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:3.6.3.1 (Mg2+-ATPase)
1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.
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PMID:A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+. 623 48

Basolateral membrane (BLM) enriched fraction was isolated from homogenized rat kidney cortex by differential centrifugation. We also obtained a fraction enriched in plasma membrane (PM). The morphology of the isolated BLM fragments was studied by transmission and freeze fracture electron microscopy. The relative specific activity of Na+-K+-ATPase was enriched 7-fold, while that of marker enzymes for PM, endoplasmic reticulum, and lysosomes was lower than in the crude homogenate. There was a 10-fold difference in the ratios of activities of Na+-k+-ATPase to Mg2+-ATPase in the BLM and in the PM enriched fractions. Kallikrein activity was determined with S-2266 substrate and by radioimmunoassay of kinin released. It was low in the BLM fraction prior to adding detergent, but Triton X-100 increased the activity 12 to 16-fold. Both free trypsin and Sepharose 4B-bound insoluble trypsin increased kallikrein activity 2- to 3-fold in both the membrane-bound and soluble fractions, probably by activating a prekallikrein. The results were interpreted that the kallikrein studied originated from the distal tubular BLM.
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PMID:Kallikrein and prekallikrein on the basolateral membrane of rat kidney tubules. 627 73

The silica microbead procedure was utilized for the isolation of plasma membrane sheets from protoplasts of a higher plant, the red beet (Beta vulgaris L.). Membrane yields, as determined by recovery of an exogenous membrane marker were approx. 75%. The plasma membrane fraction contained the enzyme marker, pH 6.5, vanadate-sensitive, K+-stimulated, Mg2+-ATPase and small amounts of mitochondria, endoplasmic reticulum, and possibly tonoplast. The silica microbead procedure was also used for the isolation of intact vacuoles from microbead-coated protoplasts.
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PMID:Evaluation of the silica microbead method for isolation of red beet protoplast plasma membrane sheets. 646 60

Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5'-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg2+-ATPase, (Na+ + K+)-ATPase, gamma-glutamyl transpeptidase, phosphodiesterase I, alkaline phosphatase and xanthine oxidase were also high (12-18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide composition in both light and heavy membranes were similar upon SDS-polyacrylamide gel electorphoresis.
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PMID:Isolation and characterization of plasma membrane from lactating bovine mammary gland. 720 55

Stimulation of pancreatic acinar cells raises [Ca2+]i via Ca2+ release from inositol-1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores, generally considered to reside within the endoplasmic reticulum (ER). However, with physiological doses of cholinergic agonists, the [Ca2+]i increase is localized to the apical (secretory) pole of the cell, leading to suggestions that zymogen (secretory) granules themselves may constitute an InsP3-sensitive Ca2+ store responsible for localized Ca2+ release. We have therefore re-investigated whether the ER in pancreatic acinar cells is capable of acting as a functional Ca2+ store in all, or only some, cellular regions. In streptolysin O-permeabilized cells, the ER accumulated up to 25 mmol of 45Ca2+ per liter ER volume by an ATP-dependent, thapsigargin-sensitive, process. This tracer Ca2+ uptake was dependent on ambient (loading) [Ca2+], as was the intra-ER free [Ca2+], assessed by imaging the fluorescence of Magfura-2 within the Ca2+ stores. Comparison of free and total intra-ER [Ca2+] indicated that 200-300 Ca2+ ions are bound within the ER lumen for every Ca2+ ion remaining free. Subcellular analysis showed that ER stores in all regions of the permeabilized cell took up Ca2+ at loading [Ca2+] between 60 nM and 1 microM. Thapsigargin released Ca2+ from stores in all cellular regions, as did InsP3. Immunofluorescence with antibodies against sarco(endo)plasmic reticulum-2b type Ca2+,Mg2+-ATPase or calreticulin confirmed that ER Ca2+ stores were present throughout the cytoplasm. In summary, these results clearly show that the endoplasmic reticulum can act as a functional Ca2+ store in all regions of the acinar cell, including the apical pole.
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PMID:The endoplasmic reticulum can act as a functional Ca2+ store in all subcellular regions of the pancreatic acinar cell. 934 20

Highly purified tonoplast and plasma membrane vesicles were isolated from microsomes of Arabidopsis thaliana by preparative free-flow electrophoresis. The most electronegative fractions were identified as tonoplast using nitrate-inhibited Mg2+-ATPase as enzyme marker. The least electronegative fractions were identified as plasma membrane using glucan-synthase II, UDPG: sterol-glucosyl-transferase, and vanadate-inhibited Mg2+-ATPase as enzyme markers. Other membrane markers, latent inosine-5'-diphosphatase (Golgi), NADPH-cytochrome-c reductase (endoplasmic reticulum) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane. Immunoblot analysis of membrane fractions by antibodies directed against tonoplast and plasma membrane proteins confirmed the nature and the purity of the isolated membranes. The cytoskeletal protein actin, which was also identified by immunoblotting, was found to be specifically attached to the plasma membrane vesicles. The structural and functional integrity of the isolated membranes from Arabidopsis thaliana is discussed in the light of results obtained for the location of receptors and enzymes, or for the determination of ligand binding activity.
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PMID:Free-flow electrophoresis for fractionation of Arabidopsis thaliana membranes. 966 77

The characteristics of the Mg2+-dependent ATPase activity from the peribacteroid membrane of pea symbiosomes was compared with that from pea root plasma membranes. Enzyme inhibitors, optimum reaction pH, substrate specificity and antibody recognition were the main parameters examined. Both the symbiosomes and the root plasma membrane were purified with an aqueous polymer two-phase system (APS). The final concentration of the APS for the purification of symbiosomes were: 6.3% w/w dextran T500, 6.3% w/w poly(ethylene glycol) 3350, 5 mM KH2PO4-K2HPO4, 5 mM KCl, 0.33 M sucrose, (pH 7.85); for the root plasma membrane was: 6.2% (w/w) dextran T500, 6.2% poly(ethylene glycol) 3350, 330 mM sucrose, 5 mM K2HPO4 and 4 mM KCl (pH 7.8). The lack of contamination of pea symbiosomes with endoplasmic reticulum, mitochondria, broken bacteroids and/or tonoplast vesicles was established. Similarly, the aqueous two-phase system used here provided a fairly enriched root plasma membrane with low cross-contamination from other sources. Both symbiosomal and root plasma membrane ATPase activities were highly specific to ATP. The symbiosome ATPase apparently corresponds to an E1E2-ATPase mechanism, similar to that found at the plasma membrane. The similarity between these two ATPases was further supported by immuno-analysis. Mg2+-ATPase of pea symbiosome and root plasma membranes were very similar, by all parameters tested.
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PMID:Comparison of Mg2+-dependent ATP hydrolase activities of pea nodule symbiosomes and of pea root plasmalemma, obtained by an aqueous polymer two-phase system. 969 83

The immunophilin FKBP12 associates with intracellular Ca2+ channels and this interaction can be disrupted by the immunosuppressant FK506. We have investigated the effect of FK506 on Ca2+ release and Ca2+ uptake in permeabilized cell types. Changes in medium free [Ca2+] were detected by the fluorescent Ca2+ indicator fluo-3 in digitonin-permeabilized SH-SY5Y human neuroblastoma cells, DT40 and R23-11 (i.e. triple inositol 1,4,5-trisphosphate (IP3) receptor knockout cells) chicken B lymphocytes and differentiated and undifferentiated BC3H1 skeletal muscle cells. 45Ca2+ fluxes were studied in saponin-permeabilized A7r5 rat smooth muscle cells. Addition of FK506 to permeabilized SH-SY5Y cells led to a sustained elevation of the medium [Ca2+] corresponding to approximately 30 % of the Ca2+ ionophore A23187-induced [Ca2+] rise. This rise in [Ca2+] was not dependent on mitochondrial activity. This FK506-induced [Ca2+] rise was related to the inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-Mg2+-ATPase (SERCA) Ca2+ pump. Oxalate-facilitated 45Ca2+ uptake in SH-SY5Y microsomes was inhibited by FK506 with an IC50 of 19 microM. The inhibition of the SERCA Ca2+ pump was not specific since several macrocyclic lactone compounds (ivermectin > FK506, ascomycin and rapamycin) were able to inhibit Ca2+ uptake activity. FK506 (10 microM) did not affect IP3-induced Ca2+ release in permeabilized SH-SY5Y and A7r5 cells, but enhanced caffeine-induced Ca2+ release via the ryanodine receptor (RyR) in differentiated BC3H1 cells. In conclusion, FK506 inhibited active Ca2+ uptake by the SERCA Ca2+ pump; in addition, FK506 enhanced intracellular Ca2+ release through the RyR, but it had no direct effect on IP3-induced Ca2+ release.
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PMID:Effects of the immunosuppressant FK506 on intracellular Ca2+ release and Ca2+ accumulation mechanisms. 1085 21

The molecular basis of transbilayer movement or flipping of phospholipids in the endoplasmic reticulum is largely unknown. To circumvent the problems inherent to studies with artificial phospholipid analogs, we studied microsomal flip-flop of endogenous phosphatidylethanolamine in yeast. The transbilayer transport of phosphatidylethanolamine was measured in reconstituted proteoliposomes derived from microsomal detergent extracts. Our results demonstrate that flipping is protease sensitive but does not require metabolic energy. Our assay is the first to use the endogenous substrate of the so-called 'flippase' to study phospholipid translocation in endomembranes and may therefore be crucial for the understanding of the catalytic properties of this elusive enzyme.
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PMID:Reconstitution of yeast microsomal lipid flip-flop using endogenous aminophospholipids. 1091 28

Phospholipid translocation (flip-flop) across membrane bilayers is typically assessed via assays utilizing partially water-soluble phospholipid analogs as transport reporters. These assays have been used in previous work to show that phospholipid translocation in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum is facilitated by specific membrane proteins (flippases). To extend these studies to natural phospholipids while providing a framework to guide the purification of a flippase, we now describe an assay to measure the transbilayer translocation of dipalmitoylphosphatidylcholine, a membrane-embedded phospholipid, in proteoliposomes generated from detergent-solubilized rat liver endoplasmic reticulum. Translocation was assayed using phospholipase A(2) under conditions where the vesicles were determined to be intact. Phospholipase A(2) rapidly hydrolyzed phospholipids in the outer leaflet of liposomes and proteoliposomes with a half-time of approximately 0.1 min. However, for flippase-containing proteoliposomes, the initial rapid hydrolysis phase was followed by a slower phase reflecting flippase-mediated translocation of phospholipids from the inner to the outer leaflet. The amplitude of the slow phase was decreased in trypsin-treated proteoliposomes. The kinetic characteristics of the slow phase were used to assess the rate of transbilayer equilibration of phospholipids. For 250-nm diameter vesicles containing a single flippase, the half-time was 3.3 min. Proportionate reductions in equilibration half-time were observed for preparations with a higher average number of flippases/vesicle. Preliminary purification steps indicated that flippase activity could be enriched approximately 15-fold by sequential adsorption of the detergent extract onto anion and cation exchange resins.
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PMID:Transbilayer movement of dipalmitoylphosphatidylcholine in proteoliposomes reconstituted from detergent extracts of endoplasmic reticulum. Kinetics of transbilayer transport mediated by a single flippase and identification of protein fractions enriched in flippase activity. 1200 Jul 68


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