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)

1. Patch-clamp recording techniques were used, to examine the effects of diazoxide on KATP currents in CRI-G1 insulin-secreting cells in the presence of non-hydrolysable nucleotides. 2. In the presence of non- or slowly-hydrolyzed ATP analogues, bathing the intracellular aspect of cell-free membrane patches diazoxide inhibited KATP channel activity. 3. Under whole-cell recording conditions, with various non-hydrolysable nucleotides present intracellularly (after dialysis), diazoxide induced KATP current activation. The largest activation occurred with Mg-adenylyl-(beta, gamma-methylene) diphosphate (Mg-AMP-PCP) present in the dialysing solution. This activation was diazoxide- and nucleotide-concentration-dependent. 4. In the absence of Mg2+, or in the presence of manganese (Mn2+) ions intracellularly, diazoxide did not induce KATP current activation, regardless of the species of nucleotide present in the pipette. 5. Intracellularly applied trypsin prevented the activation of KATP currents by diazoxide in the presence of Mg-AMP-PCP, an effect reversed by co-application of intracellular polymethylsulphonyl fluoride with the trypsin. 6. The application, by dialysis, of a CRI-G1 cell lysate, with negligible Mg-ATP, resulted in a substantial activation of the KATP current by diazoxide. 7. It is concluded that diazoxide can activate KATP channel currents by two separate pathways, one requiring a phosphorylation process, the other the presence of an intracellular protein coupled with a Mg-purine nucleotide.
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PMID:Nucleotide-dependent activation of KATP channels by diazoxide in CRI-G1 insulin-secreting cells. 142 77

An antibiotic cerulenin, (2R, 3S)-2,3-epoxy-4-oxo-7,10-trans,trans- dodecadienamide, irreversibly inhibits fatty acid synthetase from Saccharomyces cerevisiae. Three moles of cerulenin were bound to 1 mol of the enzyme with concomitant loss of its activity. Pretreatment of the enzyme with iodoacetamide reduced the amount of cerulenin bound to the enzyme. Since iodoacetamide is known to specifically bind to the cysteine residue on the condensing reaction domain, cerulenin is considered to bind to the same domain. Tryptic digestion of the [3H] cerulenin-treated enzyme gave a radioactive peptide; its amino acid composition was Asx 1, Thr 1, Ser 1, Glx 2, Pro 1, Gly 1, Ala 1, Val 1, Ile 1, and Leu 2. This composition included all the amino acids of the condensing reaction site (Thr-Pro-Val-Gly-Ala-Cys) previously reported by Kresze et al. (Eur. J. Biochem., 79, 181 [1977] except for Cys. When the enzyme was treated with [3H]cerulenin and digested successively with trypsin and carboxypeptidase P, a [3H] cerulenin-cysteine adduct was isolated as the sole product. This was identified with the adduct chemically synthesized from non-labeled cerulenin and cysteine, and its structure was elucidated by 1H-, 13C-NMR, and fast atom bombardment mass spectrometry. These results indicate that cerulenin, forming a hydroxylactam ring, reacts at its epoxide carbon (C-2 position) with the SH-group of the cysteine residue in the condensing reaction domain of yeast fatty acid synthetase.
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PMID:Binding site of cerulenin in fatty acid synthetase. 266 7

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

Specific [3H]phencyclidine (PCP)-binding studies were carried out in homogenates prepared from different regions of post-mortem human brains derived from subjects free of any neurological disease. Scatchard analysis revealed a single class of [3H]PCP-binding sites. Binding was found to be stereospecific i.e. markedly greater ability of the potent PCP agonist dexoxadrol to displace specifically bound 10 nM [3H]PCP as compared to its behaviorally inactive enantiomer levoxadrol. Stereospecific binding was abolished by preincubation of homogenate with trypsin or by heating the homogenate to 90 degrees C for 15 min. Various sigma opiates displaced specifically bound [3H]PCP binding in a rank order similar to that seen in rat brain. Thus a baseline has been established to enable future elucidation of the possible role of these sites in psychiatric illnesses.
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PMID:Characterization of specific sigma opiate/phencyclidine (PCP)-binding sites in the human brain. 631 73

[3H]Phencyclidine ( [3H]PCP) bound to crayfish abdominal muscle membranes at pH 7.4 with two affinities (Kd of 0.96 nM for 0.38 pmole/mg of protein, and 18.9 nM for 7.6 pmoles/mg of protein). Binding affinities increased at higher pH, suggesting that binding may be due mostly to the un-ionized form of [3H]PCP. This high-affinity [3H]PCP binding was sensitive to the actions of trypsin, protease, and dicyclohexylcarbodiimide, but insensitive to phospholipase A, concanavalin A,N-ethylmaleimide, and dithiothreitol. Calcium channel antagonists were most potent in inhibiting the high-affinity [3H]PCP binding with the following descending order of potencies: bepridil greater than nicardipine = diltiazem = verapamil greater than cinnarizine greater than (+)-D-600 greater than (-)-D-600 greater than 4-NO2-nifedipine greater than 2-NO2-nifedipine. The binding was also highly sensitive to several PCP analogues, antipsychotics, piperocaine , and tilorone, and moderately sensitive to d-tubocurarine, atropine, imipramine, nortryptyline , and tetracaine. Although verapamil and nifedipine inhibited the action potential of crayfish muscle, PCP did not and actually prolonged slightly the falling phase of the action potential. Although it is unlikely that the [3H]PCP binding protein in crayfish muscles is a Ca2+ channel, it is possible that it may be a K+ channel.
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PMID:Interactions of phencyclidine with crayfish muscle membranes. Sensitivity to calcium channel antagonists and other drugs. 632 63

The binding characteristics of [3H]Dynorphin A-(1-13) ([3H]Dyn A-(1-13) were examined in membrane preparations of rat heart. Saturation binding studies with increasing concentrations between 2.5 and 500 nM indicated that [3H]Dyn A-(1-13) binds to a single population of sites with a Kd of 285 nM and a Bmax of 215 pmol/mg protein. [3H]Dyn A-(1-13) binding is sensitive to trypsin treatment and it is inhibited by Zn2+ and Mg2+ with IC50 values of 159 and 310 microM, respectively. Dyn A and related peptides competes with the binding of [3H]Dyn A-(1-13) with the following order of potency: Dyn A-(1-13) > Dyn A > Dyn B > alpha-neo-endorphin > Dyn A-(1-8). The non-opioid peptides Dyn A-(2-13), Dyn A-(3-13) and Dyn A-(5-13) are as potent (Ki of 0.35, 0.44 and 0.59 microM, respectively) as Dyn A-(1-13) (Ki of 0.36 microM) in inhibiting [3H]Dyn A-(1-13) binding while Leu-enkephalin (Leu-Enk) exhibits no inhibitory effect at 100 microM. Selective ligands for kappa (kappa: U-50,488H, U-69,593), mu (mu: [D-Ala2, MePhe4, Glyol5]Enk) and delta (delta: [D-Ser2, Thr6]Leu-Enk) opioid receptors as well as for phencyclidine (PCP: MK-801, TCP) and sigma (sigma: (+)-SKF-10047, DTG, 3(+)-PPP) receptors show little or no inhibition of [3H]Dyn A-(1-13) binding at 100 microM. These results indicate that the heart contains a low affinity high capacity binding site for Dyn A and related peptides, distinct from opioid, PCP and sigma receptors.
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PMID:Characterization of non-opioid [3H]dynorphin A-(1-13) binding sites in the rat heart. 790 2

3'-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a photoaffinity analog of ATP to label potential ATP receptors in ciliated cells. Like ATP, without photoactivation, BzATP stimulated the ciliary beat frequency in tissue culture up to threefold. Irradiation of intact cells in the presence of [alpha-32P]BzATP followed by SDS-PAGE and autoradiography revealed two labeled proteins with molecular masses of 46 and 96 kDa (p46 and p96). Photolabeling of both proteins was susceptible to digestion with trypsin, implying that the labeled proteins are at least partially exposed on the extracellular surface of the plasma membrane. The dependence of 32P incorporation in both proteins on [alpha-32P]BzATP concentration was similar. Labeling of p46 but not p96 required Ca2+ or Mg2+. Various nucleotides stimulated the ciliary frequency, and inhibited the photolabeling of p46 and p96. The rank order of apparent affinity for p46 is: ATP approximately equal to ADP > GTP gamma S > ADP beta S, UTP, 2MeSATP, AMP-PNP > AMP-PCP > AMP > adenosine; for p96 it is: ADP approximately equal to ADP beta S > or = ATP >> AMP-PCP, AMP-PNP > GTP gamma S > or = AMP > 2MeSATP, UTP, adenosine. The rank of stimulation of ciliary beat frequency is: ADP beta S, UTP > or = 2MeSATP, GTP gamma S, AMP-PNP, ATP > or = ADP > AMP-PCP > adenosine > AMP. These results suggest the involvement of p46 in the stimulatory effect of extracellular ATP on the ciliary beat, as a P2 purinoceptor. On the other hand, p96 may represent a P2 purinoceptor or an ectonucleotidase.
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PMID:Extracellular ATP binding proteins as potential receptors in mucociliary epithelium: characterization using [32P]3'-O-(4-benzoyl)benzoyl ATP, a photoaffinity label. 853 Dec 2

Analysis of the proteome of Haemophilus influenzae by two-dimensional polyacrylamide gel electrophoresis on conventional Tris-glycine gels does not usually result in efficient separation of the proteins in the 5-20 kDa range, which are mainly accumulated in the lower acidic and basic regions. In order to improve the separation of the low molecular mass proteins, we used homogeneous Tricine gels of two urea concentrations in the second-dimensional separation. The Tricine gel systems allowed the efficient and reproducible separation of the proteins of the microorganism with masses between 5 and 20 kDa, however, no proteins with masses below 5 kDa could be visualized. Approximately 80 proteins migrating in the 5-25 kDa region were identified by matrix assisted laser desorption/ionization - mass spectrometry, of which 40 identified for the first time. The digestion of the low mass proteins often produced only few peptides, which were insufficient for confident identification by mass spectrometry. Therefore, the identification was occasionally achieved by a sequential digestion with two proteases, trypsin or endoproteinase Lys-C as first and carboxypeptidase P as second enzyme. The gel system described may be useful for the efficient separation of low molecular mass proteins from other organisms to construct standard maps.
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PMID:Reference map of the low molecular mass proteins of Haemophilus influenzae. 971 65

A novel mucoadhesive drug carrier system has been generated which protects a model polypeptide antigen from degradation by the most abundant intestinal proteases. The enzyme inhibitors antipain, chymostatin and elastatinal, respectively, were covalently attached to the mucoadhesive polymer sodium carboxymethylcellulose (NaCMC) and the inhibitory efficacy of the resulting polymer-inhibitor conjugates was evaluated in vitro. When these inhibitor conjugates were combined with the thiolated polymer polycarbophil-cysteine (PCP-Cys), 95.8 +/- 3.8% (mean +/- SD, n = 3) of the incorporated model antigen ovalbumin (OVA) was protected from enzymatic degradation within 90 min incubation in the presence of an artificial intestinal fluid containing the pancreatic serine proteases trypsin, chymotrypsin and elastase. Replacing the CMC-inhibitor conjugates in the dosage form by unmodified CMC significantly reduced the protective effect to 78.8 +/- 4.7% (mean +/- SD, n = 3), whereas incorporation of the model antigen in a CMC dosage form omitting PCP-Cys protected 72.5 +/- 3.2% (mean +/- SD, n = 3) of OVA from degradation within a 90 min incubation period. Further, the incorporation of PCP-Cys resulted in higher cohesiveness within the dosage form and controlled drug release of the antigen for a time period of more than 9 h. Results suggest that a delivery system combining thiolated polymer and polymer-inhibitor conjugates improves the metabolic stability of the model polypeptide antigen and may therefore be a useful tool for oral protein vaccination.
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PMID:Design and in vitro evaluation of a mucoadhesive oral delivery system for a model polypeptide antigen. 1159 93

The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E1 and E2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E2 (Ca(2+)-free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca(2+)-dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E1 form of scallop Ca-ATPase was occupied by Pi, AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca2+. These results suggest that occupation of the catalytic site on E1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of Pi on the E2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E2-P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time.
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PMID:Effect of orthophosphate, nucleotide analogues, ADP, and phosphorylation on the cytoplasmic domains of Ca(2+)-ATPase from scallop sarcoplasmic reticulum. 1464 52


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