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1.
Microsomal membrane vesicles isolated from the petals of young carnation ( Dianthus caryophyllus L. cv White Sim) flowers accumulate Ca 2+ in the presence of ATP. The specific activity of ATP-dependent uptake is ~20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca 2+ stimulation of ATP hydrolysis was not discernible above the high background of Ca 2+-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca 2+ concentration, suggesting that Ca 2+ serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca 2+ at half maximal velocity of uptake (S 0.5) was 12.5 micromolar and the Hill coefficient ( nH) was 2.5. The addition of calmodulin to membrane preparations that had been isolated in the presence of chelators did not promote ATP-dependent accumulation of Ca 2+, although this may reflect the fact that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca 2+ into membrane vesicles was unaffected by 50 micromolar ruthenium red and 5 micromolar sodium azide, indicating that uptake is primarily into vesicles of non-mitochondrial origin. By subfractionating the microsomes on a linear sucrose gradient, it was established that the ATP-dependent Ca 2+ transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca 2+ into microsomal vesicles declined by ~70%. This occurred before the appearance of petal-inrolling and the climacteric-like rise in ethylene production, parameters that denote the onset of senescence. There were no significant changes during this period in S 0.5 or nH, but Vmax for ATP-dependent Ca 2+ uptake decreased by ~40%. A similar decline in ATP-dependent uptake of Ca 2+ into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene. 相似文献
2.
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
3.
- 1.1. In the plasma membrane of mussel gill cells an ouabain insensitive, Ca2+-activated ATPase activity is present. The ATPase has high Ca2+ affinity (Kma = 0.3 μM).
- 2.2. The optimum assay conditions to evaluate the enzymatic activity of the Ca2+-stimulated ATPase at 19°C are: 120–300 mM KCl ionic strength, pH 7.0 and 2 mM ATP. As for mammalian enzymes, the Ca2+ ATPase activity is stimulated by DTT (0.5–1 mM) and it is inhibited by low concentrations of vanadate (10–50 μM) and -SH inhibitors such as PCMB and PCMBS (10 μM); the enzyme appears to be calmodulin insensitive.
- 3.3. Electrophoretic analyses of plasma membrane proteins demonstrate that: (a) Ca2+ at n-μM concentrations is necessary to activate ATP hydrolysis with consequent formation of the enzyme-phosphate complex; (b) the steady state concentration of the phosphorylated intermediate is increased in the presence of La3+; (c) the mol. wt of Ca2+ ATPase is about 140 kDa.
- 4.4. Low Ca2+ concentrations (n-μM) are sufficient to stimulate the ATP-dependent Ca2+ uptake by plasma membrane inside-out vesicles.
- 5.5. The results indicate that the Ca2+ pump present in the gill plasma membranes could be responsible for Ca2+ extrusion and therefore involved in maintaining the cytosolic Ca2+ concentration within physiological levels.
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4.
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
5.
The subcellular localization and biochemical characterization of calcium transport were studied in the unicellular green alga Mesotaenium caldariorum. Membrane fractions prepared by osmotic lysis of Mesotaenium protoplasts exhibit high rates of ATP-dependent calcium uptake. Sucrose gradient centrifugation separates two pools of activity, which display specific activities for calcium transport as high as 15 nanomoles Ca 2+ per minute per milligram of protein. Marker enzyme analysis shows that this dual distribution of calcium transport activity is similar to that of vanadate-insensitive ATPase and pyrophosphatase, activities considered to be associated with the tonoplast. Plasma membranes, endoplasmic reticulum vesicles, mitochondrial membranes, and thylakoids band at higher densities than either calcium transport fraction. Both pools of ATP-dependent calcium uptake contain two components which are not separable on sucrose gradients but can be distinguished on the basis of inhibitor sensitivity. One component is inhibited by nigericin or trimethyltin chloride (I 50 values of 3 nanomolar and 4 micromolar, respectively), while the other component is vanadate sensitive (I 50 of 25 micromolar). These results suggest that direct Ca 2+ transport and Ca 2+/H + antiport activities are present in both sucrose gradient fractions. 相似文献
6.
Ca 2+ transport activity in synaptosomal membranes has been identified as having two major components: Ca 2+-stimulated ATP hydrolysis and ATP-dependent CA 2+ uptake. Both processes exhibit similar affinities for Ca 2+ and operate maximally under identical buffer conditions. Subcellular fractionation studies revealed the Ca 2+/Mg 2+ ATPase and ATP-dependent CA 2+ uptake activities to be highest in synaptic plasma membrane fractions 1 and 2, with lesser activity in synaptic vesicles and mitochondria. Progressive treatment with Triton X-100 activated, then decreased Ca 2+/Mg 2+ ATPase, Mg 2+ ATPase and Ca 2+ ATPase. ATP-dependent Ca 2+ uptake was progressively decreased by similar treatment with Triton X-100. These studies illustrate that Ca 2+ ATPase and ATP-dependent Ca 2+ uptake may provide two important mechanisms for buffering of cytosolic Ca 2+ at the nerve terminal. These systems may function to rapidly sequester cytosolic Ca 2+ following a rise during depolarization and then extrude Ca 2+ from the terminal against a concentration gradient. This regulation of cytosolic Ca 2+, represented by two processes of the type seen in other plasma membranes, may play critical roles in calcium homeostasis in nerve cells.Footnote: Portions of this research were submitted by K. M. Garrett in partial fulfillment of requirements for the Doctor of Philosophy Degree in Pharmacology at the University of Texas Health Science Center. 相似文献
7.
1. Microsomes were isolated from rabbit fast-twitch and slow-twitch muscle and were separated into heavy and light fractions by centrifugation in a linear (0.3–2 m) sucrose density gradient. The membrane origin of microsomal vesicles was investigated by studying biochemical markers of the sarcoplasmic-reticulum membranes and of surface and T-tubular membranes, as well as their freeze-fracture properties. 2. Polyacrylamide-gel electrophoresis showed differences in the Ca 2+-dependent ATPase/calsequestrin ratio between heavy and light fractions, which were apparently consistent with their respective origin from cisternal and longitudinal sarcoplasmic reticulum, as well as unrelated differences, such as peptides specific to slow-muscle microsomes (mol.wts. 76000, 60000, 56000 and 45000). 3. Freeze-fracture electron microscopy of muscle microsomes demonstrated that vesicles truly derived from the sarcoplasmic reticulum, with an average density of 9nm particles on the concave face of about 3000/μm 2 for both fast and slow muscle, were admixed with vesicles with particle densities below 1000/μm 2. 4. As determined in the light fractions, the sarcoplasmic-reticulum vesicles accounted for 84% and 57% of the total number of microsomal vesicles, for fast and slow muscle respectively. These values agreed closely with the percentage values of Ca 2+-dependent ATPase protein obtained by gel densitometry. 5. The T-tubular origin of vesicles with a smooth concave fracture face in slow-muscle microsomes is supported by their relative high content in total phospholipid and cholesterol, compared with the microsomes of fast muscle, and by other correlative data, such as the presence of (Na ++K +)-dependent ATPase activity and of low amounts of Na +-dependent membrane phosphorylation. 6. Among intrinsic sarcoplasmic-reticulum membrane proteins, a proteolipid of mol.wt. 12000 is shown to be identical in the microsomes of both fast and slow muscle and the Ca 2+-dependent ATPase to be antigenically and catalytically different, though electrophoretically homogeneous. 7. Basal Mg 2+-activated ATPase activity was found to be high in light microsomes from slow muscle, but its identification with an enzyme different from the Ca 2+-dependent ATPase is still not conclusive. 8. Enzyme proteins that are suggested to be specific to slow-muscle longitudinal sarcoplasmic reticulum are the flavoprotėin NADH:cytochrome b5 reductase (mol.wt. 32000), cytochrome b5 (mol.wt. 17000) and the stearoyl-CoA desaturase, though essentially by criteria of plausibility. 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 protein(s) that constitute(s) the ATP-driven Ca 2+-translocator of plasma membrane enriched vesicles obtained by aqueous two-phase partitioning from leaves of Commelina communis L. has/have been solubilized and reincorporated into tightly sealed liposomes. The reconstituted Ca 2+-transport system was studied using ATP-driven 45Ca 2+ import into the proteoliposomes as a measure of activity. The detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate proved to be the most suitable and was used at 10 millimolar concentration, i.e. just above its critical micellar concentration. The presence of additional phospholipid (2 milligrams phosphatidylcholine per milliliter) and ATP (5 millimolar) improved the solubilization and/or reconstitution. The characteristics of the reconstituted system were similar to those of the plasma membrane-bound activity, including the apparent Km for Ca 2+ (5.2 micromolar), inhibition by relatively high levels of vanadate (IC 50 = 500 micromolar) and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B (IC 50 = 0.01 micromolar) and had a low apparent Km for ATP (11.4 micromolar). As in the plasma membrane vesicles, the protonophore carbonylcyanide m-chlorophenyl hydrazone did not affect Ca 2+-transport detectably in the reconstituted system. However, low levels of the Ca 2+-ionophore A 23187 instantaneously discharged 90% of the Ca 2+ associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca 2+-transport in the reconstituted system was thus primary active, through a Ca 2+-translocating ATPase. The system reported here may serve as a valuable tool for purifying the Ca 2+-ATPase and for studying structural and functional aspects of the purified enzyme. 相似文献
10.
Calcium transport into tomato ( Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10-fold by MgATP. Two ATP-dependent Ca 2+ transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca 2+. A low affinity Ca 2+ uptake system ( Km > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H +/Ca 2+ antiport. A high affinity Ca 2+ uptake system ( Km = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca 2+ transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12°C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca 2+/H + antiport activity could only by partially ascribed to an effect of low temperature on H +-ATPase activity, ATP-dependent H + transport, passive H + fluxes, or passive Ca 2+ fluxes. These results suggest that low temperature directly affects Ca 2+/H + exchange across the tomato fruit tonoplast, resulting in an apparent change in activation energy for the transport reaction. This could result from a direct effect of temperature on the Ca 2+/H + exchange protein or by an indirect effect of temperature on lipid interactions with the Ca 2+/H + exchange protein. 相似文献
11.
The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca 2+-dependent ATPase and are also able to accumulate up to 10 μmol Ca 2+ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+ a3 and an increase in the content of [ 32P]phosphoenzyme. The basic components in “calcium-oxalate preparation” from hearts are proteins with molecular weights of about 100 000 (Ca 2+-dependent ATPase) and 55 000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca 2+-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12–16 μmol P i/min per mg protein. Enzyme activity of purified Ca 2+-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca 2+-dependent ATPases isolated from heart and skeletal muscles. 相似文献
12.
Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca 2+-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH 2)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent Km of 0.2 μM Ca 2+ and a Vmax of 5.3 nmol Ca 2+/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca 2+-ATPase are: Km = 0.25 μM Ca 2+ and Vmax = 19.2 nmol P i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the Vmax of ATP-dependent calcium uptake amounts to 25 nmol Ca 2+/min per mg protein and of the Ca 2+-ATPase to 23 nmol P i/min per mg protein. The close correlation between kinetic parameters of Ca 2+-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca 2+-pump located in duodenal basolateral plasma membranes. 相似文献
13.
The ATPase activity of the chloroplast coupling factor 1 (CF 1) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca 2+ -dependent activity. The Ca 2+ dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca 2+ to Mg 2+. Both the Ca 2+ -dependent and Mg 2+ -dependent ATPase activities of heat-treated Dunaliella CF 1 are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF 1. However, when assayed under identical conditions, the Ca 2+ -dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg 2+ -dependent activity. These data are interpreted as indicating that soluble Dunaliella CF 1 can exist in a variety of conformations, at least one of which catalyzes a Ca 2+ -dependent ATPase and two or more of which catalyze an Mg 2+ -dependent ATPase. 相似文献
14.
The Ca 2+ transport system of corn ( Zea mays) leaf plasma membrane is composed of Ca 2+ pump and Ca 2+/H + antiporter driven by H + gradient imposed by a H + pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca 2+ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca 2+ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C 12E 8) was the most effective detergent for a series of extraction and functional reconstitution of the Ca 2+ pump among seven detergents examined. This was judged from activities of ATP-dependent 45Ca 2+ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C 12E 8-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca 2+-ATPase was separated from VO 43−-sensitive Mg 2+-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca 2+ uptake was measured. The liposomes reconstituted with the Ca 2+-ATPase, but not with the VO 43−-sensitive Mg 2+-ATPase, showed ATP-dependent Ca 2+ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca 2+ uptake into liposomes reconstituted with the Ca 2+-ATPase, suggesting that the Ca 2+/H + antiporter was not present in the preparation. These results indicate that the Ca 2+-ATPase actually functions as Ca 2+ pump in the corn leaf plasma membrane. 相似文献
15.
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
16.
ATP-dependent Ca 2+ uptake distinct from that of the mitochondria is found in both plasma membrane and microsomal membranes of rat kidney. Activity attributed to these fractions is enhanced by ammonium oxalate and is apparently insensitive to NaN 3. In contrast, rat kidney mitochondrial Ca 2+ uptake is blocked by NaN 3. The pH of optimal activity is significantly higher for the mitochondrial fraction. Microsomal membrane Ca 2+ uptake differs from that of the plasma membrane. Microsomal membranes are four times as active as the plasma membrane at high (5 mM) ATP levels. Apparent Km values for Mg 2+-ATP differ in the two preparations with a higher affinity for Mg 2+-ATP found in the plasma membrane Ca 2+ uptake activity of the plasma membrane preparation is readily inhibited by Na +. Sucrose gradient density fractionation indicates that the observed microsomal membrane Ca 2+ pump activity is associated with membrane vesicles derived from the endoplasmic reticulum. Ca 2+ pump activity of both plasma membrane and microsomal fraction is depressed din the adrenalectomized rat. This activity is not restored by a single natriuretic dose of aldosterone. 相似文献
17.
The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca 2+-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca 2+-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed. 相似文献
18.
Calcium is actively transported into intracellular organelles and out of the cytoplasm by Ca 2+/Mg 2+-ATPases located in the endoplasmic reticulum and plasma membranes. We studied the effects of aluminum on calcium transport in the adult rat brain. We examined 45Ca-uptake in microsomes and Ca 2+-ATPase activity in microsomes and synaptosomes isolated from the frontal cortex and cerebellum of adult male Long-Evans rats. ATP-dependent 45Ca-uptake was similar in microsomes from both brain regions. The addition of 50-800 μM AICI 3 resulted in a concentration-dependent inhibition of 45Ca-uptake. Mg 2+-dependent Ca 2+-ATPase activity was significantly lower in synaptosomes compared to microsomes in both frontal cortex and cerebellum. In contrast to the uptake studies, AICI 3 stimulated Mg 2+-dependent Ca 2+-ATPase activity in both microsomes and synaptosomes from both brain regions. To determine the relationship between aluminum and Mg 2+, we measured ATPase activity in the presence of increasing concentrations of Mg 2+ or AICI 3. Maximal ATPase activity was obtained between 3 and 6 mM Mg 2+. When we substituted AICI 3 for Mg 2+, ATPase activity was also stimulated in a concentration-dependent manner, but to a greater extent than with Mg 2+. One interpretation of these data is that aluminum acts at multiple sites to displace both Mg 2+ and Ca 2+, increasing the activity of the Ca 2+-ATPase, but disrupting transport of calcium. 相似文献
19.
Ca 2+-activated ATPase (EC 3.6.1.15) in canine cardiac sarcoplasmic reticulum was stimulated 50–80% by cyclic adenosine 3′ : 5′-monophosphate. The relationship of this stimulation to cyclic AMP-dependent membrane phosphorylation with phosphoester bands was studied. Cyclic AMP stimulation of ATPase activity was specific for Ca 2+-activated ATPase and was half-maximal at about 0.1 μM which is similar to the concentration required for half-maximal stimulation of membrane phosphorylation by endogenous cyclic AMP-stimulated protein kinase (EC 2.7.1.37). Cyclic AMP stimulation of Ca 2+-activated ATPase was calcium dependent and maximal at calculated Ca 2+ concentrations of 2.0 μM. Cyclic AMP-dependent Ca 2+-activated ATPase correlated well with the cyclic AMP-dependent membrane phosphorylation of which 80% was 20 000 molecular weight protein identified by sodium dodecyl sulfate discontinuous polyacrylamide gel electrophoresis. In trypsin-treated microsomes, cyclic AMP did not stimulate Ca 2+-activated ATPase or phosphorylation of the 20 000 molecular weight membrane protein. An endogenous calcium-stimulated protein kinase (probably phosphorylase b kinase) with an apparent Km for ATP of 0.21–0.32 mM was present and appeared to be involved in the cyclic AMP-dependent phosphorylation of the 20 000 molecular weight protein which was calcium dependent. Cyclic guanosine 3′ : 5′-monophosphate did not inhibit any of the stimulatory effects of cyclic AMP. These data suggest that the cyclic AMP stimulation of Ca 2+-activated ATPase in cardiac sarcoplasmic reticulum is mediated by the 20 000 molecular weight phosphoprotein product of a series of kinase reactions similar to those activating phosphorylase b. 相似文献
20.
Summary Ca 2+ uptake into Ehrlich ascites tumor cells was studied at 0°C in the presence of mitochondrial inhibitors, conditions that minimized complications caused by sequestration of Ca 2+ into organelles or by excretion. Under these conditions Ruthenium Red inhibited Ca 2+ uptake, but other previously implicated ions, such as P i or Mg 2+, had no effect. Valinomycin either inhibited or slightly stimulated Ca 2+ uptake depending on the presence of excess K + on the outside or inside of the cell, respectively. Nigericin inhibited Ca 2+ transport. Based on these data we propose an electrogenic uptake of Ca 2+, possibly via a Ca 2+/H + antiport mechanism.The observation that glucose inhibited Ca 2+ uptake suggested that in Ehrlich ascites tumor cells an energy-driven Ca 2+ expulsion mechanism is operative, similar to that in erythrocytes. Plasma membrane preparations of ascites tumor cells were found to contain a Ca 2+-dependent ATPase. These preparations, when incorporated into liposomes in an inside-out orientation, catalyzed an ATP-dependent uptake of Ca 2+. 相似文献
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