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)

Several mouse monoclonal antibodies to carboxypeptidase A (CPA) were prepared and purified, and their interaction with the enzyme was investigated. CPA is a well-characterized zinc-containing exopeptidase exhibiting peptidase as well as esterase activity. The antibodies obtained could be classified as follows: antibodies inhibiting mainly the peptidase activity of the enzyme, antibodies inhibiting mainly its esterase activity, antibodies affecting both activities, and antibodies which bind to the enzyme but have no marked effect on its catalytic properties. Binding constants of approximately 10(6) M-1 were obtained for most of the antibody-enzyme complexes tested. Additional information on the effect of the monoclonal antibodies on the active site of CPA was obtained by determining the change in the circular dichroism spectra of arsanilazotyrosine-248 carboxypeptidase A occurring as a result of the interaction of the enzyme with the antibodies studied. These findings suggest that CPA possesses at least three different specific antigenic sites, and that the active site of the enzyme for its peptidase activity differs from that for its esterase activity, though both sites seem to overlap to a considerable extent.
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PMID:Interaction of carboxypeptidase A with monoclonal antibodies. 620 Jul 67

This study concerned the fragmentation of beta-endorphin (beta-EP-(1-31) by synaptic membrane-bound peptidases. The peptides which accumulated during digestion of beta-endorphin by isolated synaptosomal plasma membrane preparations of rat brain were separated and isolated by high pressure liquid chromatography. Amino acid analysis of the peptide fractions indicated the formation of beta-EP-(1-21), beta-EP-(2-21) (pH 7.4), beta-EP-(18-31), beta-EP-(1-14), and beta-EP-(1-13) (pH 5.0) in addition to previously identified gamma-endorphin (beta-EP-(1-17)), alpha-endorphin (beta-EP-(1-16), and their des-tyrosine fragments (Burbach, J. P. H., Loeber, J. G., Verhoef, J., Wiegant, V. M., De Kloet, E. R., and De Wied, D. (1980) Nature 283, 96-97). The beta-endorphin fragments obtained with crude or with purified synaptosomal plasma membranes differed only quantitatively. The peptidase which converted gamma-endorphin into beta-EP-(1-16), beta-EP-(1-15), beta-EP-(1-14), and beta-EP-(1-13), was considerably active at pH 5.0 and resembled carboxypeptidase A in degrading gamma-endorphin; the activity was reduced by the carboxypeptidase A inhibitor D-phenylalanine. The data supplement previous findings and allow routes to be delineated for the conversion of beta-endorphin by brain synaptic membranes. A pathway comprising the main events in the conversion processes is proposed and is discussed in relationship to the significance of beta-endorphin as a precursor for neuropeptides with distinct central activities.
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PMID:Proteolytic conversion of beta-endorphin by brain synaptic membranes. Characterization of generated beta-endorphin fragments and proposed metabolic pathway. 627 86

The 22076-Mr Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces abuls G effectively catalyses the transfer of the N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl electrophilic group of the standard tripeptide substrate N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl-D-alanine to water. It also performs a weak beta-lactamase activity, hydrolysing penicillin into penicilloate at a very low rate. This protein consists of 212 amino acid residues in a single polypeptide chain. The N terminus is partially blocked as a result of the cyclization of the dipeptide Asn-Gly into anhydroaspartylglycine imide. The protein has been fragmented by cyanogen bromide into five fragments whose sequences have been determined via appropriate subcleavages with various proteases. The ordering of the cyanogen bromide peptide fragments has been carried out (a) by submitting the S-carboxymethylated protein to complete tryptic digestion and labelling the methionine-containing peptides thus obtained with iodo[14C]-acetamide, and (b) by submitting to limited tryptic digestion the S-[2-(4'-pyridyl)ethyl]-cysteine protein whose amino groups have been blocked by reaction with exo-cis-3,6-endoxo-delta 4-tetrahydrophthalic anhydride prior to digestion. The protein contains six cysteine residues in the form of three disulfide bridges. No homology is found by comparing this peptidase with other Zn2+-containing enzymes (carboxypeptidase A, thermolysin, carbonic anhydrase B and alcohol dehydrogenase) and several completely or partially sequenced, serine-containing D-alanyl-D-alanine-cleaving peptidases and Zn2+/serine-containing beta-lactamases.
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PMID:The complete amino acid sequence of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of streptomyces albus G. 682 89

1. Growing rats were fed either ad lib. or with six (equal) meals offered every 4 h (from 10.00 hours). Rats of each group were killed at intervals of 4 h beginning at 11.00 hours. Activities of cathepsin A (carboxypeptidase A; EC 3.4.12.2), C (dipeptidyl peptidase; EC 3.4.14.1) and D (endopeptidase D EC 3.4.23.5) were measured in liver and muscle homogenates and free amino acids in blood were determined. 2. In the rats fed ad lib. activities of carboxypeptidase A and endopeptidase D in liver and muscle showed significant variation, with maximum activity in the light period. In general, meal-feeding only caused minor differences in cathepsin activities; although significant differences occurred for carboxypeptidase A. For the later enzyme a peak in activity occurred in the dark as well as in the light period. 3. Irrespective of the feeding schedule, the lower concentration of free essential amino acids of blood occurred generally during the night period. With the controlled-feeding schedule there is an increase of essential amino acids and a slight decrease of non-essentail amino acids of blood.
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PMID:Variations through the day of hepatic and muscular cathepsin A (carboxypeptidase A; EC 3.4.12.2), C (dipeptidyl peptidase; EC 3.4.14.1) and D (endopeptidase D; EC 3.4.23.5) activities and free amino acids of blood in rats: influence of feeding schedule. 719 24

We have established the peptidase content of a P2 fraction (enriched in synaptosomes) and plasma membranes prepared from canine intestinal mucosa. Fourteen exo- and endopeptidases were assayed with fluorimetric or chromogenic substrates and identified by means of specific peptidase inhibitors. Post-proline dipeptidyl aminopeptidase IV, aminopeptidase M, and carboxypeptidase A were the most abundant exopeptidases, while aminopeptidases A and B, dipeptidyl aminopeptidase, pyroglutamyl peptide hydrolase I, and carboxypeptidase B displayed little, if any, activity. Endopeptidase 24.11 was the only endopeptidase that was detected in high amount. By contrast, proline endopeptidase exhibited a low activity, while angiotensin-converting enzyme, endopeptidase 24.15, endopeptidase 24.16, and cathepsin B and D-like activities were not detected. The catabolic rates of the two related neuropeptides, neurotensin (NT) and neuromedin N (NN), established that NN was inactivated 16 to 24 times faster than NT by plasma membrane and P2 fractions, respectively. Furthermore, the two peptides underwent qualitatively distinct mechanisms of degradation. A phosphoramidon-sensitive formation of NT(1-10) was detected as the major NT catabolite, indicating that NT was susceptible to an endoproteolytic cleavage elicited by endopeptidase 24.11. By contrast, NN was inactivated by the action of an exopeptidase at its N-terminus, leading to the formation of [des-Lys1]NN. The occurrence of this NN metabolite was prevented by bestatin and actinonin, but not by the aminopeptidase B inhibitor, arphamenine B, indicating that the release of the N-terminal residue of NN was likely due to aminopeptidase M.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential catabolic fate of neuromedin N and neurotensin in the canine intestinal mucosa. 833 46

The sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 has been analysed by proton-induced X-ray emission. It contains 1 equivalent Zn2+ per mol of protein. As derived from gene cloning and sequencing, the B. sphaericus Zn peptidase I is a two-module protein. A 100-amino-acid-residue N-terminal domain consisting of two tandem segments of similar sequences, is fused to a 296-amino-acid-residue C-terminal catalytic domain. The catalytic domain belongs to the Zn carboxypeptidase A family, the closest match being observed with the Streptomyces griseus carboxypeptidase [Narahashi (1990) J. Biochem. 107, 879-886] and with the family prototype, bovine carboxypeptidase A. The catalytic domain of the B. sphaericus peptidase I possesses, distributed along the amino-acid sequence, peptide segments, a triad His162-Glu165-His307 and a dyad Tyr347-Glu366 that are equivalent to secondary structures, the zinc-binding triad His69-Glu72-His196 and the catalytic dyad Tyr248-Glu270 of bovine carboxypeptidase A respectively. The N-terminal repeats of the B. sphaericus peptidase I have similarity with the C-terminal repeats of the Enterococcus hirae muramidase 2, the Streptococcus (now Enterococcus) faecalis autolysin and the Bacillus phi PZA and phi 29 lysozymes, to which a role in the recognition of a particular moiety of the bacterial cell envelope has been tentatively assigned. Detergents enhance considerably the specific activity of the B. sphaericus peptidase I.
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PMID:Characterization of the sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 as a member of the metallo(zinc) carboxypeptidase A family. Modular design of the protein. 850 90

Tryptase is the major protein constituent of human mast cells, where it is stored within the secretory granules as a fully active tetramer. Two tryptase genes (alpha and beta) are expressed by human mast cells at the level of mRNA and protein, each with a 30 amino acid leader sequence. Recombinant precursor forms of human alpha- and beta-tryptase were produced in a baculovirus system, purified, and used to study their processing. Monomeric beta-protryptase first is shown to be intermolecularly autoprocessed to monomeric beta-pro'tryptase at acid pH in the presence of heparin by cleavage between Arg-3 and Val-2 in the leader peptide. The precursor of alpha-tryptase has an Arg-3 to Gln-3 mutation that precludes autoprocessing. this may explain why alpha-tryptase is not stored in secretory granules, but instead is constitutively secreted by mast cells and is the predominant form of tryptase found in blood in both healthy subjects and those with systemic mastocytosis under nonacute conditions. Second, the NH2-terminal activation dipeptide on beta-pro'tryptase is removed by dipeptidyl peptidase I at acid pH in the absence of heparin to yield an inactive monomeric form of tryptase. Conversion of the catalytic portion of beta-tryptase to the active homotetramer at acid pH requires heparin. Thus, beta-tryptase homotetramers probably account for active enzyme detected in vivo. Also, processing of tryptase to an active form should occur optimally only in cells that coexpress heparin proteoglycan, restricting this pathway to a mast cell lineage.
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PMID:A novel heparin-dependent processing pathway for human tryptase. Autocatalysis followed by activation with dipeptidyl peptidase I. 861 39

Chymase, a major product of mast cell activation, is secreted as a fully active enzyme. We have prepared recombinant human prochymase and have investigated the conditions under which it may be activated by dipeptidyl peptidase I (DPP I). The gene for human chymase was cloned in a baculovirus vector and expressed in High Five insect cells, and the recombinant protein purified by heparin-agarose and gel-filtration chromatography. The purified prochymase was homogeneous by SDS/PAGE with the same molecular mass as native human chymase, and its identity confirmed by N-terminal sequence analysis and Western blotting with chymase-specific antibodies. Treatment with DPP I to remove the N-terminal dipeptide prosequence resulted in enzymatically active chymase, with substrate and inhibitor profiles very similar to those of the native human enzyme. The activation of prochymase by DPP I was strongly inhibited by heparin (IC50 = 0.5 microg ml[-1]) and histamine (IC50 = 2 mM), though these mast cell products had little effect on the action of DPP I towards a low molecular-mass substrate. The pH optimum of DPP I was also higher and narrower with prochymase. The inhibitory action of heparin was lost at NaCl or KCl concentrations sufficient to elute prochymase from a heparin agarose column. Dextran sulphate was as inhibitory as heparin, whereas chondroitin sulphate C was more than 10-fold less effective. Our findings suggest that the activation of prochymase might be restricted to the early stages of vesicle maturation, when the pH is close to neutrality and the histamine and heparin concentrations are low.
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PMID:The conversion of recombinant human mast cell prochymase to enzymatically active chymase by dipeptidyl peptidase I is inhibited by heparin and histamine. 957 89

Dipeptidyl peptidase I (DPPI) is a cysteine protease found predominantly in myelomonocytic cells, cytotoxic T-cells, and mast cells. Recent studies identify an intracellular role for mast cell-DPPI (MC-DPPI) by activating prochymase and protryptase to their mature forms. To better define MC-DPPI and to explore the possibility of extracellular roles, we purified MC-DPPI from mastocytoma cells. We found the dog C2 mastocytoma cell line to be the richest source yet described for DPPI, purifying up to 200 microg of enzyme per g of cells. Dog MC-DPPI has an Mr of approximately 175,000 and consists of four subunits, each composed of a propeptide, light chain, and heavy chain. The heavy chain is N-glycosylated and is heterogeneously processed to three different forms. NH2-terminal sequences of the heavy chain and propeptide are identical to those predicted from a cDNA clone we sequenced from a mastocytoma cDNA library. The dog cDNA-derived sequence is 86% identical to that of human DPPI. Dog mastocytoma cells incubated with 12-O-tetradecanoylphorbol-13-acetate increase expression of MC-DPPI mRNA. MC-DPPI maintains its activity for dipeptide substrates at a neutral to alkaline pH. Cells stimulated with ionophore or substance P secrete MC-DPPI in parallel with the granule-associated mediators tryptase and histamine. Thus, dog mastocytoma cells secrete DPPI that is active at the pH of extracellular fluids, suggesting that MC-DPPI may act outside the cell.
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PMID:Regulated expression, processing, and secretion of dog mast cell dipeptidyl peptidase I. 962 39

Tryptase, a serine protease, is the major protein component in mast cells. In an animal model of asthma, tryptase has been established as an important mediator of inflammation and late airway responses induced by antigen challenge. Human tryptase is notable for its tetrameric structure, requirement of heparin for stability, and resistance to endogenous inhibitors. Human protryptase was expressed as a recombinant protein in Pichia pastoris. The recombinant protein consisted of two forms of protryptase, one containing the entire propeptide and the other containing only the Val-Gly dipeptide at its amino terminus. Isolation of active recombinant tryptase required a two column purification protocol and included a heparin- and dipeptidyl peptidase I-dependent activation step. Purified recombinant tryptase migrated as a tetramer on a gel filtration column and displayed kinetic parameters identical to those of a native tryptase obtained from HMC-1 cells, a human mast cell line. Recombinant and HMC-1 tryptase exhibited comparable sensitivities to an array of protein and low-molecular-weight inhibitors, including one that is highly specific for tryptase (APC-1167). Similarly, the recombinant enzyme cleaved both alpha- and beta-chains of fibrinogen to generate fibrinogen fragments indistinguishable from those generated by HMC-1-derived tryptase. Thus, recombinant tryptase expressed in P. pastoris displays physical and enzymatic properties essentially identical to the native enzyme. This system provides a cost-effective and easy to manipulate expression system that will enable the functional characterization of this unique enzyme.
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PMID:Expression and characterization of recombinant mast cell tryptase. 1009 84


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