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)

We have developed a saturation transfer EPR (ST-EPR) method to measure selectively the rotational dynamics of those lipids that are motionally restricted by integral membrane proteins and have applied this methodology to measure lipid-protein interactions in native sarcoplasmic reticulum (SR) membranes. This analysis involves the measurement of spectral saturation using a series of six stearic acid spin labels that are labeled with a nitroxide at different carbon atom positions. A large amount of spectral saturation is observed for spin labels in native SR membranes, but not for spin labels in dispersions of extracted SR lipids, implying that the motional properties of those lipids interacting with the Ca-ATPase, i.e., the boundary or annular lipid, can be directly measured without the need for spectral subtraction procedures. A comparison of the motional properties of the restricted lipid, measured by ST-EPR, with those measured by digital subtraction of conventional EPR spectra qualitatively agree, for in both cases the Ca-ATPase restricts the rotational mobility of a population of lipids, whose rotational mobility increases as the nitroxide is positioned toward the center of the bilayer. However, the ability of ST-EPR to directly measure the motionally restricted lipid in a model-independent means provides the greater precision necessary to measure small changes in the rotational dynamics of the lipid at the protein-lipid interface, providing a valuable tool in clarifying the relationship between the physical nature of the protein-lipid interface and membrane function.
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PMID:Selective detection of the rotational dynamics of the protein-associated lipid hydrocarbon chains in sarcoplasmic reticulum membranes. 255 90

To evaluate physicochemical properties of the small intestinal basolateral cell surface during postnatal development, membranes were isolated from suckling (14-17 days) and weanling-mature (35-49 days) rabbit jejunal and ileal enterocytes at 30- to 40-fold purification (based on Na+-K+-ATPase specific activity) and with limited contamination from coisolated cellular elements. Membrane lipid analysis demonstrated age-dependent reductions and proximal to distal increases in total lipid (per milligram protein). Postnatal increases in membrane total cholesterol of jejunum (suckling vs. mature, 0.18 vs. 0.26 mumol/mg protein; P less than 0.01) and ileum (0.18 vs. 0.31 mumol/mg protein; P less than 0.01) resulted in markedly higher cholesterol-to-phospholipid molar ratios (jejunum, 0.43 vs. 0.73; ileum, 0.43 vs. 0.72 mumol/mg protein; P less than 0.01). Membranes from mature animals had higher relative sphingomeylin and phosphatidylcholine content and, in both age groups, fatty acyl saturation was increased in ileum compared with jejunum. By utilization of the fluorophores 1,6-diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid, the fluidity of basolateral membranes and liposomes prepared from extracted membrane lipid decreased markedly in mature rabbits. Arrhenius plots demonstrated higher apparent thermotropic transition temperatures of mature membrane lipid. These data therefore demonstrate significant changes in small intestinal basolateral membrane lipid composition and fluidity that occur during the weaning period. Possible relationships to ontogenesis of membrane protein function are discussed.
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PMID:Ontogeny of basolateral membrane lipid composition and fluidity in small intestine. 275 Sep 4

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.
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PMID:Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia. 282 49

The dynamic properties of plasma membrane in epidermal cells were determined by means of electron spin resonance using two kinds of doxyl stearic acid spin labeling agents: 5-DSA and 12-DSA. 5-DSA and 12-DSA are stearic acid analogues with a nitroxide radical ring at the 5th and 12th carbon positions, and these motions reflect molecular motion of lipid bilayer surrounding the hydrophilic region and the hydrophobic region, respectively. Guinea pig epidermal cells were separated into three regions of keratinocytes by Percoll density gradient centrifugation; the upper, middle, and lower epidermal cells. The order parameter S values for 5-DSA and 12-DSA incorporated into the isolated keratinocytes increased, suggesting a decrease in the plasma membrane fluidity, as cells approached the upper epidermal cell layer. The Na+,K+-ATPase activity as a plasma membrane-bound enzyme was determined in each epidermal cell region, and was found to decrease gradually as the cells approached the upper layer. Accordingly, the differentiation of epidermal cells in the keratinization process was found to be associated with a decrease in plasma membrane fluidity and with a decline of Na+,K+-ATPase activity.
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PMID:Changes of membrane fluidity and Na+,K+-ATPase activity during cellular differentiation in the guinea pig epidermis. 283 81

The effects of ethinyl estradiol, a synthetic estrogen with cholestatic properties and a propensity to alter hepatocyte and ileal brush-border membrane fluidity, on lipid structure and Na+-K+-ATPase activity of rabbit small intestinal basolateral membranes were determined. Utilizing the fluorophores 1,6-diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid, increases in fluorescence anisotropy, the reciprocal of fluidity, were found in basolateral membranes and in membrane lipid liposomes isolated from ileum. Fluidity alterations were accompanied by a marked decrease in bilayer phospholipids (0.37 vs. 0.48 mumol/mg protein; P less than 0.01) and an increase in both the cholesterol-to-phospholipid molar ratio (0.85 vs 0.61; P less than 0.02) and membrane saturated fatty acid content. Estrogen-mediated physicochemical changes were associated with a significant reduction in ileal basolateral membrane Na+-K+-ATPase specific activity (100.0 vs. 185.8 nmol Pi.min-1.mg protein-1; P less than 0.02). Control values both for fluorescence anisotropy and for Na+-K+-ATPase specific activity were restored after in vitro membrane fluidization with benzyl alcohol. The data therefore indicate that ethinyl estradiol effects on basolateral membrane lipid dynamics are confined to the ileum and are associated with inhibition of Na+-K+-ATPase activity. These structural and functional changes appear to be related, in part, to specific modifications in the availability of phospholipid after estrogen treatment.
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PMID:Estrogen modulates ileal basolateral membrane lipid dynamics and Na+-K+-ATPase activity. 283 62

Highly purified preparations of plasma membranes from control and ketoconazole-treated (1 microM, 120 h) epimastigotes of Trypanosoma cruzi have been obtained by cell disruption using abrasion with glass beads, differential centrifugation and isopycnic centrifugation in continuous, self-generating Percoll gradients. The purity of the preparation was ascertained by the specific activity 125I bound to the membranes obtained from enzymatically radiolabeled epimastigotes and by the alpha-methyl-mannoside sensitive binding of 125I-concanavalin A. The membranes form closed vesicles of 0.2-0.4 micron in diameter which display Mg2+ ATPase and acid phosphatase activities, but are devoid of 5'-nucleotidase and succinate-cytochrome c oxidoreductase; these vesicles can be strongly agglutinated by concanavalin A. The lipid order profiles of membranes from control and treated cells were compared with that present in egg phosphatidylcholine/ergosterol liposomes (84:16, mol/mol) by electron spin resonance spectroscopy of doxylstearic acid probes with the nitroxide group bound to carbon 5, 10, 12 and 16 of the stearic acid chain. Membranes from treated epimastigotes have a lipid order profile which resembles that of control plasma membranes near the polar surface (positions 5 and 10) but there is an abrupt decrease of order at position 12 and from there to the center of bilayer is highly disordered, even more than in pure lipid membranes. Consistent with these results, the leakage of L-[14C]glucose from membrane vesicles of ketoconazole-treated cells is much faster than that observed in vesicles obtained from control cells. These results indicate a strong alteration of the plasma membrane physical and biological properties due to the incubation of the parasite with the drug; this alteration is consistent with the accumulation of methylated precursors of ergosterol, which affects both lipid-lipid and lipid-protein interactions in the membrane.
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PMID:Alteration of lipid order profile and permeability of plasma membranes from Trypanosoma cruzi epimastigotes grown in the presence of ketoconazole. 284 68

Three types of proteoliposome containing mitochondrial H+-ATPase have been prepared: Mg2+-'free', one-side and two-side Mg2+-containing proteoliposomes. The ATPase activity as well as its sensitivity to oligomycin or N,N'-dicyclohexylcarbodiimide of the three proteoliposome preparations has been compared. They decreased in the order: L X (H+-ATPase)+ Mg2+ greater than L X (H+-ATPase)-+ Mg2+ greater than L X (H+-ATPase)-Mg2+. The fluidity of the proteoliposomes has also been compared by fluorescence polarization probes diphenylhexatriene (DPH) or 7-(9-anthroyloxy)stearic acid (7-AS). The degree of polarization for DPH in these proteoliposomes decreased in the order: L X (H+-ATPase) +Mg2+ greater than L X (H+-ATPase)-+ Mg2+ greater than L X (H+-ATPase)-Mg2+, while that for 7-AS: L X (H+-ATPase) +Mg2+ approximately equal to L X (H+-ATPase)-+ Mg2+ greater than L X (H+-ATPase) -Mg2+.
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PMID:Comparison of the effects of one-side- and two-side Mg2+ on the reconstitution of mitochondrial H+-ATPase. 285 63

Human red cell membrane Ca2+-stimulatable, Mg2+-dependent adenosine triphosphatase (Ca2+-ATPase) activity and its response to thyroid hormone have been studied following exposure of membranes in vitro to specific long-chain fatty acids. Basal enzyme activity (no added thyroid hormone) was significantly decreased by additions of 10(-9)-10(-4) M-stearic (18:0) and oleic (18:1 cis-9) acids. Methyl oleate and elaidic (18:1 trans-9), palmitic (16:0) and lauric (12:0) acids at 10(-6) and 10(-4) M were not inhibitory, nor were arachidonic (20:4) and linolenic (18:3) acids. Myristic acid (14:0) was inhibitory only at 10(-4) M. Thus, chain length of 18 carbon atoms and anionic charge were the principal determinants of inhibitory activity. Introduction of a cis-9 double bond (oleic acid) did not alter the inhibitory activity of the 18-carbon moiety (stearic acid), but the trans-9 elaidic acid did not cause enzyme inhibition. While the predominant effect of fatty acids on erythrocyte Ca2+-ATPase in situ is inhibition of basal activity, elaidic, linoleic (18:2) and palmitoleic (16:1) acids at 10(-6) and 10(-4) M stimulated the enzyme. Methyl elaidate was not stimulatory. These structure-activity relationships differ from those described for fatty acids and purified red cell Ca2+-ATPase reconstituted in liposomes. Thyroid hormone stimulation of Ca2+-ATPase was significantly decreased by stearic and oleic acids (10(-9)-10(-4) M), but also by elaidic, linoleic, palmitoleic and myristic acids. Arachidonic, palmitic and lauric acids were ineffective, as were the methyl esters of oleic and elaidic acids. Thus, inhibition of the iodothyronine effect on Ca2+-ATPase by fatty acids has similar, but not identical, structure-activity relationships to those for basal enzyme activity. To examine mechanisms for these fatty acid effects, we studied the action of oleic and stearic acids on responsiveness of the enzyme to purified calmodulin, the Ca2+-binding activator protein for Ca2+-ATPase. Oleic and stearic acids (10(-9)-10(-4) M) progressively inhibited, but did not abolish, enzyme stimulation by calmodulin (10(-9) M). Double-reciprocal analysis of the effect of oleic acid on calmodulin stimulation indicated noncompetitive inhibition. Addition of calmodulin to membranes in the presence of equimolar oleic acid restored basal enzyme activity. Oleic acid also reduced 125I-calmodulin binding to membranes, but had no effect on the binding of [125I]T4 by ghosts. The mechanism of the decrease by long chain fatty acids of Ca2+-ATPase activity in situ in human red cell ghosts thus is calmodulin-dependent and involves reduction in membrane binding of calmodulin.
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PMID:Action of long-chain fatty acids in vitro on Ca2+-stimulatable, Mg2+-dependent ATPase activity in human red cell membranes. 296 20

Lipid-protein interactions in (Na+,K+)-ATPase-rich membranes from the rectal gland of Squalus acanthias have been studied by using spin-labeled lipids in conjunction with electron spin resonance (ESR) spectroscopy. Lipid-protein associations are revealed by the presence of a second component in the ESR spectra of the membranes in addition to a component which corresponds very closely to the ESR spectra obtained from dispersions of the extracted membrane lipids. This second component corresponds to spin-labeled lipids whose motion is very significantly restricted relative to that of the fluid lipids in the membrane or the lipid extract. A stoichiometry of approximately 66 lipids per 265 000-dalton protein is found for the motionally restricted component of those spin-labeled lipids (e.g., phosphatidylcholine) which show least specificity for the protein. This corresponds approximately to the number of lipids which may be accommodated within the first shell around the alpha 2 beta 2 protein dimer. A selectivity of the various spin-labeled lipids for the motionally restricted component associated with the protein is found in the following order: cardiolipin greater than phosphatidylserine approximately stearic acid greater than or equal to phosphatidic acid greater than phosphatidylglycerol approximately phosphatidylcholine approximately phosphatidylethanolamine approximately androstanol.
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PMID:Spin-label studies of lipid-protein interactions in (Na+,K+)-ATPase membranes from rectal glands of Squalus acanthias. 298 12

The pH dependence and salt dependence of the lipid-protein interactions of phosphatidic acid, phosphatidylserine, and stearic acid with Na+,K+-ATPase membranes from Squalus acanthias have been studied with spin-label electron spin resonance spectroscopy, using lipids with nitroxide labels on the 14-position C atom of the sn-2 chain. For phosphatidic acid and stearic acid, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 6.6 and 8.0, respectively. In contrast, the pKa of stearic acid in the bulk lipid environment of the membrane is estimated from spin-label spectroscopy to be approximately equal to 6.6. The fraction of motionally restricted phosphatidylserine spin-label remains constant over the pH range 4.7-9.2. In the fully dissociated state the fractions of motionally restricted spin-labeled phosphatidic and stearic acids decrease with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M. For stearic acid the net decrease is comparable to that obtained on protonation, but for phosphatidic acid the decrease is considerably smaller (by approximately 55%) than that obtained on protonating the lipid. The fraction of motionally restricted phosphatidylserine spin-label varies relatively little with salt concentration up to 1 M NaCl. Direct electrostatic effects alone cannot account for the whole of the observed specificity of interaction of the two phospholipids with Na+,K+-ATPase membranes.
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PMID:Spin-label studies on the origin of the specificity of lipid-protein interactions in Na+,K+-ATPase membranes from Squalus acanthias. 299 12


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