more susceptible. However, when subtilisin was added to exogenously to a protease-deficient strain, the organism assumed the characteristics of nafcillin tolerance. Similarly, when phenylmethylsulfonyl fluoride, a serine protease inhibitor, was added to the tolerant strains, they became susceptible to nafcillin-induced lysis. The effects of nafcillin on B. subtilis were studied with both viability determinations and assay of cellular lysis. The minimum inhibitory concentrations of nafcillin tended to be higher for the protease hyperproducing strains, but these values could be reduced by the protease inhibitor. No loss of antibiotic activity was observed when nafcillin was incubated with either subtilisin or trypsin. Furthermore, protease and autolysin from B. subtilis were not modified by nafcillin. The results showed that extracellular proteases could render B. subtilis relatively tolerant to the killing and lytic effects of a cell wall antibiotic. The proteases were probably acting on the autolysins of the organism, thereby increasing tolerance to nafcillin.
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PMID:Extracellular proteases increase tolerance of Bacillus subtilis to nafcillin. 681 97

The tumor-induced marrow and red blood cell cytolysis assays have been used to explore the mechanism of cancer cell destruction of normal cells. Previously, we suggested that tumor-induced cytolysis was caused by tumor cell membrane-bound serine proteases. In this study, we have shown that concentrations of the broad-spectrum serine protease inhibitor diisopropylfluorophosphate that did not inhibit tumor cell DNA and protein synthesis completely abrogated tumor-induced red blood cell cytolysis. In addition, tumor cell membranes isolated by differential and sucrose density gradient centrifugation and characterized by electron microscopy and enzyme marker analysis were cytolytic for rat 59Fe-labeled red blood cells. The specific activity expressed as release index (%) per microgram of protein was 1.620 for the tumor cell membrane preparations as compared to 0.002 for intact Walker 256 tumor cells. Tumor cell membranes solubilized in Triton X-100 had activity in the p-toluenesulfonyl-L-arginine methyl ester assay for trypsin-like enzymes and the N-benzoyl-L-tyrosine ethyl ester assay for chymotrypsin-like enzymes. The enzyme activities demonstrated in these assays could be inhibited by N-alpha-p-tosyl-L-lysine chloromethyl ketone HCl and L-1-tosylamide-alpha-phenyl-ethyl chloromethyl ketone, respectively. Using [3H]diisopropylfluorophosphate affinity labeling of the tumor cell membrane proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we have identified membrane-bound serine protease(s) that appear to be responsible for tumor-induced marrow and red blood cell cytolysis.
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PMID:Role of tumor cell membrane-bound serine proteases in tumor-induced target cytolysis. 703 91

1. Two trypsin-like enzymes, assayed by their amidase activity with N-alpha-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) as the substrate, were isolated from the gut of the arctic fish capelin (Mallotus villosus). 2. Purification involved affinity chromatography (Benzamidine-CH-Sepharose 4B) of the 30 to 70% (NH4)2SO4 precipitation fraction of a crude extract of the gut, followed by DEAE-Sephadex chromatography, yielding two enzymes, designated Enzyme I and II. 3. Both enzymes had MW of about 28,000 as determined by SDS-electrophoresis. Their isoelectric points were 5.6-5.9 (Enzyme I) and 5.1-5.3 (Enzyme II) and they had similar amino acid composition. 4. Both enzymes were inhibited by standard trypsin inhibitors including the serine protease inhibitor phenylmethyl sulphonyl fluoride (PMSF), but not by the chymotrypsin inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK). 5. The enzymes had a pH optimum of 8-9 and their stability was not affected by CaCl2. Low pH (2.3) caused an initial rapid loss of enzyme activity, followed by relatively slow decomposition of the activity remaining after 1 hr at 4 degrees C. 6. The enzymes had an apparent temperature optimum of 42 degrees C, resulting from rapid self digestion at higher temperatures.
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PMID:Characteristics of two trypsin type isozymes isolated from the arctic fish capelin (Mallotus villosus). 708 13

Squamous cell carcinoma (SCC) antigen was tested, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, for its ability to inhibit the activity of serine proteases, i.e., trypsin, chymotrypsin and elastase. We demonstrated that the serine protease inhibitor (serpin) of SCC antigen is specific for chymotrypsin. Preincubation of chymotrypsin with recombinant SCC antigen inhibited chymotryptic digestion of gelatin and ovalbumin through the formation of sodium dodecyl sulfate-stable complexes. These findings promote understanding of the biological functions of SCC antigen as serpin in the stratification of the normal squamous cells and in the malignant behavior of the tumor cells.
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PMID:Serine protease inhibitor activity of recombinant squamous cell carcinoma antigen towards chymotrypsin, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 749 25

Pigment epithelium-derived factor (PEDF), a neurite-promoting factor, has an amino acid primary structure that is related to members of the serine protease inhibitor (serpin) family. Controlled proteolysis of native PEDF (50 kDa) with either trypsin, chymotrypsin, elastase, or subtilisin yields in each case one major limited product of 46 kDa as analyzed by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analysis of the isolated 46-kDa products indicates a favored cleavage region located toward the C-terminal end of PEDF. A proteolyzed PEDF protein reaction mixture reveals two overlapping sequences: that of the N terminus of intact PEDF and that of an internal region, consistent with cleavage of PEDF about position 382. These data indicate that PEDF protein has a globular conformation with one protease-sensitive exposed loop that contains the homologous serpin-reactive site. Cleavage within the reactive-site loop of PEDF does not cause a conformational change in the molecules (the stressed (S)-->relaxed (R) transition) and results in heat denaturation identical to its native counterpart. This lack of conformational change is also seen upon cleavage within the reactive-site loop of the noninhibitory serpin ovalbumin. Furthermore, the PEDF neurite-promoting function is not lost with cleavage of the exposed loop. Recombinant PEDF polypeptide fragments with larger truncations from the C-terminal end show neurotrophic activity. Our results clearly indicate that integrity of the PEDF homologous serpin reactive center is dispensable for neurotrophic activity. Thus, the PEDF induction of neurites must be mediated by a mechanism other than serine protease inhibition. Altogether our data indicate that PEDF belongs to the subgroup of noninhibitory serpins and that its N-terminal region confers a neurite-promoting activity to the protein. The neurotrophic active site of PEDF is separated from the serpin reactive-site loop, not only in the primary structure, but also in the folded protein structure.
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PMID:Pigment epithelium-derived factor behaves like a noninhibitory serpin. Neurotrophic activity does not require the serpin reactive loop. 759 90


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