Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:029742 (skin tryptase)
 38 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Leupeptin is a small peptide microbially derived inhibitor of certain proteolytic enzymes. Using N-alpha-benzoyl-DL-arginine 4-nitroanilide as substrate, we found a novel leupeptin-sensitive proteolytic enzyme in N-methyl-N-nitrosourea(MNU)-induced rat mammary adenocarcinoma. This enzyme was apparently different from urokinase-type plasminogen activator or cathepsin B and was present in mammary tumour at levels at least 20 times higher than those in normal mammary tissue. This enzyme was separated and purified from crude extracts of MNU-induced mammary adenocarcinoma approx. 1900-fold with 34% yield. It was a trypsin-like serine endopeptidase and had a pH optimum at 7.0. The native enzyme had an apparent M(r) of 180,000 and exhibited four isoelectric points ranging from 4.3 to 5.0. Electrophoresis of denatured enzyme, however, yielded, with reduction, a major band with an apparent M(r) of 37,500 and a minor band with an apparent M(r) of 35,500. The N-terminal 23 residues of the major band were Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Xaa12-Pro13- Val14- Gln15-Val16-Xaa17-Leu18-Xaa19-Val20- Trp21-Leu22-Pro23. These and other properties of this enzyme suggested that it most closely resembles rat skin tryptase, followed by rat peritoneal mast-cell tryptase and then by tryptases from other species. The rat, like human and mouse, may carry multiple tryptase genes, and this mammary-tumour enzyme may be an additional form of rat tryptase within a new serine-proteinase family.
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PMID:Separation, purification and N-terminal sequence analysis of a novel leupeptin-sensitive serine endopeptidase present in chemically induced rat mammary tumour. 131 62

Tryptase was purified 13,000-fold to apparent homogeneity from rat skin. The two-step procedure involved ammonium sulfate fractionation of the initial extract followed by combined sequential affinity chromatography on agarose-glycyl-glycyl-p-aminobenzamidine and concanavalin A-agarose. The purified enzyme had a specific activity toward N-benzoylarginine ethyl ester (BzArgOEt) of 170 mumol/min mg-1 and was obtained in a yield of 28% as determined by the specific substrate, H-D-Ile-Pro-Arg-p-nitroanilide. Rat skin tryptase was thermal labile, losing 50% of its activity when preincubated for 30 min at 30 degrees C. The presence of NaCl (1 M) improved thermal stability and was necessary for long-term storage. Heparin did not stabilize the enzyme against thermal denaturation, and heparin-agarose failed to bind the enzyme. Rat skin tryptase was inhibited by diisopropylphosphofluoridate, antipain, leupeptin, and aprotinin but not by alpha 1-antitrypsin, ovomucoid, or soybean or lima bean trypsin inhibitors. Substrate specificity studies using a series of tri- and tetrapeptidyl-p-nitroanilide and peptidyl-7-amino-4-methylcoumarin substrates demonstrated the existence of an extended substrate binding site. Rat skin tryptase hydrolyzed [Arg8]vasopressin, neurotensin, and the oxidized B-chain of insulin at the -Arg8-Gly9-NH2, -Arg8-Arg9-, and -Arg22-Gly23-bonds, respectively. No general proteinase activity was observed toward casein, hemoglobin, or azocoll. Rat skin tryptase had a Mr of 145,000 by gel filtration. The subunit Mr was either 34,000 or 30,000 depending on the electrophoretic technique used. Treatment of the enzyme with peptide N-glycosidase F (N-glycanase) decreased the subunit Mr by 4000. The enzyme exhibited multiple isoelectric forms (pI's of 4.5-4.9). Rat skin tryptase was found to be related statistically to other tryptases on the basis of amino acid composition. The N-terminal amino acid sequence was Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Trp12-Pro13- Trp14- Gln15-Val16-Ser17-Leu18-Arg19-Val20- --21-Asp-22Thr23-Tyr24-Typ25-, with a putative glycosylation site at residue 21. This sequence was 72-80% homologous with the N-terminus of other tryptases but only 40% homologous with that of bovine trypsin.
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PMID:Tryptase from rat skin: purification and properties. 203 67

We have expressed the 57-amino acid Kunitz domain of the Alzheimer's beta-amyloid precursor protein (APP751) as a bacterial fusion protein. The protease inhibitory properties of the purified fusion protein, BX9, were virtually identical in all respects tested to those of purified secreted APP751. Both proteins strongly inhibited pancreatic trypsin (Kis = 0.2 and 0.3 nM) and less well epidermal growth factor-binding protein (Kis = 1 and 3.5 nM), alpha-chymotrypsin (Kis = 3 and 6 nM), and the gamma-subunit of nerve growth factor (Kis = 8 and 9 M). Neither protein appreciably inhibited plasma and pancreatic kallikreins, thrombin, lung tryptase, neutrophil elastase, or cathepsin G. The remarkable similarity of the protease inhibitory profile of BX9 to that of secreted APP751 suggests that proper intramolecular disulfide bond formation has occurred in the bacterial fusion protein and leads to the conclusion that the amyloid precursor protein Kunitz domain is a relatively specific inhibitor of only a few trypsin-like arginine esterases.
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PMID:The protease inhibitory properties of the Alzheimer's beta-amyloid precursor protein. 211 13

Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the approximately 1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family.
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PMID:Human mast cell tryptase: multiple cDNAs and genes reveal a multigene serine protease family. 218 93

An antiserum was produced against a chymotryptic proteinase purified from human skin. The antiserum did not cross-react with human leukocyte cathepsin G and elastase, rat mast cell proteinase I, and human skin tryptase. Indirect immunofluorescent staining of frozen skin sections to localize the proteinase showed cytoplasmic staining of cells scattered about the papillary dermis and around blood vessels and appendages. Restaining these sections with toluidine blue revealed that the fluorescently stained cells contained metachromatically staining granules, the major distinguishing feature of mast cells. A similar correlation was found in lung tissue. Ultrastructural studies employing the ferritin bridge technique to immunologically identify the proteinase additionally localized the proteinase to mast cell granules. Biochemical and immunochemical characterization of chymotryptic activity solubilized from isolated human lung mast cells identified a chymotryptic proteinase that may be identical to the skin chymotryptic proteinase. These studies establish that human skin mast cells contain a chymotrypsin-like proteinase that is a granule constituent and provide evidence that indicates a comparable proteinase is also present in lung mast cells.
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PMID:Identification of a chymotrypsin-like proteinase in human mast cells. 242 94

The distribution of tryptase in various human tissue high-salt extracts (skin, lung, pancreas, liver, kidney, and spleen) was studied. Tryptase activity was compared with tissue histamine concentration, chymase activity, and cathepsin D, and histamine-N-methyltransferase (HMT) activities. Tryptase activity, found biochemically in tissue extracts, was localized in tissue sections by an enzyme-histochemical method using peptide 4-methoxy-2-naphthylamide substrates and Fast Garnet GBC as the chromogen. The highest levels of tryptase activity were found in lung and skin extracts. Liver, kidney, and spleen extracts displayed only a little activity. The distribution of histamine was similar to that of tryptase, whereas distributions of cathepsin D and HMT were quite different from that of tryptase. High-salt extracts of lung contained no detectable chymase activity, but in skin extracts this activity was high. Using an enzyme-histochemical method, the tryptase activity in tissue sections seemed solely to be confined to cells, which were granular and Giemsa positive after the red azo dye had been removed with Tween 20. Skin and lung sections contained the highest number of positively stained cells. The inhibition properties of tryptase, found in both tissue extracts and sections, and the substrate profile in tissue sections were identical. Human leukocyte preparation was negative for tryptase when stained enzyme-histochemically. The present results suggest that tryptase in human tissues is found only in the mast cells. The enzyme seems to be identical in the various human tissues studied because the different high-salt extracts were immunologically cross-reactive when tested with a rabbit polyclonal antibody against skin tryptase.
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PMID:Biochemical and histochemical evaluation of tryptase in various human tissues. 267 65

Capillary damage induced in sheep by intravenous infusion of Escherichia coli endotoxin, oleic acid, or air emboli causes the appearance in lung lymph of a serine protease with trypsin-like activity. The time course of the appearance of the enzyme and the extent of its activity increase indicate a close association with capillary injury. The enzyme was isolated from active lymph after a 9,000-fold purification by affinity chromatography on Reactive Blue-agarose, aprotinin-agarose, and p-amino-benzamidine-agarose columns. The protein, molecular mass of 70-75 kDa, is composed of two polypeptide chains of 31 and 43 kDa linked by disulfide bonds. Studies with synthetic peptide and thioester substrates showed preferential cleavage of substrates having two or more basic amino acids and the importance for activity of secondary enzyme-substrate interactions at sites removed from the scissile bond. The specificity of the enzyme and its pattern of sensitivity to inhibition by a series of isocoumarin derivatives distinguish it from enzymes of the clotting and complement systems and also from tissue plasminogen activator and lung and skin tryptase. The origin of the enzyme, its role in capillary damage, and its physiological function remain to be established.
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PMID:Lung lymph capillary injury-related protease. 267 41

A serine protease (Mr 70,000 to 75,000) appearing in sheep lung lymph after capillary damage induced by Escherichia coli endotoxin, oleic acid, or air emboli, was studied for its specificity toward a series of synthetic peptide and thioester substrates containing an Arg residue in the P1 position. High specificity constants (kcat/Km) were generally obtained with substrates having two or more basic amino acid residues, and with those having a Gln residues in the P2 position. Secondary enzyme-substrate interactions at sites more removed from the scissile bond are of importance, since a few peptides with two basic residues were hydrolyzed slowly, and the site of cleavage of natural peptides was influenced by the amino acid sequence beyond the immediate vicinity of the hydrolyzed bond. The properties of the enzyme and its pattern of specificity distinguish it from enzymes of the clotting cascade, from components of the complement system, and from lung and skin tryptase. The enzyme was inactivated by p-amidinophenylmethanesulfonyl fluoride and by a series of mechanism-based isocoumarin derivatives, the most potent inhibitor being 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Enzyme solutions inactivated by reaction with isocoumarin inhibitors could be completely reactivated after 30 h by treatment with hydroxylamine at neutral pH. Formation of a stable sheep lymph acyl enzyme--in contrast to thrombin and other trypsin-like enzymes--is not followed by alkylation of an active site nucleophile that leads to irreversible enzyme inactivation. The high activity toward substrates with two basic residues suggests that the enzyme may potentially function in processing of precursors of bioactive peptides.
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PMID:Substrate specificity and inhibitors of a capillary injury-related protease from sheep lung lymph. 291 36

The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z-Gly-Pro-Arg-pNA, Z-Gly-Pro-Arg-AMC, benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z-Gly-Pro-Arg-pNA or -AMC) were used. Only the Km value increased, while the Vmax value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent Km and Vmax values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.
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PMID:The allosteric effect of salt on human mast cell tryptase. 304 11

A tryptic protease with the characteristics of a mast cell tryptase was purified from dog mastocytoma cells propagated in nude mice. Partial amino acid sequence of the mastocytoma tryptase revealed unexpected differences in comparison with other mast cell and leukocyte granule protease sequences. Extraction from mastocytoma homogenates at high ionic strength, followed by gel filtration and benzamidine affinity chromatography yielded a product with several closely spaced bands (Mr 30,000-32,000) on gel electrophoresis and a single N-terminal sequence. Nondenaturing analytical gel filtration revealed an apparent Mr of 132,000, suggesting noncovalent association as a tetramer. Studies with peptide p-nitroanilides indicated pronounced substrate preferences, with P1 arginine preferred to lysine. Benzoyl-L-Lys-Gly-Arg-p-nitroanilide was the best of the substrates screened. Inhibition by diisopropyl fluorophosphate and tosyllysine chloromethyl ketone indicated that the enzyme is a serine protease. Like the tryptases of human mast cells, mastocytoma tryptic protease was inhibited by NaCl, resistant to inactivation by alpha 1-proteinase inhibitor and plasma, and stabilized by heparin. Comparison of the N-terminal 24 residues of mastocytoma tryptase revealed 80% identity with the more limited sequence reported for human lung tryptase, and surprisingly, closer homology to serine proteases of digestion and clotting than to other leukocyte granule proteases sequenced to date, including mast cell chymase. The N-terminal isoleucine is the homolog of trypsinogen Ile-16 which becomes the new N-terminus upon cleavage of the activation peptide. Thus, the tryptase N-terminus is related to the catalytic domain of activated serine proteases, and lacks the N-terminal regulatory domains found in most clotting and complement serine proteases. These findings provide further evidence that tryptases are unique serine proteases and that they may be less closely related in evolution and function than are other leukocyte granule proteases described to date.
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PMID:Dog mastocytoma tryptase: affinity purification, characterization, and amino-terminal sequence. 311 12


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