Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.1 (Mg2+-ATPase)
 1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously shown the presence of two different forms of glutathione disulfide (GSSG)-stimulated Mg2+-ATPases in human erythrocytes. We have now investigated a low-Km form of the enzyme from human erythrocytes. Purification of the enzyme was performed to apparent homogeneity involving procedures of affinity chromatography and gel filtration. The enzyme was composed of two non-identical subunits of Mr = 82K and 62K. The enzyme reconstituted into phospholipid vesicles showed both GSSG-stimulated Mg2+-ATPase activity (285 nmol Pi released/mg protein/min) and active GSSG transport activity (320 nmol GSSG/mg protein/min). The amino acid composition of the enzyme was similar to that of the enzyme purified from cytoplasmic membranes of human hepatocytes. These enzymes were immunologically cross reactive. These results indicate that this enzyme functions in the active transport of GSSG as it possibly does in hepatocytes.
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PMID:Purification and characterization of glutathione disulfide-stimulated Mg2+-ATPase from human erythrocytes. 252 29

Inside-out erythrocyte membranes attached to polycationic beads manifested glutathione disulfide (GSSG)-stimulated ATPase activity. A Lineweaver-Burk plot of the ATPase activity as a function of GSSG concentration was biphasic and gave apparent Km values of 0.13 mM and 2.0 mM. These kinetics are similar to those reported for the ATP-requiring GSSG-transport systems in human erythrocytes and for the GSSG-stimulated ATPase activity in the plasma membranes of rat hepatocytes. Erythrocyte membranes that were depleted of extrinsic proteins were solubilized in 0.5% Triton X-100. Affinity chromatography on S-hexylglutathione-Sepharose 6B, with elution by a linear gradient of S-hexyl-glutathione, resulted in the resolution of two peaks of enzyme activity. One enzyme, which was eluted at approximately 0.5 mM S-hexylglutathione, had a high affinity for GSSG (apparent Km of 150 microM) and for ATP (80 microM). The other enzyme, which was eluted at approximately 1 mM S-hexylglutathione, had a low affinity for GSSG (apparent Km of 2.0 mM) and ATP (140 microM). GSSG-independent Mg2+-ATPase, Ca2+-dependent Mg2+-ATPase and Na+, K+-dependent Mg2+-ATPase were undetectable in the fractions. Addition of Ca2+, ouabain, or vanadate neither activated nor inhibited the activities, further indicating that the enzymes are distinguishable from ion-pumping ATPases. The enzymes required GSSG for activation; reduced glutathione (GSH) was ineffective. The ATPase activity of the high-Km enzyme was inhibited by addition of p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide and was activated by treatment with dithiothreitol, whereas the ATPase activity of the low-Km enzyme was not modified by these thiol reagents. The properties of the enzymes are similar to those of ATP-dependent GSSG-transport systems in human erythrocytes, suggesting that these ATPases may function in the active transport of GSSG.
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PMID:Glutathione disulfide-stimulated Mg2+-ATPase of human erythrocyte membranes. 295 60

Phosphatidylserine (PS) containing a 7-nitrobenz-2-oxa-1, 3-diazol-4-yl- (NBD-) hexanoyl residue, like native PS, preferentially distributes into the inner membrane leaflet of human erythrocytes. In the case of NBD-PS, this preference results from two opposite active processes, an inward translocation mediated by the aminophospholipid flippase and an outward translocation mediated by an ill-defined floppase. Selective inhibition of this floppase by alkylating reagents or cationic and anionic drugs increases the extent of accumulation of NBD-PS in the inner membrane leaflet from about 70% in control cells to about 90%. Different inhibitor sensitivities of the flippase and the floppase strongly suggest that both represent different entities. The floppase was characterized in further detail by comparing inhibitory effects of various compounds on this translocase with their effects on known primary active transport systems for amphiphilic compounds. The inhibitory effects of various drugs, glutathione conjugates and GSSG on the floppase activity closely correlate with those reported for the active transport by the multidrug resistance protein (MRP) while only poorly going parallel with those for the active transport by the low affinity pump for glutathione conjugates and the multidrug resistance MDR1 P-glycoprotein. The NBD-phospholipid floppase activity of the erythrocyte is thus probably a function of MRP.
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PMID:Evidence for a role of the multidrug resistance protein (MRP) in the outward translocation of NBD-phospholipids in the erythrocyte membrane. 965 91

The active outward translocation of phospholipid analogues from the inner to the outer membrane leaflet of human erythrocytes by the multi-drug resistance protein MRP1 (ABCC1) depends on intracellular reduced glutathione (GSH). Entrapment of ATP and increasing amounts of GSH inside resealed ghosts prepared from erythrocytes resulted in an up to six-fold increase of the translocation rate. Entrapped oxidized glutathione (GSSG) acted inhibitory but produced stimulation after addition of the disulphide-reducing reagent dithioerythritol. Modification of GSH by esterification of the C-terminal carboxylate of Gly, removal of the N-terminal Glu or substitution of the SH group by an anionic S-dicarboxyethyl or sulphonate group abolished stimulation. The effect of S-alkylation of GSH depended on the length of the alkyl group. S-methyl GSH was somewhat more effective than GSH, but maximal stimulation was similar. S-butyl GSH acted poorly stimulatory while S-hexyl GSH was essentially ineffective. Analyses of the kinetic data of translocation revealed K(m) values for GSH and methyl-GSH of respectively 7.4 +/- 2.4 and 4.9 +/- 1.1 mmol l(-1). At high GSH levels and defined constant ATP levels using an ATP-regenerating system, the Km for ATP of the outward translocation was 0.16 +/- 0.02 mmol l(-1). In the same system lacking GSH, the Km for ATP of the inward translocation by the aminophospholipid flippase was 0.53 +/- 0.23 mmol l(-1).
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PMID:ATP and GSH dependence of MRP1-mediated outward translocation of phospholipid analogs in the human erythrocyte membrane. 1457 45

Cytotoxicity by unconjugated bilirubin involves disturbances of membrane structure, excitotoxicity and cell death. These events were reported to trigger elevated free radicals production and impairment of calcium homeostasis, and to result in loss of cell membrane integrity. Therefore, this study was designed to investigate whether interaction of clinically relevant concentrations of free unconjugated bilirubin with synaptosomal membrane vesicles could be linked to oxidative stress, cytosolic calcium accumulation and perturbation of membrane function. Synaptosomal vesicles were prepared from gerbil cortical brain tissue and incubated with purified bilirubin (<or=1 microM), for 4 h at 37 degrees C. Intracellular concentrations of reactive oxygen species (ROS) and calcium were determined by dichlorofluorescin and BAPTA fluorescent probes, respectively. Membrane protein and lipid oxidation were evaluated by immunocytochemistry and phosphatidylserine exposure by annexin V binding. Levels of reduced and oxidized glutathione (GSH and GSSG, respectively), as well as activities of Mg(2+)-ATPase aminophospholipid translocase (flippase) and Na(+),K(+)-ATPase, were also measured. Our results showed that bilirubin induced oxidative stress, due to a rise in lipid (>or=10%, P<0.05) and protein oxidation (>or=20%, P<0.01), ROS content (approximately 17%, P<0.01), and a decrease in GSH/GSSG ratio (>30%, P<0.01). In addition, synaptosomes exposed to bilirubin exhibited increased externalization of phosphatidylserine (approximately 10%, P<0.05), together with decreased flippase and NA(+),K(+)-ATPase (>or=15%, P<0.05) activities, events that were accompanied by enhanced intracellular calcium levels ( approximately 20%, P<0.01). The data obtained point out that interaction of unconjugated bilirubin with synaptosomal membrane vesicles leads to oxidative injury, loss of membrane asymmetry and functionality, and calcium intrusion, thus potentially contributing to the pathogenesis of encephalopathy by hyperbilirubinemia.
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PMID:A link between hyperbilirubinemia, oxidative stress and injury to neocortical synaptosomes. 1547 95