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1.
A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.  相似文献   

2.
Ca2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca2+ ATPase isoforms, which are all selectively inhibited by nanomolar concentrations of thapsigargin. Using ATP/Mg2+-dependent 45Ca2+ transport in rat brain microsomes, tissue sections, and permeabilized cells, as well as Ca2+ imaging in living cells we distinguish two ER Ca2+ pools in the rat CNS. Nanomolar levels of thapsigargin blocked one component of brain microsomal 45Ca2+ transport, which we designate as the thapsigargin-sensitive pool (TG-S). The remaining component was only inhibited by micromolar thapsigargin, and thus designated as thapsigargin resistant (TG-R). Ca2+ ATPase and [32P]phosphoenzyme assays also distinguished activities with differential sensitivities to thapsigargin. The TG-R Ca2+ uptake displayed unique anion permeabilities, was inhibited by vanadate, but was unaffected by sulfhydryl reduction. Ca2+ sequestered into the TG-R pool could not be released by inositol-1,4,5-trisphosphate, caffeine, or cyclic ADP-ribose. The TG-R Ca2+ pool had a unique anatomical distribution in the brain, with selective enrichment in brainstem and spinal cord structures. Cell lines that expressed high levels of the TG-R pool required micromolar concentrations of thapsigargin to effectively raise cytoplasmic Ca2+ levels. TG-R Ca2+ accumulation represents a distinct Ca2+ buffering pool in specific CNS regions with unique pharmacological sensitivities and anatomical distributions.  相似文献   

3.
The effect of inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] and caffeine on Ca2+ release from digitonin-permeabilised bovine adrenal chromaffin cells was examined by using the Ca2+ indicator fura-2 to monitor [Ca2+]. Permeabilised cells accumulated Ca2+ in the presence of ATP and addition of either Ins(1,4,5)P3 or caffeine released 17% or 40-50%, respectively, of the accumulated Ca2+, indicated by sustained rises in [Ca2+] in the cell suspension. Prior addition of Ins(1,4,5)P3 had no effect on the magnitude of the response to a subsequent addition of caffeine. The response to Ins(1,4,5)P3 was prevented by prior addition of caffeine or CaCl2, indicating that the Ins(1,4,5)P3 response was blocked by elevated [Ca2+]. The responses were essentially identical in the presence of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, indicating that the Ca2+ release was not from mitochondria or secretory granules and that a proton gradient was not required for Ca2+ accumulation into the Ins(1,4,5)P3- or caffeine-sensitive stores. Ca2+ release from the caffeine-sensitive store was selectively blocked by ryanodine. The Ins(1,4,5)P3-sensitive store was emptied by thapsigargin, which had no effect on caffeine responses. These data suggest that permeabilised chromaffin cells possess two distinct nonoverlapping Ca2+ stores sensitive to either Ins(1,4,5)P3 or caffeine and support previous conclusions that these stores possess different Ca2(+)-ATPases.  相似文献   

4.
The functional confirmation of availability of Ca2+ transport initially-active systems in the embryo cells of loach Misgurnus fossilis L. has been obtained. Using thapsigargin, the specific inhibitor of endoplasmic reticulum of Ca2+, Mg(2+)-ATPase, this enzyme activity was divided into thapsigargin-sensitive (actually endoplasmic reticulum Ca2+, Mg(2+)-ATPase) and thapsigargin-insensitive (plasma membrane Ca2+, Mg(2+)-ATPase) constituents. The Ca(2+)-independent Mg(2+)-dependent ATPase activity makes above 39.7% of the common Ca2+, Mg(2+)-ATPase activity of embryo loach. The periodic changes of Ca2+, Mg(2+)-ATPase activity (except for the changes of plasma membrane Ca2+, Mg(2+)-ATPase activity) were found out, which coincide with periodic [Ca2+]i oscillations during the synchronous divisions of loach blastomers embryos.  相似文献   

5.
Annexin 7 mobilizes calcium from endoplasmic reticulum stores in brain   总被引:1,自引:0,他引:1  
Mobilization of intracellular calcium from inositol-1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum (ER) stores plays a prominent role in brain function. Mice heterozygous for the annexin A7 (Anx7) gene have a profound reduction in IP3 receptor function in pancreatic islets along with defective insulin secretion. We examined IP3-sensitive calcium pools in the brains of Anx7 (+/-) mice by utilizing ATP/Mg(2+)-dependent (45)Ca(2+) uptake into brain membrane preparations and tissue sections. Although the Anx7 (+/-) mouse brain displayed similar levels of IP3 binding sites and thapsigargin-sensitive (45)Ca(2+) uptake as that seen in wild-type mouse brain, the Anx7 (+/-) mouse brain Ca(2+) pools showed markedly reduced sensitivity to IP3. A potent and saturable Ca(2+)-releasing effect of recombinant ANX7 protein was demonstrated in mouse and rat brain membrane preparations, which was additive with that of IP3. We propose that ANX7 mobilizes Ca(2+) from an endoplasmic reticulum-like pool, which can be recruited to enhance IP3-mediated Ca(2+) release.  相似文献   

6.
An explanation of the complex effects of hormones on intracellular Ca2+ requires that the intracellular actions of Ins(1,4,5)P3 and the relationships between intracellular Ca2+ stores are fully understood. We have examined the kinetics of 45Ca2+ efflux from pre-loaded intracellular stores after stimulation with Ins(1,4,5)P3 or the stable phosphorothioate analogue, Ins(1,4,5)P3[S]3, by simultaneous addition of one of them with glucose/hexokinase to rapidly deplete the medium of ATP. Under these conditions, a maximal concentration of either Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 evoked rapid efflux of about half of the accumulated 45Ca2+, and thereafter the efflux was the same as occurred under control conditions. Submaximal concentrations of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 caused a smaller rapid initial efflux of 45Ca2+, after which the efflux was similar whatever the concentration of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 present. The failure of submaximal concentrations of Ins(1,4,5)P3 and Ins(1,4,5)P3[S]3 to mobilize fully the Ins(1,4,5)P3-sensitive Ca2+ stores despite prolonged incubation was not due either to inactivation of Ins(1,4,5)P3 or to desensitization of the Ins(1,4,5)P3 receptor. The results suggest that the size of the Ins(1,4,5)P3 sensitive Ca2+ stores depends upon the concentration of Ins(1,4,5)P3.  相似文献   

7.
The distribution of binding sites for the calcium-mobilizing second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was investigated in subcellular fractions of bovine adrenal cortex. The [3H]Ins(1,4,5)P3-binding capacity was enriched in the microsomal fraction, which contained a single class of high affinity binding sites with a Kd of 21.6 +/- 3.0 nM. The specific [3H]Ins(1,4,5)P3 binding appeared to be sharply pH dependent and was inhibited by millimolar concentrations of ATP. Upon fractionation of microsomes on sucrose density gradient there was a clearcut separation of the Ins(1,4,5)P3 receptor-containing fractions from those enriched in specific endoplasmic reticulum markers such as sulfatase C activity or RNA content. The microsomes enriched in Ins(1,4,5)P3-binding sites were of lower density than the endoplasmic reticulum and co-purified partly with the plasma membrane. In addition, Ins(1,4,5)P3-sensitive 45Ca2+ uptake into the microsomes was maximal in the lighter fractions. This distinction between Ins(1,4,5)P3-binding sites and endoplasmic reticulum-derived microsomes was confirmed upon fractionation according to their electrophoretic mobilities by free flow electrophoresis. These results indicate that in adrenal cortical cells, the source of Ca2+ mobilized by Ins(1,4,5)P3 upon stimulation with an agonist is not located in the endoplasmic reticulum. Our data support the hypothesis that a specialized vesicular organelle, distinct from endoplasmic reticulum and in close apposition with the plasma membrane, is involved in intracellular Ca2+ homeostasis.  相似文献   

8.
The sarcoplasmic reticulum Ca2(+)-ATPase of skeletal muscle has two high affinity calcium sites, one of fast access ("f" site) and one of slow access ("s" site). In addition to Ca2+ these sites are able to interact with other cations like Mg2+ or K+. We have studied with a stopped-flow method the modifications produced by Mg2+ and K+ on the kinetics of the intrinsic fluorescence changes produced by Ca2+ binding to and dissociation from the Ca2(+)-ATPase of sarcoplasmic reticulum. The presence of Mg2+ ions (K1/2 = 0.5 mM at pH 7.2) leads to the appearance of a rapid phase in the Ca2+ binding, which represents half of the signal amplitude at optimal Mg2+. The presence of K+ greatly accelerates both the Ca2+ binding and the Ca2+ dissociation reactions, giving, respectively, a 4- and 8-fold increase of the rate constant of the induced fluorescence change. K+ ions also increase the rate of the 45Ca/40Ca exchange reaction at the s site measured by rapid filtration. These results lead us to build up a model for the Ca2(+)-binding mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase in which Mg2+ and K+ participate at particular steps of the reaction. Moreover, we propose that, in the absence of Ca2+, this enzyme may be the pathway for monovalent ion fluxes across the sarcoplasmic reticulum membrane.  相似文献   

9.
Recent evidence indicates that unesterified arachidonic acid functions as a mediator of intracellular Ca2+ mobilization by inducing Ca2+ release from the endoplasmic reticulum of pancreatic islet beta cells in a manner closely similar to that of inositol 1,4,5-trisphosphate. To test the generality and explore the mechanism of this phenomenon we have examined the effects of arachidonic acid on calcium accumulation and release by hepatocyte subcellular fractions enriched in endoplasmic reticulum (microsomes). At concentrations above 0.017 mumol/mg microsomal protein, arachidonate induced rapid (under 2 min) 45Ca2+ release from microsomes that had been preloaded with 45Ca2+. Arachidonate also suppressed microsomal 45Ca2+ accumulation when present during the loading period, as reflected by reduction both of 45Ca2+ accumulation at steady state and of the rate of uptake. Neither the cyclooxygenase inhibitor indomethacin nor the lipoxygenase/cyclooxygenase inhibitor BW755C suppressed arachidonate-induced 45Ca2+ release, indicating that this effect was not dependent upon oxygenation of the fatty acid to metabolites. The long-chain unsaturated fatty acids oleate and linoleate were less potent than arachidonate in inducing 45Ca2+ release, and the saturated fatty acid stearate did not exert this effect. Albumin prevented 45Ca2+ release by arachidonate, presumably by binding the fatty acid. As is the case for inositol 1,4,5-trisphosphate, the ability of arachidonate to induce 45Ca2+ release was dependent on the ambient free Ca2+ concentration. Arachidonate did not influence microsomal membrane permeability or Ca2+-ATPase activity and may exert its effects on microsomal Ca2+ handling by activation of a Ca2+ extrusion mechanism or by dissociating Ca2+ uptake from Ca2+-ATPase activity.  相似文献   

10.
Capacitative Ca(2+) entry (CCE) refers to the influx of Ca(2+) through plasma membrane channels activated on depletion of endoplasmic-sarcoplasmic reticulum Ca(2+) stores. We utilized two Ca(2+)-sensitive dyes (one monitoring cytoplasmic free Ca(2+) and the other free Ca(2+) within the sarcoplasmic reticulum) to determine whether adult rat ventricular myocytes exhibit CCE. Treatments with inhibitors of the sarcoplasmic endoplasmic reticulum Ca(2+)-ATPases were not efficient in releasing Ca(2+) from stores. However, when these inhibitors were coupled with either Ca(2+) ionophores or angiotensin II (an agonist generating inositol 1,4,5 trisphosphate), depletion of stores was observed. This depletion was accompanied by a significant influx of extracellular Ca(2+) characteristic of CCE. CCE was also observed when stores were depleted with caffeine. This influx of Ca(2+) was sensitive to four inhibitors of CCE (glucosamine, lanthanum, gadolinium, and SKF-96365) but not to inhibitors of L-type channels or the Na(+)/Ca(2+) exchanger. In the whole cell configuration, an inward current of approximately 0.7 pA/pF at -90 mV was activated when a Ca(2+) chelator or inositol (1,4,5)-trisphosphate was included in the pipette or when Ca(2+) stores were depleted with a Ca(2+)-ATPase inhibitor and ionophore. The current was maximal at hyperpolarizing voltages and inwardly rectified. The channel was relatively permeant to Ca(2+) and Ba(2+) but only poorly to Mg(2+) or Mn(2+). Taken together, these data support the existence of CCE in adult cardiomyocytes, a finding with likely implications to physiological responses to phospholipase C-generating agonists.  相似文献   

11.
The Ca2+, Mg2(+)-ATPase of the myometrium sarcolemma purified by the method of affinity chromatography on calmodulin sepharose is reconstituted into azolectin liposomes in the functionally active form by means of cholate dialysis. The ATPase-dependent accumulation of 45Ca is shown on the obtained model system. It makes up 95% of the total accumulation and may decrease to 43% under the effect of 0.8 microM A23187. Ca2+, Mg2(+)-ATPase reconstituted into azolectin liposomes is in the high affinity to Ca2+; Km for Ca2+ is equal to 0.88 +/- 0.22 microM, calmodulin practically does not change it. The highest activity of the reconstituted enzyme is observed at pH 7.0, temperature 50 degrees C, the Mg-ATP concentration 1-2 mM. The Km for substrate is 0.45 +/- 0.02 mM.  相似文献   

12.
Kinetic properties of Ca2+, Mg2+-ATPases membranes from acinar cells of rat submandibular salivary glands have been investigated. It was found that kinetics of ATP hydrolysis dependent on Ca2+, Mg2+-ATPases corresponds to the first-order reaction during first 2 min. It was found that the initial velocity of the reaction (V0), maximal amount of the reaction product (Pmax) and characteristic time of the reaction (T) comprised 1.8 +/- 0.4 and 1.6 +/- 0.2 mmole Pi/min per 1 mg protein, 7.5 +/- 1.3 and 1.4 +/- 0.2 mmole Pi/mg protein and 4.1 +/- 0.7 min and 1.1 +/- 0.1 for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. High- and low-affinity sites of ATP and Ca2+-binding in Ca2+-ATPases from plasma and endoplasmic reticulum membranes were identified. Negative cooperation in ATP binding to Ca2+-ATPase from plasma membrane and a positive cooperation for Ca2+-ATPase from endoplasmic reticulum has been found. Ca2+ binding to low-affinity sites of both Ca2+-ATPases showed no cooperation, while Ca2+ binding to high-affinity sites showed the positive cooperation. Using the Hill's coordinates we have found the values of the Mg2+ Michaelis constant (K(Mg)) which yielded 3.89 x 10(-5) and 3.80 x 10(-5) mole/l for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. It is supposed that obtained data are important for further studies of molecular and membrane mechanisms involved in the regulation of intracellular calcium signalling and secretion by salivary acinar cells.  相似文献   

13.
ATP-dependent Ca2+ accumulation was measured in pig aortic microsomal fractions containing plasmalemma and endoplasmic reticulum. In vesicles sonicated with histamine, to allow access to internally located receptor sites, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), added to activate externally located guanine-nucleotide-transducing proteins, caused a concentration-dependent decrease in steady-state Ca2+ accumulation that was reversed by guanosine 5'-[beta-thio]diphosphate. In the presence of p[NH]ppG, sonication with histamine produced a concentration-dependent inhibition of Ca2+ accumulation that could be antagonized by the H1 antagonist mepyramine, but not by the H2 antagonist cimetidine. The inhibition of steady-state Ca2+ accumulation could have resulted from an inhibition of ATP-dependent Ca2+ uptake or a stimulation of Ca2+ release. We observed, however, that p[NH]ppG plus histamine stimulated, rather than inhibited, Ca2(+)-ATPase activity. We concluded that p[NH]ppG and histamine acted together to increase Ca2+ permeability. In support of this, p[NH]ppG accelerated efflux of Ca2+ from passively loaded vesicles sonicated with, but not without, histamine. The effect of p[NH]ppG was unlikely to be due to Ins(1,4,5)P3 (and hence release from endoplasmic-reticulum vesicles), since addition of Ins(1,4,5)P3 to vesicles sonicated with histamine did not alter steady-state Ca2+ accumulation. Our results therefore suggest that histamine and p[NH]ppG increased the permeability of the plasmalemma vesicles and may thus model the process of receptor-mediated Ca2+ entry into intact cells.  相似文献   

14.
Pumiliotoxins (PTX) A, B, and 251D, members of a new class of indolizidine alkaloids isolated from the skin of poison frogs of the family Dendrobatidae, inhibit Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from frog and rat hind-limb muscles. PTX-B and PTX-A appear to be relatively specific inhibitors of Ca2+-ATPase; PTX-A is much less potent than PTX-B. PTX-251D is a potent inhibitor of Ca2+-ATPase, and was also found to inhibit Na+, K+, and Mg2+-ATPases in rat brain synaptosomes. Caffeine and verapamil, two drugs known to affect calcium translocation, are very weak inhibitors of the Ca2+-ATPase. The Ki values for inhibition of the Ca2+-ATPase of rat and frog sarcoplasmic reticulum by PTX-B were comparable and ranged between 22 and 36 microM. Inhibition of calcium-dependent ATPase in sarcoplasmic reticulum by pumiliotoxin-B is noncompetitive with calcium and is not readily reversible. Based on structure-activity profiles, it is concluded that inhibition of Ca2+-ATPase by the indolizidine alkaloids is responsible for the alkaloid-elicited prolongation of twitch in intact muscle.  相似文献   

15.
Isolated membrane vesicles from pig stomach smooth muscle (antral part) were subfractionated by a density gradient procedure modified in order to obtain an efficient extraction of extrinsic proteins. By using this method in combination with digitonin-treatment, an endoplasmic reticulum fraction contaminated with maximally 10 to 20% of plasma membranes was isolated, together with a plasma membrane fraction containing at most 30% endoplasmic reticulum. The endoplasmic reticulum and plasma membrane fractions differed in protein composition, reaction to digitonin, binding of wheat germ agglutinin, activities of marker enzymes and in the characteristics of the Ca2+ uptake. The Ca2+ uptake by the endoplasmic reticulum was much more stimulated by oxalate than the uptake by plasma membranes. Both fractions showed a (Ca2+ + Mg2+)-ATPase activity, but the largest amount of this enzyme was present in the plasma membranes. The study of the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPase by polyacrylamide gel electrophoresis revealed two phosphoproteins one of 130 kDa and one of 100 kDa (Wuytack, F., Raeymaekers, L., De Schutter, G. and Casteels, R. (1982) Biochim. Biophys. Acta 693, 45-52). The 130 kDa enzyme was predominant in the fraction enriched in plasma membrane whereas the distribution of the 100 kDa polypeptide correlated with the endoplasmic reticulum markers. The 130 kDa ATPase was the main 125I-calmodulin binding protein detected on nitrocellulose blots of proteins separated by gel electrophoresis. The (Ca2+ + Mg2+)-ATPase activity of the plasma membranes was higher than the (Na+ + K+)-ATPase activity, suggesting that the Ca2+ extrusion from these cells depends much more on the activity of the (Ca2+ + Mg2+)-ATPase than on Na+-Ca2+ exchange.  相似文献   

16.
Highly purified pig myocardium sarcolemma vesicles possess the Ca2+,Mg2+-ATPase activity (4.1 mumol Pi/mg protein/hour) and induce the ATP-dependent accumulation of 45Ca2+ (6.0 nmol/mg protein/min). This reaction is not stimulated by oxalate; Ca2+ are released from the vesicles by saponin and Na+ treatment, which suggests that Ca2+ transport against the concentration gradient is induced by myocardium sarcolemma vesicles and not by sarcoplasmic reticulum fragments. The phorbol ester possessing a biological activity of a growth-promoting factor and activating membrane-bound protein kinase C stimulates the Ca2+,Mg2+-ATPase activity and the ATP-dependent accumulation of Ca2+, whereas its counterpart devoid of biological activity does not influence Ca2+ transport. Polymixin B, a specific inhibitor of protein kinase C, prevents the activating effect of phorbol esters on Ca2+ accumulation inside the vesicles. It is suggested that the ATP-dependent transport of Ca2+ in myocardium sarcolemma is controlled by Ca2+-phospholipid-dependent phosphorylation catalyzed by protein kinase C.  相似文献   

17.
J Barańska 《FEBS letters》1989,256(1-2):33-37
It has been shown that the ATP-dependent incorporation of [14C]serine into phosphatidylserine in rat liver mitochondrial and microsomal fractions is prevented by EGTA. On the other hand, at low (microM) Ca2+ concentrations, serine incorporation is strongly stimulated by ATP and Mg2+. This stimulatory effect is reduced by calcium ionophore A23187. It is therefore suggested that the ATP-dependent process is that of serine base-exchange reaction, stimulated by endogenous Ca2+ accumulated inside the microsomal vesicles by Ca2+,Mg2+-ATPase. The mitochondrial activity can be accounted for by contamination by the endoplasmic reticulum.  相似文献   

18.
Vanadate and vanadyl have many insulin-mimetic effects on cellular metabolism and also have been shown to alter cellular Ca2+ fluxes. In this report, vanadate and vanadyl, like insulin, are shown to inhibit the plasma membrane (Ca2+ + Mg2+)-ATPase/Ca2+ transport system as well as Ca2+ transport by endoplasmic reticulum from rat adipocytes. Ca2+ transport by the endoplasmic reticulum was inhibited half-maximally (I50) by vanadate and vanadyl at concentrations of 30 and 33 microM, respectively. Inhibition of the plasma membrane Ca2+ transport by vanadate and vanadyl was less sensitive, with I50 values of 144 and 92 microM, respectively. These I50 values for plasma membrane Ca2+ transport were similar when measured under conditions of calmodulin-stimulated and non-calmodulin-stimulated Ca2+ transport. The predominant effect of both ions on the kinetic parameters of Ca2+ transport was a substantial decrease in the Vmax by 43-46% for both transport systems. An increase in intracellular Ca2+ following the inhibition of the (Ca2+ + Mg2+)-ATPase/Ca2+ pump in the plasma membrane and endoplasmic reticulum by these vanadium ions may result, at least in part, in the observed insulin-mimetic alterations in cellular metabolism.  相似文献   

19.
Rough endoplasmic reticulum membranes, purified from isolated rat pancreatic acini stimulated by carbachol, had a decreased Ca2+ content and increased (Ca2+ + Mg2+)-ATPase activity. Ca2+ was regained and ATPase activity reduced to control levels only after blockade by atropine. The (Ca2+ + Mg2+)-ATPase was activated by free Ca2+ (half-maximal at 0.17 microM; maximal at 0.7 microM) over the concentration range which occurs in the cell cytoplasm. Pretreatment with EGTA, at a high concentration (5 mM), inhibited ATPase activity which, our results suggest, was due to removal of a bound activator such as calmodulin. The rate of (Ca2+ + Mg2+)-ATPase actively declined during the 10-min period over which maximal active accumulation of Ca2+ by membrane vesicles occurs. In the presence of ionophore A23187, which released actively accumulated Ca2+ and stimulated the (Ca2+ + Mg2+)-ATPase, this time-dependent decline in activity was not observed. Our data provide evidence that the activity of the Ca2+-transporting ATPase of the rough endoplasmic reticulum is regulated by both extra and intravesicular Ca2+ and is consistent with a direct role of this enzyme in the release and uptake of Ca2+ during cholinergic stimulation of pancreatic acinar cells.  相似文献   

20.
The rate of Mg2+, Ca2+-ATPase reaction and ATP-dependent Ca2+ accumulation in a preparation of plasma membranes from brain synaptosomes increases 60 min following whole-body X-irradiation of rats with a dose of 0.21 C/kg, a calcium sensitivity of both processes being increased. A unidirectional change in their kinetics indicates the early radiosensitivity of Ca2+ transfer systems in the brain synaptosome membranes. There is an increase in the availability of SH-groups of membrane preparation proteins for SH-reagents and in the sensitivity of Mg2+, Ca2+-ATPase reaction and ATP-dependent Ca2+ accumulation to trifluoperazine, a calmodulin inhibitor. Both processes lose their ability to be activated by exogenous calmodulin. It is suggested that at an early stage of radiation affection, a change occurs in the molecular organization of the ATPase-calmodulin membrane complex in plasma membranes of rat brain synaptosomes.  相似文献   

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