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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Regional myocardial ischemia was produced in anesthetized pigs by occluding the left anterior descending coronary artery. Mitochondria were prepared from both normally perfused and ischemic myocardium after 2 h of occlusion. Mitochondria from the ischemic area exhibited an 89% increase in cholesterol content from 32.7 +/- 1.9 (control) to 62.0 +/- 0.47 (ischemic) nmol/mg protein with no change in either total phospholipid content or in membrane fatty acid composition. This increase in mitochondrial membrane cholesterol was accompanied by an increase in membrane microviscosity as indicated by increased fluorescence polarization using the fluorescent membrane probe, 1,6-diphenyl-1,3,5-hexatriene. In these same experiments the Arrhenius plot discontinuity temperature of oligomycin-sensitive adenosinetriphosphatase (ATPase) activity fell from 20.0 to 14.2 degrees C. Our results suggest that, during the myocardial ischemic process in pigs, there is an intracellular redistribution of free cholesterol that produces a marked increase in mitochondrial membrane cholesterol content. This appears to produce an altered mitochondrial membrane lipid bilayer packing, resulting in increased membrane microviscosity and, possibly, altered inner membrane ATPase function. Intracellular cholesterol redistribution may thus contribute to the cell membrane damage that occurs during the myocardial ischemic process.
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PMID:Mitochondrial cholesterol content and membrane properties in porcine myocardial ischemia. 646 Dec 57

The temperature behaviour of the fluorescent probes--1,6- diphenyl-1,3,5-hexatriene (DPH) and pyrene--in the outer and intracellular membranes differs markedly. Fluorescence polarization of DPH in microsomal preparations of duck salt gland and bovine brain enriched with Na,K-ATPase decreases nonmonotonously with an increase in temperature, the critical temperature being coincident with that for Na,K-ATPase. In preparations of sarcoplasmic reticulum intracellular membranes enriched with Ca-ATPase, the DPH fluorescence polarization changes linearly with temperature. Studies of the lipid composition of membrane preparations demonstrated that intracellular membranes are more fluid than outer plasma membranes. At the same time, these membranes reveal a critical temperature for another parameter that characterizes the changes in the hydrophobic volume of the bilayer, i.e., the degree of pyrene excimerization. The Arrhenius plots for this reaction coincide with that for Ca-ATPase in the same preparations.
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PMID:Diphenylhexatriene and pyrene as tools for characterization of biological membranes. 647 33

The isolation of basolateral membranes from rat proximal colonic epithelial cells is described. Cells were harvested using a technique combining chelation of divalent cations with mechanical dissociation. After homogenization, differential centrifugation yielded a 'crude' membrane fraction which was further purified using sucrose density centrifugation. The final membrane fraction was enriched 10-14-fold over homogenate in ouabain-sensitive sodium-potassium dependent adenosine triphosphatase and ouabain-sensitive potassium-dependent phosphatase specific activities. SDS-polyacrylamide gel electrophoresis of this membrane revealed at least 18 protein bands with molecular weights of 14600-200000. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, free cholesterol and fatty acids were the major lipid components of this membrane. The predominant fatty acids were palmitic (16:0), oleic (18:1), stearic (18:0) and linoleic (18:2) acid. Membranes and their liposomes were studied, using the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH), by steady-state fluorescence polarization. The fluorescence anisotropy was greater in the intact membranes compared to their liposomes, indicating greater fluidity in the liposomes. Compositional studies suggested that the high fluidity of this membrane was due to its low ratios of protein/lipid (w/w), cholesterol/phospholipid (mol/mol), and sphingomyelin/phosphatidylcholine (mol/mol).
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PMID:Isolation and partial characterization of basolateral membranes from rat proximal colonic epithelial cells. 683 Jul 71

The characteristics of the interaction between N-(3-pyrene)maleimide (PMI) and the sarcoplasmic reticulum (SR) membranes were investigated, and the sites of labeling with PMI on Ca(2+)-transporting ATPase were identified. PMI was dissolved in the membrane lipids before reacting with the ATPase protein. The measurement of resonance energy transfer from PMI to 1-(dimethylaminophenyl)-6-phenyl-1,3,5-hexatriene revealed that the pyrene moiety of PMI stayed in the lipid layer after it had been covalently attached to the ATPase molecule. PMI-labeled SR membranes at an average labeling density of 1 mol PMI/mol ATPase were digested with trypsin, and the labeled peptides were purified through a series of reversed-phase HPLC procedures on C18 and C4 columns. The amino acid analysis of the purified peptides revealed multiple cysteine residues mainly distributed over the C-terminal half of the cytoplasmic domain of the ATPase molecule as the targets of PMI. This implied that PMI molecules mediated cross-linking between the cytoplasmic domain of the ATPase molecule and the membranes. The distortion of the structure of the former due to this cross-linking may explain the uncoupling of ATP-splitting from Ca(2+)-transport caused by PMI.
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PMID:Sites of labeling with N-(3-pyrene)maleimide on Ca(2+)-transporting ATPase of the sarcoplasmic reticulum. 759 54

Physical-chemical-activity relationship of aromatic hydrocarbons (n = 10) and alkyl acetates (n = 16) with respect to their in vitro effects on synaptosomal membranes was studied. Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase) activity and membrane fluidity, which was determined using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene, were used as potential indicators of neuronal cell toxicity. The potency of inhibition for the enzyme (IC50), the potency of increasing membrane fluidity (IC12.5), and n-octanol/water partition coefficient (P) were all determined experimentally for 26 solvents. Correlation analyses were made on aromatic hydrocarbons and on alkyl acetates. There were linear relationships between log P and pIC50 (log1/IC50) values, and between log P and pIC12.5 (log1/IC12.5) values, indicating that the hydrophobicity of the solvents determines their toxic ability to affect membrane environment; the more hydrophobic the solvents are, the more toxic they are. A direct linear relationship between Na(+)-K(+)-ATPase activity pIC50 and membrane fluidity pIC12.5 values was also shown. This predictive correlation suggests a similar mechanism of membrane surface interaction govering both processes that are common to the test solvents. The present results confirm the importance of the lipid environment of neuronal membranes in maintaining the normal function of membrane-bound protein.
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PMID:Physical-chemical-activity relationship of organic solvents: effects on Na(+)-K(+)-ATPase activity and membrane fluidity in mouse synaptosomes. 786 56

Previous studies have implicated glucocorticoids as an important factor in the postnatal maturational increase in proximal tubule volume absorption, Na+/H+ antiporter, Na(HCO3)3 symporter, and Na(+)-K(+)-ATPase activity. The present study examined whether glucocorticoids are also a potentially important factor in the maturational decrease in proximal tubule phosphate transport. Renal BBMs were prepared from neonatal rabbits who received dexamethasone (10 micrograms/100 g body weight) or vehicle. Brush-border membrane vesicles from dexamethasone-treated neonates had a lower rate of Na-phosphate cotransport than controls (50.8 +/- 3.6 versus 29.2 +/- 2.6 pmol 32P(i)/10 s/mg protein, p < 0.001). This decrease was due to a decrease in the Vmax with no change in the affinity of the transporter for phosphate. The dexamethasone-induced decrease in BBM Na-phosphate transport was not due to a reduction in transporters as assayed by phosphate-protectable Na-dependent equilibrium binding of phosphonoformic acid. Dexamethasone treatment caused an increase in the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium-1,6-diphenyl-1,3,5-hexatriene (i.e. a decrease in membrane fluidity). Brush-border membranes from dexamethasone-treated neonates had a decrease in sphingomyelin and an increase in phosphatidylcholine and phosphatidylinositol content but no change in cholesterol or total phospholipid content. These data are consistent with glucocorticoids playing a role in the postnatal maturational decrease in proximal tubule phosphate transport by altering membrane characteristics.
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PMID:Maturational effects of glucocorticoids on neonatal brush-border membrane phosphate transport. 804 84

The structure-toxicity relationship of monoketones, a class of organic solvents widely used in industry, was investigated with respect to their in vitro effects on synaptosomal membrane proteins. The toxic parameters used were Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase), a well-known marker enzyme often used as a membrane toxicity model, and 3H-dihydroalprenolol (3H-DHA)-labeled beta-adrenergic receptor binding that has been shown to be vulnerable to solvent-induced changes in membrane fluidity. In vitro treatments with 12 kinds of monoketones (carbon chain length from 3-10) dose-dependently inhibited both 3H-DHA binding to mouse synaptosomes and Na(+)-K(+)-ATPase activity. The potency of inhibition (IC50) for both the two parameters was linearly related to n-octanol/water partition coefficient and synaptosome/buffer partition coefficient of the test compounds. Additions of monoketones did not significantly alter the number of 3H-DHA binding sites but markedly decreased their affinity. In each monoketone, the IC50 values for 3H-DHA binding and Na(+)-K(+)-ATPase activity were generally within the same range. The anisotropy of fluorescence probe 1,6-diphenyl-1,3,5-hexatriene-labeled synaptosomal membranes was dose-dependently decreased by the monoketones, implying increased membrane fluidity. These results indicate that increasing lipophilicity of monoketones results in increased solvent penetration of synaptic membrane preparations, leading to conformational changes in membrane structure and increased ability to inhibit both neuroreceptor binding and enzyme activity. The present data confirm the importance of the lipid micro-environment of membranes in maintaining the normal functions of membrane-bound proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structure-toxicity relationship of monoketones: in vitro effects on beta-adrenergic receptor binding and Na(+)-K(+)-ATPase activity in mouse synaptosomes. 827 28

The fluorescent polyunsaturated parinaric acid (PnA) incorporated in sarcoplasmic reticulum membranes (SR) was used to probe the initial stages of membrane lipid peroxidation. The experimental set up of the PnA assay was investigated by means of several peroxidation initiators to ascertain peroxidation conditions. This assay in SR is particularly useful to evaluate the membrane susceptibility to peroxidation and to ascertain suitable conditions (concentration of initiators and cofactors) to challenge peroxidation in each preparation under study. On the basis of the PnA assay, Fe2+/ascorbate was selected among the different initiator systems to assess the effect of lipid peroxidation upon biochemical and biophysical parameters of SR membranes. Under mildly controlled conditions at 25 degrees C, the lipid degradative process, as detected by fatty acid analysis, decreases the Ca2+ uptake (up to about 50% of control) and reduces the Ca2+ pump efficiency (Ca2+/ATP ratio) up to about 58% of control, without inactivation the ATPase enzyme turnover. The effect of lipid peroxidation on the SR bilayer organization is dependent either on the extent of lipid peroxidation or on the depth of the bilayer as probed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and by intramolecular excimerization of 1,3-di(1-pyrenyl)propane. It is concluded that the effect of mild lipid peroxidation on Ca2+ pump activity is partially exerted through the alteration of physical properties in the lipid phase or lipid-protein interfaces.
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PMID:Lipid peroxidation in sarcoplasmic reticulum membranes: effect on functional and biophysical properties. 838 29

Perturbations of rat brain synaptic plasma membrane (SPM) bilayer structure and Na+/K(+)-ATPase activity were correlated for drugs that are structurally related and exhibit similar toxicological side effects but belong to different pharmacological classes. Na+/K(+)-ATPase IC50 values decrease linearly with increasing octanol/water partition coefficients (log-log plot) for a series of dimethylethylamine-containing drugs (i.e., chlorpromazine, amitriptyline, imipramine, doxepin, and diphenhydramine), emphasizing hydrophobicity in inhibition. However, nortriptyline and desipramine are 1.2 log units less hydrophobic than their N-methylated parent drugs but more potent inhibitors. To investigate this, bilayer surface structure was examined by the binding of the fluorophore 1-anilinonaphthalene-8-sulfonic acid (ANS) to SPMs. The dissociation constant and wavelength maximum of ANS are invariant with drug binding; however, the limiting fluorescence intensity of ANS (F infinity) is increased. Such data indicate that these cationic drugs bind to the membrane surface, increasing the number but not the polarity of ANS binding sites by cancelling charge at anionic phospholipid groups. More importantly, there is a close linear correlation between the concentrations of drugs necessary to increase F infinity by 40% and the IC50 values, with full compensation for the N-demethylated drugs. This correlation implies that drug-induced increases in SPM-bound ANS fluorescence are a better predictor of Na+/K(+)-ATPase inhibition than are octanol/water partition coefficients and that electrostatic interactions are also involved in inhibition. Furthermore, it points toward similar mechanisms of biomembrane surface interaction governing both inhibition and fluorescence change that are common to these drugs. K(+)-dependent p-nitrophenylphosphatase activity is inhibited with the same potency as Na+/K(+)-ATPase activity, indicating that inhibition may involve drug interaction near the K+ binding sites. Furthermore, chlorpromazine, diphenhydramine, and dimethylaminopropyl chloride alter K(+)-activation of K(+)-dependent p-nitrophenylphosphatase, progressing from noncompetitive through mixed to competitive inhibition as their hydrophobicity changes, and these mechanisms are consistent with steric hindrance of K+ binding. In contrast to the ANS data, decreases in 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy induced by these drugs do not correlate with Na+/K(+)-ATPase inhibition, and drug N-demethylation enhances inhibition without altering anisotropy; both findings indicate that Na+/K(+)-ATPase activity is not predominantly influenced by changes in bulk fluidity. Taken together, these data suggest that electrostatic interactions at the biomembrane surface between the protonated amino group of the drug and anionic groups on the enzyme and/or phospholipids near the K+binding sites are crucial to inhibition and that drug hydrophobicity modulated the number and orientation of these interactions.
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PMID:Interaction of tricyclic drug analogs with synaptic plasma membranes: structure-mechanism relationships in inhibition of neuronal Na+/K(+)-ATPase activity. 839 17

The Ca(2+)-ATPase activity of human red cells was studied on calmodulin-free membrane fragments after previous incubation with Mg2+ and vanadate. In the presence of EGTA (5 mM), the activity was slightly affected by either ion alone. However, when added together, both Ca2+ affinity and Vmax were increased up to levels found with calmodulin (0.3 microM). This synergistic activation was not abolished by proteinase inhibitors (iodoacetamide, 10 mM; leupeptin, 200 microM; pepstatin A, 100 microM; phenylmethanesulfonyl fluoride, 100 microM), neomycin (200 microM), washing with EDTA (5 mM) or by both incubating and washing with delipidized serum albumin (1 mg/ml). During preincubation under optimal Mg2+ and vanadate conditions, the replacement of K+ by Na+ or Li+ was without effect. Co2+ or Zn2+ (10 mM) could not substitute for Mg2+, whereas Mn2+ almost replaced it at equimolar amounts. By contrast, addition of ATPMg (2 mM) decreased the activation by about one-half. Like calmodulin, pretreatment with Mg2+ plus vanadate also increased the affinity for ATP and elicited appearance of a second (low) affinity site (apparent Km = 120 microM). The fluorescence depolarization of 1,6-diphenyl- and 1-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating with Mg2+, vanadate or Mg2+ plus vanadate. The results show that Mg2+ and vanadate are acting neither via proteolysis or fatty acid production nor by facilitating phospholipid metabolism or altering membrane fluidity. They may be enhancing the Ca(2+)-ATPase activity by stabilizing the E1 conformer or promoting an enzyme conformation which facilitates the E2-E1 transition.
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PMID:Synergistic activation of the human red cell calcium ATPase by magnesium and vanadate. 849 54


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