UNIPROT:P15088 - FACTA Search

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)

113Cd and 31P NMR have been used to investigate the interactions of inhibitors with the metal ion of bovine carboxypeptidase A, using 113Cd as a replacement for the native zinc atom. In the absence of inhibitor and over the pH range 6-9, no 113Cd resonance is visible at room temperature. Upon lowering the temperature to 270 K, however, a broad resonance can be seen at 120 ppm. These results are discussed in terms of possible sources for this resonance modulation. Binding of low molecular weight inhibitors containing potential metal-coordinating moieties results in the appearance of a sharp 113Cd resonance. These inhibitors all bind to the metal ion, a fact which is reflected in the chemical shift of the cadmium resonance and, for L-phenylalanine phosphoramidate phenyl ester, by two-bond 113Cd-31P spin-spin coupling of 30 Hz in the 31P resonance of the bound inhibitor. For inhibitors that coordinate to the metal ion via oxygen, the 113Cd chemical shift is in the range 127-137 ppm, whereas for sulfur coordination there is a downfield shift of approximately 210 ppm. The complexes of 113Cd-substituted carboxypeptidase A with the D and L isomers of thiolactic acid are distinguished by a difference of 11 ppm in the chemical shift of their cadmium resonances. The enzyme complex formed with the macromolecular inhibitor from potatoes, which fills the S1 and S2 subsites, shows one or possibly two closely spaced broad 113Cd resonances. Both the chemical shift and the line width of the 113Cd resonances of the [113Cd]carboxypeptidase-inhibitor complexes give valuable structural and dynamic information about the enzyme active site.
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PMID:On the coordination of inhibitors to the metal ion of carboxypeptidase A. A 113Cd and 31P NMR study. 378 76

Three thioamide peptides in which the oxygen atom of the scissile peptide bond is replaced by sulfur (denoted by (= S)) were synthesized and found to be good, convenient substrates for carboxypeptidase A. The thioamide bond absorbs strongly in the ultraviolet region, and enzymatic hydrolysis is monitored easily using a continuously recording spectrophotometric assay. The reaction follows Michaelis-Menten kinetics with kcat values of 68, 9.0, and 3.7 sec-1 and Km values of 0.83, 0.81, and 0.53 mM for Z-Glu-Phe(= S)-Phe, Z-Gly-Ala(= S)-Phe, and Z-Phe(= S)-Phe, respectively. Activities of the thioamides and their oxygen amide analogs were determined with a series of metal-substituted carboxypeptidases. The Cd(II), Mn(II), Co(II), and Ni(II) enzymes exhibit 30%-35%, 60%-85%, 150%-190%, and 40%-55% of the Zn(II) enzyme activity with the amide substrates; this compares with 240%-970%, 0%-15%, 340%-840%, and 30%-140% of the Zn(II) activity, respectively, with the thioamides. The activity of the Cu(II) and Hg(II) enzymes is less than 3% toward all substrates. Cadmium, a thiophilic metal, yields an enzyme which is exceedingly active with the thioamides; the kcat/Km values are 2.4-9.7-fold higher than with Zn(II) carboxypeptidase. In contrast, Mn(II), which has a relatively low affinity for sulfur, yields an enzyme with correspondingly low activity toward the thioamides. The results are consistent with a mechanism for peptide bond hydrolysis in which the metal atom interacts with the substrate carbonyl atom during catalysis.
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PMID:Thioamide substrate probes of metal-substrate interactions in carboxypeptidase A catalysis. 380 99

1. Rat and guinea-pig lung tissues were incubated for 20 min at 37 degrees C in Krebs-Ringer phosphate buffer at pH 7.4, or in Tyrode-Tris buffer at pH 8.2, and the release of histamine produced by adding different concentrations of compound 48/80 to the incubation medium was determined.2. At pH 7.4, increasing concentrations of 48/80 increased the release of histamine from the rat lung, with a tendency towards a maximum. No release of histamine from guinea-pig lung was observed at this pH. At pH 8.2, histamine release occurred both from rat and guinea-pig lung, and was proportional to the logarithm of the concentration of compound 48/80.3. Histamine release from rat lung by 20 mug/ml. of 48/80 decreased when the pH was raised from 7.4 to 8.2; but the release caused by 1 mg/ml. of 48/80 increased both in rat and guinea-pig lung as the pH was raised.4. 2-4-Dinitrophenol (DNP) inhibited the release of histamine from rat lung by a concentration of 20 mug/ml. of 48/80; the inhibition was prevented by glucose. DNP did not affect histamine release from rat or guinea-pig lung by a concentration of 1 mg/ml. of 48/80 and enhanced the release when the pH was raised from 7.4 to 8.2.5. 1 mg/ml. of 48/80 did not inhibit the enhanced oxygen consumption produced by DNP in the isolated rat diaphragm.6. Iodoacetic acid (IAA) or a Ca/Mg-free medium inhibited the release of histamine by 20 mug/ml. of 48/80 from rat lung but not the release produced by 1 mg/ml. in either rat or guinea-pig lung.7. The degranulation of rat mesentery mast cells caused by 20 mug/ml. of compound 48/80 was inhibited by DNP. The degranulation evoked by 1 mg/ml. of 48/80 was also sensitive to this inhibitor; in this instance, however, the metachromatic staining reaction of the mesentery mast cells was greatly diminished.8. It is concluded that two processes of histamine release by compound 48/80 occur in rat lung. One, dependent on cell metabolism, involves, mast cell granule secretion. The other, independent of cell metabolism, seems to consist of a simple exchange reaction between histamine and compound 48/80, and this is the only one occurring in guinea-pig lung.
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PMID:Mechanisms of histamine release by compound 48-80. 418 29

Activities of all known forms of bovine carboxypeptidase A's (alpha having 307, beta having 305, and gamma having 300 amino acids) are essentially the same in solution under given conditions. However, activities in the crystals differ. The A(alpha) crystals elongated along the a axis (a) have unit cell parameters a = 51.41 A, b = 59.89 A, c = 47.19 A, and beta = 97 degrees 35', (b) show about [unk] of the activity of the dissolved enzyme, and (c) have the same color of the arsanilazo Tyr 248 derivative in the crystalline and solution states, namely red at pH 8.2 and yellow at pH 7.4. The A(gamma) crystals elongated along the b axis (a) have unit cell parameters a = 50.9 A, b = 57.9 A, c = 45.0 A, and beta = 94 degrees 40', (b) show [unk] of the activity of the dissolved enzyme, and (c) show, in the arsanilazo Tyr 248 derivative, yellow crystals and red solution at pH 8.2. Although the detailed three-dimensional structure is known for the A(alpha) form described above, the structure of the A(gamma) form is as yet undertermined. A reasonable hypothesis is that the major part of the differences in crystal behaviors is due to differences in intermolecular (crystal-packing) interactions. In particular the movement of Tyr 248 may be somewhat hindered by these intermolecular contacts in the A(gamma) crystals, and in other crystalline forms which are elongated along the b axis. The movement observed in the x-ray diffraction studies, of the OH group of Tyr 248 by 12 A when the very slowly hydrolyzed substrate Gly-Tyr is bound to A(alpha) crystals, appears to be largely unhindered by intermolecular interactions. Examination of a three-dimensional space-filling structural model of the carboxypeptidase A molecule reveals that the phenolic oxygen of Tyr 248 can approach within 2 A of the Zn cofactor. This approach requires a movement by about 6 A of the polypeptide chain in the general region of Tyr 248. Moreover, the position of Tyr 248 when bonded to Zn can just be seen in the electron density map of the crystal structure at a level which, averaged over many unit cells, suggests some 15-25% of the enzyme is in this form at pH 7.4 and 4 degrees in the crystals of the x-ray diffraction study. It is probable that when the Zn-Tyr 248 bond is present the enzyme is catalytically inactive.
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PMID:Enzymatic activities of carobxypeptidase A's in solution and in crystals. 452 Dec 5

By means of the mixed anhydride procedure the benzyl alpha-ketoside of N5-acetyl-D-neuraminic acid was linked to L-glycine, L-glutamic acid and L-phenylalanine. Hydrogenolytic cleavage of the benzyl group resulted in the corresponding free N5-acetyl-beta-D-neuraminoylpeptides. This new class of compounds is no substrate for Vibrio cholerae sialidase. The enzyme does not split the benzyl alpha-ketosides of N5-acetyl-D-neuraminoylpeptides nor is its activity inhibited by these compounds. The results strongly support the assumption that in sialidase substrates the carboxy group must be located close to the ketosidic oxygen. N-(N5-acetyl-beta-D-neuraminoyl)-L-phenylalanine was readily hydrolysed by carboxypeptidase A from bovine pancreas.
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PMID:On the specificity of sialidase. Synthesis and properties of N5-acetyl-beta-D-neuraminoylpeptides - AcNeu-Gly-OH, AcNeu-Glu-OH, AcNeu-Phe-OH - and the corresponding alpha-ketosides. 613 32

The catalytic role of the metal ion in bovine carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) is investigated by application of cryoenzymologic and electron paramagnetic resonance methods with use of the Co2+-reconstituted enzyme. Incorporation of 17O into oxygen-donor ligands induces a substantial change in the spin-lattice relaxation probability of the paramagnetic ion. While a change in spin-lattice relaxation is observed for the free Co2+-enzyme in 17O-enriched water, no change is observed for the enzyme complexed to glycyl-L-tyrosine. These results are consistent with x-ray crystallographic studies showing that the metal-bound water molecule in the active site is displaced upon binding of the peptide inhibitor. A change in spin-lattice relaxation of the Co2+ ion in the mixed anhydride, acyl-enzyme intermediate formed with the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate is observed when 17O is enriched either into water or into the carbonyl oxygen position of the scissile bond of the substrate. Since the protein supplies three amino acid side chains as ligands to the metal ion, these results indicate that the metal ion is altered from a tetra-coordinate species in the free enzyme to a penta-coordinate species in the acyl-enzyme reaction intermediate. In addition, the results provide structural support for our assignment of ionization of a metal-bound water molecule in rate-limiting deacylation (Makinen, M. W., Kuo, L. C., Dymowski, J. J., and Jaffer, S. (1979) J. Biol. Chem. 254, 356-366) and affirm that the metal-hydroxide species is the nucleophile responsible for the breakdown of the mixed anhydride reaction intermediate of carboxypeptidase A.
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PMID:Hydrolysis of esters by carboxypeptidase A requires a penta-coordinate metal ion. 627 27

We have previously established a relationship between mast cell degranulation and hypoxic pulmonary vasoconstriction (HPV). In the present study, we investigated the possible role of slow-reacting substance of anaphylaxis (SRS-A) in the mediation of HPV. In 18 conscious sheep, pulmonary artery pressure, pulmonary arterial wedge pressure, and cardiac output were measured for the calculation of pulmonary vascular resistance (PVR) along with arterial oxygen tension (PaO2) while breathing room air and while breathing 13% O2 (balance, N2). Before and during 13% O2 breathing (pretreatment), Group 1 received an intravenous infusion of cromolyn sodium (3 mg/kg-1/min-1) and Group 2 was infused with FPL-57231, a SRS-A antagonist (2 mg/kg-1/min-1); Groups 3 and 4 received infusions of cromolyn sodium or FPL-57231 after induction of HPV. During 13% O2 breathing (mean PaO2, 47 mmHg), mean PVR increased to 190% of baseline. Pretreatment with cromolyn sodium prevented HPV, whereas infusion of cromolyn sodium after induction of HPV failed to reverse it; FPL-57231 both prevented HPV (pretreatment) and reversed it when infused after induction of HPV. Pretreatment with the prostaglandin synthetase inhibitor indomethacin (2 mg/kg) 1 h before the experiment failed to modify the FPL-57231-induced reversal of hypoxic vasoconstriction, thus excluding the release of inhibitory prostaglandins by this compound. We conclude that cromolyn sodium prevented HPV, presumably by inhibiting the release of SRS-A, which mediates pulmonary vasoconstriction directly or indirectly through other mechanisms.
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PMID:Does slow-reacting substance of anaphylaxis mediate hypoxic pulmonary vasoconstriction? 640 64

Successful management of adverse drug reactions requires early identification and prompt treatment of anaphylaxis, whether due to immunoglobulin (Ig) E- or non-IgE-mediated mechanisms of mast cell mediator release. Acute therapy is directed toward enhancement of oxygenation and maintenance of normotension. Requisite measures include the use of epinephrine, oxygen, and adequate fluid replacement; in some instances, vasopressors or corticosteroid drug therapy may be warranted. Emergency measures may be needed to maintain the airway. Although the offending drug is usually discontinued, a necessary drug for which there is no satisfactory alternative occasionally may be continued without danger of further anaphylaxis as long as therapy is not interrupted. Other nonemergent adverse drug reactions requiring an early decision include accelerated urticarial and late maculopapular eruptions, in both of which the patient may tolerate a necessary drug with schedule manipulation. Differentiation of an adverse drug reaction from problems unrelated to the drug is essential so that needed medication is not inappropriately discontinued. Good management also requires anticipation of adverse reactions whenever a therapeutic program is instituted. Familiarity with the drug groups most commonly responsible for immunologic reactions is helpful, as is knowledge of satisfactory alternatives for these drugs in the presence of known hypersensitivity. An adverse reaction can often be minimized through use of established protocols for premedication. Desensitization, when essential, may be achieved for most drugs with graduated dosage schedules and maintained through continued administration of the drug. Identification to avoid inadvertent exposure to agents that have caused immunologic reactions in the past is essential.
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PMID:Management of adverse drug reactions. 649 Nov 4

The crystal structures of zinc-free carboxypeptidase A (apocarboxypeptidase A) and the complex of glycyl-L-tyrosine with apocarboxypeptidase A are described and compared to the corresponding structures of the zinc-containing enzyme. Only small conformational changes in the zinc ligands accompany removal of the metal. Interactions between the tyrosine residue of glycyl-L-tyrosine and apocarboxypeptidase A are similar to those observed in the complex with the holoenzyme. However, in the absence of zinc, the carbonyl oxygen of the glycyl moiety now receives a hydrogen bond from the side chain of arginine-127. Although not as yet observed, a similar shift of the carbonyl oxygen of a susceptible bond from the zinc to arginine-127 could stabilize tetrahedral intermediates generated during the hydrolysis of substrates by carboxypeptidase.
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PMID:Crystallographic studies on apocarboxypeptidase A and the complex with glycyl-L-tyrosine. 658 Jun 31

Benoxaprofen (BXP) is a nonsteroidal anti-inflammatory drug which causes cutaneous phototoxicity. Because the in vivo phototoxicity may involve photosensitized damage to mast cell membranes, the mechanism for photosensitized damage was studied in a single model system, the red blood cell. Oxygen-dependent and oxygen-independent mechanisms for membrane disruption were detected. Oxygen-dependent lysis was not quenched by superoxide dismutase and was quenched only at high sodium azide concentrations. A two-step mechanism is proposed involving initial photodecarboxylation of BXP to form a lipophilic photoproduct which subsequently photosensitizes membrane damage. Human serum albumin at 0.03% totally inhibited BXP-photosensitized lysis.
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PMID:Benoxaprofen photosensitization of cell membrane disruption. 669 24

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