Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.16.2 (PCP)
 3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A reactive ATP analog, N6-(6-bromoacetamidohexyl)-AMP-PCP, reacted specifically with the ATP inhibitory site of rabbit skeletal muscle phosphofructokinase without affecting the active site. Modification resulted in the incorporation of 1.01 mol of the reagent per mol of enzyme subunit. The modified enzyme was insensitive to allosteric inhibition by ATP and to activation by AMP at pH 7.2, where the native enzyme exhibits allosteric kinetic behavior. These observations demonstrate that we had succeeded in obtaining PFK fixed in the T state. Using the kinetic parameters of this modified enzyme, the kinetic properties of native enzyme can be quantitatively accounted for by the allosteric model of Monod-Wyman-Changeux. Further, the reagent was shown to have reacted with a specific cysteine residue near or at the ATP inhibitory site, and the sequence around the cysteine was determined as Cys-Lys-Asp-Phe-Arg.
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PMID:Analysis of the allosteric properties of rabbit muscle phosphofructokinase by means of affinity labeling with a reactive ATP analog. 16 Apr 16

The pH dependence of kinetic parameters and inhibitor dissociation constants for the adenosine cyclic 3',5'-monophosphate dependent protein kinase reaction has been determined. Data are consistent with a mechanism in which reactants selectively bind to enzyme with the catalytic base unprotonated and an enzyme group required protonated for peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) binding. Binding of the peptide apparently locks both of the above enzyme residues in their correct protonation state. MgATP preferentially binds fully ionized and requires an enzyme residue (probably lysine) to be protonated. The maximum velocity and V/KMgATP are pH independent. The V/K for Ser-peptide is bell-shaped with pK values of 6.2 and 8.5 estimated. The pH dependence of 1/Ki for Leu-Arg-Arg-Ala-Ala-Leu-Gly is also bell-shaped, giving pK values identical with those obtained for V/KSer-peptide, while the Ki for MgAMP-PCP increases from a constant value of 650 microM above pH 8 to a constant value of 4 mM below pH 5.5. The Ki for uncomplexed Mg2+ obtained from the Mg2+ dependence of V and V/KMgATP is apparently pH independent.
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PMID:Chemical mechanism of the adenosine cyclic 3',5'-monophosphate dependent protein kinase from pH studies. 282 Apr 83

In this paper we report that while 55% of the total post-proline dipeptidyl-aminopeptidase activity in guinea-pig brain is associated with the soluble fraction of the cells, the remaining activity is widely distributed throughout the particulate fractions. A significant portion of this particulate activity is, however, associated with a synaptosomal membrane fraction. The specific activity of this enzyme rose as the synaptosomal membrane fraction was prepared from a synaptosomal fraction and had previously risen at the synaptosomal fraction was prepared from a postmitochondrial pellet. The synaptosomal membrane post-proline dipeptidyl-aminopeptidase was released from the membrane by treatment with Triton X-100 and partially purified by chromatography on Sephadex G-200. By contrast with the soluble enzyme the partially purified solubilised synaptosomal membrane post-proline dipeptidyl-aminopeptidase was not inhibited by 1.0 mM p-chloromercuribenzoate, 1.0 mM N-ethylmaleimide or 0.5 mM puromycin but was inhibited by 0.5 mM bacitracin. The partially purified solubilised enzyme was capable of releasing His-Pro from His-Pro-Val, His-Pro-Leu, His-Pro-Phe and His-Pro-Tyr and of releasing Gly-Pro from Gly-Pro-Ala but could not release Arg-Pro from Arg-Pro-Pro or from Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin). It was also unable to release Pro-Pro from Pro-Pro-Gly or Glp-Pro from Glp-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-MetNH2 (eledoisin). Using [Pro-3H]thyroliberin we show that the membrane-bound enzyme converts His-ProNH2, produced by the action of the synaptosomal membrane pyroglutamate aminopeptidase, to His-Pro thus competing with the spontaneous cyclisation of His-ProNH2 to His-Pro diketopiperazine. Purified preparations of synaptosomal membrane pyroglutamate aminopeptidase were used to generate His-ProNH2, which could then be converted to His-Pro by the presence of the partially purified synaptosomal membrane post-proline dipeptidyl-aminopeptidase. This preparation was free of contaminating post-proline cleaving endopeptidase, carboxypeptidase P, aminopeptidase P, prolyl carboxypeptidase or proline dipeptidase.
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PMID:Post-proline dipeptidyl-aminopeptidase from synaptosomal membranes of guinea-pig brain. A possible role for this activity in the hydrolysis of His-ProNH2, arising from the action of synaptosomal membrane pyroglutamate aminopeptidase on thyroliberin. 286 1

The ubiquitin-histone H2A conjugate, uH2A, of the protozoan Tetrahymena pyriformis was isolated by gel chromatography and octadecylsilyl-silica chromatography, from the fractions on the chromatographic purification of histone H2A [Fusauchi, Y. & Iwai, K. (1983) J. Biochem. 93, 1487-1497]. The uH2A showed an amino acid composition corresponding to the sum of an equimolar mixture of two protozoan H2A variants and protozoan free ubiquitin. N- and C-terminal sequencing of the uH2A, by Edman degradation and carboxypeptidase P digestion, showed a branched structure having two N-terminals, those of the H2A and ubiquitin components, and one C-terminal, that of each H2A variant component. Further structural analyses of the uH2A, by tryptic digestion of citraconylated uH2A and of a ubiquitinated BrCN fragment, showed that the ubiquitin C-terminal Gly-Gly is linked to the epsilon-amino group of either Lys-123, 125, or 126 in the H2A sequence, and that the ubiquitin sequence is similar to that of calf thymus but differs at least in the sequence of residues 12-27. The deducted structure was compared with the only known uH2A structure, that of calf thymus, with special reference to the branched site.
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PMID:Tetrahymena ubiquitin-histone conjugate uH2A. Isolation and structural analysis. 299 68

We have examined the interaction of the nicotinic acetylcholine receptor with decidium diiodide, a bisquaternary analogue of ethidium containing 10 methylene groups between the endocyclic and trimethylamino quaternary nitrogens. Decidium inhibits mono-[125I]iodo-alpha-toxin binding, inhibits agonist-elicited 22Na+ influx in intact cells, augments agonist competition with mono-[125I]iodo-alpha-toxin binding, and enhances [3H]phencyclidine (PCP) binding to a noncompetitive inhibitor site. These effects occur over similar concentration ranges (half-maximum effects between 0.1 and 0.4 microM). Thus, decidium binds to the agonist site and converts the receptor to a desensitized state exhibiting increased affinity for agonist and heterotropic inhibitors. These properties are similar to metaphilic antagonists characterized in classical pharmacology. At higher concentrations decidium associates directly with the noncompetitive inhibitor site identified by [3H]phencyclidine binding. Dissociation constants of decidium at this site in the resting and desensitized states are determined to be 29 and 1.2 microM, respectively. Analysis of fluorescence excitation and emission maxima reveal that binding to both the agonist and noncompetitive inhibitor sites is associated with approximately 2-fold enhancement of fluorescence. The excitation maximum for decidium bound at the agonist site appears at 490 nm while that for decidium bound at the noncompetitive inhibitor site appears at 530 compared to 480 nm in buffer. These results suggest that decidium experiences a more hydrophobic environment upon binding to the nicotinic acetylcholine receptor sites, particularly to the noncompetitive inhibitor site. Fluorescence energy transfer between N'-fluorescein isothiocyanate-lysine-23 alpha-toxin (FITC-toxin), and decidium is not detected when each is bound to one of the two agonist sites on the receptor. This allows a minimal distance to be estimated between fluorophores. In contrast, energy transfer is observed between decidium nonspecifically associated with the membrane or with nonspecific sites and the FITC-toxin at the agonist sites.
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PMID:Decidium. A novel fluorescent probe of the agonist/antagonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor. 365 51

Fumarases in the mitochondrial and cytosolic fractions of rat liver were separately purified and crystallized. These two fumarases were not distinguishable in physicochemical, catalytic, or immunochemical properties. The sequences of seven amino acids in the C-terminal portions of the two fumarases were shown using carboxypeptidase P to be identical, i.e.-Val-Asp-Glu-Thr-Ala-Leu-Lys-. The amino acid sequence of the N-terminal portion of the mitochondrial fumarase was determined by the Edman method as Ala-Gln-Gln-Asn-Phe-Glu-Ile-Pro-Asp-, but that of the cytosolic fumarase could not be determined by the Edman method, since the N-terminal amino acid was blocked. The N-terminal amino acid of the cytosolic fumarase was identified as N-acetyl-alanine by analysis of the acidic amino acid produced by digestion of the enzyme protein with pronase E, carboxypeptidase A and B. Then the sequence of five amino acids in the N-terminal portion was determined by analyzing the acidic peptide obtained by limited proteolysis of the enzyme protein with carboxypeptidase A as Ac-Ala-Ser-Gln-Asn-Ser-. Peptide mapping of the tryptic peptides obtained from the mitochondrial and cytosolic fumarases showed no difference in the amino acid sequences of the two except in their N-terminal portions. The turnover rates of the mitochondrial and cytosolic fumarases were determined by injecting L-[U-14C]leucine into rat and following the decay of specific radioactivity incorporated into immunoprecipitates from the partially purified enzyme. The half-life of the cytosolic fumarase was estimated as 4.8 days from the decay curve of its specific radioactivity. The decay curve of the specific radioactivity of the mitochondrial fumarase, obtained after a single injection of L-[U-14]leucine, was quite unusual: its specific radioactivity remained constant for about 7 days after pulse labeling, and then decreased exponentially with a half-life of 9.7 days. Similar amounts of cytosolic and mitochondrial fumarase were found in the livers of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp, respectively. Similar subcellular distributions of the enzyme were also found in the kidney, heart, and skeletal muscle of rats, and in hepatoma cells (AH-109A). However, in rat brain no fumarase activity was detected in the cytosolic fraction. Two putative precursor polypeptides of rat liver fumarase were synthesized when rat liver RNA was translated in vitro in a rabbit reticulocyte lysate system.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanism of synthesis and localization of mitochondrial and cytosolic fumarases in rat liver. 381 85

The interaction of phencyclidine (PCP) with its specific receptor sites in the central nervous system has been further characterized. Kinetic association and dissociation rate constants of 2.9 X 10(6) M-1 and 4.8 X 10(-1) min-1 were determined, yielding a kinetic KD of 1.6 X 10(-7) M, in agreement with the KD previously determined at equilibrium. Permissible separation time of 13 s was calculated from the kinetic data, well above the actual separation time of less than 10 s in the rapid filtration assay. Presoaking of filters in 0.01% poly-L-lysine eliminated displacable [3H]PCP adsorption to filter material. Binding data obtained via centrifugation assays was identical to that obtained with the rapid filtration method. Stereospecificity of the PCP receptor was demonstrated by the finding that (+)-ketamine is four-fold more potent than (-)-ketamine in displacing specifically bound [3H]PCP. Several proteolytic enzymes including trypsin, papain and thermolysin potently inactivated PCP receptors. Detailed regional distribution studies showed highest density of PCP receptors in subicular cortex and hippocampus, intermediate levels in hypothalamus, striatum, frontal cortex and cerebellum, lower levels in brainstem and spinal cord, and negligible levels in corpus callosum, a white-matter control area. Benzomorphan opiates with PCP-like behavioral effects interact with the PCP receptor. These data support the pharmacological relevance of the PCP receptor site as demonstrated by the rapid filtration method.
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PMID:Specific binding of [3H]phencyclidine in rat central nervous tissue: further characterization and technical considerations. 629 64

Urinary acidification is widely used to increase the excretion rate of PCP in abusers. Various acidifying techniques were used and compared with regard to efficacy in lowering pH, side effects, and patient acceptability. On the basis of our findings and data from routine monitoring with test tapes, we would recommend the following acidifications procedures as efficacious and reasonably well tolerated: Ammonium chloride, 4 gm. per day, 1 gm. q.i.d., with sufficient water or cranberry juice. Lysine dihydrochloride, 6 gm. per day, 2 gm. t.i.d., with sufficient water or cranberry juice. Lysine hydrochloride, 8 gm. per day, 2 gm. q.i.d., with water or cranberry juice. Cranberry juice, 18 or more oz. per day alone, or plus lysine, ammonium chloride, or ascorbic acid.
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PMID:Urinary acidifiers in phencyclidine detoxification. 642 7

Lysine 318 in the conserved sequence SXXXGXGKS of bacteriophage T7 gene 4A' protein was mutated to an alanine to understand the effect of this substitution on the helicase and primase activities. The dTTPase activity of 4A'/K318A mutant protein was much lower than that of 4A', and both Km and kcat values were affected. The Km of the mutant protein was 3-5-fold higher, and the kcat was about 100-fold lower, than that of 4A'. The mutation did not affect the ability of 4A'/K318A to assemble into hexamers or bind DNA in the presence of MgdTTP. Interestingly, the mutant protein does not bind DNA in the presence of MgdTMP-PCP. The reduced dTTPase activity, however, decreased the helicase activity of the mutant protein to an undetectable level, whereas its primase activity was only 1.5-2.5-fold lower. When 4A'/K318A mutant protein was mixed with 4A', heterooligomers were formed and the helicase and the DNA-dependent dTTPase activities of 4A' were inhibited, but the DNA-independent activity actually increased. The extent of decrease in activities upon heterooligomer formation depended both on the length of time 4A' and 4A'/K318A proteins were incubated and on the concentration of the mutant protein. In addition, the decrease in the dTTPase activity was observed only when the two proteins were incubated in the absence of MgdTTP and DNA, conditions under which both proteins form unstable hexamers. Even though 4A'/K318A does not bind a 30-mer DNA in the presence of MgdTMP-PCP, heterooligomers were capable of binding DNA with the same stoichiometry as 4A'. Protein-DNA cross-linking experiments with (dT)30 and poly(5-BrdU) showed that DNA interacts with five and perhaps all six subunits of 4A'. Therefore, unless heterooligomer restores the ability of the mutant protein to bind DNA in the presence of MgdTMP-PCP, these results suggest that the DNA can bind 4A' by interacting with a few subunits. However, a fully active hexamer is required for both the helicase and the single-stranded M13 DNA-dependent dTTPase activities.
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PMID:The K318A mutant of bacteriophage T7 DNA primase-helicase protein is deficient in helicase but not primase activity and inhibits primase-helicase protein wild-type activities by heterooligomer formation. 801 49

The complete amino acid sequences of the alpha-subunits of pea (Pisum sativum L.) seed and root lectin, the C-terminal amino acids of the beta-subunits of pea seed lectin, and most of the sequence of the beta-subunit of pea root lectin were determined. In contrast to earlier reports it was shown that the beta-subunits of both seed isolectins end at Asn-181. The alpha 1 subunits end at Gln-241 (major fraction) or Lys-240 (minor fraction), whereas the alpha 2 subunits end at Ser-239, Ser-238, Ser-237 or Thr-236. psl cDNA clones from seed are identical to psl cDNA clones from root, and root PSL is identical to seed PSL2, ending at Ser-239, Ser-238 or Ser-237. It seems that the presence of Lys-240 is the sole determinant of the charge difference between pea isolectins. PSL1 can be converted into PSL2 by carboxypeptidase P from Penicillium janthinellum. These results confirm that PSL from roots is encoded by the same gene as PSL from seeds. Thus, it seems that, next to an Asn-X specific protease responsible for the processing at positions 181/182 and 187/188, a carboxypeptidase is responsible for the conversion of PSL1 and PSL2, which is probably the final processing product.
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PMID:Pea (Pisum sativum L.) seed isolectins 1 and 2 and pea root lectin result from carboxypeptidase-like processing of a single gene product. 811 Oct 28


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