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)

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

A hypertrehalosaemic neuropeptide from the corpora cardiaca of the blowfly Phormia terraenovae has been isolated by reversed-phase h.p.l.c., and its primary structure was determined by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal pyroglutamate residue. The C-terminus was also blocked, as indicated by the lack of digestion when the peptide was incubated with carboxypeptidase A. The octapeptide has the sequence pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 and is clearly defined as a novel member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of peptides. It is the first charged member of this family to be found. The synthetic peptide causes an increase in the haemolymph carbohydrate concentration in a dose-dependent fashion in blowflies and therefore is named 'Phormia terraenovae hypertrehalosaemic hormone' (Pht-HrTH). In addition, receptors in the fat-body of the American cockroach (Periplaneta americana) recognize the peptide, resulting in carbohydrate elevation in the blood. However, fat-body receptors of the migratory locust (Locusta migratoria) do not recognize this charged molecule, and thus no lipid mobilization is observed in this species.
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PMID:Isolation and structure of a novel charged member of the red-pigment-concentrating hormone-adipokinetic hormone family of peptides isolated from the corpora cardiaca of the blowfly Phormia terraenovae (Diptera). 238 78

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

Histamine inhibits superoxide anion (O-2) production from human neutrophils stimulated by N-formylmethionyl-leucyl-phenylalanine (FMLP). The effects of histamine are dose-dependent and competitively antagonized by cimetidine. When passively sensitized rat serosal mast cells and human neutrophils are mixed together, O-2 production from FMLP-activated granulocytes is significantly reduced, following mast cell degranulation by acetylcholine. These inhibitory effects can be counteracted by cimetidine. Exposure of non-sensitized rat mast cells to FMLP-stimulated human neutrophils causes histamine release. These results suggest bidirectional control mechanisms between mast cells and neutrophils, that further stress the role of histamine in regulating inflammatory processes.
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PMID:Mast cell and neutrophil interactions: a role for superoxide anion and histamine. 240 74

A new 28 amino acid peptide, we recently isolated from the venom of the bumblebee Megabombus pennsylvanicus. has been characterized. The peptide, Met-Cys-Ile-Cys-Lys-Asn-Gly-Lys-Pro-Leu-Pro-Gly-Phe-Ile-Gly-Lys-Ile-Cys- Arg-Lys-Ile-Cys-Met-Met-Gln-Gln-Thr-His(NH2), has been named bumblebee mast cell degranulating (MCD) peptide due to its ability to degranulate rat peritoneal mast cells, and its resemblance to the bee venom MCD peptide. Bumblebee MCD peptide, unlike bombolitins, the other mast cell degranulating heptadecapeptides of bumblebee venom, is not lytic and releases histamine at a dose as low as 0.05 micrograms/ml (1.6 X 10(-8) M).
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PMID:Amino acid sequence of bumblebee MCD peptide: a new mast cell degranulating peptide from the venom of the bumblebee Megabombus pennsylvanicus. 242 Dec 65

Human neutrophils, having been activated by the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP), evoke histamine release from rat serosal mast cells. The release is dependent on FMLP concentration and it can be inhibited by disodium cromoglycate and by a flavonoid, silymarin, which displays free radical scavenging properties. Silymarin inhibition of neutrophil-mediated histamine release is dose-dependent. These results further stress the concept of a neutrophil-mast cell interaction, which may be involved in inflammatory processes.
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PMID:FMLP-activated neutrophils evoke histamine release from mast cells. 242 73

We have investigated certain aspects of the mechanism whereby substance P triggers secretion of 5-hydroxytryptamine (5-HT) from rat peritoneal mast cells in vitro. Substance P-induced release of 5-HT was inhibited following pretreatment of rat peritoneal cells with 0.01-1.0 units/ml neuraminidase; secretion induced by anti-IgE antibody was inhibited by pretreatment with 1.0 units/ml but not by lower concentrations of enzyme. Addition of the sialic acid-rich substances N-acetyl-neuraminlactose (up to 1.0 mM) and mucin (up to 1.0 mg/ml) to substance P in free solution failed to block the activity of the neuropeptide. Limulin, a sialic acid-specific lectin, failed to block substance P-induced secretion of 5-HT, but was found to possess intrinsic non-lytic secretory activity (at 5-20 micrograms/ml). Release of 5-HT induced by limulin was independent of that induced by substance P. A range of octapeptides incorporating the C-terminal sequence Gly-Ser-Phe-Phe, but differing in degree of cationicity and positioning of cationic residues in the four N-terminal positions, were tested for their capacity to antagonise the mast cell-triggering activity of substance P. A peptide incorporating two lysine residues at the N-terminus was found to have partial substance P antagonist activity; no effects on IgE-mediated secretion were observed.
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PMID:The mast cell response to substance P: effects of neuraminidase, limulin, and some novel synthetic peptide antagonists. 242 85

Pretreatment of rat peritoneal mast cells, human basophils, bone marrow-derived mouse mast cells (BMMC) and mouse mast cell line PT-18 cells with 1 microgram/ml pertussis toxin (PT) failed to inhibit immunoglobulin E (IgE)-dependent histamine release from the cells. In BMMC and PT-18 cells, even 20-hr incubation of the cells with 1 microgram/ml PT, which ADP-ribosylates more than 97% of 41 kDa, alpha-subunit of Ni in the cells, failed to affect the IgE-dependent release of histamine or arachidonate. The results indicate that GTP-binding protein, Ni, is not involved in the transduction of triggering signals induced by cross-linking of IgE receptors. In contrast, pretreatment of rat mast cells with 1 ng/ml to 0.1 microgram/ml PT for 2 hr inhibited histamine release induced by compound 48/80 in a dose-dependent manner. A similar pretreatment with PT inhibited thrombin-induced histamine release from BMMC and N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced histamine release from human basophils in a similar dose-dependent fashion. However, even 20 hr of incubation of sensitized BMMC with 1 microgram/ml PT failed to inhibit either thrombin-induced or antigen-induced breakdown of phosphatidylinositides (PI), i.e., the formation of inositol triphosphate and diacylglycerol, Quin-2 signal, and the release of arachidonic acid. The results indicate that the inhibition of thrombin-induced histamine release by PT-treatment is not due to the inhibition of PI-turnover, and that Ni is not involved in thrombin-induced or antigen-induced (IgE-dependent) hydrolysis of phosphatidylinositides in mast cells.
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PMID:Effects of ADP-ribosylation of GTP-binding protein by pertussis toxin on immunoglobulin E-dependent and -independent histamine release from mast cells and basophils. 243 30


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