Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ability of plasma proteinase inhibitors to inactivate human chymase, a chymotrypsin-like proteinase stored within mast cell secretory granules, was investigated. Incubation with plasma resulted in over 80% inhibition of chymase hydrolytic activity for small substrates, suggesting that inhibitors other than alpha 2-macroglobulin were primarily responsible for chymase inactivation. Depletion of specific inhibitors from plasma by immunoadsorption using antisera against individual inhibitors established that alpha 1-antichymotrypsin (alpha 1-AC) and alpha 1-proteinase inhibitor (alpha 1-PI) were responsible for the inactivation. Characterization of the reaction between chymase and each inhibitor demonstrated in both cases the presence of two concurrent reactions proceeding at fixed relative rates. One reaction, which led to inhibitor inactivation, was about 3.5 and 4.0-fold faster than the other, which led to chymase inactivation. This was demonstrated in linear titrations of proteinase activity which exhibited endpoint stoichiometries of 4.5 (alpha 1-AC) and 5.0 (alpha 1-PI) instead of unity, and SDS gels of reaction products which exhibited a banding pattern indicative of both an SDS-stable proteinase-inhibitor complex and two lower Mr inhibitor degradation products which appear to have formed by hydrolysis within the reactive loop of each inhibitor. At inhibitor concentrations approaching those in plasma where inhibitor to chymase concentration ratios were in far excess of 4.5 and 5.0, the rate of chymase inactivation by both serpin inhibitors appeared to follow pseudo-first order kinetics. The "apparent" second order rate constants of inactivation determined from these data were about 3000-fold lower than the rate constants reported for human neutrophil cathepsin G and elastase with alpha 1-AC and alpha 1-PI, respectively. This suggests that chymase would be inhibited about 650-fold more slowly than these proteinases when released into plasma. These studies demonstrate that although chymase is inactivated by serpin inhibitors of plasma, both inhibitors are better substrates for the proteinase than they are inhibitors. This finding along with the slow rates of inactivation indicates that regulation of human chymase activity may not be a primary function of plasma.
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PMID:Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors. 259 76

The neuronal membrane protein which binds the K+-channel ligands dendrotoxin, mast cell degranulating peptide, and beta-bungarotoxin was purified from rat brain membranes. When analysed on 10% SDS gel electrophoresis, the purified protein contained two peptides: the toxin-binding subunit of apparent Mr 90,000 and another peptide of Mr 38,000. Neuraminidase treatment reduced the Mr of the toxin-binding subunit to 70,000. Glycopeptidase F gave a further reduction to Mr 65,000. In contrast, the peptide of Mr 38,000 showed no change in Mr upon treatment with neuraminidase and/or glycopeptidase F. It is concluded that the toxin-binding subunit of the dendrotoxin-binding protein, a presumptive K+ channel, is a sialated membrane protein with a peptide core of, at most, Mr 65,000.
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PMID:Enzymatic deglycosylation of the dendrotoxin-binding protein. 270 49

A carboxypeptidase activity was recently identified in highly purified human lung mast cells and dispersed mast cells from skin. Using affinity chromatography with potato-tuber carboxypeptidase inhibitor as ligand, mast cell carboxypeptidase was purified to homogeneity from whole skin extracts. The purified enzyme yielded a single staining band of approximately 34,500 D on SDS-PAGE. Carboxypeptidase enzyme content estimated by determination of specific activity, was 0.5, 5, and 16 micrograms/10(6) mast cells from neonatal foreskin, adult facial skin, and adult foreskin, respectively. Human mast cell carboxypeptidase resembled bovine carboxypeptidase A with respect to hydrolysis of synthetic dipeptides and angiotensin I, but was distinguished from carboxypeptidase A in its inability to hydrolyze des-Arg9 bradykinin. The amino acid composition of human mast cell carboxypeptidase was similar to the composition of rat mast cell carboxypeptidase. The amino-terminal amino acid sequence of mast cell carboxypeptidase demonstrated 65% positional identity with human pancreatic carboxypeptidase B, but only 19% with human carboxypeptidase A. Thus, human mast cell carboxypeptidase is a novel member of the protein family of zinc-containing carboxypeptidases, in that it is functionally similar but not identical to bovine carboxypeptidase A, but has structural similarity to bovine and human pancreatic carboxypeptidase B.
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PMID:Human mast cell carboxypeptidase. Purification and characterization. 270 24

A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.
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PMID:Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells. 271 18

The topography of chloroplast cytochromes f and b6 was probed with proteases carboxypeptidase A (CpA), trypsin, and Staph, aureus V8. The cytochrome and its proteolytic products were detected by heme stain and, in most experiments, by immunoreaction. In thylakoids, the only protease that significantly affected the intactness of cytochrome f was CpA that caused a small (delta Mr = -1-2000) decrease in the apparent molecular weight. In SDS-treated thylakoids, both trypsin and V8 degraded cytochrome f. The inferred topography of cytochrome f., with the COOH-terminus on the stromal (n) side, one membrane-spanning alpha-elix near the COOH-terminus, and most of the Cyt f mass on the lumen (p) side, is consistent with that previously inferred by others. Cytochrome b6 was not sensitive to CpA, but was more sensitive to trypsin and V8 protease than cytochrome f, cytochrome b-559, or the 17 kDa OEC extrinsic protein. Trypsin caused a small decrease in size of cytochrome b6, which was observed using whole protein antibody as a single smaller band (delta Mr approximately 2000) or two smaller discrete bands (delta Mr = -1000 and 2500, respectively) which, unlike the untreated protein, did not react with antibody generated to a peptide mimicking Asp-5-Gln-14 near the NH2-terminus. These shortened tryptic fragments were attributed to cleavage after R-10 and K-23 near the NH2-terminus, implying an orientation with the NH2-terminus on the stromal side of the membrane. The sensitivity of cytochrome b6 toward this trypsin cleavage was increased if the membranes were first incubated with CpA, showing that the NH2-terminal region of cytochrome b6 is masked by the COOH-terminal domain of one or more thylakoid proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topography of the chloroplast cytochrome b6: orientation of the cytochrome and accessibility of the lumen-side interhelix loops. 276 55

A growth factor acting synergistically with IL-3 on thiol-sensitive "mucosal type" bone marrow-derived mast cell lines, and therefore termed mast cell growth enhancing activity, is present in PWM stimulated spleen cell conditioned medium. Mast cell growth enhancing activity can be partially purified and completely separated from IL-3, IL-4, and IL-5, and for the most part from IL-6 and GM-CSF using strong cation exchange and Procion red affinity chromatography. Mast cell growth enhancing activity binds to Con A-Sepharose and can be digested with trypsin and chymotrypsin. It shows a Mr ranging from 37 to 43 kDa under nonreducing SDS-PAGE and a main isoelectric point ranging from 6.2 to 7.3.
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PMID:Partial purification of a mast cell growth-enhancing activity and its separation from IL-3 and IL-4. 278 57

Three different procarboxypeptidases A and two different procarboxypeptidases B have been isolated for the first time, in a pure and native state, from human pancreatic extracts. These proteins were purified in one or two quick steps by anion-exchange HPLC. All these forms have been biochemically characterized. Two of the procarboxypeptidases A, the A1 and A2 forms, are obtained in a monomeric state while the other, the A3 form, is obtained as a binary complex of a procarboxypeptidase A with a proproteinase E. This complex is stable in aqueous buffers at various ionic strengths and develops carboxypeptidase A and proteinase E activities in the presence of trypsin. The A1 and A2 forms show clear differences in electrophoretic mobility in SDS/polyacrylamide gels, isoelectric point, proteolytic activation process with trypsin and susceptibility to thermal denaturation. In contrast, these properties are similar in the A1 and A3 (binary complex) forms. On the other hand, with respect to the properties listed above, the B1 and B2 forms differ from each other mainly in isoelectric point. An overall comparison of the above properties reveals the unusual character of the A2 form, midway between the other A and B forms. N-terminal extended sequence analysis carried out on these proenzymes confirm that they constitute different isologous forms.
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PMID:Purification and properties of five different forms of human procarboxypeptidases. 292 Jul 28

A rat hybridoma producing a high-affinity IgG2a monoclonal antibody (B3B4) directed against against the murine lymphocyte IgE receptor (Fc epsilon R) was established by using purified Fc epsilon R from Fc epsilon R+ murine hybridoma B cells as immunogen. The monoclonal and polyclonal anti-Fc epsilon R inhibited the binding of IgE to the murine lymphocyte Fc epsilon R and were also used to isolate the Fc epsilon R. B3B4 specifically recognized only the 49-Kd Fc epsilon R on murine B lymphocyte as determined by immunoprecipitation and SDS-PAGE analysis. In addition to its reaction with intact Fc epsilon R, B3B4 also recognized Fc epsilon R fragments that were present in the culture media of Fc epsilon R+ hybridoma cells. The predominant fragments isolated were 38 Kd and 28 Kd by SDS-PAGE analysis. When tested for reactivity with other cell types, B3B4 was highly specific for murine B lineage cells in that it did not significantly react with Fc epsilon R on macrophages and T cells and, in addition, did not react with the high affinity mast cell Fc epsilon R. B3B4 completely blocked IgE rosetting, and a reciprocal inhibition of binding was seen in a dose-dependent fashion between IgE and B3B4, indicating a close proximity of the IgE and B3B4 binding sites. Saturation binding analysis indicated that the Fab' fragment of B3B4 bound to twice as many sites/cell as IgE, suggesting that there are two identical B3B4 determinants per 49-Kd Fc epsilon R or that the IgE binding site is formed by the association of at least two 49-Kd Fc epsilon R. However, unlike IgE, neither B3B4 nor F(ab')2-B3B4 nor Fab'-B3B4 were very effective in causing Fc epsilon R upregulation on murine hybridoma B cells; in fact, B3B4 prevented this upregulation when added in combination with IgE. These results suggest that a site-specific interaction provided only by IgE may be essential for ligand-specific upregulation. Both polyclonal and monoclonal antibodies will be useful in further studies concerning the functional relationship between the membrane Fc epsilon R and the soluble Fc epsilon R fragments.
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PMID:Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE. 295 Jan 71

Colony-stimulating factors (CSFs) produced by two simian virus 40(SV40) transformed macrophage cell lines (BAM1 and BAM3), and three hybrids (HM3-11, HM3-12, and HM3-14) derived from fusion between BAM3 and a Chinese hamster cell line (hs222-16) were examined. HM3-11 and HM3-14 produce two molecular species of CSF, which are not found in the conditioned media from cultures of BAM1 and BAM3 or lipopolysaccharide (LPS), phorbolmyristate-acetate (PMA), and zymosan-stimulated BAM3. HM3-12, which is classified into another group in terms of CSF secretion, does not produce these two CSFs. On the basis of various criteria, one of these CSF species (peak 1-CSF) was characterized as a macrophage-colony-stimulating factor (M-CSF). The other CSF (peak 2-CSF) induced a group of bone marrow cells in granulocytes and macrophages as well as growth of a mast cell line, IC2. This CSF has an apparent molecular weight of 18,000, estimated by SDS-polyacrylamide gel electrophoresis. Unlike interleukin 3 (IL3) from WEHI-3 cells, the growth factor activity of peak 2-CSF binds to DEAE-Sephacel. Thus, peak 2-CSF is similar to a granulocyte-macrophage colony-stimulating factor (GM-CSF) rather than to IL3. The anti L cell CSF serum does not inhibit the CSF activity in Chinese hamster fibroblast conditioned medium, and the IC2 cells do not respond to Chinese hamster lung conditioned medium (CHLCM), suggesting that peak 1- and peak 2-CSF are of mouse origin.
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PMID:Properties of colony-stimulating factors produced by macrophage cell lines and hybrid cells. 302 9

The capacity of purified tryptase from human lung mast cells to metabolize human fibrinogen, fibrin, and plasminogen was evaluated. Tryptase (5 micrograms/ml) inactivated the thrombin-induced clotting activity of fibrinogen (100 micrograms/ml) with essentially similar t 1/2 values of 4.6 min in the absence of heparin and 5.8 min in the presence of heparin (20 micrograms/ml) that were not appreciably different than with lysine-Sepharose-purified plasmin (5 micrograms/ml). Fibrinogen treated with tryptase together with heparin lost all detectable clotting activity by 4 hr at 37 degrees C, whereas fibrinogen treated with tryptase alone resulted in destruction of only 80% of fibrinogen clotting equivalents after 16 hr. Tryptase alone was observed to cleave only the alpha-chains of fibrinogen by electrophoresis of tryptase-treated, denatured, and reduced fibrinogen in polyacrylamide gradient gels. Tryptase together with heparin cleaved first the alpha-chain and then the beta-chain, the latter cleavage corresponding to complete loss of fibrinogen clotting activity by 4 hr. No fibrinogen fragments with anticoagulant activity were generated by tryptase. In contrast, plasmin left no residual clotting activity after 4 hr of incubation and generated fibrinogen fragments with anticoagulant activity. Plasmin sequentially cleaved the alpha, beta, and gamma subunits of fibrinogen. Tryptase alone (6 micrograms/ml) or together with heparin (20 micrograms/ml) failed to activate plasminogen (0.6 mg/ml) after a 60-min incubation at 37 degrees C. Addition of urokinase to tryptase-treated or untreated plasminogen resulted in essentially identical plasmin activities (0.32 and 0.34 U/ml, respectively), indicating that tryptase neither activates nor destroys plasminogen. Tryptase (700 ng) also failed to substantially solubilize cross-linked fibrin (2.6 micrograms) or the corresponding amount of fibrinogen bound to plastic microtiter plates with or without heparin. The failure to solubilize fibrinogen and, possibly, fibrin is consistent with the observation that the apparent m.w. by SDS polyacrylamide gel electrophoresis of unreduced fibrinogen is not appreciably altered by prior treatment with tryptase, even though cleavage of alpha-and beta-chains is revealed after reduction. Fibrinogenolysis by tryptase complements other mast cell mediators with anticoagulant properties such as heparin and suggests a significant prevention of coagulation by activated mast cells.
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PMID:The fibrinogenolytic activity of purified tryptase from human lung mast cells. 316 48


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