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1.
The Mg 2+-dependency of Ca 2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg 2+- and Ca 2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg 2+ with a Km of 13 μ M in the absence or presence of 1 μ M free Ca 2+. At free Mg 2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg 2+ -independent, but is strongly stimulated by submicromolar Ca 2+ concentrations Km 0.25 μM, Vmax 24 μmol P i/h per mg protein). The Ca 2+-stimulated ATP hydrolysis strongly decreases at higher Mg 2+ concentrations. The Ca 2+-stimulated Mg 2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg 2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca 2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca 2+-pumping in renal basolaterals and on Ca 2+-ATPase in erythrocyte ghosts suggest that the Ca 2+-pumping enzyme requires Mg 2+. In contrast, a role of the Ca 2+-stimulated Mg 2+-independent ATP hydrolysis in active Ca 2+ transport across basolateral membranes is rather unlikely. 相似文献
2.
The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca 2+-ATPase activity and ATP-dependent Ca 2+-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). ( Na+ + K+)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca 2+-transport in fraction II ( 8.2 ± 0.3 nmol Ca 2+/min per mg protein ) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca 2+-transport activity ( 0.9 ± 0.1 nmol Ca 2+/min per mg protein ). The distribution of high-affinity Ca 2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca 2+-transport. The activity of Na +/Ca 2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca 2+-transport system, the Na +/Ca 2+ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum. 相似文献
3.
Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca 2+-ATPase, ATP-dependent Ca 2+ transport and Na +/Ca 2+ exchange have been studied with special emphasis on the relative transport capacities of the two Ca 2+ transport systems. The kinetic parameters of Ca 2+-ATPase activity in digitonin-treated membranes are: K
m
=0.11 m Ca 2+ and V
max=81±4 nmol P i/min·mg protein at 37°C. ATP-dependent Ca 2+ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca 2+/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca 2+ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca 2+ transport, a stoichiometry of 0.7 mol Ca 2+ transported per mol ATP is found for the Ca 2+-ATPase. In the presence of 75 mm Na + in the incubation medium ATP-dependent Ca 2+ uptake is inhibited 22%. When Na + is present at 5 mm an extra Ca 2+ accumulation is observed which amounts to 15% of the ATP-dependent Ca 2+ transport rate. This extra Ca 2+ accumulation induced by low Na + is fully inhibited by preincubation of the vesicles with 1 mm ouabain, which indicates that (Na +–K +)-ATPase generates a Na + gradient favorable for Ca 2+ accumulation via the Na +/Ca 2+ exchanger. In the absence of ATP, a Na + gradient-dependent Ca 2+ uptake is measured which rate amounts to 5% of the ATP-dependent Ca 2+ transport capacity. The Na + gradient-dependent Ca 2+ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na +/Ca 2+ exchange system for Ca 2+ is between 0.1 and 0.2 m Ca 2+, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca 2+ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca 2+-ATPase system exceeds the capacity of the Na +/Ca 2+ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca 2+ homeostasis of rat kidney cortex cells. 相似文献
4.
Basolateral plasma membrane vesicles of rat small intestinal epithelium accumulate calcium through an ATP-dependent pumping system. The activity of this system is highest in duodenum and decreases towards the ileum. This distribution along the intestinal tract is similar as the active calcium absorption capacity of intact intestinal epithelial segments. ATP-dependent calcium uptake in basolateral membrane vesicles from duodenum and ileum increased significantly after repletion of young vitamin D-3-deficient rats with 1α,25-dihydroxy-vitamin D-3. Ca 2+-ATPase activity in duodenal basolateral membranes increased to the same extend as ATP-dependent calcium transport, but (Na + + K +)-ATPase activity remained unaltered. 相似文献
5.
The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum ( D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg 2+-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca 2+ + Mg 2+-ATPase) of relatively low activity. Mg 2+-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca 2+ + Mg 2+)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg 2+-ATPase activity revealed apparent Km values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg 2+-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg 2+-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca 2+ + Mg 2+)-ATPase was 13.7 nmol 32P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol 32P/mg/min. Characterization of (Ca 2+ + Mg 2+)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent Kms for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca 2+ + Mg 2+)-ATPase activity was stimulated by potassium with an apparent Km of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca 2+ + Mg 2+)-ATPase are similar to those of the (Ca 2+ + Mg 2+)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca 2+ + Mg 2+)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme. 相似文献
6.
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
8.
Previous studies have identified a calmodulin-stimulated ATP-dependent Ca2+ pump as the major Ca2+ efflux pathway in enterocytes. Here, we developed methods to quantify the number of Ca2+ pumps in basolateral and intracellular membranes from porcine duodenum. By the use of a pig strain with a genetic defect in renal 1 alpha-hydroxylase, we were able to investigate the influence of 1,25(OH)2D3-deficiency on the number of Ca(2+)-ATPases in porcine duodenum. The amount of Ca(2+)-ATPase in isolated basolateral membranes was 5.5 +/- 0.7 micrograms/mg protein, while the Vmax of ATP-dependent Ca2+ transport into inside-out resealed basolateral membrane vesicles was 2.6 +/- 0.4 nmol/mg protein per min. From these data we estimated roughly about 95 x 10(3) plasma membrane Ca2+ pump sites per enterocyte. In addition, the amount of intracellular Ca(2+)-ATPase in microsomal fractions was 0.41 +/- 0.02 microgram/mg protein. Comparison of these parameters between control and rachitic animals showed that Ca2+ pump capacities in both basolateral membranes and microsomal fractions of porcine duodenum are not influenced by 1,25(OH)2D3-deficiency. In conclusion, stimulatory effects of 1,25(OH)2D3 on intestinal Ca2+ transport most likely result from specific effects on apical influx and facilitation of cytosolic Ca2+ diffusion by Ca(2+)-binding proteins and not from an increase in Ca2+ pumping capacity in basolateral membranes. 相似文献
9.
Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps. The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent. In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, β,γ-methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent Km of 325.5 μM and a Vmax of 0.0669 nmol/mg protein per min for 13-OXO-SG and a Km of 169 μM and a Vmax of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps. The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid. 相似文献
10.
The Mg 2+-dependency of Ca 2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg 2+- and Ca 2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg 2+ with a Km of 13 μ M in the absence or presence of 1 μ M free Ca 2+. At free Mg 2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg 2+ -independent, but is strongly stimulated by submicromolar Ca 2+ concentrations Km = 0.25 μM, Vmax = 24 μmol P i/h per mg protein). The Ca 2+-stimulated ATP hydrolysis strongly decreases at higher Mg 2+ concentrations. The Ca 2+-stimulated Mg 2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg 2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca 2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca 2+-pumping in renal basolaterals and on Ca 2+-ATPase in erythrocyte ghosts suggest that the Ca 2+-pumping enzyme requires Mg 2+. In contrast, a role of the Ca 2+-stimulated Mg 2+-independent ATP hydrolysis in active Ca 2+ transport across basolateral membranes is rather unlikely. 相似文献
11.
Addition of luteinizing hormone releasing hormone (LHRH) in vitro (10 –5–5×10 –9 M) to murine pituitary membranes resulted in a dose-related decrease in Ca 2+-ATPase activity within 15 min. Inhibitory effects of LHRH (10 –7 M) occurred after 90 sec, and appeared maximal by 120 sec. Eadie-Hofstee analysis at 10 –7 M LHRH, at varying [Ca 2+] free, resulted in a K
m=0.89±0.06 M and a V
max=18.8±0.71 nmol/mg per 2 min, compared to a K
m=0.69±0.06 M and a V
max=32.8±1.21 nmol/mg per 2 min for controls. Pre-incubation for 5 min with LHRH antagonist (10 –8 M) significantly attenuated (50%) the inhibitory effects of 10 –7 M LHRH on pituitary Ca 2+ ATPase activity with a K
m=0.97±0.24 M and a V
max=28.1±2.8 nmol/mg per 2 min. The addition of LHRH (10 –7 M) to pituitary homogenates significantly increased luteinizing hormone (LH) release already at 10 and up to 40 sec compared to basal LH release. Systemic administration of 50 ng LHRH (i.p.), significantly ( P<0.05) reduced pituitary Ca 2+-ATPase after 30, 60 and 90 min, with a return to control levels by 120 min. Pituitary LH content was reduced slightly at 15 min, but was increased significantly at 90 and 120 min post-treatment. Plasma LH levels were elevated by 5 min, reached a peak by 15 min and returned to control within 60 min. The present findings indicate that LHRH receptor activation may influence cytosolic Ca 2+ transport through effects on membrane Ca 2+-ATPase activity. These actions may regulate LHRH-induced synthesis, storage and release of LH from pituitary gonadotropes. 相似文献
12.
Highly purified sarcolemmal membranes were prepared from pig heart homogenates by differential and density gradient centrifugations. The membrane fragments exhibit ATP-dependent Ca 2+-transport and Na +/Ca 2+-exchange activities. ATP-dependent Ca 2+-transport ( KCa2+0.5 = 0.3 μM; Vmax = 4.6 nmol Ca 2+?mg protein ?1 ?min ?1)_is not stimulated by oxalate. Ca 2+-uptake is also not supported by p-nitrophenylphosphate. Preincubation of sarcolemma with MgATP, calmodulin and catalytic subunit of cyclic AMP-dependent protein kinase stimulates active Ca 2+-transport 1.8-fold. The effects of calmodulin and catalytic subunit are potentiating rather than additive. A large portion of the Ca 2+ additionally accumulated after prephosphorylation of membranes is exchangable for Na + via the Na +/Ca 2+-exchange system. 相似文献
13.
The dependence of the Ca 2+-ATPase activity of sarcoplasmic reticulum vesicles upon the intravesicular concentration of calcium accumulated after active uptake was studied. The internal calcium concentration was modified by addition of the ionophore A23187 at the steady state of accumulation. About half of the calcium accumulated could be released at low ionophore concentration without any concomitant activation of the Ca 2+-ATPase. This population of calcium might consist of calcium free in the lumen of the vesicles or bound to the bilayer at sites which do not interact with the ATPase activity. At higher concentrations of ionophore (above 1.75 nmol A23187/mg protein) the release of calcium activated this enzyme. This phenomenon was independent of the extravesicular calcium concentration and might be explained by assuming second species of calcium ions bound to the inner side of the membrane and in close functional interaction with the Ca 2+-ATPase. 相似文献
14.
Procedures were developed for measurement of Na +/Ca 2+ exchange in resealed plasma membrane vesicles from postmortem human brain. The vesicle preparation method permits use of stored frozen tissue with minimal processing required prior to freezing. Vesicles prepared in this manner transport Ca 2+ in the presence of a Na + gradient. The kinetic characteristics of the Na +/Ca 2+ exchange process were determined in membrane vesicles isolated from hippocampus and cortex. The K act for Ca 2+ was estimated to be 32 M for hippocampal and 17 M for cortical tissue. The maximal rate of Ca 2+ uptake (V max) was 3.5 nmol/mg protein/15 sec and 3.3 nmol/mg protein/15 sec for hippocampal and cortical tissue, respectively. Exchange activity was dependent on the Na + gradient, and was optimal in the high pH range. Therefore, membranes in which Na +-dependent o Ca 2+ transport activity is preserved can be isolated from postmortem human brain and could be used to determine the influence of pathological conditions on this transport system. 相似文献
15.
Lung surfactant is synthesized in lung epithelial type II cells and stored in the lamellar bodies prior to its secretion onto the alveolar surface. The lamellar bodies, like other secretory organelles, maintain an ATP-dependent pH gradient that is sensitive to inhibitors of H +-ATPase. This report shows that the ATPase activity of lamellar bodies is enriched in a fraction prepared from lamellar bodies that were disrupted after isolation. The apparent Vmax for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent Km for ATP was approximately 50 μM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) (all inhibitors of vacuolar-type H +-ATPase) and vanadate (inhibitor of phosphoenzyme-type ATPase). Besides, the activity could also be inhibited with diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and Ca 2+. Two proteins (of approximately 45 kDa and 17 kDa) of this fraction showed acid-stable phosphorylation with ATP. The labeling of proteins with ATP (-γ- 32P) could be chased with unlabelled ATP, suggesting that phosphorylation and dephosphorylation of these proteins is associated with the ATPase activity. Our results on inhibition characteristics of the enzyme activity suggest that besides a vacuolar type H +-ATPase, the lamellar bodies also contain a phosphoenzyme type ATPase that is sensitive to inhibitors of vacuolar type H +-ATPase. 相似文献
16.
Two types of ATP-dependent calcium (Ca 2+) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca 2+ uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO 3−. The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg 2+. Like the tonoplast H+-ATPase activity, vanadate-insensitive Ca2+ accumulation was stimulated by 20 millimolar Cl− and inhibited by 10 micromolar 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid or 50 micromolar N,N′-dicyclohexylcarbodiimide. This Ca2+ transport system had an apparent Km for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca2+ transport was abolished by compounds that eliminated a pH gradient and Ca2+ dissipated a pH gradient (acid inside) generated by the tonoplast-type H+-ATPase. These results provide compelling evidence that a pH gradient generated by the H+-ATPase drives Ca2+ accumulation into right-side-out tonoplast vesicles via a Ca2+/H+ antiport. This transport system was saturable with respect to Ca2+ (Km apparent = 14 micromolar). The Ca2+/H+ antiport operated independently of the H+-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca2+ accumulation. Ca2+ transport by this system may be one major way in which vacuoles function in Ca2+ homeostasis in the cytoplasm of plant cells. 相似文献
17.
The presence of an Na +/Ca 2+ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na + gradient-dependent Ca 2+ uptake and the inhibition of ATP-dependent Ca 2+ accumulation by Na +. The presence of 75 mM Na + in the uptake solution reduces ATP-dependent Ca 2+ transport by 45%, despite the fact that Na + does not affect Ca 2+-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na + inhibition of ATP-dependent Ca 2+ uptake to 20%, apparently by preventing accumulation of Na + in the vesicles realized by the Na +-pump. It was concluded that high intravesicular Na + competes with Ca 2+ for intravesicular Ca 2+ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca 2+ transport shows a sigmoidal dependence on the Na + concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na + is between 15 and 20 mM. Uptake of Ca 2+ in the absence of ATP can be enhanced by an Na + gradient (Na + inside > Na + outside). This Na + gradient-dependent Ca 2+ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca 2+ of this system is below 1 μM. In contrast to the ATP-dependent Ca 2+ transport, there is no significant difference in Na + gradient-dependent Ca 2+ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca 2+ uptake is 5-times greater than the Na +/Ca 2+ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na +/Ca 2+ exchange mechanism is not subject to regulation by 1α,25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na +/Ca 2+ exchange system while it doubles the ATP-driven Ca 2+ pump activity. 相似文献
18.
Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca 2+-activated Mg 2+-ATPase and Ca 2+ transport activities. Membrane vesicles that were exposed to oxalate as a Ca 2+-trapping agent accumulated Ca 2+ in the presence of Mg 2+ and ATP. The for Ca 2+ uptake was 33 nmol/mg protein per h, and the values for Ca 2+ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca 2+ ionophore A23187, added initially, completely inhibited net Ca 2+ uptake and, if added later, caused the release of Ca 2+ previously accumulated. A Ca 2+-activated ATPase was present in the same membrane vesicles which had a of 1.5 μmol/mg protein per h at free Ca 2+ concentration of 10 μM. This Ca 2+-ATPase had values of 4.5 μM and 110 μM for Ca 2+ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca 2+ by the vesicles. The Ca 2+-ATPase activity was insensitive to ouabain. Both Ca 2+ transport and Ca 2+-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca 2+ transport activity and a Ca 2+-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin. 相似文献
19.
Abstract: Activation of the Ca 2+/Mg 2+ ATPase associated with highly purified Torpedo synaptic vesicles results in 45Ca 2+ uptake. The accumulated 45Ca 2+ is released by hypoosmotic buffer and by the Ca 2+ ionophore A23187. Density-gradient centrifugation and permeation chromatography reveal that vesicular acetylcholine and the membrane-bound 45Ca 2+ co-migrate, thus implying that 45Ca 2+ is transported into cholinergic vesicles. ATP-dependent 45Ca 2+ uptake follows saturation kinetics, with KmCa2+= 50 μM, KmATP= 5 μM, and V max= 3 ± 0.3 nmol Ca 2+/mg protein/min. Treatment of the vesicles with mersalyl, dicyclohexyl-carbodiimide, and quercetin leads to inactivation of the Ca 2+/Mg 2+ ATPase and to comparable inhibition of 45Ca 2+ transport. Ruthenium red and ouabain have no effect on either of these activities. Nigericin in the presence of external K + is a potent inhibitor of 45Ca 2+ translocation, whereas gramicidin activates transport. The proton translocator carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and FCCP + the ionophore valinomycin partially inhibit 45Ca 2+ transport. By contrast, the above ionophores do not affect Ca 2+/Mg 2+ ATPase activity. Tentative mechanisms for ATP-dependent Ca 2+ transport into cholinergic synaptic vesicles and the physiological significance of this process are discussed. 相似文献
20.
Summary Previous work by this and other laboratories has shown that glucagon administration stimulates calcium uptake by subsequently
isolated hepatic mitochondria. This stimulation of hepatic mitochondrial Ca 2+ uptake by in vivo administration of glucagon was further characterized in the present report. Maximal stimulation of mitochondrial Ca 2+ accumulation was achieved between 6–10 min after the intravenous injection of glucagon into intact rats. Under control conditions,
Ca 2+ uptake was inhibited by the presence of Mg 2+ in the incubation medium. Glucagon treatment, however, appeared to obliterate the observed inhibition by Mg 2+ of mitochondrial Ca 2+ uptake. Kinetic experiments revealed the usual sigmoidicity associated with initial velocity curves for mitochondrial calcium
uptake. Glucagon treatment did not alter this sigmoidal relationship. Glucagon treatment significantly increased the V max for Ca 2+ uptake from 292±22 to 377±34 nmoles Ca 2+ /min per mg protein (n=8) but did not affect the K 0.5, (6.5–8.6 μM). Since the major kinetic change in mitochondrial Ca 2+ uptake evoked by glucagon is an increase in V max, the enhancement mechanism is likely to be an increase either in the number of active transport sites available to Ca 2+ or in the rate of Ca 2+ carrier movement across the mitochondrial membranes. 相似文献
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