Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The effects of N-1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide(SLM) on the pellet height response and ATPase activity of glycerinated Triton X-100 extracted cilia of Tetrahymena pyriformis have been studied. Preincubation of cilia with SLM caused complete inhibition of the pellet height response and an initial increase in ATPase activity followed upon longer exposure to SLM by inhibition of ATPase. The effect of SLM on extracted 30S dynein was the reverse of that for whole cilia: ATPase activity was increased when 30S dynein was added to a mixture of ATP and SLM and inhibited when the 30S dynein was preincubated with SLM. The activity of 14S dynein was only inhibited by SLM. Electron spin resonance spectra of ciliary axonemes that had reacted with SLM for various times showed that much of the covalently bound SLM was strongly immobilized even after 1 min of reaction, when ATPase activity increased twofold. The proportion of strongly immobilized label increased with longer times of reaction. Addition of ATP to SLM-labeled axonemes caused a small decrease in the height of the spectral peak corresponding to strongly immobilized label as compared with that of weakly immobilized label, indicating an increase in rotational freedom of some covalently bound label. The results suggest that ATP causes a conformation change affecting a sulfhydryl group(s) involved in the mechanochemical system. It was also shown that beta,gamma-methylene ATP(AMP-PCP) is an inhibitor of dynein ATPase. This analogue of ATP is not hydrolyzed by whole cilia or by the extracted dyneins and does not cause a pellet height response. With Mg2+ as divalent cation, AMP-PCP inhibits 30S dynein more than it inhibits 14S dynein; with Ca2+, the inhibition of 30S dynein is reduced, and there is no inhibition of 14S dynein. Under conditions where AMP-PCP inhibited 30S dynein ATPase it was much less effective than ATP in protecting against the loss of ATPase activity by SLM. Although SLM inhibited Mg2+-activated 14S and 30S dyneins in solution, it did not inhibit ciliary ATPase activity. These results support the view that at least 2 SH groups are involved in ciliary motility and that their reactivity to SH reagents depends on whether the dyneins are in situ or have been extracted.
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PMID:Effect of spin-labeled maleimide on 14S and 30S dyneins in solution and on demembranated ciliary axonemes. 19 83

Isolated sarcotubular membranes (SR) from skeletal muscle bound 3.7 nmol of beta, gamma-methylene [8-3H]ATP (AMP-PCP) per mg of membrane protein. Only one class of binding site was identified and the dissociation constant (K) for this site was 1.5 X 10(-5) M. Addition of 0.05% Triton X-100 increased the number of binding sites to 5.7 nmol/mg. ATP and ADP competitively inhibited AMP-PCP binding. The dissociation constants for ATP and ADP were 3.5 X 10(-5) M and 3.3 X 10(-6) M, respectively. Since this data was obtained in the presence of 5 mM EDTA, it was established that the sarcoplasmic reticulum has a high affinity for the metal free forms of ATP, ADP, and AMP-PCP. Magnesium concentrations in excess of 1 X 10(-4) M inhibited AMP-PCP binding. Lower concentrations of magnesium had little effect on AMP-PCP binding. The effect of calcium on AMP-PCP binding was biphasic. Calcium concentration between 1 X 10(-6) and 1 X 10(-4) M inhibited AMP-PCP binding. Inhibition was maximal at 1 X 10(-5) M. Calcium concentration above 1 X 10(-4) M facilitated analogue binding. Possible sites of magnesium and calcium actions are discussed.
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PMID:Effect of calcium and magnesium on binding of beta, gamma-methylene ATP to sarcoplasmic reticulum. 86 83

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.
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PMID:Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells. 131 16

1. Actions of histamine on the voltage-dependent Ba2+(Ca2+) currents (IBa, ICa) were investigated using the whole-cell patch-clamp technique on dispersed smooth muscle cells from the rabbit saphenous artery. 2. Histamine (half-maximal dose, EC50 = 530 nM) augmented the IBa evoked by a brief depolarizing pulse (100 ms duration; to +10 mV from a holding potential of -80 mV) in a concentration-dependent manner. The maximum augmentation was obtained with 30 microM-histamine (1.29 times control). This augmentation of IBa was inhibited by the H3-antagonist, thioperamide (Ki = 30 nM, slope of the Schild plot = 1.0), but not by H1- or H2-antagonists (mepyramine or diphenhydramine, or cimetidine, respectively). 3. An H3-agonist, R alpha-methylhistamine (EC50 = 93 nM), also augmented IBa in a concentration-dependent manner at a holding potential of -80 mV and the maximum augmentation (1.25 times control) was obtained with 10 microM. This augmentation was also inhibited by thioperamide, but not by the above H1- and H2- antagonists. 4. Intracellularly applied 500 microM-guanosine 5'-triphosphate (GTP) enhanced, but 1 mM-guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) abolished, the histamine-induced augmentation of IBa. When one of the non-hydrolysable GTP analogues, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; greater than 5 microM), guanylyl-imidodiphosphate (GMP-PNP; 200 microM) or guanylyl (beta, gamma-methylene)-diphosphonate (GMP-PCP; 1 mM) was intracellularly applied, the IBa amplitude evoked without the application of histamine was not affected, but the excitatory effect of histamine on IBa was reversed to an inhibition. Pre-treatment with pertussis toxin (PTX: 300 ng/ml and 3 micrograms/ml) did not modify the histamine-induced responses in the absence or presence of GTP gamma S. 5. 4 beta-Phorbol 12,13-dibutylate (PDBu) increased the amplitude of IBa. However, this action of PDBu was not enhanced by the application of GTP (500 microM) in the pipette, but additional application of histamine further increased the amplitude of IBa. Pre-treatment with a potent non-selective protein kinase inhibitor, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H-7; 100 microM), did not modify the histamine-induced current augmentation or inhibition observed in the presence or absence of intracellular GTP gamma S.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Histamine H3-receptor activation augments voltage-dependent Ca2+ current via GTP hydrolysis in rabbit saphenous artery. 131 41

Sarcoplasmic reticulum (SR) vesicles, prepared from rabbit skeletal muscle, were characterized by functional and binding assays and incorporated into planar lipid bilayers. Single-channel activity was recorded in an asymmetric calcium buffer system and studied under voltage clamp conditions. Under these experimental conditions, a large conductance (100 pS in 50 mM Ca2+ trans) divalent cation selective channel displaying high ruthenium red and low Ca2+ sensitivity was identified. This pathway has been previously described as the Ca(2+)-release channel of the SR of skeletal muscle. We now report that in the presence of a Mg-ATP complex, the Ca2+ sensitivity of the open probability of this channel is increased. Furthermore, we show that micromolar cis Sr2+ concentrations also activated the Ca(2+)-release channel. The open probability of the Sr(2+)-activated channel was increased in the presence of a 2 mM Mg-ATP complex and adenine nucleotides on the cytoplasmic face of the Ca(2+)-release channel. These results were confirmed by isotopic flux measurements using passively 45Ca(2+)-loaded vesicles. In the latter case, the presence of extravesicular AMP-PCP (the nonhydrolysable ATP analog) enhanced the percentage of 45Ca2+ release induced either by Ca2+ or Sr2+ activation. In conclusion our findings emphasize the fact that the divalent cation activation of the Ca(2+)-release channel may be induced by Ca2+ and Sr2+, but not by Ba2+, in the presence of adenine nucleotides. Furthermore, they support the view that in situ Ca2+ and Mg-ATP complexes are involved in modulating the gating mechanism of this specific pathway.
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PMID:Functional sensitivity of the native skeletal Ca(2+)-release channel to divalent cations and the Mg-ATP complex. 131 62

In NGF-treated PC12 cells, nicotine-induced K+ release was measured with a K(+)-sensitive microelectrode. The K+ outflow via nicotinic ACh receptor cation channels was inhibited by various psychotomimetic sigma ligands in the sequence of PCP, dextromethorphan >> DTG, MK 801, (+)SKF10047 >> (+)3-PPP. The K+ release was not affected by the neuroleptic sigma ligand haloperidol nor by the calcium antagonist nifedipine. The results suggest that psychotomimetic sigma ligands inhibit nicotine-stimulated K+ flux by interacting with nicotinic, rather than via sigma 2 receptors.
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PMID:Inhibitory effects of psychotomimetic sigma ligands on nicotine-induced K+ flux from differentiated PC12 cells. 133 53

1. Phencyclidine (PCP) block of Ca2+ channel current in enzymatically dissociated neurones from the CA1 region of the adult guinea-pig hippocampus was studied using whole-cell voltage clamp techniques. Ca2+ channel current was recorded with 3 mM-Ba2+ as the charge carrier. Na+ currents were blocked with tetrodotoxin and K+ currents were eliminated by using tetraethylammonium and N-methyl-D-glucamine as the predominant extracellular and intracellular cations, respectively. 2. Peak Ca2+ channel current evoked by depolarization from -80 to -10 mV was reduced in a use-dependent fashion by PCP. The apparent forward and reverse rate constants for block at the depolarized voltage were 10(6) s-1 M-1 and 11-14 s-1, respectively. These values were at least 60 times faster than the corresponding rates at the resting voltage. The steady-state block produced by PCP increased in a concentration-dependent fashion with an IC50 of 7 microM. Other dissociative anaesthetic drugs were substantially weaker inhibitors of the current (tiletamine > dizocilpine (MK-801) > ketamine). 3. The Ca2+ channel current recorded under identical conditions in rat dorsal root ganglion neurones was less sensitive to blockade by PCP (IC50, 90 microM). 4. PCP block of the hippocampal Ca2+ channel current occurred in a voltage-dependent fashion with the fractional block decreasing at positive membrane potentials. Analysis indicated that the PCP blocking site senses 56% of the transmembrane electric field. 5. Analysis of tail currents recorded at -80 mV demonstrated that PCP does not affect the voltage-dependent or time-dependent activation or deactivation of the Ca2+ channel current. 6. The rate and extent of inactivation of the Ca2+ channel current was maximal at -10 mV and diminished at more positive potentials. Experiments with Ba(2+)-free external solution demonstrated that inactivation of the Ca2+ channels is largely voltage-dependent and is not affected by Ba2+ influx. 7. PCP markedly increased the apparent extent of inactivation of the Ca2+ channel current during prolonged voltage steps. This increase in apparent inactivation was more pronounced at depolarized potentials. Inactivation at -10 mV proceeded in two exponential phases; PCP had little effect on the fast decay phase and caused a moderate speeding of the slow decay phase. Although block of the activated state evolved on the same time scale as inactivation, the apparent rate of inactivation was not increased in a concentration-dependent fashion by PCP indicating that the block does not occur by a conventional open channel mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Phencyclidine block of calcium current in isolated guinea-pig hippocampal neurones. 133 8

Rat brain cortex synaptosomes pre-incubated with [3H]norepinephrine were used (1) to provide evidence that part of the NMDA receptors mediating stimulation of norepinephrine (NE) release are located on the noradrenergic varicosities themselves, (2) to characterize these receptors and (3) to examine whether ethanol specifically inhibits the NMDA-evoked NE release via a presynaptic site of action. In synaptosomes superfused with Mg(2+)-free Krebs-Henseleit solution, NMDA (2-min exposure) stimulated tritium overflow in a concentration- and glycine-dependent manner. The stimulatory effect of NMDA was not altered by tetrodotoxin but was abolished by omission of Ca2+ from the superfusion fluid and was considerably reduced in the presence of 1.2 mM Mg2+. DL-(E)-2-Amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849; a competitive NMDA receptor antagonist) produced a parallel shift of the concentration-response curve for NMDA to the right, whereas dizocilpine (MK-801; an antagonist at the phencyclidine, PCP, recognition site of the NMDA-gated ion channel) reduced the maximum effect of NMDA. Ethanol inhibited the NMDA-evoked tritium overflow in a concentration-dependent manner. In contrast, in synaptosomes superfused with Ca(2+)-free Krebs-Henseleit solution containing 15 mM K+ throughout, ethanol did not affect the tritium overflow evoked by 2 min introduction of 75 microM Ca2+ into the superfusion fluid. This Ca(2+)-evoked overflow was also not altered by tetrodotoxin and dizocilpine, but was inhibited by the inorganic Ca2+ channel antagonist Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Presynaptic site of action underlying the ethanol-induced inhibition of norepinephrine release evoked by stimulation of N-methyl-D-aspartate (NMDA) receptors in rat cerebral cortex. 135 86

Effects of endotoxin administration on ryanodine receptor in canine cardiac junctional sarcoplasmic reticulum (SR) were studied. The results show that the Bmax for [3H]ryanodine binding to cardiac junctional SR was decreased by 25% (8 +/- 0.38 vs 6 +/- 0.41 pmole/mg protein for control and endotoxic, respectively; (P less than 0.01) while the kd (13.7 +/- 1 nM for control vs 13.2 +/- 2 nM for endotoxic) was unaffected 4 hr following endotoxin administration. Ca2+ activated [3H]ryanodine binding in both groups sigmoidally but the Vmax for Ca2+ activation was decreased by 24% (P less than 0.05) while the S0.5 and the Hill coefficient values remained unchanged after endotoxin injection. Caffeine, ATP, and AMP-PCP activated while calmodulin, SKF-525A, ruthenium red, and Mg2+ inhibited [3H]ryanodine binding in both groups but the A0.5 (concentration requires for half-maximum activation) and the I50 (concentration requires for half-maximum inhibition) for the above-mentioned activators and inhibitors, respectively, were unaffected during endotoxin shock. Digestion of cardiac SR isolated from control dogs with phospholipase A2 inhibited [3H]ryanodine binding and the inhibition was reversed completely by the addition of phosphatidylserine. Addition of phosphatidylserine to cardiac SR isolated from endotoxic dogs stimulated [3H]ryanodine binding and the stimulation represents a complete reversal of the inhibition caused by endotoxin administration. Based on these findings together with previous observation that phospholipase A2 activity is activated during endotoxin shock, it is concluded that endotoxin administration decreases the number of ryanodine receptor in canine cardiac junctional SR and the decrease in ryanodine receptor is associated with a mechanism involving a modification of membrane lipid microenvironment in response to phospholipase A2 activation.
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PMID:Altered ryanodine receptor of canine cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock. 138 10

The (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum was labeled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It was shown that a single cysteine residue (Cys-344) was labeled on the ATPase, with a 25% reduction in steady-state ATPase activity and no reduction in the steady-state rate of hydrolysis of p-nitrophenyl phosphate. The fluorescence intensity of the labeled ATPase was sensitive to pH, consistent with an effect of protonation of a residue of pK 6.8. Fluorescence changes were observed on binding Mg2+, consistent with binding to a single site of Kd 4 mM. Comparable changes in fluorescence intensity were observed on binding ADP in the presence of Ca2+. Binding of AMP-PCP produced larger fluorescence changes, comparable to those observed on phosphorylation with ATP or acetyl phosphate. Phosphorylation with P(i) also resulted in fluorescence changes; the effect of pH on the fluorescence changes was greater than that on the level of phosphorylation measured directly using [32P]P(i). It is suggested that different conformational states of the phosphorylated ATPase are obtained at steady state in the presence of Ca2+ and ATP and at equilibrium in the presence of P(i) and absence of Ca2+.
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PMID:Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum with 4-(bromomethyl)-6,7-dimethoxycoumarin: detection of conformational changes. 138 23


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