UNIPROT:P15088 - FACTA Search

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Query: UNIPROT:P15088 (mast cell)
14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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. The L-type Ca2+ current was recorded in guinea-pig ventricular myocytes by the patch clamp technique in the whole-cell configuration. The modification of the current by intracellular application of proteases was studied. 2. During the first phase of action, trypsin, an endopeptidase, increased the amplitude of Ca2+ current about 3-fold. 3. Thereafter, there was a drastic slowing of the inactivation time course of the enhanced Ca2+ current. The half-time of inactivation increased from a control value of about 25 ms to values larger than 200 ms. 4. Cell dialysis with carboxypeptidase A, an exopeptidase, also enlarged the amplitude of Ca2+ current, but did not affect the kinetics of Ca2+ current. Leuaminopeptidase did not modify the Ca2+ current. 5. The hypothesis that Ca2+ channels are affected by the protease is supported by the fact that alterations of the extracellular Na+ or K+ concentration did not influence the modification of the membrane current. Another argument for the involvement of Ca2+ channels is that the modified membrane current could be blocked by inorganic and organic Ca2+ channel blockers (e.g. 10 microM-Cd2+, 100 microM-La3+ or 1 microM-D600). 6. Although the actions of trypsin and maximal concentrations of isoprenaline on the amplitude of the Ca2+ current were not additive, the slowing of inactivation by trypsin occurred independently from beta-adrenergic stimulation. 7. The effect of trypsin on the Ca2+ current could not be blocked by intracellular 5'-adenylyl-imidodiphosphate (AMP-PNP) or Rp-adenosine 3'5'-monothionophosphate (Rp-cAMPS), both of which are known to suppress the cyclic AMP-dependent phosphorylation of the Ca2+ channel. 8. It was concluded that trypsin may directly modify the membrane protein which forms the Ca2+ channel. Since the increment in peak Ca2+ current resembled the action of cyclic AMP-dependent phosphorylation, it may be related to the removal of a 'chemical' inactivation gate which is normally controlled by phosphorylation. The slowing of the time course of Ca2+ current inactivation by trypsin could be due to a modification of the voltage-dependent inactivation gate. Alternatively, the endopeptidase might remove an internal Ca2+ binding site normally responsible for Ca2+-dependent inactivation.
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PMID:Modification of L-type calcium current by intracellularly applied trypsin in guinea-pig ventricular myocytes. 285 49

The liquid-state 113Cd NMR data of carboxypeptidase A in the presence and absence of inhibitors obtained by Gettins (Gettins, P. (1986) J. Biol. Chem. 261, 15513-15518) are analyzed in terms of whether the inhibitors displace water from Cd2+ upon binding to the protein. This question is addressed by applying the single crystal data and the methods introduced by Honkonen and Ellis (Honkonen, R. S., and Ellis, P. D. (1984) J. Am. Chem. Soc. 106, 5488-5497). Calculations based upon these data demonstrate that displacement of water by a carboxyl group should lead to significant shielding of a 113Cd resonance by approximately 100 ppm. Since the observed 113Cd chemical shifts for carboxypeptidase A are modest and deshielding (12-17 ppm), it is argued that the chemical shifts imply that water is not displaced from the Cd2+ center upon binding of inhibitors to carboxypeptidase A. Rather, the Cd2+ ion increases its coordination number from five to six upon binding of the inhibitor.
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PMID:113Cd NMR of Cd2+-substituted carboxypeptidase. Support for a hexa-coordinate metal ion in the presence of inhibitors. 291 45

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

The carboxypeptidase A purified from goat pancreas has been found to have a molecular weight of 34,600 +/- 300. The enzyme is a zinc-protein and the molar ratio of zinc to enzyme protein is 1:1. Removal of zinc yields an inactive apocarboxypeptidase A. The loss of activity of the native enzyme and restoration of the activity of the apoenzyme run parallel with the zinc content of the protein, thus showing the essentiality of zinc for the enzymatic activity. The exact role of zinc in the enzyme catalysed hydrolysis of the acylpeptides has been investigated after preparing metallo proteins by substituting the zinc of carboxypeptidase A with Co2+, Mn2+, Ni2+, Fe2+, Cd2+, Hg2+, and Cu2+ and determining the kinetic parameters of such metalloproteins. These studies indicate that the metal ion is involved in both binding the substrate and polarising the peptide bond.
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PMID:Role of metal ions in goat carboxypeptidase A-catalysed hydrolysis of acyl peptides. 650 15

Voltage-gated whole-cell currents were recorded from cultured microglial cells which had been developed in the presence of the macrophage/microglial growth factor granulocyte/macrophage colony-stimulating factor. Outward K+ currents (IK) were most prominent in these cells. IK could be activated at potentials more positive than -40 mV. Half-maximal activation of IK was achieved at -13.8 mV and half-maximal inactivation of IK was determined at -33.8 mV. The recovery of IK from inactivation was described by a time constant of 7.9 sec. For a tenfold change in extracellular K+ concentration the reversal potential of IK shifted by 54 mV. Extracellularly applied 10 mM tetraethylammonium chloride reduced IK by about 50%, while 5 mM 4-aminopyridine almost completely abolished IK. Several divalent cations (Ba2+, Cd2+, Co2+, Zn2+) reduced current amplitudes and shifted the activation curve of IK to more positive values. Charybdotoxin (IC50 = 1.14 nM) and noxiustoxin (IC50 = 0.89 nM) blocked IK in a concentration-dependent manner, whereas dendrotoxin and mast cell degranulating peptide had no effect on the current amplitudes. The outward K+ currents showed a frequency dependence when depolarizing pulses were applied at a frequency of 1 Hz. A frequency-independent outward current (IK') characterized by the same activation behavior as IK was detected. IK' was blocked completely by 10 nM charybdotoxin or by 10 nM noxiustoxin. In contrast to its effect on IK, 10 mM tetraethylammonium chloride did not reduce IK'.
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PMID:Properties of voltage-gated potassium currents of microglia differentiated with granulocyte/macrophage colony-stimulating factor. 856 50

Perturbed angular correlation of gamma-rays (PAC) spectroscopy has been used to investigate the angiotensin-I-converting enzyme (ACE) of rabbit lung. By substituting the zinc ions in ACE with excited 111mCd2+ ions, analysis of PAC spectra gave directly the percentage of cadmium ions bound to ACE. The result of the analysis was a dissociation constant of about 1 microM for the cadmium-ACE complex, and a stoichiometry of two moles cadmium/mole enzyme. Cadmium binding is thus about two orders of magnitude weaker than zinc binding to ACE but two orders of magnitude stronger than cobalt binding. PAC spectra monitor the nuclear quadrupole interaction (NQI) for 111mCd. The NQI for ACE exhibits very low frequencies in the PAC spectra with a rather large spectral broadening. In the presence of the inhibitor ramiprilat, the frequencies increase but the spectral broadening is about the same as for ACE without inhibitor. When the inhibitor captopril is added, very high frequencies are obtained consistent with sulfur binding, but now with a narrower distribution of NQI's. A simple molecular orbital analysis of the obtained NQI's has been performed, using a coordination sphere of two His, one Glu residue and a solvent ligand, equivalent to the zinc ligands in thermolysin and carboxypeptidase. The calculated spectral parameters could be modelled with the measured parameters if the solvent ligand is H2O in free ACE, carboxylate from ramiprilat in the ACE-ramiprilat complex and a mercapto group in the ACE-captopril complex. The coordination geometry for cadmium carboxypeptidase obtained by X-ray diffraction gives a calculated set of NQI parameters consistent with the measured parameters for cadmium in the captopril-ACE complex using a mercapto group as the solvent ligand. However, for ACE and its complex with ramiprilat, a significant distortion of the cadmium geometry for carboxypeptidase A had to be adopted in order to calculate NQI's close to the experimental values.
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PMID:Effect of inhibitors on the coordination geometries of cadmium at the metal sites in angiotensin-I-converting enzyme. 857 35

1. The properties of voltage-gated potassium currents were studied in acutely isolated rat hippocampal pyramidal cells from area CA1 and CA3 at postnatal ages of day 6-8, 9-14, and 26-29 (P6-8, P9-14, and P26-29) with the use of the whole cell version of the patch-clamp technique. 2. The outward current pattern of all cells under investigation could be separated in a fast transient A current (IA) and a delayed rectifier-like current (IK). 3. In both preparations, IA activated and inactivated rapidly. Vh describing steady-state inactivation was -84.5 mV in CA3 cells and -85.5 mV in CA1 cells. The activation behavior was characterized by Vh = -23.8 mV in CA3 cells and -27.2 mV in CA1 cells. The removal of inactivation was monoexponential both in CA1 and CA3 neurons with time constants of 32.1 and 28.5 ms, respectively. IA was insensitive to tetraethylammonium (TEA), dendrotoxin (300 nM), and mast cell degranulating peptide (200 nM), but could be blocked with 5 mM 4-aminopyridine (4-AP) by approximately 80%. In both preparations, A currents did not depend on Ca2+ influx. 4. Delayed rectifier currents (IK) in CA1 and CA3 pyramidal neurons decayed along a double exponential time course. Steady-state inactivation was described by Vh = -79.5 mV in CA3 cells and -76.0 mV in CA1 cells. The activation curves were characterized by midpoints of -3.8 mV in CA3 cells and of -1.4 mV in CA1 cells. The removal of inactivation was monoexponential in CA1 and CA3 neurons with time constants of 210.3 and 202.4 ms, respectively. All kinetic properties were identical for the differentially decaying components of IK. In CA1 cells IK was blocked by TEA at +30 mV with an IC50 of 0.98 mM. In CA3 cells the corresponding IC50 value was 1.05 mM. About 20% of IK were insensitive to TEA. IK was partially blocked by approximately 30% with 100 microM 4-AP. Mast cell degranulating peptide (100-200 nM) and dendrotoxin (50-300 nM) had no effect on IK. 6. Perfusion of charybdotoxin (30 nM), Cd2+ (300 microM), La3+ (10 microM), or Ca(2+)-free solutions resulted in the isolation of a small noninactivating outward current component. Around 10% of IK appeared to be Ca2+ dependent in CA1 neurons. In CA3 pyramidal cells Ca(2+)-dependent outward currents seemed to be somewhat larger with approximately 20%. 7. In CA1 as well as in CA3 cells, the kinetic and pharmacological properties of IA and IK remained stable during postnatal development. However, the contribution of IA and IK to the whole cell current varied with age. IA was more prominent in CA1 cells of age group P6-8 than in age-matched CA3 cells. CA3 cells had smaller A currents and larger delayed rectifier currents than CA1 pyramidal cells. Current densities of IA and IK were analyzed during development to assess changes in the expression of these currents. With increasing postnatal age, the expression of IA was downregulated in both preparations. This effect was more pronounced in CA3 than in CA1 cells. In contrast, IK was upregulated during the same developmental period. This increase in the expression of IK was with approximately 300% much larger in CA1 cells than in CA3 cells with only approximately 50%.
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PMID:Comparison of voltage-dependent potassium currents in rat pyramidal neurons acutely isolated from hippocampal regions CA1 and CA3. 859 91

PAC spectra (perturbed angular correlation of gamma-rays) of cadmium-substituted carboxypeptidase A (CPD) show that the enzyme in solution imposes a flexible, pH- and chloride-dependent coordination structure on the metal site, in contrast to what is found in the crystalline state. A much more restricted coordination geometry occurs for the steady-state peptide intermediates of Bz-Gly-l-Phe and Bz-Gly-Gly-l-Phe in solution, suggesting that substrate binding locks the structure in a rigid conformation. The results further indicate that the peptide intermediate has a six-coordinated metal coordination geometry with an OH- ligand at the solvent site and a carbonyl oxygen at an additional ligand site. In marked contrast, conformational rigidity is not induced by the inhibitor/poor substrate Gly-L-Tyr nor by the products of high turnover substrates, Bz-Gly, Bz-Gly-Gly, and L-Phe. These results are consistent with an intact scissile peptide bond in the enzyme-substrate complex of Bz-Gly-L-Phe and Bz-Gly-Gly-L-Phe. A single nuclear quadrupole interaction (NQI) is observed for the crystalline state of the enzyme between pH 5.7 and pH 9.4. This NQI agrees with calculations based on the metal coordination geometry for cadmium in crystalline CPD derived from X-ray diffraction studies. A single broad distribution of NQIs is observed for CPD in sucrose solutions and 0.1 M NaCl at pH values below 6.5. This NQI (NQI-1') has parameters very close to those for the crystalline state. The enzyme metal site, characterized by this NQI, is converted into two new enzyme metal sites over the pH range of 6.5-8.3. The metal coordination sphere of one of these has a NQI (NQI-1) with parameters similar to those at lower pH values (NQI-1') while the other NQI (NQI-2) is characterized by markedly different NQI parameters. Angular overlap model (AOM) calculations indicate that the coordination sites giving NQI-1' and NQI-1 both have a metal-bound water molecule while the coordination site giving NQI-2 has a metal-bound hydroxide ion. PAC results at pH 8.3-10.5 indicate that in this pH range the two metal coordination geometries related to NQI-1 and NQI-2 occur in a pH independent ratio of 2:1, with the one with the water ligand being the most abundant species. The observed pH-independent equilibrium between the two different metal coordination geometries for cadmium can be explained by an equilibrium between tautomeric forms of a hydrogen bond between the Glu-270 carboxyl group and the metal-bound water (Glu-270 COO-...(HOH)M <==> Glu-270 COOH...(OH-)M) being slow on the time scale of a PAC experiment, i.e., slower than 0.5 micros. We finally suggest that NQI-1' observed at low pH reflects an enzyme species containing a metal-coordinated water molecule and the protonated carboxyl group of Glu-270.
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PMID:Structure and dynamics of the metal site of cadmium-substituted carboxypeptidase A in solution and crystalline states and under steady-state peptide hydrolysis. 929 72

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