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)

We have investigated the viscosity of carboxypeptidase A catalyzed Bz-Gly-Phe hydrolysis at pH 7.5 (Tris) and 0.5 mol.l-1 NaCl over the range 10-100 mp, varied by addition of glycerol or sucrose. In contrast to previous reports of strong viscosity effects on the corresponding Cbz-Ala-Ala-Ala hydrolysis, both the catalytic constant and the Michaelis constant are virtually independent of viscosity over the 10-fold range investigated. Furthermore, the CD spectra of carboxypeptidase A in the high-viscosity media point to no change in the alpha-helix and beta-sheet structure in these media. The data are compatible either with a compacter, more rigid enzyme-substrate structure or with a more prominent role of intramolecular nuclear reorganization compared to protein reorganization for Bz-Gly-Phe than for Cbz-Ala-Ala-Ala. These views can be given a preciser frame in terms of stochastic chemical rate theory.
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PMID:Substrate specificity of solvent viscosity effects in carboxypeptidase A catalyzed peptide hydrolysis. 200 84

Various N-alpha-hydroxyalkyl derivatives of N-acyl amino acids and di- and tripeptides were prepared by hydrolysis or aminolysis of N-acyl 5-oxazolidinones. The stability of these derivatives was studied in aqueous solution as a function of pH. The compounds were all degraded quantitatively to their parent N-acylated amino acid or peptide and aldehyde but with vastly different rates. At pH 7.4 and 37 degrees C the half-lives of decomposition ranged from 4 min to 1500 hr. The structural factors influencing the stability included both steric and polar effects within the acyl and N-alpha-hydroxyalkyl moieties as well as within the amino acid attached to the N-alpha-hydroxyalkylated N-acyl amino acid. Whereas the N-benzyloxycarbonyl (Z) derivatives of the dipeptides Gly-L-Leu and Gly-L-Ala were readily hydrolyzed by carboxypeptidase A, the N-hydroxymethylated compounds, i.e., Z-Gly(CH2OH)-Leu and Z-Gly(CH2OH)-Ala, were resistant to cleavage by the enzyme as revealed by their similar rates of decomposition in the presence or absence of the enzyme at pH 7.4 and 37 degrees C. The results suggest that N-alpha-hydroxyalkylation of a peptide bond protects not only this bond but also an adjacent peptide bond against proteolytic cleavage. Since the N-alpha-hydroxyalkyl derivatives are readily bioreversible, undergoing spontaneous hydrolysis at physiological pH, this prodrug approach promises to overcome the enzymatic barrier to absorption of various peptides.
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PMID:Prodrugs of peptides. 9. Bioreversible N-alpha-hydroxyalkylation of the peptide bond to effect protection against carboxypeptidase or other proteolytic enzymes. 205 17

The role of the C-terminal Phe882-Ala883 residues of bacteriophage T7 RNA polymerase in specific transcription has been investigated by means of site-directed mutagenesis. A mutant enzyme that lacks the C-terminal Phe882-Ala883 residues, denoted the "foot" mutant, has been cloned and overproduced, and the effects of the deletion on promoter recognition, initiation, and elongation have been determined. Gel retardation assays and DNase I footprinting show that the foot mutant specifically recognizes and binds to T7 promoters, although this binding appears to be approximately 30-fold weaker than that of the wild-type enzyme. Transcription assays using oligonucleotide templates that contain the consensus T7 promoter show a dramatic decrease in transcriptional activity for the foot mutant. With templates whose coding region begins CCC..., the mutant synthesizes poly(G) products even in the presence of all four nucleotides. The synthesis of poly(G) products from such templates has previously been observed for the wild-type enzyme when GTP is the sole nucleotide present in the reaction and is thought to occur by a novel mechanism involving slippage of the RNA chain 3' to 5' relative to the template [Martin, C.T., Muller, D.K., & Coleman, J.E. (1988) Biochemistry 27, 3966-3974]. These data suggest that the loss in transcriptional activity by the foot mutant results from a severe decrease in processivity as well as catalytic efficiency of the enzyme. Removal of the C-terminal Phe and Ala residues from the wild-type enzyme with carboxypeptidase A generates the phenotype of the mutant precisely, proving that all of the properties of the foot mutant derive from the loss of the Phe-Ala-COOH moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Processivity of T7 RNA polymerase requires the C-terminal Phe882-Ala883-COO- or "foot". 205 36

Sonicates of mouse bone marrow-derived mast cells (BMMC) differentiated in vitro and of mouse serosal mast cells differentiated in vivo contained small but approximately equal amounts of aminopeptidase activity, as determined by cleavage of leucine-beta-naphthylamide and resolution of the reaction products by reverse-phase high-performance liquid chromatography. Aminopeptidase activity was exocytosed from antigen-activated, IgE-sensitized BMMC in proportion to the secretory granule enzyme beta-hexosaminidase, thereby localizing approximately 60% of the total cell-associated aminopeptidase activity to the secretory granules of the mast cells. A prominent secretory granule location for aminopeptidase was confirmed by activity measurement in subcellular fractions of disrupted BMMC. The secretory granule aminopeptidase had a pH optimum of 6.0-8.0 and a Km of 0.36 +/- 0.06 mM (mean +/- SD; n = 3) for leucine-beta-naphthylamide. When various amino acid beta-naphthylamides were used as substrates, the preference of the secretory granule enzyme was Ala greater than Leu greater than Phe much greater than Arg much greater than Asp = Tyr. Most of the aminopeptidase activity that was exocytosed from calcium ionophore-activated BMMC was bound to 35S-labeled proteoglycans in complexes of greater than 1 x 10(7) kDa as defined by exclusion during Sepharose CL-2B gel-filtration chromatography. We postulate that the amino-peptidase in the mast cell protease/proteoglycan complexes allows the removal of N-terminal amino acids from peptides that are generated by the action of mast cell endopeptidases.
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PMID:Identification of aminopeptidase activity in the secretory granules of mouse mast cells. 206 74

The neutral protease tryptase has been isolated from a human mast cell line, HMC-1. The HMC-1 line was established from the peripheral blood of a patient with mast cell leukemia and maintained as continuously proliferating clones in vitro and as solid mast cell tumors in nude mice. HMC-1-derived tryptase was purified by sequential chromatography on Dowex 1, DEAE 5 PW, and heparin-agarose. Purified tryptase has an apparent molecular weight of 150,000, as determined by molecular sieve HPLC, but migrates as a doublet of bands of 32/35,000 on SDS-PAGE gels. Maximal enzymatic activity was observed at pH 8.5. Cleavage of tosyl-L-arginine methyl ester by purified tryptase was inhibited by dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone 2 HCl, HgCl2, tosyl-L-lysine chloromethyl ketone, leupeptin, and PMSF but not by benzamidine, aprotinin, tosyl-L-phenyl-alanine chloromethyl ketone, soybean trypsin inhibitor, human plasma, ovomucoid inhibitor, or lima bean trypsin inhibitor. Microsequencing of purified tryptase yielded an amino terminal sequence that was identical to that previously reported for human pituitary-derived tryptase.
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PMID:Purification of tryptase from a human mast cell line. 211 May 91

Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.
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PMID:Purification and properties of recombinant rat catalase produced in Escherichia coli. 220 16

Crystallographic studies suggest that Arg-127 is a key amino acid in the hydrolysis of peptides and esters by carboxypeptidase A. The guanidinium group of Arg-127 is hypothesized to stabilize the oxyanion of the tetrahedral intermediate formed by the attack of water on the scissile carbonyl bond. We have replaced this amino acid in rat carboxypeptidase A1 with lysine (R127K), methionine (R127M), and alanine (R127A), in order to define the role of Arg-127 in carboxypeptidase catalyzed hydrolysis. The wild-type and mutant enzymes were expressed in yeast and purified. Kinetic studies show that Arg-127 substitution decreases kcat for both ester and amide substrates, whereas Km is relatively unchanged; for R127M and R127A this corresponds to a 6 kcal/mol decrease in transition state stabilization of the rate-limiting step. The binding affinity for the phosphonate transition state analog, Cbz-Phe-Ala(P)-OAla, was decreased by 5.4 kcal/mol, whereas binding affinity for the ground state inhibitor, DL-benzylsuccinic acid, was decreased by only 1.7 kcal/mol for R127M. Electrostatic calculations employing a finite difference solution to the Poisson-Boltzmann equation predict that the positive charge of Arg-127 should stabilize the transition state by 6-8 kcal/mol. Therefore, the experimental and theoretical data suggest that the primary role of Arg-127 is stabilization of the transition state through electrostatic interaction with the oxyanion.
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PMID:Arginine 127 stabilizes the transition state in carboxypeptidase. 224 16

The protease inhibitor alpha-1-antichymotrypsin, which binds to chymotrypsin-like enzymes in a sodium dodecyl sulfate-resistant manner, has been shown recently to be both a normal constituent of brain and an integral component of the neuritic plaques that form in Down's syndrome and Alzheimer's disease. We have now identified in rat brain a Mr 25,000 alpha-1-antichymotrypsin-binding protein classified as a chymotrypsin-like protease by its inhibitor profile and substrate specificity. Release of 125I-labeled breakdown products from bands containing the protease in substrate-linked polyacrylamide gels was examined in parallel with hydrolysis of tetrapeptide chromogenic substrates in vitro to establish conditions under which the Mr 25,000 protease was the only activity being measured in vitro. The protease was completely membrane associated but was extractable using 1 M MgCl2; prior extraction of detergent- and low ionic strength-soluble proteins from membranes was used to increase its specific activity. The formation of sodium dodecyl sulfate-resistant bonds between human alpha-1-antichymotrypsin and the protease (kassoc = 2.9 X 10(6) M-1 s-1) was used to titrate the concentration of free protease solubilized from membranes. The protease cleaved both succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and methoxy-succinyl-Ala-Ala-Pro-Met-p-nitroanilide, the latter being of interest because cleavage after a methionine residue is predicted to generate the amino terminus of the neuritic plaque component beta-amyloid from its precursor protein. In fact, the solubilized protease degraded 90% of membrane-associated beta-amyloid precursor protein detected by Western blot analysis. The protease was kinetically distinct from both chymotrypsin and cathepsin G in direct comparisons and did not match kinetic values published for the rat mast cell proteases against comparable substrates; we therefore refer to the protease with the descriptive acronym clipsin (for chymotrypsin-like protease). Proteases similar to and potentially identical to clipsin were detected by enzymography in other organs from rat (most notably spleen and adult lung). The enzyme in brain was distinguished by a narrow window of elevated activity surrounding postnatal day 5, which was 12-14-fold higher than levels in day 1 or adult brain. Because independent lines of evidence suggest that a brain chymotrypsin-like protease may be involved in the etiology of Down's syndrome and Alzheimer's disease, clipsin is discussed as a candidate for such a role.
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PMID:Clipsin, a chymotrypsin-like protease in rat brain which is irreversibly inhibited by alpha-1-antichymotrypsin. 230 81

The posttranslational modification of proteins by amino acids has been described in a variety of biological systems. These reactions occur at low levels in intact sciatic nerves of rats but are increased 10-fold following nerve injury and during subsequent regeneration of the nerve. While it has been shown in brain and liver that the site of addition of Arg is to the N-terminus, there is no information on the location at which the other amino acids add on to targeted proteins nor the site of addition of Arg in regenerating nerves. In the present study, we have used manual micro-Edman degradation combined with HPLC, and digestion with carboxypeptidase A and B to determine the site of addition of various amino acids to targeted proteins. Of the 3H-labelled amino acids incorporated posttranslationally into proteins of regenerating sciatic nerves (Arg, Lys, Leu, Phe, Val, Ala, Pro and Ser), only [3H]Arg was found to be present at the N-terminus. To determine whether amino acid additions were occurring at the C-terminus, proteins modified by two of the amino acids incorporated in greatest amounts (Lys and Leu) were incubated with specific carboxypeptidases. [3H]Leucine was not liberated following incubation with carboxypeptidase, suggesting that Leu is not added at the C-terminus of modified proteins. Under similar conditions, some [3H]Lys was liberated, but in amounts not significantly different from controls incubated without carboxypeptidase, indicating a non-specific degradation of Lys modified proteins rather than a specific release of Lys from the C-terminus. These experiments show that in regenerating sciatic nerves of rats, Arg is the only amino acid added posttranslationally to the amino terminus of target proteins, and that Leu, and probably Lys, are not conjugated to proteins at the C-terminus.
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PMID:The site of amino acid addition to posttranslationally modified proteins of regenerating rat sciatic nerves. 233 81

O-[[(1R)-[[N-(Phenylmethoxycarbonyl)-L-alanyl]amino]ethyl] hydroxyphosphinyl]-L-3-phenyllacetate [ZAAP(O)F], an analogue of (benzyloxycarbonyl)-Ala-Ala-Phe or (benzyloxycarbonyl)-Ala-Ala-phenyllactate, binds to carboxypeptidase A with great affinity (Ki = 3 pM). Similar phosphonates have been shown to be transition-state analogues of the CPA-catalyzed hydrolysis [Hanson, J. E., Kaplan, A. P., & Bartlett, P. A. (1989) Biochemistry 28, 6294-6305]. In the present study, the structure of the complex of this phosphonate with carboxypeptidase A has been determined by X-ray crystallography to a resolution of 2.0 A. The complex crystallizes in the space group P2(1)2(1)2(1) with cell dimensions a = 61.9 A, b = 67.2 A, and c = 76.2 A. The structure of the complex was solved by molecular replacement. Refinement of the structure against 20,776 unique reflections between 10.0 and 2.0 A yields a crystallographic residual of 0.193, including 140 water molecules. The two phosphinyl oxygens of the inhibitor bind to the active-site zinc at 2.2 A on the electrophilic (Arg-127) side and 3.1 A on the nucleophilic (Glu-270) side. Various features of the binding mode of this phosphonate inhibitor are consistent with the hypothesis that carboxypeptidase A catalyzed hydrolysis proceeds through a general-base mechanism in which the carbonyl carbon of the substrate is attacked by Zn-hydroxyl (or Zn-water). An unexpected feature of the bound inhibitor, the cis carbamoyl ester bond at the benzyloxycarbonyl linkage to alanine, allows the benzyloxycarbonyl phenyl ring of the inhibitor to interact favorably with Tyr-198. This complex structure is compared with previous structures of carboxypeptidase A, including the complexes with the potato inhibitor, a hydrated keto methylene substrate analogue, and a phosphonamidate inhibitor. Comparisons are also made with the complexes of thermolysin with some phosphonamidate inhibitors.
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PMID:Crystal structure of the complex of carboxypeptidase A with a strongly bound phosphonate in a new crystalline form: comparison with structures of other complexes. 238 84


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