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1.
The (K +,Mg 2+)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N 2 without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co 2+ > Mg 2+ > Mn 2+ > Zn 2+ > Ca 2+) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg 2+, the enzyme was further activated by monovalent cations (K +, NH 4+, Rb + Na +, Cs +, Li +). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a Km of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K + approached simple Michaelis-Menten kinetics, with a Km of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K +, and preferred Mn 2+ to Mg 2+. The results demonstrate that the (K +,Mg 2+)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots. 相似文献
2.
The ATPase activity of a membrane fraction from soybean ( Glycine max L.) root and callus cells, presumed to be enriched in plasma membrane, has been characterized with respect to ion stimulation, pH requirement, and nucleotide specificity. The enzyme from both sources was activated by divalent cations (Mg 2+ > Mn 2+ > Zn 2+ > Ca 2+ > Sr 2+) and further stimulated by monovalent salts. Preparations from root cells were stimulated by monovalent ions according to the sequence: K + > Rb + > Choline + > Na + > Li + > NH 4+ > Cs + > tris +. Membrane preparations from callus cells showed similar stimulatory patterns except for a slight preference for Na + over K +. No synergism between K + and Na + was found with preparations from either cell source. 相似文献
3.
When assayed in the presence of azide, NO 3− was shown to be a specific inhibitor of a proton-translocating ATPase present in corn ( Zea mays L. cv WF9 × M017) root microsomal membranes. The distribution of the NO 3−-sensitive ATPase on sucrose gradients and its general characteristics are similar to those previously reported for the anion-stimulated H +-ATPase of corn roots believed to be of tonoplast origin. An ATPase inhibited by 20 μ m vanadate and insensitive to molybdate was also identified in corn root microsomal membranes which could be largely separated from the NO 3−-sensitive ATPase on sucrose gradients and is believed to be of plasma membrane origin. Inasmuch as both ATPase most likely catalyze the efflux of H + from the cytoplasm, our objective was to characterize and compare the properties of both ATPases under identical experimental conditions. The vanadate-sensitive ATPase was stimulated by cations (K + > NH 4+ > Rb + > Cs + > Li + > Na + > choline +) whereas the NO 3−-sensitive ATPase was stimulated by anions (Cl − > Br − > C 2H 3O 2− > SO 42− > I − > HCO 3− > SCN −). Both ATPases required divalent cations. However, the order of preference for the NO 3−-sensitive ATPase (Mn 2+ > Mg 2+ > Co 2+ > Ca 2+ > Zn 2+) differed from that of the vanadate-sensitive ATPase (Co 2+ > Mg 2+ > Mn 2+ > Zn 2+ > Ca 2+). The vanadate-sensitive ATPase required higher concentrations of Mg:ATP for full activity than did the NO 3−-sensitive ATPase. The kinetics for Mg:ATP were complex for the vanadate-sensitive ATPase, indicating positive cooperativity, but were simple for the NO 3−-sensitive ATPase. Both ATPases exhibited similar temperature and pH optima (pH 6.5). The NO 3−-sensitive ATPase was stimulated by gramicidin and was associated with NO 3−-inhibitable H + transport measured as quenching of quinacrine fluorescence. It was insensitive to molybdate, azide, and vanadate, but exhibited slight sensitivity to ethyl-3-(3-dimethylaminopropyl carbodiimide) and mersalyl. Overall, these results indicate several properties which distinguish these two ATPases and suggest that under defined conditions NO 3−-sensitive ATPase activity may be used as a quantitative marker for those membranes identified tentatively as tonoplast in mixed or nonpurified membrane fractions. We feel that NO 3− sensitivity is a better criterion by which to identify this ATPase than either Cl − stimulation or H + transport because it is less ambiguous. It is also useful in identifying the enzyme following solubilization. 相似文献
4.
Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca 2+ and Mg 2+ and the role of these changes in SR Ca 2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N 2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca 2+] <1 µM, ß-adrenergic stimulation increased luminal Ca 2+ activation of single RyR channels, decreased luminal Mg 2+ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg 2+. At cytoplasmic [Ca 2+] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg 2+ and Ca 2+ inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca 2+ activation site were not affected by ß-adrenergic stimulation.Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca 2+ binding to the luminal Ca 2+ site and decreasing its affinity for luminal Mg 2+ and 2) decreasing affinity of the low-affinity Ca 2+/Mg 2+ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter. 相似文献
5.
Microsomal vesicles from 24-hour-old radish ( Raphanus sativus L.) seedlings accumulate Ca 2+ upon addition of MgATP. MgATP-dependent Ca 2+ uptake co-migrates with the plasma membrane H +-ATPase on a sucrose gradient. Ca 2+ uptake is insensitive to oligomycin, inhibited by vanadate (IC 50 40 micromolar) and erythrosin B (IC 50 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca 2+ uptake is insensitive to protonophores. These results indicate that Ca 2+ transport in these microsomal vesicles is catalyzed by a Mg 2+-dependent ATPase localized on the plasma membrane. Ca 2+ strongly reduces ΔpH generation by the plasma membrane H +-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca 2+ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca 2+ uptake into plasma membrane vesicles. The Ca 2+-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca 2+-induced decrease of ΔpH generation by the plasma membrane H +-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H +-ATPase, is accelerated by MgATP-dependent Ca 2+ uptake, indicating that the decrease of ΔpH generation induced by Ca 2+ reflects the efflux of H + coupled to Ca 2+ uptake into plasma membrane vesicles. It is therefore proposed that Ca 2+ transport at the plasma membrane is mediated by a Mg 2+-dependent ATPase which catalyzes a nH +/Ca 2+ exchange. 相似文献
6.
Two methods for preparing membrane fractions from barley ( Hordeum vulgare cv California Mariout 72) roots were compared in order to resolve reported differences between the characteristics of the plasma membrane ATPase of barley and that of other species. When microsomal membranes were prepared by a published procedure and applied to a continuous sucrose gradient, the membranes sedimented as a single broad band with a peak density of 1.16 grams per cubic centimeter (g/cm 3). Activities of NADH cytochrome (Cyt) c reductase, Ca 2+-ATPase, and Mg 2+-ATPase were coincident and there was little ATP-dependent proton transport anywhere on the gradient. When the homogenization procedure was modified by increasing the pH of the buffer and the ratio of buffer to roots, the microsomal membranes separated as several components on a continuous sucrose gradient. A Ca 2+-phosphatase was at the top of the gradient, NADH Cyt c reductase at 1.08 g/cm 3, a peak of ATP-dependent proton transport at 1.09 to 1.12 g/cm 3, a peak of nitrate-inhibited ATPase at 1.09 to 1.12 g/cm 3, and of vanadate-inhibited ATPase at 1.16 g/cm 3. The Ca 2+-phosphatase had no preference for ATP over other nucleoside di- and tri-phosphates and was separated from the vanadate-inhibited ATPase on a sucrose gradient; approximately 70% of the Ca 2+-phosphatase was removed from the microsomes by washing with 150 millimolar KCl. The vanadate-sensitive ATPase required Mg 2+, was highly specific for ATP, and was not affected by the KCl wash. These results show that barley roots have a plasma membrane ATPase similar to that of other plant species. 相似文献
7.
Heart sarcolemma has been shown to contain an ATPase hydrolizing system which is activated by millimolar concentrations of divalent cations such as Ca 2+ or Mg 2+. Although Ca 2+-dependent ATPase is released upon treating sarcolemma with trypsin, a considerable amount of the divalent cation dependent ATPase activity was retained in the membrane. This divalent cation dependent ATPase was solubilized by sonication of the trypsin-treated dog heart sarcolemma with 1% Triton X-100. The solubilized enzyme was subjected to column chromatography on a Sepharose-6B column, followed by ion-exchange chromatography on a DEAE cellulose column. The enzyme preparation was found to be rather labile and thus the purity of the sample could not be accurately assessed. The solubilized ATPase preparations did not show any cross-reactivity with dog heart myosin antiserum or with Na + + K + ATPase antiserum. The enzyme was found to be insensitive to inhibitors such as ouabain, verapamil, oligomycin and vanadate. The enzyme preparation did not exhibit any Ca 2+-stimulated Mg 2+ dependent ATPase activity. Furthermore, the low affinity of the enzyme for Ca 2– (Ka = 0.3 mM) rules out the possibility of its involvement in the Ca 2+ pump mechanism located in the plasma membrane of the cardiac cell. 相似文献
8.
Sealed microsomal vesicles were prepared from corn ( Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000 g microsomal fraction onto a 10% dextran T-70 cushion. The Mg 2+-ATPase activity of the sealed vesicles was stimulated by Cl − and NH 4+ and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO 3−, was insensitive to K +, and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide. Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg2+, was stimulated by Cl−, inhibited by NO3−, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg2+ ≥ Mn2+ > Co2+) and were inhibited by high concentrations of Ca2+. The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F1F0-ATPase and from those of the K+-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase. 相似文献
9.
The association of K +-stimulated, Mg 2+-dependent ATPase activity with plasma membranes from higher plants has been used as a marker for the isolation and purification of a plasma membrane-enriched fraction from cauliflower ( Brassica oleraceae L.) buds. Plasma membranes were isolated by differential centrifugation followed by density gradient centrifugation of the microsomal fraction. The degree of purity of plasma membranes was determined by increased sensitivity of Mg 2+-ATPase activity to stimulation by K + and by assay of approximate marker enzymes. In the purified plasma membrane fraction, Mg 2+-ATPase activity was stimulated up to 700% by addition of K +. Other monovalent cations also markedly stimulated the enzyme, but only in the presence of the divalent cation Mg 2+. Ca 2+ was inhibitory to enzyme activity. ATPase was the preferred substrate for hydrolysis, there being little hydrolysis in the presence of ADP, GTP, or p-nitrophenylphosphate. Monovalent cation-stimulated activity was optimum at alkaline pH. Enzyme activity was inhibited nearly 100% by AgNO 3 and about 40% by diethylstilbestrol. 相似文献
10.
ATP and the divalent cations Mg 2+ and Ca 2+ regulated K + stimulation of the Ca 2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K +-stimulated ATPase activity of the Ca 2+ pump by two mechanisms. First, ATP chelated free Mg 2+ and, at low ionized Mg 2+ concentrations, K + was shown to be a potent activator of ATP hydrolysis. In the absence of K + ionized Mg 2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K + the concentration of ionized Mg 2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K +-stimulation. With a saturating concentration of ionized Mg 2+, stimulation by K + was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K + increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold.Activation of K +-stimulated ATPase activity by Ca 2+ was maximal at anionized Ca 2+ concentration of approx. 1 μM. At very high concentrations of either Ca 2+ or Mg 2+, basal Ca 2+-dependent ATPase activity persisted, but the enzymic response to K + was completely inhibited. The results provide further evidence that the Ca 2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme. 相似文献
11.
A membrane fraction enriched with a magnesium-dependent, monovalent cation-stimulated ATPase was isolated from red beet ( Beta vulgaris L.) storage roots by a combination of differential centrifugation, extraction with KI, and sucrose density gradient centrifugation. This fraction was distinct from endoplasmic reticulum, Golgi, mitochondrial, and possibly tonoplast membranes as determined from an analysis of marker enzymes. The ATPase activity associated with this fraction was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg 2+ and K +. The activity was substrate specific for ATP and had a temperature optimum near 40°C. Kinetics with Mg:ATP followed a simple Michaelis-Menten relationship. However the kinetics of K +-stimulation were complex and suggestive of negative cooperativity. When monovalent cations were present at 2.5 millimolarity, ATPase was stimulated in the sequence K + > Rb + > Na + > Li + but when the concentration was raised to 50 millimolarity, the sequence changed to K + ≥ Na + ≥ Rb + > Li. The activity was not synergistically stimulated by combinations of Na + and K +. The enzyme was insensitive to NaN 3, oligomycin, ouabain, and sodium molybdate but sensitive to N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and sodium vanadate. Based on the similarity between the properties of this ATPase activity and those from other well characterized plant tissues, it has been concluded that this membrane fraction is enriched with plasma membrane vesicles. 相似文献
12.
The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two-phase partitioning. Its purity was examined with marker enzymes, Mg 2+-dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH-cytochrome c reductase, as well as the sensitivity of Mg 2+-dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid-chromic acid. Mg 2+- or Ca 2+-dependent ATPases were associated with the plasma membrane. Ca 2+-dependent ATPase was activated at physiological cytoplasmic concentrations of Ca 2+ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg 2+-dependent ATPase. The isolated plasma membrane vesicles were mostly right side-out. To test for H +-translocation, right side-out vesicles were inverted; 27% of vesicles were inside-out after treatment with Triton X-100. The inside-out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca 2+. The reduced fluorescence was recovered with the addition of 10 mmol/L NH 4Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca 2+-dependent ATPase might pump H + out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification. 相似文献
13.
A chicken pectoralis muscle membrane fraction enriched in a Mg 2+- or Ca 2+-activated (‘basic’) ATPase was obtained by sucrose gradient centrifugation. Enzymatic properties of the ‘basic’ ATPase were determined and used to localize its enzymatic activity in situ by ultrastructural cytochemistry. The enzyme was activated by Mg 2+ or Ca 2+ but not by Sr 2+, Ba 2+, Co 2+, Ni 2+ or Pb 2+. It was present in a membranous fraction with a buoyant density of 1.10-1.12 (24–27.5% (w/w) sucrose). ‘Basic’ ATPase activity had a sedimentation pattern similar to the putative plasma membrane enzymes, 5′-nucleotidase and leucyl β-naphthylamidase, but different from that of sarcoplasmic reticulum Ca 2+ ATPase. Also unlike sarcoplasmic reticulum Ca 2+ ATPase, ‘basic’ ATPase was resistant to N-ethylmaleimide and aldehyde fixatives, was active in a medium containing a high Ca 2+ concentration (3 mM), and was lost when exposed to Triton X-100 or deoxycholate. In cytochemical studies, a low Pb 2+ concentration was used to capture the enzymatically released phosphate ions. Under conditions which eliminated interfering (Na + + K +) ATPase and sarcoplasmic reticulum Ca 2+ ATPase activities, electron-dense lead precipitates were present at the plasmalemma and T-system membranes. These studies suggest that ‘basic’ ATPase activity is associated with plasmalemma and T-system membranes of skeletal muscle. 相似文献
14.
Conditions for the dissociation and reassembly of the multi-subunit vacuolar proton-translocating ATPase (H +-ATPase) from oat roots ( Avena sativa var Lang) were investigated. The peripheral sector of the vacuolar H +-ATPase is dissociated from the membrane integral sector by chaotropic anions. Membranes treated with 0.5 molar KI lost 90% of membrane-bound ATP hydrolytic activity; however, in the presence of Mg 2+ and ATP, only 0.1 molar KI was required for complete inactivation of ATPase and H +-pumping activities. A high-affinity binding site for MgATP (dissociation constant = 34 micromolar) was involved in this destabilization. The relative loss of ATPase activity induced by KI, KNO 3, or KCl was accompanied by a corresponding increase in the peripheral subunits in the supernatant, including the nucleotide-binding polypeptides of 70 and 60 kilodaltons. The order of effectiveness of the various ions in reducing ATPase activity was: KSCN > KI > KNO 3 > KBr > K-acetate > K 2SO 4 > KCl. The specificity of nucleotides (ATP > GTP > ITP) in dissociating the ATPase is consistent with the participation of a catalytic site in destabilizing the enzyme complex. Following KI-induced dissociation of the H +-ATPase, the removal of KI and MgATP by dialysis resulted in restoration of activity. During dialysis for 24 hours, ATP hydrolysis activity increased to about 50% of the control. Hydrolysis of ATP was coupled to H + pumping as seen from the recovery of H + transport following 6 hours of dialysis. Loss of the 70 and 60 kilodalton subunits from the supernatant as probed by monoclonal antibodies further confirmed that the H +-ATPase complex had reassembled during dialysis. These data demonstrate that removal of KI and MgATP resulted in reassociation of the peripheral sector with the membrane integral sector of the vacuolar H +-ATPase to form a functional H + pump. The ability to dissociate and reassociate in vitro may have implications for the regulation, biosynthesis, and assembly of the vacuolar H +-ATPase in vivo. 相似文献
15.
The leucine zipper, EF hand–containing transmembrane protein 1 ( Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca 2+ and K + homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca 2+ fluorophore and 45Ca 2+-based assays, we demonstrate directly that Letm1 is a Ca 2+ transporter, with apparent affinities of cations in the sequence of Ca 2+ ≈ Mn 2+ > Gd 3+ ≈ La 3+ > Sr 2+ >> Ba 2+, Mg 2+, K +, Na +. Kinetic analysis yields a Letm1 turnover rate of 2 Ca 2+/s and a K m of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca 2+/2 H + antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na +/Ca 2+ exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H +-dependent Ca 2+ transport mechanism identified in intact mitochondria. 相似文献
16.
Cardiac plasma membrane Ca 2+/Mg 2+ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca 2+ or Mg 2+ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca 2+/Mg 2+ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca 2+/Mg 2+ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca 2+-pump ATPase or sarcolemmal Ca 2+-pump ATPase. On the other hand, the cardiac Ca 2+/Mg 2+ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca 2+-pump ATPase or plasma membrane Ca 2+-pump ATPase. Furthermore, the immune serum inhibited the Ca 2+/Mg 2+ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca 2+/Mg 2+ ecto-ATPase antibodies indicated the localization of Ca 2+/Mg 2+ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca 2+/Mg 2+ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca 2+/Mg 2+ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle. 相似文献
17.
To understand the function and membrane origin of ionophore-stimulated ATPases, the activity of nigericin-stimulated ATPase was characterized from a low-density microsomal fraction containing sealed vesicles of autonomous tobacco ( Nicotiana tabacum Linnaeous cv. Wisconsin no. 38) callus. The properties of KCl-stimulated, Mg-requiring ATPases (KCl-Mg,ATPase) were similar in the absence or presence of nigericin. Nigericin (or gramicidin) stimulation of a KCl-Mg,ATPase activity was optimum at pH 6.5 to 7.0. The enzyme was inhibited completely by N,N′-dicyclohexylcarbodiimide (10 μ m), tributyltin (5 μ m), and partially by vanadate (200 μ m), but it was insensitive to fusicoccin and mitochondrial ATPase inhibitors, such as azide (1 m m) and oligomycin (5 μg/ml). The ATPase was more sensitive to anions than cations. Cations stimulated ATPase activity with a selectivity sequence of NH 4+ > K +, Rb +, Cs +, Na +, Li + > Tris +. Anions stimulated Mg, ATPase activity with a decreasing sequence of Cl − = acetate > SO 42− > benzene sulfonate > NO 3−. The anion stimulation was caused partly by dissipation of the electrical potential (interior positive) by permeant anions and partly by a specific ionic effect. Plant membranes had at least two classes of nigericin-stimulated ATPases: one sensitive and one insensitive to vanadate. Many of the properties of the nigericin-sensitive, salt-stimulated Mg,ATPase were similar to a vanadate-sensitive plasma membrane ATPase of plant tissues, yet other properties (anion stimulation and vanadate insensitivity) resembled those of a tonoplast ATPase. These results support the idea that nigericin-stimulated ATPases are mainly electrogenic H + pumps originated in part from the plasma membrane and in part from other nonmitochondrial membranes, such as the tonoplast. 相似文献
18.
Altered cytosolic Ca 2+ is implicated in the aetiology
of many diseases including diabetes but there are
few studies on the mechanism(s) of the altered Ca 2+
regulation. Using human lymphocytes, we studied
cytosolic calcium (Ca i) and various Ca 2+ transport
mechanisms in subjects with Type 2 diabetes
mellitus and control subjects. Ca 2+-specific fluorescent
probes (Fura-2 and Fluo-3) were used to
monitor the Ca 2+ signals. Thapsigargin, a potent and
specific inhibitor of the sarco(endo)plasmic reticulum
Ca 2+-ATPase (SERCA), was used to study Ca 2+-
store dependent Ca 2+ fluxes. Significant ( P < 0.05)
elevation of basal Ca i levels was observed in
lymphocytes from diabetic subjects. Ca i levels were
positively correlated with fasting, plasma glucose
and HbAlc. There was also a significant ( P < 0.05)
reduction in plasma membrane calcium (PMCA)
ATPase activity in diabetic subjects compared to
controls. Cells from Type 2 diabetics exhibited an
increased Ca 2+ influx (as measured both by Fluo-3
fliorescence and C45a assays) as a consequence of
of thapsigargin-mediated Ca 2+ store depletion. Upon
addition of Mn 2+ (a surrogate of Ca 2+), the fura-2
fluorescence decayed in an exponential fashion and
the rate and extent of this decline was steeper and
greater in cells from type 2 diabetic patients. There
was also a significant ( P < 0.05) difference in the
Na +/Ca 2+ exchange activity in Type 2 diabetic
patients, both under resting conditions and after challenging the cells with thapsigargin, when the
internal store Ca 2+ sequestration was circumvented.
Pharmacological activation of protein kinase C
(PKC) in cells from patients resulted in only partial
inhibition of Ca 2+ entry. We conclude that cellular
Ca 2+ accumulation in cells from Type 2 diabetes
results from (a) reduction in PMCA ATPase activity,
(b) modulation of Na +/Ca 2+ exchange and (3)
increased Ca 2+ influx across the plasma membrane. 相似文献
19.
A mitochondria-free membrane fraction prepared from rat myometrium accumulated 45Ca 2+ in the presence of oxalic acid and ATP. The rate of transport of Ca 2+ into the membranous vesicles was increased by greater than 50% in the presence of 3′,5′-cyclic AMP, but not by 2′,3′-cyclic AMP or 5′-AMP. Membrane ATPase activity was stimulated by cyclic AMP in a manner similar to Ca 2+-transport. ATPase activity was stimulated by Mg 2+; slight additional stimulation was obtained in the presence of Na + and K + but not in the presence of Ca 2+. Despite the cyclic AMP sensitivity of membrane ATPase activity, the absence of any effect of inhibitors of Ca 2+-transport suggest it has little to do with Ca 2+ accumulation by the membranes.Cyclic AMP-induced increase in Ca 2+-transport and membrane ATPase activity was duplicated in vivo by incubating uteri in 10 −4 M isoproterenol prior to membrane isolation. Isoproterenol has been previously shown to increase myometrial cyclic AMP levels, and changes in Ca 2+-transport by cell membranes in relation to intracellular cyclic AMP levels may be the mechanism through which hormones modulate uterine contractility. 相似文献
20.
Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils of Bryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as Mg 2+ATP-driven transport of 45Ca 2+. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca 2+-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca 2+-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K +, Mg 2+-ATPase and the Ca 2+-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling.Abbreviations CAM
calmodulin
- CCCP
carbonylcyanide m-chlorophenylhydrazone
- IC 50
concentration giving 50% inhibition
- PM
plasma membrane
- rER
rough endoplasmic reticulum
- sER
smooth endoplasmic reticulum
- FC
fusicoccin
- U 3+U 3
the two PM-rich upper phases obtained after phase partitioning of microsomal membranes
The authors wish to thank the Deutsche Forschungsgemeinschaft, Bonn, Germany, and the Fonds der Chemischen Industrie, Frankfurt, Germany (literature provision) for financial support. 相似文献
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