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)

Pig kidney microvillar proteins were extracted with octyl beta-glucoside and reconstituted in liposomes prepared from microvillar lipids of known composition. Four peptidases, namely endopeptidase (EC 3.4.24.11), aminopeptidases N (EC 3.4.11.2) and A (EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), were shown to be reconstituted. At lipid/protein ratios greater than 4:1, about half the detergent-solubilized protein and nearly all of the activity of the four peptidases were reconstituted. Dissolution of the liposomes with Triton X-100 did not increase the activity of any of these peptidases, a result consistent with an asymmetric, 'right-side-out', orientation of these enzymes. When purified, endopeptidase was subjected to the same procedure; the two amphipathic forms of the enzyme (the detergent form and the trypsin-treated detergent form) were fully reconstituted. The amphiphilic form, purified after toluene/trypsin treatment, failed to reconstitute. Electron microscopy of microvilli showed that the appearance of the surface particles was profoundly altered by treatment with papain. Before treatment, the microvilli were coated with particles of stalk lengths ranging from 2.5 to 9 nm. After papain treatment nearly all the particles had stalks of 2-3 nm. Reconstituted microvillar proteins in liposomes showed the same heterogeneity of stalk length. In contrast, liposomes containing reconstituted endopeptidase revealed a very homogeneous population of particles of stalk length 2 nm. Since the smallest dimension of a papain molecule is 3.7 nm, the ability of papain, and other proteinases of similar molecular size, to release microvillar enzymes is crucially affected by the length of the junctional peptide that constitutes the stalk of this type of membrane protein.
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PMID:Proteins of the kidney microvillar membrane. Reconstitution of endopeptidase in liposomes shows that it is a short-stalked protein. 634 16

Pyruvate carboxylase from Pseudomonas citronellolis is composed of non-identical subunits which include a larger biotin-containing polypeptide (alpha) of Mr = 65,000, and a smaller biotin-free polypeptide (beta) of Mr = 54,000. We have investigated these two polypeptides by analyzing their amino acid composition, cyanogen bromide peptide maps, and immunochemistry. The results showed that the subunits of the enzyme have quite different properties. Antibodies prepared against the polypeptides were used as probes of the catalytic functions of the subunits. Immunotitration studies indicated that only anti-alpha inhibited enzyme activity. The antibiotin fraction of this antibody population was removed by passage through biotin-Sepharose (anti-alpha'). Titration curves using anti-alpha' showed identical inhibition when total pyruvate carboxylase activity, ATP/Pi exchange activity, and pyruvate/oxalacetate exchange activity were measured, suggesting that both active sites are located on the alpha polypeptide. The arrangement of the subunits in the quaternary structure was investigated by means of the surface probe carbonic anhydrase linked to toluene isocyanate, and by partial digestion experiments with trypsin, chymotrypsin, and pronase. The results indicated that the alpha polypeptides are on the outside of the molecule and the beta polypeptides are the internal subunits.
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PMID:Characterization of the subunit structure of pyruvate carboxylase from Pseudomonas citronellolis. 679 93

A metallo-endoproteinase was purified from mouse kidney. The enzyme was solubilized from the 100 000 g sediment of kidney homogenates with toluene and trypsin, and further purified by fractionation with (NH4)2SO4. DEAE-cellulose chromatography and gel filtration. The molecular weight of the metalloproteinase was estimated by gel filtration on Sepharose 6B to be 270 000--320 000. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of 2-mercaptoethanol, a single major protein with a mol.wt. of 81 000 was observed. Thus the active enzyme is an oligomer, probably a tetramer. It is a glycoprotein and has an apparent isoelectric point of 4.3. Kidney homogenates and purified preparations of the metalloproteinase degraded azocasein optimally at pH 9.5 and at I 0.15--0.2. The activity was not affected by inhibitors of serine proteinases (di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride), cysteine proteinases (4-hydroxymercuribenzoate, iodoacetate), aspartic proteinases (pepstatin) or several other proteinase inhibitors from actinomycetes (leupeptin, antipain and phosphoramidon). Inhibition of the enzyme was observed with metal chelators (EDTA, EGTA, 1,10-phenanthroline), and thiol compounds (cysteine, glutathione, dithioerythritol, 2-mercaptoethanol). The metalloproteinase degraded azocasein, azocoll, casein, haemoglobulin and aldolase, but showed little or no activity against the synthetic substrates benzoylarginine 2-naphthylamide, benzoylglycylarginine, benzyloxycarbonylglutamyltyrosine or acetylphenylalanyl 2-naphthyl ester. This metalloproteinase from mouse kidney appears to be distinct from previously described kidney proteinases.
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PMID:Purification and characterization of a metallo-endoproteinase from mouse kidney. 704 88

The possibility of synthesizing stable alkyl analogues of acyl trypsins by introducing the alkyl residue by means of active-site-directed sulfonic acid esters was studied. Nine amidino- or guanidino-substituted sulfonic acids of different geometries and their methyl esters were prepared. The time-dependent inhibition of bovine trypsin by these esters, indicating modification at the active site of the enzyme, was followed. With the exception of p-guanidinobenzenesulfonic acid methyl ester, all the esters acted as irreversible inhibitors. The site of methylation, Ser-195 or His-57 (chymotrypsinogen numbering), was determine by analyzing for O-methylserine and methylhistidines. With four of the esters indications of a possible formation of, at most, 0.1 residue of O-methylserine per inactivated trypsin molecule were obtained. tau-Methylhistidine (but no pi-methylhistidine) was, however, always observed as the main product of the modification reaction. A further product, hitherto not yet described in active site methylations of serine proteinases, was pi, tau-dimethylhistidine (1,3-dimethylhistidine). The failure of an attempted synthesis of the N-acetyl-ethanolamine ester of p-toluene-sulfonic acid reported in the literature is shown to be due to the high instability of this ester.
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PMID:Formation of pi, tau-dimethylhistidine on alkylation of trypsin with active-site-directed sulfonic acid methyl esters. 753 86

To determine whether the measurement of specific markers of inflammatory cells in peripheral blood might be used to detect the inflammatory activity in the airways in asthma induced by toluene diisocyanate (TDI), we measured the levels of eosinophil cationic protein (ECP), histamine and tryptase in peripheral blood before and during inhalation challenge with TDI or methacholine in two groups of subjects who exhibited or did not exhibit an asthmatic reaction after exposure to toluene diisocyanate in the laboratory. When the subjects developed a late asthmatic reaction after exposure to TDI, they showed an increase in their ECP serum levels. By contrast, there were no significant changes in serum ECP levels after exposure to TDI in the control group or after methacholine challenge in either group. Tryptase levels in serum were not detectable before or during inhalation challenge with TDI or methacholine. There was no significant increase in plasma histamine levels during inhalation challenge with TDI or methacholine. These results suggest that eosinophils are 'activated' in subjects who develop a late asthmatic reaction after exposure to TDI and that the measurement of ECP levels in peripheral blood may be a useful marker to monitor airway inflammation.
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PMID:Eosinophil cationic protein (ECP), histamine and tryptase in peripheral blood before and during inhalation challenge with toluene diisocyanate (TDI) in sensitized subjects. 798 22

Pancreatic-derived proteases play a central role in the pathogenesis of ischemic intestinal injury. We postulated that exocrine blockade by pretreatment with a long-acting somatostatin analogue, octreotide acetate, would attenuate ischemic mucosal injury. Sprague-Dawley rats received subcutaneous octreotide (10 micrograms/kg/d) for 6 days by means of surgically implanted infusion ports. In a group of sham control rats, splanchnic blood flow (portal vein Doppler measurement) and duodenal trypsin activity (p-toluene sulfonyl-L-arginine methyl ester assay) were determined. In a separate experiment, pretreated animals were subjected to 60 minutes of superior mesenteric artery ischemia alone or followed by 30 minutes of reperfusion. Gross extent of hemorrhagic necrosis and microscopic injury (rank analysis) were assessed by a blinded observer. Pretreatment with octreotide reduced intraluminal duodenal trypsin activity by 46% without affecting portal blood flow. However, octreotide pretreatment significantly attenuated the microscopic depth of injury during ischemia and the extent of gross injury during reperfusion. It appears that somatostatin may have an adjuvant role in the prevention or progression of intestinal ischemic injury.
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PMID:Somatostatin attenuates ischemic intestinal injury. 809 72

In order to identify the endogenous protease associated with stratum corneum (SC) desquamation, we examined properties of proteases in the stratum corneum of normal human skin. SC were obtained by tape stripping, washed in toluene and then dried. The proteolytic activity in SC was measured using peptidyl 4-methyl-coumaryl-7-amides (MCAs). The SC was dispersed uniformly in the reaction mixture with dimethylformamide and Triton X-100 and incubated with the peptidyl MCAs. The protease in the SC hydrolysed both Boc-Phe-Ser-Arg-MCA and Boc-Gln-Ala-Arg-MCA (substrates for trypsin) very effectively. The hydrolytic activity was inhibited by the serine protease inhibitors diisopropyl fluorophosphate (DFP), aprotinin, antipain and leupeptin, but not by chymostatin, a chymotrypsin inhibitor. These results show that one or more trypsin-like serine protease is present in the SC of normal human skin. Casein-acrylamide electrophoresis showed that the molecular weight of this serine protease was about 30 kDa. We have previously shown that cells dissociate from human SC sheets in a detergent mixture (N,N-dimethyldodecylamine oxide and sodium lauryl sulphate). This cell dissociation was inhibited by aprotinin and leupeptin. In addition, the proteolytic activity in the outer SC was higher than that in the inner SC, and the activity in the SC of scaly skin induced by SLS treatment was higher than that of untreated skin. These results strongly suggest that the trypsin-like serine protease described here is involved in SC desquamation.
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PMID:Detection and characterization of endogenous protease associated with desquamation of stratum corneum. 821 86

Sera from 29 patients who had reacted to a platelet (27) or packed red cell (2) transfusion and from 5 patients who had received platelets without reacting were collected over a 13-month period. The sera were assayed for total IgE, and IgE specific for ethylene oxide, phthalic anhydride, hexamethylene diisocyanate, methylene diphenyl diisocyanate, toluene diisocyanate, and mast cell tryptase. Three patients with reactions had elevated total IgE levels, and specific IgE was positive for hexamethylene diisocyanate in 2 of 28 (7.1%), methylene diphenyl diisocyanate in 1 of 29 (3.4%), and toluene diisocyanate in 14 of 27 (51.9%). No positives were found in patients without reactions and no patients had an elevated tryptase level. It is unlikely that anti-plasticizer hypersensitivity was responsible for the transfusion reactions, but the prevalence and significance of such antibodies in both the hospital population and the general population would merit further investigation.
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PMID:Anti-plasticizer specific IgE is present in the serum of transfused patients. 871 85

A 30 kDa protein was purified from pig liver cytosol by using ATP-Sepharose and Green A column chromatography. The partial amino acid sequences of the protein (95 amino acid residues) had no similarity with any proteins recorded in data banks. The protein was able to form a stable complex with unfolded dihydrofolate reductase (DHFR). The spontaneous refolding of chemically denatured DHFR was arrested by the 30 kDa protein. This inhibition presumably results from the formation of a stable complex between the 30 kDa protein and DHFR. Bound DHFR could be released from the protein with ATP. The protein also showed protease resistance in an ATP-dependent manner. Incubation of the 30 kDa protein with 5 mM ATP resulted in its resistance to V8 protease or to trypsin treated with 1-chloro-4-phenyl-3-L-toluene-p-sulphonamidobutan-2-one. Divalent cations enhanced the ATP-protection effect. CD analysis of the 30 kDa protein showed that ATP induced an increase in the beta-pleated sheet content and a decrease in the alpha-helix content of the 30 kDa protein. These results suggest that the 30 kDa protein, a novel cytosolic protein, might have an affinity for ATP, a chaperonin activity, and and an ATP-protection effect against some proteases in vivo.
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PMID:Novel 30 kDa protein possessing ATP-binding and chaperone activities. 929 Nov 33

A trypsin-like serine proteinase was purified from the incubation medium of rat brain slices by gelatin zymography. The purification consisted of ammonium sulfate precipitation, benzamidine-Sepharose 6B affinity chromatography, and carboxymethyl-cellulose and gel filtration chromatographies. The gelatinolytic activity, identified at 22 kDa (P22) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, was eluted as one active peak throughout the purification, and the final preparation gave a single protein peak on reverse-phase HPLC. Diisopropyl fluorophosphate, benzamidine, p-toluenesulfonyl-L-lysine chloromethyl ketone, and aprotinin completely inhibited the activity of P22, whereas phenanthroline, p-toluene-sulfonyl-L-phenylalanine chloromethyl ketone, and elastinal did not. P22 efficiently digested the extracellular matrix proteins laminin and type IV collagen. P22 produced an increase in intracellular Ca2+ concentration in A172 glioblastoma, which was desensitized through prior stimulation with protease-activated receptor-2 agonist peptide SLIGKV, indicating that P22 can stimulate protease-activated receptor-2. Rat brain penetration injury induced gelatinolytic activity in the lesioned area whose molecular size was consistent with that of P22. These results indicated that on incubation of rat brain slices, a trypsin-like serine proteinase was secreted into the medium that was capable of digesting extracellular matrix and stimulating protease-activated receptor-2. It is suggested that the gelatinolytic activity induced by brain injury might be that of P22.
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PMID:Purification and characterization of a trypsin-like serine proteinase from rat brain slices that degrades laminin and type IV collagen and stimulates protease-activated receptor-2. 1073 32


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