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1.
Closed vesiculate preparations of pig myometrium sarcolemma (predominantly with inside-out orientation) are characterized by passive permeability for Ca2+. The kinetics of Ca2+ release from the vesicles is exponential. Using the grapho-analytical subtraction method, the kinetic parameters of this reaction were determined. Myometrium sarcolemma contains endogenous Ca2+-calmodulin-dependent protein kinase and phosphoprotein phosphatase which is inhibited by sodium o-vanadate. The Ca2+-calmodulin-dependent phosphorylation stimulates passive Ca2+ release from sarcolemmal vesicles. In the course of phosphorylation the capacity of the pool providing for rapid Ca2+ release increases by 61%, the initial rate of Ca2+ release showing a 28% increase. Trifluoroperazine, an inhibitor of Ca2+-calmodulin-dependent processes, eliminates the activating effect of phosphorylation on the rate of Ca2+ release from sarcolemmal vesicles. 相似文献
2.
The effects of Li+ on Na-Ca exchange in bovine cardiac sarcolemmal vesicles were examined. The initial rate of Na(+)-dependent Ca2+ uptake and efflux was inhibited by Li+ in a dose dependent manner. The initial rate of Na(+)-dependent Ca2+ uptake was inhibited 49.8 +/- 2.9% (S.E.) (n = 6) in the presence of Li+ compared to activity in external K+ or choline+. Kinetic analysis indicated that Li+ increased the Km for Ca2+ (96.3 microM) compared to K+ and choline+ (25.5 and 22.9 microM respectively) while Vmax (1.4, 1.2 and 1.1 nmol Ca2+/mg protein/sec respectively) remained unchanged. Li+ did not alter the experimentally derived stoichiometry of the exchange reaction of 3 Na+ for 1 Ca2+. 相似文献
3.
The apparent values of intravesicular volume (45 microliter/mg of protein), maximal capacity of adsorbed calcium binding on the inner surface of the vesicles (4.5 nmol/mg of protein) and dissociation constants for the Ca2+-binding site complexes (36 microM) were determined from the analysis of peculiarities of passive transport of 45Ca2+ into cow myometrium sarcolemmal vesicles. The kinetics of passive efflux of ionized Ca2+ from the vesicles is described by a two-phase exponential curve. Dilution of the vesicles with a dilution medium is associated with a rapid efflux of ionized Ca2+ from the intravesicular space resulting in dissociation of the Ca2+-binding site complexes on the inner surface of the vesicles and, correspondingly, in the passage from a rapid to the slow phase of Ca2+ efflux from the vesicles which is limited by the dissociation of the Ca2+-binding site complexes. The values of the apparent rate constants for the transmembrane transfer of Ca2+ and dissociation of the Ca2+-binding site complexes (0.73 and 0.02 min-1, respectively) and the permeability of sarcolemmal vesicles for the cation (10(-15) mol of Ca2+/cm2.s) were determined. Alkalinization of the dilution medium stimulates 45Ca2+ release from the vesicles. The blockers of passive Co2+ and Mn2+ transport injected into the vesicles inhibit the efflux of 45Ca2+ from the vesicles. The data obtained were used to analyze the role of sarcolemma in the Ca2+ control of myometrium contraction. 相似文献
4.
Chlorotetracycline (CTC) fluorescence is shown to be a competent and quantitative measure of the free internal calcium concentration, [Ca2+]i, obtained by ATP supported active uptake by bovine cardiac sarcolemmal (SL) vesicles. The fluorescence response of CTC to [Ca2+]i is calibrated by pre-equilibrating the vesicles with known Ca2+ concentrations and then diluting into a Ca2+-free medium containing CTC. The experiments show that CTC comes into equilibrium with the internal Ca2+ more rapidly than the latter can passively leak from the vesicles. The amplitude of the fluorescence increase is proportional to the Ca2+ concentration with which the vesicles are pre-equilibrated. This constitutes a calibration procedure for the use of CTC fluorescence as a quantitative measure of the free internal Ca2+ concentrations achieved in active transport. This method is applied to the determination of the average free Ca2+ concentrations achieved in ATP-energized uptake with sarcolemmal vesicles. Under optimal conditions an initial rate of 13 mM/min (37 nmol/mg/min) is observed. Uptake reaches a maximum corresponding to 70 mM (179 nmol/mg). Half-maximal values are obtained after 5 min of reaction. The mechanism of the CTC response to free internal Ca2+ concentration is discussed and is compared with measurements of vesicle-associated 45Ca2+. 相似文献
5.
Adenine nucleotide stimulation of Ca2+-induced Ca2+ release in sarcoplasmic reticulum 总被引:28,自引:0,他引:28
G Meissner 《The Journal of biological chemistry》1984,259(4):2365-2374
Rabbit skeletal muscle sarcoplasmic reticulum was fractionated into a "Ca2+-release" and "control" fraction by differential and sucrose gradient centrifugation. External Ca2+ (2-20 microM) caused the release of 40 nmol of 45Ca2+/mg of protein/s from Ca2+-release vesicles passively loaded at pH 6.8 with an internal half-saturation Ca2+ concentration of 10-20 mM. Ca2+-induced Ca2+ release had an approximate pK value of 6.6 and was half-maximally inhibited at an external Ca2+ concentration of 2 X 10(-4) M and Mg2+ concentration of 7 X 10(-5) M. 45Ca2+ efflux from control vesicles was slightly inhibited at external Ca2+ concentrations that stimulated the rapid release of Ca2+ from Ca2+-release vesicles. Adenine, adenosine, and derived nucleotides caused stimulation of Ca2+-induced Ca2+ release in media containing a "physiological" free Mg2+ concentration of 0.6 mM. At a concentration of 1 mM, the order of effectiveness was AMP-PCP greater than cAMP approximately AMP approximately ADP greater than adenine greater than adenosine. Other nucleoside triphosphates and caffeine were minimally effective in increasing 45Ca2+ efflux from passively loaded Ca2+-release vesicles. La3+, ruthenium red, and procaine inhibited Ca2+-induced Ca2+ release. Ca2+ flux studies with actively loaded vesicles also indicated that a subpopulation of sarcoplasmic reticulum vesicles contains a Ca2+ permeation system that is activated by adenine nucleotides. 相似文献
6.
Sarcolemmal vesicles were prepared from bovine cardiac muscle by differential and discontinuous sucrose density gradient centrifugation. Na+/K+-ATPase was purified 33-fold to a specific activity of 53 +/- 0.5 (12) mumol Pi X mg-1 X h-1, binding sites for strophantin 20-fold to a density of 56.3 +/- 5.3 (14) pmol/mg and that for the calcium antagonist nitrendipine 5.5-fold to a density of 0.72 +/- 0.07 (6) pmol/mg. The specific activity of the Na+/Ca2+ exchanger was 61.1 +/- 3.7 (6) nmol/mg. The vesicles had an intravesicular volume of 20 +/- 4 (4) microliter/mg and 56.9 +/- 6 (4)% of the vesicles were right-side-out oriented. Several peptides of the purified membranes were phosphorylated in the presence of Mg . ATP and EGTA. Most of the radioactive phosphate was incorporated into a peptide with an apparent molecular mass of 22 kDa. Denaturation of the membranes at 100 degrees C changed the mobility of this peptide to 15 kDa and 11 kDa. This peptide could not be distinguished from a sarcoplasmic reticulum peptide of similar molecular mass. The phosphorylation of the sarcolemmal peptide was stimulated by Ca2+/calmodulin, cAMP and the catalytic subunit of cAMP-dependent protein kinase. A comparison of the phosphorylation of sarcolemmal membranes with that of sarcoplasmic reticulum showed that Ca2+/calmodulin stimulated in each membrane, the phosphorylation of the 22-kDa peptide and a 44-kDa peptide, and in the sarcoplasmic reticulum the phosphorylation of an additional peptide of 55-kDa. Ca2+/calmodulin-dependent phosphorylation of a 55-kDa peptide could not be demonstrated in sarcolemma, regardless if sarcolemmal membranes were incubated together with sarcoplasmic reticulum or if the phosphorylation was carried out in the presence of purified cardiac myosin light chain kinase or phosphorylase kinase. 'Depolarization' induced Ca2+ uptake which was measured according to Bartschat, D.K., Cyr, D.L. and Lindenmayer, G.E. [(1980) J. Biol. Chem. 255, 10044-10047] was 5 nmol/mg protein. This uptake was not enhanced after preincubation of the vesicles with Mg . ATP or Mg . ATP and cAMP-dependent protein kinase. The value of 5 nmol/mg protein is in agreement with the theoretical amount of Ca2+ which can be accumulated by the bovine cardiac sarcolemma in the absence of a driving force other than the Ca2+ gradient. The potassium-stimulated Ca2+ uptake was not blocked by the organic Ca2+ channel blockers. Prolonged incubation of Mg . ATP with sarcolemmal vesicles in the presence of various ATPase inhibitors led to the hydrolysis of ATP. The liberated phosphate precipitated with Ca2+ in the presence of LaCl3. These precipitates amounted to an apparent Ca2+ uptake ranging from 50 to over 1000 nmol/mg. The results suggest that potassium-stimulated Ca2+ uptake of bovine cardiac sarcolemmal vesicles is not enhanced in the presence of ATP or by phosphorylation of a 22-kDa peptide. 相似文献
7.
Ca2+-calmodulin-dependent phosphorylation and passive transport of Ca2+ in the myocardial sarcolemma
Highly purified vesicles of rabbit myocardium sarcolemma with predominant inside-out orientation possess the Ca2+-calmodulin-dependent protein kinase activity. At optimal concentrations of calmodulin (0.5 microM) and Ca2+ (0.1 mM), the activity of protein kinase is 0.21 nmol 32P X min X mg of protein. The Km(app) value for ATP is 3.0 X 10(-6) M, V = 0.27 nmol 32P X mg of protein X min. Endogenous Ca2+-calmodulin-dependent protein kinase phosphorylates four protein substrates in sarcolemmal vesicles (Mr = 145, 22, 11.5, and 6-8 KD). Studies with passive efflux of Ca2+ from the SL vesicles showed that the Ca2+-calmodulin-dependent phosphorylation of protein components of sarcolemma inhibits this reaction. 相似文献
8.
Inhibition of Na+/Ca2+ exchanger activity in cardiac and skeletal muscle sarcolemmal vesicles by monoclonal antibody 44D7 总被引:3,自引:0,他引:3
Monoclonal antibodies 44D7 and 4F2 inhibited specifically the Na+-dependent Ca2+ fluxes characteristic of the Na+/Ca2+ exchanger in cardiac and skeletal muscle sarcolemmal vesicles. Preincubation of membrane vesicles with monoclonal antibody 44D7 inhibited 90% of the Na+-dependent Ca2+ uptake measured in the first 10 s of the reaction and 50% of that measured after 60 s. Ca2+/calmodulin-dependent ATPase activity and ATP-dependent Ca2+ uptake by sarcolemmal vesicles were not affected by monoclonal antibody 44D7 whereas the Na+-dependent release of accumulated Ca2+ was inhibited. In the presence of the 44D7 antigen isolated from human kidney, monoclonal antibody 44D7 could no longer inhibit Na+-dependent Ca2+ fluxes. The distribution of 4F2 antigenic activity in the isolated muscle membrane fractions correlated with that of Na+/Ca2+ exchanger activity; cardiac and skeletal muscle sarcolemmal vesicles expressed higher levels of the antigen than skeletal muscle transverse tubule membrane, while no antigen could be detected in sarcoplasmic reticulum membranes. Our results suggest that monoclonal antibodies 44D7 and 4F2 interact either directly with the Na+/Ca2+ exchanger molecules or with some other protein(s) responsible for the regulation of this activity in the heart and skeletal muscle. 相似文献
9.
Treatment of canine cardiac sarcolemmal vesicles with phospholipase D resulted in a large stimulation (up to 400%) of Na+-Ca2+ exchange activity. The phospholipase D treatment decreased the apparent Km (Ca2+) for the initial rate of Nai+-dependent Ca2+ uptake from 18.2 +/- 2.6 to 6.3 +/- 0.3 microM. The Vmax increased from 18.0 +/- 3.6 to 31.5 +/- 3.6 nmol of Ca2+/mg of protein/s. The effect was specific for Na+-Ca2+ exchange; other sarcolemmal transport enzymes ((Na+, K+)-ATPase; ATP-dependent Ca2+ transport) are inhibited by incubation with phospholipase D. Phospholipase D had little effect on the passive Ca2+ permeability of the sarcolemmal vesicles. After treatment with 0.4 unit/ml of phospholipase D (20 min, 37 degrees C), the sarcolemmal content of phosphatidic acid rose from 0.9 +/- 0.2 to 8.9 +/- 0.4%; simultaneously, Na+-Ca2+ exchange activity increased 327 +/- 87%. It is probable that the elevated phosphatidic acid level is responsible for the enhanced Na+-Ca2+ exchange activity. In a previous study (Philipson, K. D., Frank, J. S., and Nishimoto, A. Y. (1983) J. Biol. Chem. 258, 5905-5910), we hypothesized that negatively charged phospholipids were important in Na+-Ca2+ exchange, and the present results are consistent with this hypothesis. Stimulation of Na+-Ca2+ exchange by phosphatidic acid may be important in explaining the Ca2+ influx which accompanies the phosphatidylinositol turnover response which occurs in a wide variety of tissues. 相似文献
10.
Membrane vesicles were prepared from Artemia nauplii (San Francisco Bay variety) 45 h after hydration of the dry cysts. Na+-loaded vesicles accumulated up to 10 nmol Ca2+/mg protein when diluted 50-fold into 160 mM KCl containing 15 microM CaCl2. Practically no accumulation of Ca2+ was observed if the vesicles were diluted into 160 mM NaCl instead of KCl, or if they were treated with monensin, a Na+ ionophore, for 30 s prior to addition of CaCl2 to the KCl medium. These observations indicate that the Artemia vesicles exhibit Na-Ca exchange activity. The velocity of Ca2+ accumulation by the vesicles in KCl was stimulated 2.6-fold by the K+ ionophore valinomycin, suggesting that the exchange system is electrogenic, with a stoichiometry greater than 2Na+ per Ca2+. Km,Ca and Vmax values were 15 microM and 7.5 nmol/mg protein.s, respectively. Exchange activity in the Artemia vesicles was inhibited by benzamil (IC50 approximately equal to 100 microM) and by quinacrine (IC50 approximately equal to 250 microM), agents that also inhibit exchange activity in cardiac sarcolemmal vesicles. Unlike cardiac vesicles, however, exchange activity in Artemia was not stimulated by limited proteolysis, redox reagents, or intravesicular Ca2+. This indicates that the two exchange systems are regulated by different mechanisms. Vesicles were prepared from Artemia at various times after hydration of the dry cysts and examined for exchange activity. Activity was first observed at approximately 10 h after hydration and increased to a maximal value by 30-40 h; hatching of the free swimming nauplii occurred at 18-24 h. The results suggest that hatching Artemia nauplii might be a particularly rich source of mRNA coding for the Na+-Ca2+ exchange carrier. 相似文献
11.
Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs. 总被引:2,自引:0,他引:2
Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux. 相似文献
12.
Vesicular sarcolemmal preparations isolated from rat hearts were characterized by high total ATPase (4.32 +/- 0.57 mumol/min per mg), adenylate cyclase (121 +/- 11 pmol/min per mg) and creatine kinase (1.73 +/- 0.35 mumol/min per mg) activities as well as Na-Ca exchange specific to sodium. ATPase activity was inhibited with digitoxigenin by 50-70% and was not changed by ouabain, ionophore A23187 or oligomycin. Sarcolemmal vesicles bound [3H]digitoxigenin and [3H]ouabain in isotonic medium in the presence of Pi and Mg2+. The number of binding sites for hydrophobic digitoxigenin (N = 237 pmol/mg) was several-times higher than that for hydrophilic ouabain (N = 32.7 pmol/mg). These data show that sarcolemmal preparations were not significantly contaminated by mitochondria and sarcoplasmic reticulum and consisted mostly of inside-out vesicles. Incubation of these vesicles with 45Ca2+ (0.5-10 mM) led to penetration of the latter into the vesicles with the following binding characteristics: number of binding sites (N = 20.5 +/- 4.6 nmol/mg, Kd approximately equal to 2.0 mM). Ca2+ binding to the inner surface of vesicles was proved by the following facts: (1) Ca2+ ionophore A23187 increased slightly total intravesicular Ca2+ content but markedly accelerated Ca2+ efflux along its concentration gradient; (2) gramicidin and osmotic shock showed a similar accelerating effect. Ca2+ efflux from the vesicles along its concentration gradient ([Ca2+]i/[Ca2+]e = 2.0 mM/0.1 microM) was inhibited by Mn2+, Co2+, and verapamil when they acted inside the vesicles. The rate of Ca2+ efflux was hyperbolically dependent on intravesicular Ca2+ concentration (Km approximately equal to 2.9 mM). These data reveal that Ca2+ efflux from sarcolemmal vesicles is controlled by Ca2+ binding to the sarcolemmal membrane. Ca2+ efflux from the vesicles was stimulated 1.7--times after incubation of vesicles with 0.2 mM MgATP or MgADP and 15-times after treatment with 0.2 mM adenylyl beta, gamma-imidodiphosphate. Enhancement in the rate of Ca2+ efflux correlated with the increase in the intravesicular Ca2+ content. ATP-stimulated Ca2+ efflux was suppressed by verapamil and was nonmonotonically dependent upon the transmembrane potential created by the K+ concentration gradient in the presence of valinomycin, Ca2+ efflux being slower at extreme values of membrane potential (+/- 80 mV). 相似文献
13.
A simple method is suggested to determine kinetic characteristics of the Ca2+ active transport systems in the smooth muscle. The use of this method has shown that the initial rate of Ca2+ accumulation in the myometrium mitochondria (57.5 nmol per 1 mg of protein/1 min) is 50 times higher than in the sarcolemma vesicles. The calcium capacity of mitochondria (254 nmol per 1 mg of protein) also exceeds essentially (36 times) that of the membrane vesicles. Meanwhile, the Ca2+-transporting systems of these two subcellular structures practically do not differ from each other in the magnitude of the cation semiaccumulation period (4-7 min). 相似文献
14.
Ca2+ transport and Ca2+-dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands. 总被引:5,自引:0,他引:5
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Ca2+ transport across mammary-gland Golgi membranes was measured after centrifugation of the membrane vesicles through silicone oil. In the presence of 2.3 microM free Ca2+ the vesicles accumulated 5.8 nmol of Ca2+/mg of protein without added ATP, and this uptake was complete within 0.5 min. In the presence of 1 mM-ATP, Ca2+ was accumulated at a linear rate for 10 min after the precipitation of intravesicular Ca2+ with 10 mM-potassium oxalate. ATP-dependent Ca2+ uptake exhibited a Km of 0.14 microM for Ca2+ and a Vmax. of 3.1 nmol of Ca2+/min per mg of protein. Ca2+-dependent ATP hydrolysis exhibited a Km of 0.16 microM for Ca2+ and a Vmax. of 10.1 nmol of Pi/min per mg of protein. The stoichiometry between ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase varied between 0.3 and 0.7 over the range 0.03-8.6 microM-Ca2+. Both Ca2+ uptake and Ca2+-stimulated ATPase were strongly inhibited by orthovanadate, which suggests that the major mechanism by which Golgi vesicles accumulate Ca2+ is through the action of the Ca2+-stimulated ATPase. However, Ca2+ uptake was also decreased by the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone), indicating that it may occur by other mechanisms too. The effect of CCCP may be related to the existence of transmembrane pH gradients (delta pH) in these vesicles: the addition of 30 microM-CCCP reduced delta pH from a control value of 1.06 to 0.73 pH unit. Golgi vesicles also possess a Ca2+-efflux pathway which operated at an initial rate of 0.5-0.57 nmol/min per mg of protein. 相似文献
15.
Vesicular preparations of sarcolemma isolated from rat myocardium possessed high ATPase (4.32 +/0 0.57 micromole/min per mg), adenylate cyclase (121 +/- 11 pmole/min per mg) and creatine kinase (1.74 +/- 0.35 micromole/min per mg) activities and a Na-Ca exchange activity specific for sodium. The ATPase activity was inhibited by digitoxigenin by 50-70% and was not changed by ouabain, EGTA, ionophore A23187 and oligomycin, thus showing the absence of mitochondrial and sarcoplasmic reticulum contaminations in the sarcolemmal preparations. The preparations consisted mostly of closed inside-out vesicles. The preparation was used to study the mechanism of Ca2+ penetration across the sarcolemmal membrane. For this purpose the vesicles were load with 45Ca2+, which relatively slowly diffused from the medium into the vesicles, and which was bound to the binding sites inside the vesicles (n = 20.5 +/- 4.6 nmoles per mg of protein, Kd approximately equal to 1.8 +/- 0.21 mM). The transmembrane movement of Ca2+ was demonstrated by the following findings: 1) the ionophore A23187 only insignificantly increased the total vesicular Ca2+ content, but strongly accelerated Ca2+ efflux from the vesicles along its concentration gradient; 2) gramicidin and osmotic shock caused a similar acceleration of Ca2+ efflux. Ca2+ efflux from these vesicles along Ca2+ concentration gradient was studied under conditions, when the extravesicular Ca2+ content was lowered due to its binding to EGTA and by dilution. The gradient of Ca2+ concentration was from 2.0 mM inside to approximately 0.1 micro M outside. The rate of 45Ca2+ efflux depended hyperbolically on the intravesicular Ca2+ efflux from the vesicles was inhibited by Mn2+, Co2+ and verapamil when they acted from the inside of the vesicles. An increase in ionophore A23187 concentration increased the efflux of Ca2+ hyperbolically and enhanced only the maximal rate of the efflux. It is concluded that the passive permeability of Ca2+ across the sarcolemmal membrane along its concentration gradient is controlled by Ca2+ binding to the membrane. 相似文献
16.
Junctional sarcoplasmic reticulum (SR) vesicles isolated from back muscles of normal and malignant hyperthermia susceptible (MHS) pigs were phosphorylated by addition of MgATP in the presence of 5 mM Ca2+ and 1 microM calmodulin (CaM). The major site of phosphorylation was a 60 kDa protein both in normal and MHS SR. The maximal amount of phosphorylation in MHS SR (5 pmol P/mg SR) was significantly lower than that in the normal SR (12 pmol P/mg SR). The phosphorylated 60 kDa protein was spontaneously dephosphorylated both in normal and MHS SR. Ca2+ release from the passively loaded SR was induced by a Ca2+-jump, and monitored by stopped-flow fluorometry using chlorotetracycline. In the absence of preincubation with MgATP, no significant difference was found in any of the kinetic parameters of Ca2+ release between normal and MHS SR. Upon addition of 20 microM MgATP to the passively loaded SR to phosphorylate the 60 kDa protein, the initial rate of Ca2+ release in normal SR significantly decreased from 659 +/- 102 to 361 +/- 105 nmol Ca2+/mg SR per s, whereas in MHS SR the rate decreased from 749 +/- 124 to 652 +/- 179 nmol Ca2+/mg SR per s. Addition of 20 microM adenosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppA) did not significantly alter the initial rate of Ca2+ release both in normal and MHS SR. These results suggest that the previously reported higher Ca2+ release rate in MHS SR (Kim et al. (1984) Biochim. Biophys. Acta 775, 320-327) is at least partly due to the reduced extent of the Ca2+/CaM-dependent phosphorylation of the 60 kDa protein. Two-dimensional gel electrophoresis study showed that amount of a protein with Mr = 55,000 was significantly lower in MHS SR than in normal SR suggesting that the abnormally lower amount of 55 kDa protein would cause the lower amount of phosphorylation of the 60 kDa protein in MHS SR. 相似文献
17.
Characterization of an active transport system for calcium in inverted membrane vesicles of Escherichia coli. 总被引:6,自引:0,他引:6
The energy-dependent uptake of calcium by inverted membrane vesicles of Escherichia coli was investigated. Methods for preparation and storage of the vesicles were devised to allow for the maximal activity and stability of the calcium transport system. The pH and temperature optima for the reaction were observed to occur at pH 8.0 AND 30 DEGREES, RESPECTIVELY. The eft was found that the extent of the reaction depended on the presence of phosphate or oxalate. Phosphate was found to enter the vesicles at a rate slower than that of calcium. A Ca2+:Pi ratio of approximately 1.5 was found, suggesting formation of Ca3(PO4)2. Monovalent cations stimulated calcium uptake, with the order of effectiveness being K+ is greater than Na+ is greater than Li+ is greater than NH4+. Inhibition was found with certain divalent cations, but these also inhibited the electron transport chain. Of the divalent cations examined only Mg2+ and Sr2+ inhibited calcium transport without a corresponding inhibition of respiration. Calcium transport exhibited biphasic Kinetics, with a low affinity system and a high affinity system. The low affinity system showed a Km of 0.34 mM and a Vmax of 85 nmol/min/mg of protein. The kinetic constants of the high affinity system were 4.5 muM and 2 nmol/min/mg of protein. The energy for calcium transport could be derived from the electron transport chain by oxidation of NADH, D-lactate, and succinate, in order of their effectiveness. Respiration-driven calcium transport was inhibited by inhibitors of the electron transport chain and by uncouplers of oxidative phosphorylation. ATP could also be used to supply enerty for calcium transport. The ATP-driven reaction was inhibited by inhibitors of the Mg2+ATPase and by an antiserum prepared against that protein, demonstrating that that enzyme is involved in the utilization of ATP for active transport in inverted vesicles. 相似文献
18.
Inositol 1,4,5-trisphosphate-triggered Ca2+ release from bovine adrenal medullary secretory vesicles 总被引:3,自引:0,他引:3
The effect of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and calcium ionophore A23187 on Ca2+ release from bovine adrenal medullary secretory vesicles and microsomes was examined. Ins(1,4,5)P3 released 3.5 nmol of Ca2+/mg protein from secretory vesicles and 1.5 nmol of Ca2+/mg protein from microsomes as measured by a Ca2(+)-selective electrode. However, A23187 promoted Ca2+ uptake into vesicles while releasing Ca2+ from microsomes. Ins(1,4,5)P3-induced Ca2+ release from secretory vesicles was rapid, but the released Ca2+ was absorbed within 3 min during which the Ins(1,4,5)P3-releasable pools were refilled. The in situ calcium content of secretory vesicle measured by atomic absorption spectrometry was 112 +/- 6.3 nmol/mg protein indicating the potential importance of secretory vesicles as an intracellular Ca2+ store. The high Ca2(+)-buffering capacity of secretory vesicles is presumed to be due to the high Ca2(+)-binding capacity of chromogranin A, the major intravesicular protein, which has calsequestrin-like properties. 相似文献
19.
A novel mitochondrial Ca2+ release phenomenon is reported. When rat liver mitochondria (oxidizing succinate) are allowed to accumulate Ca2+ in excess of 40 nmol/mg protein and are then treated with excess EGTA, a fraction of the accumulated cation is rapidly (approximately 1 nmol/s/mg protein) released. The size of the released fraction is an apparent function of the extramitochondrial Ca2+ concentration at the time of EGTA addition and can attain a maximal value of approximately 30 nmol/mg protein. Release is inhibited by ruthenium red (I50 approximately 50 pmol/mg protein) and is not dependent on the presence of Na+ or K+ in the medium. During the period of rapid release, O2 consumption is inhibited, membrane potential increases, and apparent H+ accumulation occurs at a ratio of approximately 2H+ per Ca2+ released. It is proposed that this chelator-induced Ca2+ release occurs by reverse uniport with H+ back diffusion to the matrix space providing charge movement compensation. 相似文献
20.
We have investigated the kinetic and thermodynamic properties of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum under conditions that result in a single transport cycle. Simultaneous addition of ATP and EGTA to sarcoplasmic reticulum vesicles, preincubated with calcium, resulted in a transient of intermediate species. In the presence of saturating Ca2+ levels, total E-P species reached a maximum of 2.3 nmol/mg at 100 ms, followed by a monoexponential decay with kobs = 3.6 s-1. The data are interpreted in terms of Ca2+ sequestration, either by occlusion as Ca2+ in the phosphorylated enzyme or chelation by EGTA. Maximum Ca2+ uptake was 8.3 nmol/mg with the release of 4.4 nmol/mg Pi. The ratio of Ca2+ uptake to Pi release approached 1.9 over a wide [Ca2+] range. Equilibrium Ca2+ binding, in the absence of ATP, showed a K0.5 of 0.88 microM with a Hill coefficient of 1.9. The Ca2+ concentration dependence of Ca2+ uptake during single-cycle catalysis showed a 10-fold enhanced affinity (K0.5 = 0.06 microM) and was noncooperative (nH = 0.9). Quench with excess EGTA (greater than 2 mM) decreased Ca2+ uptake to 1 nmol/mg, indicating an "off" rate of Ca2+ from high affinity sites that exceeds 100 s-1. The ATP concentration dependence for a single-cycle catalysis showed an apparent K0.5 of 1.1 microM, similar to that for ATP equilibrium binding. It is proposed that enzyme phosphorylation proceeds only following binding of a second calcium ion to externally oriented sites whose intrinsic affinity is in the same range as the calcium dependence of a single-cycle turnover. 相似文献