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1.
To evaluate Ca2+-uptake in sarcoplasmic reticulum in the hypertrophied rabbit heart, microsomes were prepared from myocardium of rabbits with experimentally induced aortic stenosis. A significant reduction of microsomal Ca2+-uptake was observed in hypertrophied left ventricle, 195±10 compared to 280±18 nmol/mg found in control animals. A similar pattern was observed for the Ca2+-stimulated ATPase (30±9 and 59±10 nmol/min/mg resp.). A minimal activity difference of the microsomal marker enzyme rotenone-insensitive NADPH cyt. reductase was found (7.77±0.05 and 8.17±0.11 nmol/min/mg resp.). The specific activity of the latter enzyme was 5–6 fold increased in microsomes compared to homogenates in both animal groups, which excludes the possibility of increased amounts of contaminant or nonfunctional protein in sarcoplasmic reticulum prepared from hypertrophied myocardium. In addition the yield of microsomal protein did not differ significantly. Maximal phosphorylation by exogenous cyclic AMP and protein kinase increased Ca2+-uptake in both microsomal preparations (to 287±27 and 375±26 nmol/mg resp. for hypertrophied and control hearts), but Ca2+-transport rate found in pathological hearts remained lower than in controls. These findings indicate that impairment of Ca2+-metabolism in the hypertrophied heart is based on a defective Ca2+-pump. 相似文献
2.
Bovine spermatozoa, maturing in the caudal epididymis, apparently have a low content of mobilizable Ca2+ (6 ± 1 nmoles cells) in situ, but will accumulate added Ca2+ when simply diluted into isotonic media. It is suggested that the low concentrations of Ca2+ (0.5 ± 0.1 mM) and O2 present in epididymal fluids prevent the accumulatin of Ca2+ by sperm prior to ejaculation. Although the seminal plasma that surrounds ejaculated sperm contains 9 ± 1 mM Ca2+, washed ejaculated sperm also have a low Ca2+ content (7 ± 1 nmoles cells). Moreover, in contrast to epididymal sperm, washed ejaculated sperm are incapable of accumulating Ca2+ supplied exogenously. Evidence is presented that bovine seminal fluids contain a component that is added to the surface membranes of the sperm at ejaculation which prevents or delays the active uptake of Ca2+ by these cells. 相似文献
3.
Lanthanum (0.25 mM) does not penetrate into fresh or Mg2+-depleted cells, whereas it does into ATP-depleted or , into cells containing more than 3 mM calcium, or cells stored for more than 4 weeks in acid/citrate/dextrose solution. In fresh cells loaded with calcium, extracellular lanthanum blocks the active Ca2+-efflux completely and inhibits (ATP phosphohydrolase, EC 3.6.1.3) activity to about 50%. In Mg2+-depleted cells Ca2+-Ca2+ exchange is inhibited by lanthanum. Ca2+-leak is unaffected by lanthanum up to 0.25 mM concentration; higher lanthanum concentrations reduce leak rate. In NaCl medium Ca2+-leak ± S.D. amounts to of cells per min, whereas in KCl medium to of cells per min at 2.5 mM [Ca2+]e and 0.25 mM [La3+]e pH 7.1.Lanthanum inhibits Ca2+-dependent rapid K+ transport in ATP-depleted and propranolol-treated red cells, i.e. whenever intracellular calcium is below a critical level. The inhibition of the rapid K+ transport can be attributed to protein-lanthanum interactions on the cell surface, since lanthanum is effectively detached from the membrane lipids by propranolol.Lanthanum at 0.2–0.25 mM concentration has no direct effect on the morphology of red cells. The shape regeneration of Ca2+-loaded cells, however, is blocked by lanthanum owing to Ca2+-pump inhibition. Using lanthanum the transition in cell shape can be quantitatively correlated to intracellular Ca2+ concentrations. 相似文献
4.
Ca2+-transport and its energy consumption were studied in intact human red cells loaded with Ca2+ by the aid of the ionophore A23187.After the complete elimination of the ionophore the passive Ca2+-permeability of the membrane returned to its normal low value, except when the intracellular Ca2+-concentration was higher than 3 mM or the ATP level fell below 100 μM. Within these limits the rate of Ca2+-extrusion was independent of the cellular ATP content but was greatly enhanced by increasing [Ca2+]i and reached a plateau at about 1 mM intracellular Ca2+-concentration. The maximum rate of Ca2+-efflux was about 85 μmol/l of cells per min at 37°C, pH 7.4. The activation energy of active Ca2+-extrusion was found to be 15 200 cal/mol, and the optimum pH in the suspension was 7.7.Ca2+-efflux was not connected with the counter-transport of cations.The Ca2+-pump was not affected by ouabain or oligomycin and only partial inhibition could be achieved by the SH-reagents: ethacrynic acid, and or with propranolol and ruthenium red. An 80 to 95% inhibition of the active Ca2+-extrusion was brought about by 50–250 μM lanthanum, which in the above concentrations caused no aggregation or haemolysis. The inhibition of the Ca2+-pump by lanthanum was found to be reversible, the site of inhibition being at the external surface of the cell membrane.To examine the energy consumption of the Ca2+-extrusion, ATPase activity was assessed by measuring inorganic phosphate liberation in Ca2+-loaded red cells the metabolism of which was inhibited by iodoacetamide + Na+-tetrathionate. Ca2+-activated ATPase activity connected with the Ca2+-pump was distinguished from other Ca2+-ATPase by using the non-penetrating inhibitor, lanthanum. The molar ratio of Ca2+-transported per ATP split was found to be . 相似文献
5.
Amphiphilic, cationic Polymyxin B is shown to displace Ca2+ from ‘gas dissected’ cardiac sarcolemma in a dose-dependent, saturable fashion. The Ca2+ displacement is only partially reversible, 57% and 63%, in the presence of 1 mM or 10 mM Ca2+, respectively. Total Ca2+ displaced by a non-specific cationic probe, lanthanum (La3+), at maximal displacing concentration (1 mM) was membrane protein. At 0.1 mM, Polymyxin B displaced 42% of the total La3+-displaceable Ca2+ or protein. 5 mM Polymyxin displaced Ca2+ in amounts equal to those displaced by 1 mM La3+. Pretreatment of the membranes with neuraminidase (removal of sialic acid) and protease leads to a decrease in La3+-displaceable Ca2+ but to an increase in the fraction displaced by 0.1 mM Polymyxin from 42% to 54%. Phospholipase D (cabbage) treatment significantly increased the La3+-displaceable Ca2+ to protein (), a gain of 0.055 nmol. All of this phospholipid specific increment in bound Ca2+ was displaced by 0.1 mM Polymyxin B. The results suggest that Polymyxin B will be useful as a probe for phospholipid Ca2+-binding sites in natural membranes. 相似文献
6.
In reconstituted human red blood cells a difference was found in (Ca2+ + Mg2+)-ATPase activity and in Ca2+ efflux at 37°C, depending on the side of the membrane at which the monovalent cations K+ and Na+ were placed. Under the conditions used, (Ca2+ + Mg2+)-ATPase activity and Ca2+ efflux was highest when K+ (, mean of four experiments) was at the inside and Na+ (130 mM) at the outside of the ghost membrane. 相似文献
7.
Ulrich Quast Alex M. Labhardt Valerie M. Doyle 《Biochemical and biophysical research communications》1984,123(2):604-611
The kinetics of the Quin 2-Ca2+ interaction have been studied using stopped-flow fluorimetry. Mixing the Quin 2-Ca2+ complex with a large excess of EGTA, EDTA or MgCl2 resulted in first order dissociation kinetics. The observed dissociation rate increased slightly with increasing EGTA concentration yielding a limiting value of 83±4 s?1 for the dissociation rate constant (k?) at pH 7.2, 37°C, ± 3mM Mg2+. The temperature dependence of the dissociation was weak (activation energy = 22±1 kJ/mol) and around neutral pH the pH dependence was negligible. The association reaction was too fast to be monitored directly. From this and the instrument dead-time, the second order rate constant k+ was estimated to be ≥109 M?1s?1, in agreement with the calculation from . These data should be useful in evaluating the potential of Quin 2 to measure fast intracellular Ca2+ transients. 相似文献
8.
Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of 45Ca2+. Ca2+ accumulation ratios greater than or approximately equal to 104 are readily attained. For ATP-dependent Ca2+ uptake, V is 1.5 mmol · 1?1 · min?1 at 27°C (approx. 0.9 nmol · mg?1 protein · min?1), [Ca2+] is 0.18 μM, [ATP] is 30–60 μM, the Ca2+ uptake rate depends on [Ca2+]2 and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 ± 1.4 kcal/mol and the pH optimum is approx. 6.9. 相似文献
9.
Vítor M.C. Madeira 《生物化学与生物物理学报:生物膜》1984,769(2):284-290
Uptake of Ca2+ by sarcoplasmic reticulum in the presence of oxalate displays biphasic kinetics. An initial phase of normal uptake is followed by a second phase coincident with precipitation of calcium oxalate inside the vesicles. The precipitation rate induced by actively transported Ca2+ is depressed by increasing the added Ca2+ concentration. This correlates linearly with the reciprocal of precipitation rate. Therefore, a maximal limit rate could be extrapolated at zero Ca2+ (). The rate of precipitation, also a function of added amount protein, gives a linear correlation in a double reciprocal plot. Thus, it was possible to estimate the maximal precipitation rate occurring at infinite protein concentration (). With the combined extrapolated values a maximal expected precipitation rate could be calculated (). Kinetics of calcium oxalate precipitation was studied in the absence of calcium uptake and empirical equations relating the rate of precipitation with the added Ca2+ were established. Entering in the equations, an internal free Ca2+ concentration of approx. 2.5 mM was estimated. Additionally, it is shown that the ionophore X-537A does not supress the Ca2+ uptake, if added during the oxalate-dependent phase, albeit the uptake proceeds at a slower rate after the release of approx. 70 nmol Ca2+/mg protein. This amount presumably equals the internal free Ca2+ not sequestered by oxalate, producing a maximal concentration approx. 14 mM. Taking into account low affinity binding of internal binding sites and the transmembrane Ca2+ gradients built up during the uptake of Ca2+, values of free Ca2+ ranging from 3 to 6 mM, approaching those estimated by the precipitation analysis, could be estimated. 相似文献
10.
11.
lumisomes are membrane-bounded bioluminescent vesicles which produce light when the lumisomal membrane is made permeable to Ca2+. During studies of Ca2+ transport we found that lumisomes can be made permeable to Ca2+ by establishing a Na+ gradient with the higher Na+ concentration being on the inside of the lumisomal membrane. No other cation will substitute for Na+ on the inside but any of several monovalent cations can be used to maintain electroneutrality external to the lumisomes. This Na+ gradient dependent Ca2+ transport appears not to involve active transport and occurs on a millisecond time scale suggesting that it is rapid enough to account for the onset of bioluminescence in . 相似文献
12.
The antibiotic A23187 carries Ca2+ across Müller-Rudin membranes made from and . The conductance of the membranes is not increased by the Ca2+-transport. The flux depends linearly on Ca2+ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4–5 between pH 6 and pH 8. Maximal Ca2+-fluxes of about were found. A counter transport of H+ could not be detected.The complex formation between A23187 and Ca2+ in egg phosphatidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2 : 1 complex. Optical absorption measurements on single phosphatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187. 相似文献
13.
effects of toxaphene on Ca2+-ATPase activity and 45Ca2+-uptake were studied in mitochondrial fractions of heart, kidney and liver tissues of rat. Mitochondrial fractions were prepared by the conventional centrifugation method. Ca2+-ATPase activity was determined by measuring the inorganic phosphate liberated during ATP hydrolysis. Toxaphene inhibited Ca2+-ATPase in a concentration dependent manner in all the three tissues. Substrate activation kinetics, with heart, kidney and liver tissue fractions, revealed that toxaphene inhibited Ca2+-ATPase activity non-competetively by decreasing the maximum velocity of the enzyme without affecting the enzyme-substrate affinity. Toxaphene also inhibited mitochondrial 45Ca2+-uptake in the three selected tissues in a concentration dependent manner. These results indicate that toxaphene is an inhibitor of mitochondrial Ca2+-ATPase and calcium transport in heart, kidney and liver tissues of rat. 相似文献
14.
Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca2+ transport in this preparation were characterized and compared to those of (Ca2+ + Mg2+)-ATPase activity. The time course of Ca2+ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8–11.5 nmol Ca2+/mg protein after 3–4 min of incubation. The rate of Ca2+ transport was increased by higher oxalate concentrations with a near linear rate of uptake at 20 mM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca2+/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent values of 100 μM and 7 μM for ATP concentration ranges above and below 50 μM, respectively. The apparent values for Mg2+ and Ca2+ were 132 μM and 0.36–0.67 μM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal ()-ATPase. The presence of potassium was required for maximum Ca2+ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca2+ transport and ( activity was transport and ( activity were both inhibited 10–20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 μM sulfonate. The results indicate that Ca2+ transport in adipocyte endoplasmic reticulum is mediated by a and suggest an important role for endoplasmic reticulum in control of intracellular Ca2+ distribution. 相似文献
15.
An increased cytoplasmic Ca2+ concentration ([Ca2+]i) has been implicated in the pathogenesis of cystic fibrosis. We compared the [Ca2+]i levels of normal and cystic fibrosis peripheral blood lymphocytes and Epstein-Barr virus-transformed lymphoblasts using quin 2, an internally trapped indicator. The [Ca2+]i levels of normal and cystic fibrosis cells were not significantly different. The ionophore-releasable intracellular Ca2+ stores were also comparable in both types of individual. 相似文献
16.
Do Han Kim Frank A. Sreter S.Tsuyoshi Ohnishi John F. Ryan James Roberts Paul D. Allen Laszlo G. Meszaros Bozena Antoniu Noriaki Ikemoto 《生物化学与生物物理学报:生物膜》1984,775(3):320-327
The time-course of Ca2+ release from sarcoplasmic reticulum isolated from muscles of normal pigs and those of pigs susceptible to malignant hyperthermia were investigated using stopped-flow spectrophotometry and arsenazo III as a Ca2+ indicator. Several methods were used to trigger Ca2+ release: (a) addition of halothane (e.g., 0.2 mM); (b) an increase of extravesicular Ca2+ concentration ([Ca02+]); (c) a combination of (a) and (b), and (d) replacement of ions (potassium gluconate with choline chloride) to produce membrane depolarization. The initial rates of Ca2+ release induced by either halothane or Ca2+ alone, or both, are at least 70% higher in malignant hyperthermic sarcoplasmic reticulum than in normal. The amount of Ca2+ released by halothane at low [Ca02+] in malignant hyperthermic sarcoplasmic reticulum is about twice as large as in normal sarcoplasmic reticulum. Membrane depolarization led to biphasic Ca2+ release in both malignant hyperthermic and normal sarcoplasmic reticulum, the rate constant of the rapid phase of Ca2+ release induced by membrane depolarization being significantly higher in malignant hyperthermic sarcoplasmic reticulum () than in normal (). Thus, all types of Ca2+ release investigated (a, b, c and d) have higher rates in malignant hyperthermic sarcoplasmic reticulum than normal sarcoplasmic reticulum. These results suggest that the putative Ca2+ release channels located in the sarcoplasmic reticulum are altered in malignant hyperthermic sarcoplasmic reticulum. 相似文献
17.
M Crompton M Hediger E Carafoli 《Biochemical and biophysical research communications》1978,80(3):540-546
The effect of inorganic phosphate on the accumulation of Ca2+ by heart mitochondria has been reinvestigated. Inorganic phosphate has no effect on the initial rate of Ca2+ uptake supported by respiration on either ascorbate tetramethylenephenylene diamine, pyruvate malate, or glutamate malate, although it does increase the final amount of Ca2+ accumulated; evidence suggests that the latter phenomenon requires phosphate influx via the phosphate carrier. It is concluded that the earlier reports that phosphate augments the initial rate of Ca2+ influx reflects an effect of phosphate on succinate oxidation, which was employed in the previous studies, rather than an Ca2+ transport itself. 相似文献
18.
Dolores M. Shoback Edward M. Brown 《Biochemical and biophysical research communications》1984,123(2):684-690
We employed the calcium (Ca++)-sensitive, intracellular dye QUIN-2 to examine the role of cytosolic Ca++ in the stimulation of PTH release by high extracellular potassium (K+) concentrations. Addition of 55 mM KCl to cells incubated with 115 mM NaCl and 5 mM KCl lowered cytosolic Ca++ at either low (0.5 mM) extracellular Ca++ (from 194±14 to 159±9 nM, p<.01, N=6) or high (1.5 mM) extracellular calcium (from 465±38 to 293±20 nM, p<.01, N=10). This reduction in cytosolic Ca++ was due to high K+ and not to changes in tonicity since addition of 55 mM NaCl was without effect while a similar decrease in cytosolic Ca++ occurred when cells were resuspended in 60 mM NaCl and 60 mM KCl. PTH release was significantly (p<.01) greater at 0.5 and 1.5 mM Ca++ in QUIN-2-loaded cells incubated with 60 mM NaCl and 60 mM KCl than in those exposed to 115 mM NaCl and 5 mM KCl. In contrast to most secretory cells, therefore, stimulation of PTH release by high K+ is associated with a decrease rather than an increase in cytosolic Ca++. 相似文献
19.
Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 μmol Pi per h per mg protein. It is suggested that this activity is related to the measured Ca2+ transport which was characterized by values for ATP and Ca2+ of and , respectively. Phosphorylation of plasma membranes with [γ-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of and from the catalytic subunit of of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells. 相似文献
20.
The uptake and release of Ca2+ by sarcoplasmic reticulum fragments and reconstituted ATPase vesicles was measured by a stopped-flow fluorescence method using chlortetracycline as Ca2+ indicator.Incorporation of the Ca2+ transport ATPase into phospholipid bilayers of widely different fatty acid composition increases their passive permeability to Ca2+ by several orders of magnitude. Therefore in addition to participating in active Ca2+ transport, the ()-activated ATPase also forms hydrophilic channels across the membrane. The relative insensitivity of the permeability effect of ATPase to changes in the fatty acid composition of the membrane is in accord with the suggestion that the Ca2+ channels arise by protein-protein interaction between four ATPase molecules. The reversible formation of these channels may have physiological significance in the rapid Ca2+ release from the sarcoplasmic reticulum during activation of muscle. 相似文献