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)

The interaction of the tertiary amine drugs chlorpromazine and dibucaine in their cationic form with carboxyl groups at the membrane surface is studied at concentrations relevant to anesthesia. Spin-labeled stearic acid is used both to provide the carboxyl groups and to monitor binding and ionization behavior in egg lecithin liposomes. Membrane anesthetic concentrations are spectrophotometrically obtained. They are shown to determine the drug influence on carboxyl groups at the membrane surface, independently of aqueous concentrations. The intramembrane association constants (related to the usual aqueous phase ones through the partition coefficient) of the drugs with fatty acids are determined. The same value (10(2) M-1) is obtained for both drugs, suggesting that it is approximately the same for all tertiary amine local anesthetics. pH titrations of anesthetic-treated spin-labeled membranes are performed. The observed shifts in the fatty acid pK are higher than can be produced assuming uniform distribution of the drug in the membrane surface, implying that there is an increased affinity of local anesthetics for superficial carboxyl. This affinity could account for the resting block of voltage-gated Na+ channels. Under these considerations, local anesthetic binding sites at voltage-gated Na+ channels and at sarcoplasmic reticulum Ca(2+)-ATPase are proposed.
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PMID:Carboxyl groups at the membrane interface as molecular targets for local anesthetics. 974 84

We have examined lipid peroxidation (LPO) and fatty acid acyl chain dynamics in synaptosomal membranes isolated from aged rat (Fischer 344 x Brown Norway F1 hybrids) brains, correlating these results with measurements of enzymatic activity of the synaptic plasma membrane Ca2(+)-ATPase (PMCA). Calcium-dependent ATPase activity in these membranes exhibits progressive decreases with a maximal loss of activity with age of approximately 35%. The sensitivity of this membrane-bound ion transporter to the lipid composition of the surrounding membrane, as well as the high abundance of oxidatively sensitive polyunsaturated fatty acyl chains in synaptosomal membranes, suggests that this age-related loss in catalytic turnover may result from LPO-mediated protein modification and/or changes in the physical structure of the bilayer. However, high-performance liquid chromatography analysis of 2,4-dinitrophenylhydrazone derivatives reveals no significant age-related increases in the content of reactive aldehydes (malondialdehyde, formaldehyde, acetaldehyde or acetone) which comprise breakdown products of lipid peroxidation. Electron paramagnetic resonance measurements employing 5- and 12-stearic acid spin labels with the nitroxide reporter groups at two depths in the bilayer were used to assess the fatty acyl chain dynamics (fluidity) of synaptosomal membranes. The resulting spectra demonstrate anisotropic lipid dynamics of two populations of lipids, i.e. lipids in direct association with membrane proteins (boundary lipids) and bulk lipids that do not directly associate with proteins. The nanosecond dynamics of both lipid populations is unaltered with age indicating that any compositional changes occurring with age are insufficient to result in alterations in bilayer fluidity relevant to PMCA activity. Thus, the observed age-related decline in PMCA activity may be explained by direct modification of membrane protein.
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PMID:Decrease in Ca-ATPase activity in aged synaptosomal membranes is not associated with changes in fatty acyl chain dynamics. 986 36

Addition of an amphipathic bee venom peptide, melittin, to sarcoplasmic reticulum (SR) vesicles isolated from rabbit skeletal muscles resulted in a fast (<1 min) blue shift in the fluorescence maximum of the melittin--SR membrane complex. Over the following 45 min the position of the fluorescence maximum did not change, but the fluorescence intensity of the melittin--SR membrane complex decreased by approximately 35% with rate constant 0.14 min-1. Melittin rapidly quenched the isotropic signal in the EPR spectrum of spin-labeled stearic acid added to SR membranes. Further changes in the spectral parameters of the spin probe bound to SR membranes in the presence of melittin indicated an increase of the viscosity of the probe microenvironment (empiric parameter T/eta was decreased by approximately 35% with rate constant 0.11 min-1). The surface potential of SR membranes measured using a pH-sensitive dye, neutral red, decreased after melittin addition from -60 to -30 mV. It was demonstrated with the use of a cross-linking agent, cupric o-phenanthroline, that melittin induced slow aggregation of Ca-ATPase protein in SR membranes; the content of enzyme in the monomeric form decreased with rate constant 0.14 min-1. It is concluded that melittin binds rapidly to SR membranes, inducing slow changes in Ca-ATPase conformation and oligomeric state as well as structural transitions in the lipid bilayer of SR membranes.
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PMID:Characteristics of the interaction of melittin with sarcoplasmic reticulum membranes. 1039 87

The ATP-generated proton pumping across tonoplast vesicles from chilling-sensitive Boro rice (Oryza sativa L. var. Boro) cultured cells was markedly decreased by chilling at 5 degrees C for 3 d. The membrane fluidity of core hydrophobic and surface hydrophilic regions of the lipid bilayer was measured by steady-state fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium 1,6-diphenyl-1,3,5-hexatriene and by electron spin resonance spectroscopy of 16- and 5-doxyl stearic acid, respectively. The fluidity of the surface region of the lipid bilayer of the tonoplast vesicles decreased by chilling. The fluidity of the surface region of the liposomes and the proton pumping across the reconstituted proteoliposomes with tonoplast H+-ATPase decreased with increasing content of the glycolipids. The proton pumping across chimera proteoliposomes was reduced by chilling only when it was reconstituted in the presence of tonoplast glycolipids from chilled Boro cells. These data suggest that the reduction in ATP-generated proton pumping across the tonoplast by chilling is due to the decrease in the fluidity of the surface region of the lipid bilayer of the tonoplast, which is caused by the changes in glycolipids.
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PMID:Mechanism of the chilling-induced decrease in proton pumping across the tonoplast of rice cells. 1096 40

Protein-lipid interactions are studied in normal and denervated electrocytes from Electrophorus electricus (L.). Structural modifications of the lipid micro-environment encircling integral membrane proteins in membrane fractions presenting Na(+),K(+)-ATPase activity are investigated using ESR spectroscopy of stearic acid spin labeled at the 14th carbon (14-SASL). The microsomal fraction derived from the innervated electric organ exhibits, on a discontinuous sucrose gradient, a bimodal distribution of the Na(+),K(+)-ATPase activity, bands a and b. Band b is almost absent in microsomes from the denervated organ, and band a', with the same density as band a has lower Na(+),K(+)-ATPase activity. Band a' presents a larger ratio of protein-interacting lipids than band a. Analysis of the lipid stoichiometry at the protein interface indicates that denervation causes at least a twofold average decrease on protein oligomerization. Physical inactivity and denervation have similar effects on protein-lipid interactions. Denervation also influences the selectivity of proteins for fatty acids. Experiments in decreasing pH conditions performed to verify the influence of stearic acid negative charge on protein interaction revealed that denervation produces loss of charge selectivity. The observed modifications on molecular interactions induced by denervation may have importance to explain modulation of enzyme activity.
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PMID:Denervation alters protein-lipid interactions in membrane fractions from electrocytes of Electrophorus electricus (L.). 1140 87

Phospholamban is a cardiac regulatory protein that, in its monomeric form, inhibits the Ca(2+)-ATPase. Lipid-protein interactions with a synthetic variant of phospholamban, in which all cysteine residues are replaced with alanine, have been studied by spin-label electron spin resonance (ESR) in different lipid host membranes. Both the stoichiometry and selectivity of lipid interactions were determined from the two-component ESR spectra of phospholipid species spin-labeled on the 14 C atom of the sn-2 chain. The lipid stoichiometry is determined by the oligomeric state of the protein and the selectivity by the membrane disposition of the positively charged residues in the N-terminal section of the protein. In dimyristoylphosphatidylcholine (DMPC) membranes, the stoichiometry (N(b)) is 7 lipids/monomer for the full-length protein and 4 for the transmembrane section (residues 26-52). These stoichiometries correspond to the dimeric and pentameric forms, respectively. In palmitoyloleoylphosphatidylcholine, N(b) = 4 for both the whole protein and the transmembrane peptide. In negatively charged membranes of dimyristoylphosphatidylglycerol (DMPG), the lipid stoichiometry is N(b) = 10-11 per monomer for both the full-length protein and the transmembrane peptide. This stoichiometry corresponds to monomeric dispersion of the protein in the negatively charged lipid. The sequence of lipid selectivity is as follows: stearic acid > phosphatidic acid > phosphatidylserine = phosphatidylglycerol = phosphatidylcholine > phosphatidylethanolamine for both the full-length protein and the transmembrane peptide in DMPC. Absolute selectivities are, however, lower for the transmembrane peptide. A similar pattern of lipid selectivity is obtained in DMPG, but the absolute selectivities are reduced considerably. The results are discussed in terms of the integration of the regulatory species in the lipid membrane.
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PMID:Lipid-protein interactions with cardiac phospholamban studied by spin-label electron spin resonance. 1271 59

Effects of the nitric oxide donors S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) on Na+,K+-ATPase-rich membrane fragments purified from pig kidney outer medulla were studied using intrinsic fluorescence and ESR of spin-labeled membranes. These S-nitrosothiols differently affected the intrinsic fluorescence of Na+,K+-ATPase: GSNO induced a partial quenching, whereas SNAP produced no alteration. Quenching can be due to a direct modification of exposed tryptophan residues or to an indirect effect caused by reactions of nitrogen oxide reactive species with other residues or even with the membrane lipids. Pre-incubation of Na+,K+-ATPase with 0.4mM GSNO resulted in a modest inhibition of ATPase activity (about 24%) measured under optimal conditions. Stearic acid spin-labeled at the 14th carbon atom (14-SASL) was used to investigate membrane fluidity and the protein-lipid interface. SNAP slightly increased the mobility of bulk lipids from Na+,K+-ATPase-rich membranes, but did not change the fraction of bulk to protein-interacting lipids. Conversely, treatment with GSNO extinguished the ESR signals from 14-SASL, indicating generation of free radicals with high affinity for the lipid moiety. Our results demonstrated that membranes influence bioavailability of reactive nitrogen species and bias the activity of different S-nitrosothiols.
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PMID:Influence of nitric oxide donors on the intrinsic fluorescence of Na+,K+-ATPase and effects on the membrane lipids. 1591 45

The activation of H(+)-ATPase solubilized from plasma membrane of rice (Oryza sativa L. var Nipponbare) culture cells was examined by the exogenous addition of various phospholipids, free fatty acids, glycerides, polar head groups of phospholipids and molecular species of phosphatidylcholine (PC). H(+)-ATPase activity appeared to be stimulated by phospholipids in the following order: asolectin > phosphatidylserine > PC > lysophosphatidylcholine > phosphatidylglycerol, and maximal ATPase activation was noted at around 0.05 to 0.03% (w/v) of asolectin or molecular species of PC. Polar head groups such as glycerol, inositol, and serine only slightly activated ATPase activity or not at all, while ethanolamine and choline had no effect. Activation was dependent on the degree of saturation or unsaturation of the fatty acyl chain and its length. The activity decreased with increase in the length of fatty acyl chain from dimyristoryl(14:0)-PC to distearoyl(18:0)-PC and the degree of unsaturation from dioleoyl(18:1)-PC to dilinolenoyl(18:3)-PC. Maximum activation was observed when PC possessing 1-myristoyl(14:0)-2-oleoyl(18:1) or 1-oleoyl-2-myristoyl was added to the reaction mixture. These data show that the activation of plasma membrane H(+)-ATPase by PC depends on a combination of saturated (myristic acid 14:0, palmitic acid 16:0, and stearic acid 18:0) and unsaturated (oleic acid 18:1, linoleic acid 18:2, and arachidonic acid 20:4) fatty acids at the sn-1 and sn-2 positions of the triglycerides.
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PMID:Mechanism for the Activation of Plasma Membrane H-ATPase from Rice (Oryza sativa L.) Culture Cells by Molecular Species of a Phospholipid. 1666 56

The effect of inclusion of various C18 fatty acids with 0-2 double bonds in either cis or trans configuration on Lactobacillus rhamnosus GG survival was analysed in simulated gastric juice at pH 2.5. The incorporation of Tween 80 (1 g l-1) in the growth media enhanced subsequent survival of stationary-phase cultures up to 1000-fold following 90 min acid exposure compared with controls grown without Tween 80. There was a significant (P<0.05) increase in bacterial content of oleic acid [C18:1 (9c), up to 55-fold] after growth of bacteria in MRS supplemented with Tween 80. The inclusion of various C18 fatty acids in the growth media revealed that only oleic and vaccenic acids [C18:1 (11t)] had protective effects on the survival of Lb. rhamnosus GG when exposed to the acidic environment. Comparative analysis with other lactobacilli indicated that all strains exhibited increased survival when grown in the presence of Tween 80. Further work with a neomycin-resistant mutant with 48% of the F0F1-ATPase activity of the parent indicated that the Tween 80 effect was independent of the complex. The mechanisms behind the effect of fatty acid protection were investigated and proton permeability assays showed that cultures grown in the presence of Tween 80 had higher extracellular pH than controls. Furthermore, there was a significant reduction of oleic acid and a significant increase in stearic acid (C18:0) (P<0.05) content of bacterial cells following exposure of Tween 80-supplemented cultures to simulated gastric juice. Overall, the data suggest that probiotic lactobacilli can use an exogenous oleic acid source to increase their acid survival and the underlying mechanism most likely involves the ability of increased membrane oleic acid to be reduced by H+ to stearic acid.
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PMID:Growth of probiotic lactobacilli in the presence of oleic acid enhances subsequent survival in gastric juice. 1718 58

Efflux pump (e.g., P-gp, MRP1, and BCRP) inhibition has been recognized as a strategy to overcome multi-drug resistance and improve drug bioavailability. Besides small-molecule inhibitors, surfactants such as Tween 80, Cremophor EL, several Pluronics, and Vitamin E TPGS (TPGS 1000) are known to modulate efflux pump activity. Competitive inhibition of substrate binding, alteration of membrane fluidity, and inhibition of efflux pump ATPase have been proposed as possible mechanisms. Focusing on TPGS 1000, the aim of our study was to unravel the inhibitory mechanism by comparing the results of inhibition experiments in a Caco-2 transport assay with data from electron spin resonance (ESR) and from ATPase activity studies. ESR results, on Caco-2 cells using 5-doxyl stearic acid (5-SA) as a spin probe, ruled out cell membrane fluidization as a major contributor; change of membrane fluidity was only observed at surfactant concentrations 100 times higher than those needed to achieve full efflux inhibition. Concurrently, TPGS 1000 inhibited substrate induced ATPase activity without inducing significant ATPase activity on its own. By investigating TPGS analogues that varied by their PEG chain length, and/or possessed a modified hydrophobic core, transport studies revealed that modulation of ATPase activity correlated with inhibitory potential for P-gp mediated efflux. Hence, these results indicate that ATPase inhibition is an essential factor in the inhibitory mechanism of TPGS 1000 on cellular efflux pumps.
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PMID:Mechanism of inhibition of P-glycoprotein mediated efflux by vitamin E TPGS: influence on ATPase activity and membrane fluidity. 1736 62


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