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 tested four synthetic substances for their histochemical value to demonstrate the catalytic activities of chymase or tryptase in mast cells in sections of human gut. Both Suc-Ala-Ala-Phe-4 methoxy-2-naphthylamide (MNA) and N-acetyl-L-methionine-alpha-naphthyl ester (alpha-N-O-Met) reacted with chymase but not tryptase in mast cells. Conversely, D-Val-Leu-Arg-MNA and Z-Ala-Ala-Lys-MNA were hydrolyzed by mast cell tryptase but not chymase. These results were confirmed by use of two inhibitors of chymotrypsin-like activity, chymostatin and Z-Gly-Leu-Phe-chloromethyl ketone (CK) and two inhibitors of trypsin-like activity, Tos-Lys-CK and D-Val-Leu-Arg-CK. Excellent staining reactions were obtained on cryostat sections of unfixed or aldehyde-fixed tissues and on paraffin sections of Carnoy-fixed tissues. For chymase, however, Suc-Ala-Ala-Phe-MNA is preferred on cryostat sections because it is more specific. On paraffin sections alpha-N-O-Met is preferred because other cells are not then stained. For tryptase, Z-Ala-Ala-Lys-MNA was more selective and more specific and is the preferred general purpose substrate on cryostat sections of aldehyde-fixed tissues and for paraffin sections. D-Val-Leu-Arg-MNA is the preferred substrate for cryostat sections of unfixed tissue. Only a limited number of mast cells showed a reaction for chymase, and these occurred mainly in the submucosa. All mast cells, however, gave a reaction for tryptase, and we recommend the use of either substrate for this enzyme for routine detection of mast cells in human tissues. Double staining for the two main mast cell proteases is most conveniently undertaken on paraffin sections of Carnoy-fixed tissues using MNA substrates for tryptase and alpha-N-O-Met for chymase.
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PMID:Enzyme histochemical discrimination between tryptase and chymase in mast cells of human gut. 264 38

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

Cobalt(II) arsanilazotyrosine-248 carboxypeptidase A has been characterized through 1H NMR spectroscopy. The ability of the azoenzyme to form binary and ternary complexes with L- and D-phenylalanine and azide has been investigated. Comparison with the 1H NMR results obtained on unmodified cobalt(II) carboxypeptidase provides direct information on the specific effect of the presence of the azo group on the reactivity of the system. Marked differences in the interaction with D-phenylalanine have been observed, and structural inferences are drawn.
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PMID:A 1H NMR study of cobalt(II) arsanilazocarboxypeptidase A. 272 28

Electronic absorption, circular dichroic (CD), and magnetic circular dichroic (MCD) spectra have been determined for complexes of cobalt(II)-substituted carboxypeptidase A and five reversible inhibitors. Three of the inhibitors, N-(1-carboxy-5-butyloxycarbonylaminopentyl)-L-phenylalanine, (I); (R,S)-2-benzyl-4-oxobutanoic acid, (III); and 2-benzyl-4-oxo-5,5,5-trifluoropentanoic acid, (IV) are mechanism-based inhibitors. Another, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine, (II), is a tight binding, slowly hydrolyzed substrate. The fifth, phosphoramidon, (V), is a mechanism-based inhibitor of thermolysin, and may also bind to carboxypeptidase in a mechanism-based mode. The absorption and CD spectra of the enzyme-inhibitor complexes all differ from the spectrum of the free enzyme and from each other. The MCD spectra indicate that the tetrahedral coordination geometry of cobalt, which is distorted in the free enzyme, is also distorted in the inhibitor complexes, although to various degrees. The complexes of I and III are spectrally similar despite being structurally dissimilar, and that of IV, whose structure resembles III, is spectrally distinct, indicating that I and III, but not IV, may perturb the metal in nearly the same way. The absorption spectrum of IV is identical to that, at high pH, of Co(II)carboxypeptidase in which Glu-270 has been modified by a carbodiimide reagent, possibly pointing to a common perturbation of this residue. The absorption and CD spectra of II are similar to those of the catalytic intermediate that precedes the rate-limiting step in peptide hydrolysis [D. S. Auld, A. Galdes, K. F. Geoghegan, B. Holmquist, R. Martinelli, and B. L. Vallee, Proc. Natl. Acad. Sci. USA 81, 4675-4681 (1984)]. Since II is a substrate, the steady-state bound species that it generates may therefore be a true productive intermediate rather than a nonproductive mimic of an intermediate. The spectra of the complexes with II and V differ considerably despite structural similarities. The negative CD ellipticity of the free enzyme is reversed in sign in the presence of V, a phenomenon previously observed with complexes of Co(II)carboxypeptidase and dipeptides. This resemblance may result from a similar interaction of cobalt with the phosphoramidate group of phosphoramidon and the N-terminal amine of dipeptides. The spectra of reversible, mechanism-based inhibitors permit general structural predictions about true intermediates but require caution when used for assigning precise conformation and ligands of bound catalytic species.
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PMID:Effects of mechanism-based reversible inhibitors on the metal environment of cobalt(II)carboxypeptidase A: an electronic spectral study. 274 19

Analogues of tri- and tetrapeptide substrates of carboxypeptidase A in which the scissile peptide linkage is replaced with a phosphonate moiety (-PO2--O-) were synthesized and evaluated as inhibitors of the enzyme. The inhibitors terminated with either L-lactate or L-phenyllactate [designated (O) Ala and (O) Phe, respectively] in the P1' position. Transition-state analogy was shown for a series of 14 tri- and tetrapeptide derivatives containing the structure RCO-AlaP-(O)Ala [RCO-AP(O)A, AP indicates the phosphonic acid analogue of alanine] by the correlation of the Ki values for the inhibitors and the Km/kcat values for the corresponding amide substrates. This correlation supports a transition state for the enzymatic reaction that resembles the tetrahedral intermediate formed upon addition of water to the scissile carbonyl group. The inhibitors containing (O) Phe at the P1' position proved to be the most potent reversible inhibitors of carboxypeptidase A reported to date: the dissociation constants of ZAFP(O)F, ZAAP(O)F, and ZFAP(O)F are 4, 3, and 1 pM, respectively. Because of the high affinity of these inhibitors, their dissociation constants could not be determined by steady-state methods. Instead, the course of the association and dissociation processes was monitored for each inhibitor as its equilibrium with the enzyme was established in both the forward and reverse directions. A phosphonamidate analogue, ZAAPF, in which the peptide linkage is replaced with a -PO2-NH- moiety, was prepared and shown to hydrolyze rapidly at neutral pH (t1/2 = 20 min at pH 7.5). This inhibitor is bound an order of magnitude less tightly than the corresponding phosphonate, ZAAP(O)F, a result that contrasts with the 840-fold higher affinity of phosphonamidates for thermolysin [Bartlett, P. A., & Marlowe, C. K. (1987) Science 235, 569-571], a zinc peptidase with a similar arrangement of active-site catalytic residues.
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PMID:Phosphonate analogues of carboxypeptidase A substrates are potent transition-state analogue inhibitors. 279

A reversed-phase high-performance liquid chromatographic (HPLC)-based method was developed to follow the time course of the hydrolytic action of pancreatic carboxypeptidase A on oligopeptide substrates of the general formula benzoyl(glycyl)n-L-Phe, n being in the range 1-5. The proposed procedure is sensitive at the nanomolar level of substrate, but also allows the kinetics of substrate hydrolysis to be investigated over a wide range of concentrations. The prior quenching of the carboxypeptidase activity and the use of isocratic conditions for the rapid separation of the substrates and their products (in less than 8 min for the slowest one), allow the automation of the procedure, which could become an easy and versatile alternative to the commonly used spectrophotometric methods. A comparative evaluation of the use of a spectrophotometric method to measure carboxypeptidase A activity on the same substrates indicated that the latter is not valid for long oligopeptides (n greater than or equal to 2) at concentrations higher than 5 mM owing to an interference of a physico-chemical nature. For benzoylglycyl-L-Phe (n = 1), the apparent kinetic parameters were calculated by means of the HPLC method and by a well established spectrophotometric procedure, and both yielded similar values.
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PMID:Kinetic analysis of the carboxypeptidase A hydrolysis of oligopeptides by reversed-phase high-performance liquid chromatography. 280 95

Studies of azide inhibition of peptide hydrolysis catalyzed by cobalt(II) carboxypeptidase A identify two anion binding sites. Azide binding to the first site (KI = 35 mM) inhibits peptide hydrolysis in a partial competitive mode while binding at the second site (KI = 1.5 M) results in competitive inhibition. The cobalt electronic absorption spectrum is insensitive to azide binding at the first site but shows marked changes upon azide binding to the second site. Thus, azide elicits a spectral change with new lambda max (epsilon M) values of 590 (330) and 540 nm (190) and a KD of 1.4 M, equal to the second kinetic KI value for the cobalt enzyme, indicating that anion binding at the weaker site involves an interaction with the active-site metal. Remarkably, in the presence of the C-terminal products of peptide or ester hydrolysis or carboxylate inhibitor analogues, anion (e.g., azide, cyanate, and thiocyanate) binding is strongly synergistic; thus, KD for azide decreases to 4 mM in the presence of L-phenylalanine. These ternary complexes have characteristic absorption, CD, MCD, and EPR spectra. The absorption spectra of azide/carboxylate inhibitor ternary complexes with Co(II)CPD display a near-UV band between 305 and 310 nm with epsilon M values around 900-1250 M-1 cm-1. The lambda max values are close to the those of the charge-transfer band of an aquo Co(II)-azide complex (310 nm), consistent with the presence of a metal azide bond in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of anions with the active site of carboxypeptidase A. 283 74

A glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme, a zinc-metallo peptidyl dipeptidase, was esterified with p-[N,N-bis(chloroethyl)amino]phenylbutyryl-L-[U-14C]proline (chlorambucyl-L-[U-14C]-L-proline), an affinity label for this enzyme (Harris, R.B., and Wilson, I.B. (1983) J. Biol. Chem. 258, 1357-1362). The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase high performance liquid chromatography (HPLC) contained the bound radiolabel. This active-site peptide (Mr = 16,000) was digested with trypsin and the labeled peptide formed (T-2) was further degraded with thermolysin. The thermolytic peptides were resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained (Th-1, Mr = 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu... The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of phenylthiohydantoin-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]proline in confirmation of our earlier findings. The sequence determined is homologous in 5 residues with the corresponding sequences of bovine carboxypeptidase A and B, two other mammalian zinc proteases. There is little sequence homology with thermolysin, a bacterial zinc protease that also contains an essential active-site glutamic acid residue.
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PMID:Sequencing of an active-site peptide of angiotensin I-converting enzyme containing an essential glutamic acid residue. 285 12

The opioid peptide antagonist, ICI 174864 ([allyl]2-Tyr-alpha-amino-isobutyric acid (Aib)-Aib-Phe-Leu-OH), can produce analgesic effects in mice. The present study explored the possibility that ICI 174864 1) may have affinity and agonist efficacy at mu receptors and/or 2) may form a carboxypeptidase degradation product in vivo that possesses mu agonist activity. In vitro, ICI 174864 (10(-7) to 10(-4) M) inhibited the twitch in the electrically stimulated mouse vas deferens (ED50 = 90 microM) and guinea pig ileum (ED50 greater than 10(-4) M). The in vitro partial agonist activity of ICI 174864 was due to interaction with delta and not mu receptors because the apparent dissociation constant for naloxone using ICI 174864 as the agonist was similar to the apparent dissociation constant for the interaction of naloxone with delta and not mu receptors. Thus, ICI 174864 is a weak partial agonist at delta receptors with little affinity or efficacy at mu receptors. The incubation of ICI 174864 with carboxypeptidase A generated a peptide, LY281217 [(allyl)2-Tyr-Aib-Aib-Phe-OH], which was a more potent agonist in the mouse vas deferens and guinea pig ileum than ICI 174864. The agonist activity of LY281217 was due to interaction with mu and not delta receptors because LY281217 was approximately 100-fold more potent than ICI 174864 as an agonist in the guinea pig ileum, the apparent dissociation constant for naloxone using LY281217 as the agonist was similar to the apparent dissociation constant for the interaction of naloxone with mu receptors and the delta selective antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Opioid agonist activity of ICI 174864 and its carboxypeptidase degradation product, LY281217. 287 70

Cu(II)-substituted carboxypeptidase A catalyzes the hydrolysis of oligopeptides and their depsipeptide (ester) analogues. Stopped-flow fluorescence assays demonstrate that relative to the zinc enzyme the Cu enzyme can have kcat/Km values up to 24% toward esters but only up to 2.5% toward the corresponding peptides. Adding Zn(II) to the copper enzyme reveals a slow exchange process that correlates with an increase in peptidase activity and with changes in the Cu(II) electron paramagnetic resonance spectra. Low concentrations of 1,10-phenanthroline (OP) (0.1-2.5 microM) markedly increase activity toward furanacryloyl-Phe-Phe (up to 8% of the zinc enzyme), but higher concentrations inhibit, resulting in complete inhibition at 0.8 mM OP. The non-metal-binding, hydrophobic analogues m- and p-phenanthroline are only activators of peptide hydrolysis, even at 1 mM. Activation is likely due to a modifier binding to a hydrophobic locus and either displacing an inhibitory peptide binding mode or inducing a conformational change in the active site.
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PMID:Activity of copper-substituted carboxypeptidase A toward oligopeptides and depsipeptides. 301 5


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