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)

This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.
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PMID:Development of a method for the incorporation of substitution-inert metal ions into proteins. Site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with cobalt(III). 4 71

Thin sections in mouse mast cells and thymic cells are stained with cobalt thiocyanate a compound known to form insoluble complexes with organic bases. Chromatin, nucleolus, ribosomes and mast cell granules are contrasted. Different blockade reactions and enzymatic digestions indicate the staining corresponds to the basic protein amino-groups. The silver methenamine reaction stains the same cellular structures. However, the specificity control reactions show the staining mainly corresponds to protein sulphydryle groups and in a lesser extent to aldehyde and polyanions.
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PMID:Cobalt thiocyanate as a stain for basic proteins and other organic bases on thin sections. 7 73

Cobalt(III)bovine carbonic anhydrase B was prepared by the oxidation of the cobalt(II) enzyme with hydrogen peroxide and was purified by affinity chromatography. The oxidation reaction is inhibited by specific inhibitors of carbonic anhydrase. The inhibition is explained by the fact that the Co(II)-enzyme . inhibitor complex cannot be directly oxidized by hydrogen peroxide, but has to dissociate to give free Co(II) enzyme which is then oxidized. The Co(III) ion in Co(III) carbonic anhydrase cannot be directly substituted by zinc ions. It can be reduced by either dithionite or BH-4 ions to give, first, their complexes with the Co(II) enzyme, and upon their removal, a fully active Co(II) enzyme. Cyanide and azide bind to cobalt(III) carbonic anhydrase with similar rate constants of 0.060 +/- 0.005 and 0.070 +/- 0.007 M-1 S-1 respectively. These rates are faster than those found for Co(III) inorganic complexes. The Co(III) ion in both Co(III) carbonic anhydrase and Co(III) carboxypeptidase A was found to be diamagnetic, indicating a near octahedral symmetry.
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PMID:Kinetic and magnetic properties of cobalt(III) ion in the active site of carbonic anhydrase. 10 27

Catalytically inactive, exchange-inert Co(III)-carboxypeptidase A has been prepared by reaction of Co(II)-carboxypeptidase A with the active-site-directed oxidizing agent m-chloroperbenzoic acid. Co(III)-carboxypeptidase A, isolated by affinity gel filtration chromatography, has the same amino acid composition and molecular weight as the starting material and contains 0.95 g-atom/mol of cobalt and 0.01 g-atom/mol of zinc. Its electron paramagnetic resonance, circular dichroic, magnetic circular dichroic, and visible absorption spectra are consistent with those of octahedral Co(III) model complexes. Co(III)-caboxypeptidase A is essentially devoid of catalytic activity toward both peptide and ester substrates of the native enzyme, and stopped-flow fluorescence studies with dansylated substrates show that it binds peptides, but not esters. Furthermore, the protein does not react with either type of substrate to yield a single turnover. The implications of these findings to the mechanism of action of carboxypeptidase A are discussed in the light of the "metal-carbonyl" and "metal-hydroxide" hypotheses. Since Co(III)-carboxypeptidase A does not bind esters, inner-sphere coordination to the metal appears to be necessary for ester binding. All attempts to prepare Co(III)-carboxypeptidase A by treatment of Co(II)-carboxypeptidase A with hydrogen peroxide according to previously published procedures (Kang, E.P., Storm, C.B., & Carson, F.W. (1975) J. Am. Chem. Soc. 97, 6723) have been unsuccessful, and the present results do not confirm earlier reports that Co(III)-carboxypeptidase A exhibits esterase activity or that its activity is dependent on the method of preparation of the precursor Co(II)-carboxypeptidase A (Jones, M.M., Hunt, J.B., Storm, C.B., Evans, P.S., Carson, F.W. & Pauli, W.J. (1977) Biochem. Biophys. Res. Commun. 75, 253). These findings call for a reexamination of mechanistic conclusions based on the assumption that Co(III)-carboxypeptidase A is an active esterase.
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PMID:Enzymatically inactive, exchange-inert Co(III)-carboxypeptidase A: role of inner sphere coordination in peptide and ester catalysis. 21 Jul 89

A group of active-site metal coordinating inhibitors of zinc proteases (carboxypeptidase A, thermolysin, Bacillus cereus neutral protease, and angiotensin-converting enzyme) have been synthesized and their properties investigated. Their general structures are R-SH and R-NH-PO2(O phi)H, where-S- or -O- serve as metal ligands and R refers to an amino acid or peptide group designed to interact with substrate recognition sites. These inhibitors can be extremely potent; thus, N-(2-mercaptoacetyl)-D-phenylalanine, e.g., inhibits carboxypeptidase A with a Kiapp of 2.2 x 10(-7) M. The spectral response of cobalt(II)-substituted thermolysin or carboxypeptidase A to the sulfur-containing inhibitors signals the direct interaction of the mercaptan with the metal. An S leads to Co(II) charge transfer band is generated near 340 nm and is detected by absorption, circular dichroism, and magnetic circular dichroism. The cobalt(II) spectra indicate both inner sphere coordination with sulfur and 4-coordination in the enzyme-inhibitor complex. Thus, the metal undergoes a simple substitution reaction, the inhibitor most likely displacing water at the fourth coordination site.
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PMID:Metal-coordinating substrate analogs as inhibitors of metalloenzymes. 23 May 2

The magnetic circular dichroic (MCD) spectra of cobalt(II) sugstituted metalloenzymes have been studied and compared to a series of four-, five-, and six-coordinate cobalt(II) model complexes previously examined (T. A. Kaden et al. (1974), Inorg. Chem. 13, 2582). The MCD spectra of cobalt substituted carboxypeptidase A, procarboxypeptidase ta, and thermolysin are consistent with earlier deductions of tetrahedral coordination from absorption spectra and also with X-ray structure analysis. Inhibitors fail to alter their MCD spectra significantly. The MCD spectra of cobalt alkaline phosphatase and carbonic anhydrase are more complex and their pH dependence and alteration by inhibitors are discussed in terms of known cobalt(II) models.
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PMID:Magnetic circular dichroic spectra of cobalt(II) substituted metalloenzymes. 23 52

Properties of carboxypeptidase A of cultured skin fibroblasts from control and cystic fibrosis patients were studied using alpha-N-carbobenzoxy-L-glutamyl-L-tyrosine as substrate. Carboxypeptidase A was inhibited by thiomersal, cyanide, iodoacetate and N-ethylmaleimide in a similar manner for control and cystic fibrosis fibroblasts. Both trypsin and dithiothreitol treatment activated the enzyme, but 1,10-phenanthroline inhibited only in the presence of dithiothreitol. Both Zn2+ and Co2+ reversed this inhibition. Trypsin treatment of carboxypeptidase A produced a form of the enzyme having a higher KM value for both control and cystic fibrosis fibroblasts. Dithiothreitol treatment of control fibroblasts resulted in a form with similar properties to the trypsin activated form, but cystic fibrosis fibroblasts yielded a variant form with even higher KM and Vmax values. Since other properties were similar, it seems likely that this difference reflected binding of a molecule to the enzyme rather than of a defect in the enzyme.
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PMID:Carboxypeptidase A activity of cultured skin fibroblasts and relationship to cystic fibrosis. 66 47

Human carboxypeptidase A has been isolated from activated pancreatic juice by means of affinity chromatography employing the competitive inhibitor benzylsuccinic acid as an affinity ligand. The structural and functional features of the human and bovine enzymes are quite analogous. The molecular weights of human and bovine carboxypeptidases A are virtually identical, their amino acid compositions are similar, both contain 1 g-atom of zinc/mole, and the activities of both are restored by addition of zinc to the apoenzyme. The inhibition of human carboxypeptidase by chelating agent is reversed by either dilution or addition of a metal such as Cu2+. When other metals are substituted for the native zinc, peptidase activity of the human metallocarboxypeptidases follows the order: cobalt greater than nickel greater than manganese greater than cadmium, while the sequence for esterase activities is: manganese greater than cobalt = cadmium greater than nickel. The latter sequence differs from that observed for the bovine enzyme. Human carboxypeptidase A crystallizes after dialysis at low ionic strength. Hydrolysis of the dipeptide carbobenzoxyglycyl-L-phenylalanine and of the ester benzoylglycyl-L-alpha-hydroxy-beta-phenyllactate exhibits kinetic anomalies, but that of their longer homologues does not. Chemical modifications with tyrosine reagents alters esterase and peptidase activities. The affinity chromatographic method here described should greatly facilitate future studies of this enzyme from human and other sources.
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PMID:Purification and crystallization of human carboxypeptidase A. 93 22

1. Current or voltage clamp recordings from CA3 neurones of the adult rat hippocampal slice were performed to study the inactivation properties of a slow outward K+ current identified as the delayed rectifier (IK). 2. In current clamp experiments, burst firing evoked from resting membrane potential by intracellular current injection was reduced or blocked by conditioning hyperpolarizing pre-pulses of 20-40 mV amplitude. This effect was inhibited by tetraethylammonium (TEA; 20 mM) but was unaffected by Cs+ (3 mM), 4-aminopyridine (4-AP; 2 mM), carbachol (30-50 microM), mast cell degranulating peptide (MCDP; 300 nM), thyrotrophin releasing hormone (TRH; 1 microM) or by a Ca(2+)-free solution containing Mn2+ or Co2+ (2 mM). 3. Single-electrode voltage clamp experiments were carried out on neurones superfused with Ca(2+)-free solution, containing tetrodotoxin (TTX; 1 microM), Mn2+ or Co2+ (2 mM), 4-AP (2 mM), Cs+ (3 mM) and carbachol (30 microM). Step depolarizations from a holding potential of -55 mV activated an outward current which reached a plateau after 200 ms, followed by an outward tail current. Such an outward current had the characteristics of IK. 4. The outward currents were significantly potentiated by conditioning hyperpolarizing pre-pulses suggesting the IK was reduced by a voltage-dependent inactivation process. Removal of inactivation was a function of the amplitude of the conditioning hyperpolarizing pre-pulse. At a holding potential of -55 mV removal of inactivation was time dependent with a time constant of 211 ms. High K+ (12.5 or 21.5 mM) solutions did not affect the inactivation characteristics of IK. 5. Tetraethylammonium (20 mM) or low concentrations of Ba2+ (0.1 mM) readily depressed the outward current without significantly affecting the inactivation process. Dendrotoxin (200 nM) also depressed such a slow current but, in addition, increased the inactivation process of IK. 6. It is suggested that removal of inactivation of IK by hyperpolarization can modulate cell excitability by fully restoring the ability of IK to inhibit burst firing of CA3 hippocampal neurones.
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PMID:Inactivation characteristics of a sustained, Ca(2+)-independent K+ current of rat hippocampal neurones in vitro. 133 65

1H NMR spectroscopy of the isotropically shifted signals in cobalt carboxypeptidase, CoCPD, permits a direct and selective detection of protons belonging to the residues liganded to the metal. The chemical shift of these protons in the free enzyme and enzyme-inhibitor complexes with changing pH monitors the state of ionization of the ligands directly and of other residues in the active center indirectly. The 1H NMR spectrum of CoCPD at pH 6 shows three well-resolved isotropically shifted signals in the downfield region at 62 (a), 52 (c), and 45 (d) ppm which have been assigned to the NH proton of His-69 and to the C-4 H's of His-69 and His-196, respectively. Titration of signal a with pH is characterized by a pKa of 8.8 which is identical to that seen in prior electronic absorption and kinetic studies. The fact that the signal reflecting the NH of His-69 is still observed at pH 10 and no major shifts occur for the signals reflecting the C-4 H's indicates the alkaline pKa in carboxypeptidase A catalysis, pKEH, cannot be ascribed to ionization of the histidyl NH of either His-69 or His-196. Binding of L-Phe shifts this pKa to 7.7 while not greatly perturbing the downfield 1H NMR signals that reflect the ligation shell of the cobalt coordination sphere. These results indicate the pKa of 8.8 in CoCPD and the pKa of 7.7 in the CoCPD.L-Phe adduct reflect ionization of the same group.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:pH-dependent properties of cobalt(II) carboxypeptidase A-inhibitor complexes. 156 40


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