An assay for sarcoplasmic reticulum Ca2(+)-ATPase activity in muscle homogenates

A spectrophotometric method is described for the determination of sarcoplasmic reticulum (SR) Ca2(+)-ATPase activity (EC 3.1.6.38) in unfractionated muscle homogenates. Conditions were established that give maximal SR Ca2(+)-ATPase activity, while eliminating Ca2(+)-dependent myofibrillar ATPase activity and reducing Ca2(+)-independent or background ATPase activity. High [Ca2+] (20 mM) could be used to selectively inhibit the SR Ca2+ ATPase. Identification of the Ca2(+)-dependent ATPase activity in muscle homogenates as being SR Ca2+ ATPase was based on a comparison of several parameters using homogenate material and purified SR. The following parameters were compared and found to be the same in homogenate and SR: activation and inactivation between 0 and 20 mM Ca2+, temperature dependence, sensitivity toward Triton X-100, and the maximal level of inhibition of ATPase activity achieved by an antibody specific for SR Ca2+ ATPase. The method is illustrated with the analysis of homogenates prepared from freeze-dried muscle fibers and thin sections of muscles typically used in microscope analyses as well as an analysis of freshly prepared homogenates from various types of muscle, which shows a good correlation over a wide range between SR specific Ca2(+)-uptake and -ATPase activities. In addition, a simple, easily constructed cuvette is described which allows the analysis of less than 5 micrograms of tissue (wet weight) in a volume of 25 microliters.     

Improved expression and characterization of Ca2+-ATPase and phospholamban in High-Five cells

The Ca2+-ATPase accounts for the majority of Ca2+ removed from the cytoplasm during cardiac muscle relaxation. The Ca2+-ATPase is regulated by phospholamban, a 52 amino acid phosphoprotein, which inhibits Ca2+-ATPase activity by decreasing the apparent affinity of the ATPase for Ca2+. To study the physical mechanism of Ca2+-ATPase regulation by phospholamban using spectroscopic and kinetic experiments, large amounts of both proteins are required. Therefore, we developed a Ca2+-ATPase and phospholamban preparation based on the baculovirus-insect cell expression system using High-Five insect cells to produce large amounts of microsomal vesicles that contain either Ca2+-ATPase expressed alone or Ca2+-ATPase co-expressed with phospholamban. The expressed proteins were characterized using immunofluorescence spectroscopy, Ca2+ -ATPase activity assays, Ca2+ uptake and efflux assays, and Western blotting. Our purification method yields 140 mg of microsomal protein per liter of infection (1.7 x 10(9)cells), and the Ca2+-ATPase and phospholamban account for 16 and 1.4%, respectively, of the total microsomal protein by weight, yielding a phospholamban:Ca2+-ATPase ratio of 1.6:1, similar to that observed in native cardiac SR vesicles. The enzymatic properties of the expressed Ca2+-ATPase are also similar to those observed in native cardiac SR vesicles, and when co-expressed with phospholamban, the Ca2+-ATPase is functionally coupled to phospholamban similar to that observed in cardiac SR vesicles.     

Ca2+-overload inhibits the cardiac SR Ca2+-calmodulin protein kinase activity

There is increasing evidence to suggest that Ca2+-calmodulin dependent protein kinase (CaMK) regulates the sarcoplasmic reticulum (SR) function and thus plays an important role in modulating the cardiac performance. Because intracellular Ca2+-overload is an important factor underlying cardiac dysfunction in a heart disease, its effect on SR CaMK was examined in the isolated rat heart preparations. Ca2+-depletion for 5 min followed by Ca2+-repletion for 30 min, which is known to produce intracellular Ca2+-overload, was observed to attenuate cardiac function as well as SR Ca2+-uptake and Ca2+-release activities. Attenuated SR function in the heart was associated with reduced CaMK phosphorylation of the SR Ca2+-cycling proteins such as Ca2+-release channel, Ca2+-pump ATPase, and phospholamban, decreased CaMK activity, and depressed levels of SR Ca2+-cycling proteins. These results indicate that alterations in cardiac performance and SR function following the occurrence of intracellular Ca2+-overload may partly be due to changes in the SR CaMK activity.     

Modification of the (Ca2+ + Mg2+)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.     

Temperature dependence of the Ca2+-ATPase (SERCA2) in the ventricles of tuna and mackerel

Recent physiological studies on the cardiovascular performance of tunas suggest that the elevated heart rates of these fish may rely on increased use of intracellular sarcoplasmic reticulum (SR) Ca2+ stores. In this study, we compare the cellular cardiac performance in endothermic tunas (bluefin, albacore, yellowfin) and their ectothermic sister taxa (mackerel) in response to acute temperature change. The cardiac sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) plays a major role during cardiac excitation-contraction (E-C) coupling, transporting Ca2+ from the cytosol into the lumen of the SR and thus promoting the relaxation of the muscle. Measurements of oxalate-supported Ca2+ uptake in SR-enriched ventricular vesicles indicated that tunas were capable of sustaining a rate of Ca2+ uptake that was significantly higher than the mackerel. Among tunas, the cold-tolerant bluefin had the highest rates of SR Ca2+ uptake and ATPase activity. The differences among Ca2+ uptake and ATP hydrolysis rates do not seem to result from intrinsic differences between the SERCA2 present in the different tunas, as shown by their similar temperature sensitivities and similar values for activation energy. Western blots reveal that increased SERCA2 protein content is associated with the higher Ca2+ uptake and ATPase activities seen in bluefin ventricles compared with albacore, yellowfin, and mackerel. We hypothesize that a key step in the evolution of high heart rate and high metabolic rate in tunas is increased activity of the SERCA2 enzyme. We also suggest that high levels of SERCA2 in bluefin tuna hearts may be important for retaining cardiac function at cold temperatures.     

A direct colorimetric assay for Ca2+ -stimulated ATPase activity

A simple and rapid colorimetric assay for measuring the high affinity Ca2+-ATPase activity in subcellular fractions is presented. With this method a one-step addition of a malachite green/molybdate/polyvinyl alcohol reagent to the assay mixture at the end of the incubation period is all that is required for the spectrophotometric quantification of the phosphomolybdate-malachite green complex. The presence of polyvinyl alcohol allows the quantification of released phosphate without having to separate it from protein. We have validated this assay by characterizing the high affinity Ca2+-ATPase activity in isolated rat liver microsomes. Comparable Ca2+-ATPase activities in rat liver microsomes and adipocyte plasma membranes were found when measured with this colorimetric assay and an isotopic assay. This method is applicable to the measurement of other types of ATPase activities.     

Comparison of ATPase activity of cardiac sarcoplasmic reticulum fraction from rat and dog

The purpose of the present study was to compare the ATPase activities of cardiac SR in two species in which the different intrinsic myocardial contractility can only partially be explained by the different properties of cardiac myosins. In cardiac SR isolated from rat heart, the total ATPase activity was 1512.5 +/- 23.3 nmol Pi/mg protein/min, nearly four times as high as in dog cardiac SR (408.8 +/- 28.9 nmol Pi/mg protein/min). The Ca2+-activated ATPase in rat cardiac SR represented only 23.8% of the total ATPase activity, while in dog cardiac SR it was approximately 50% of the total. Thus, the specific Ca2+-activated ATPase was nearly two times higher in the cardiac SR of the rat than in that of the dog. This higher rate of ATP hydrolysis in rat cardiac SR may be, at least in part, responsible for the increased intensity and shorter duration of the active state in the rat myocardium. Polyacrylamide gel electrophoresis of SR showed that the relative amount of Ca2+-pump protein was two times higher in dog heart, similar to the percentage of Ca2+-activated ATPase activity. At the same time, the specific Ca2+-activated ATPase activity and the relative amount of Ca2+ pump protein in both the rat and dog cardiac SR were inversely related.     

Comparison of (Ca2+-Mg2+)-ATPase and Ca2+-ATPase in rat cardiac sarcolemma

The calcium dependency of the Ca2+-pump ATPase of rat cardiac sarcolemma was investigated in the presence and absence of EGTA and EDTA in combination with two free Mg2+-ion concentrations. The results showed: that Mg2+-ions are not essential for the turnover of the Ca2+-pump ATPase; that the Ca2+-affinity is regulated by the concentration of the calcium-chelator complex present in the medium; that (Ca2+-Mg2+)-ATPase and Ca2+-ATPase are probably expressions of the same Ca2+-pump ATPase in the plasma membrane of the cell.     

Diabetic alterations in cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban protein expression.

Diabetic cardiomyopathy has been suggested to be caused by abnormal intracellular Ca2+ homeostasis in the myocardium, which is partly due to a defect in calcium transport by the cardiac sarcoplasmic reticulum (SR). In the present study, the underlying mechanism for this functional derangement was investigated with respect to SR Ca2+-ATPase and phospholamban (the inhibitor of SR Ca2+-ATPase). The maximal Ca2+ uptake and the affinity of Ca2+-ATPase for Ca2+ were decreased, and exogenous phosphorylation level of phospholamban was higher in streptozotocin-induced diabetic rat SR. Levels of both mRNA and protein of phospholamban were significantly increased in the diabetic hearts, whereas those of SR Ca2+-ATPase were significantly decreased. Consequently, the relative phospholamban/Ca2+-ATPase ratio was 1.88 in the diabetic hearts, and these changes were correlated with changes in the rates of SR Ca2+ uptake. However, phosphatase pretreatment of phospholamban for dephosphorylation of the sites phosphorylated in vivo did not change the levels of subsequent phospholamban phosphorylation in either control or diabetic rat hearts. The above data indicated that the increased phospholamban phosphorylation was not due to autonomic dysfunction but possibly due to increased phospholamban expression. These findings suggest that reduction of the SR Ca2+-ATPase level would contribute to decreased rates of SR Ca2+ uptake and that this function is further impaired by the enhanced inhibition by phospholamban due to its increased expression in the diabetic heart.     

白细胞介素-2对大鼠心肌Ca2+ATPase和Na+ /K+ATPase的影响

为了探讨IL－2对心肌细胞内钙影响的可能机制,用光学法检测心肌肌浆网Ca^2 ATPase的活性,以及细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性。结果：（1）用IL－2（10、40、200、800U/ml）灌流心脏后,其肌浆网Ca^2 ATPase的活性随IL－2浓度的升高而增强;（2）在ATP浓度为0.1-4mmol/L时,Ca^2 ATPase的活性随ATP浓度的升庙则增强,由IL－2（200U／ml）灌流后的心脏获得肌浆网（SR）,其Ca^2 ATPase的活性对ATP的反应强于对照组;（3）在[Ca^2 ]为1－40μmol/L时,心脏SR Ca^2 ATPase的活性随[Ca^2 ]增加而增强,而IL－2灌流心脏后分离的SR,其Ca^2 ATPase活性在[Ca^2 ]升高时没有明显改变;（4）用nor-BNI(10nmol/L）预处理5min后,IL－2（200U／ml）灌流后不再使SR Ca^2 ATPase的活性增强;（5）用PTX（5mg/L）预处理后,IL－2对SR Ca^2 ATPase的影响减弱;（6）用磷脂酶C（PLC）抑制剂U73122（5μmol/L）处理后,IL－2不再使SR Ca^2 ATPase活性增高;（7）用IL－2直接处理从正常大鼠分离的SR后,对SR Ca^2 ATPase活性无明显影响;（8）IL－2灌流后,对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase活性没有显著。上述结果表明,IL－2灌流心脏后使心肌肌浆网Ca^2 ATPase的活性增加,心肌细胞膜上的κ－阿片受体及其下游的G蛋白和PLC介导了IL－2的作用。尽管IL－2提高SR Ca^2 ATPase对ATP的反应性,但却抑制SR Ca^2 ATPase对钙离子的敏感性。IL－2对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性无明显影响。     

Analysis of sarcoplasmic reticulum Ca2+ transport and Ca2+ ATPase enzymatic properties using mouse cardiac tissue homogenates

Recent studies have focused on developing transgenic mouse models to explore the physiological roles of sarcoplasmic reticulum (SR) calcium handling proteins. The goal of this study was to develop methodology to measure SR Ca2+ transport function and enzymatic properties of SR Ca2+ ATPase (SERCA) in individual mouse hearts. We describe here the procedures to specifically measure SR Ca2+ uptake, the formation and decomposition of SERCA phosphoenzyme intermediate (E-P) in mouse cardiac homogenates. The specificity of SERCA enzymatic activity in cardiac homogenates was established by (a) the selective inhibition of SERCA enzyme by inhibitor-thapsigargin, and (b) comparison of the kinetic parameters of SERCA activity between homogenates and isolated microsomes. Here we show that the apparent affinity of SERCA for Ca2+ and ATP, the time to reach steady-state levels of E-P, and the rate of E-P decomposition (turnover rate of SERCA enzyme) are similar in homogenates and microsomes. These studies demonstrate that SERCA Ca2+ transport and enzymatic properties can be accurately measured in mouse cardiac tissue homogenates. Additionally, we show that frozen cardiac homogenates can be used without significant loss of enzymatic activity. In conclusion, we have developed and established the methods to employ tissue homogenates to study SR Ca2+ transport function in individual mouse hearts.     

Membrane crystals of Ca2+-ATPase in sarcoplasmic reticulum of fast and slow skeletal and cardiac muscles

Crystalline arrays of Ca2+ transport ATPase develop in sarcoplasmic reticulum membranes after treatment with Na3VO4 in a calcium-free medium [ Dux , L. and Martonosi , A. (1983) J. Biol. Chem. 258, 2599-2603]. The proportion of vesicles containing Ca2+-ATPase crystals in microsome preparations isolated from rat muscle of different fiber types (semimembranosus, levator ani, extensor digitorum longus, diaphragm, soleus, and heart) correlates well with the Ca2+-ATPase content and Ca2+-modulated ATPase activity. This implies that the concentration of Ca2+-ATPase in sarcoplasmic reticulum membranes of fast and slow skeletal or cardiac muscles differs only slightly, and the low Ca2+ transport activity of 'sarcoplasmic reticulum' preparations isolated from slow-twitch skeletal and cardiac muscles is due to the presence of large amount of non-sarcoplasmic-reticulum membrane elements. This is in accord with the relatively small differences in the density of 8.5-nm intramembranous particles seen by freeze-etch electron microscopy in sarcoplasmic reticulum of red and white muscles. The dimensions of the Ca2+-ATPase crystal lattice are similar in sarcoplasmic reticulum membranes of different fiber types; therefore if structural differences exist between 'isoenzymes' of Ca2+-ATPase, these are not reflected in the crystal-lattice.     

Tricyclohexyltin hydroxide effects on cationic and substrate activation kinetics of beta-adrenergic-stimulated cardiac Ca2+-ATPase

Previous studies from this laboratory have indicated that tricyclohexyltin hydroxide (Plictran) is a potent inhibitor of both basal- and isoproterenol-stimulated cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase, with an estimated IC-50 of 2.5 X 10(-8) M. The present studies were initiated to evaluate the mechanism of inhibition of Ca2+-ATPase by Plictran. Data on substrate and cationic activation kinetics of Ca2+-ATPase indicated alteration of Vmax and Km by Plictran (1 and 5 X 10(-8) M), suggesting a mixed type of inhibition. The beta-adrenergic agonist isoproterenol increased Vmax of both ATP- and Ca2+-dependent enzyme activities. However, the Km of enzyme was decreased only for Ca2+. Plictran inhibited isoproterenol-stimulated Ca2+-ATPase activity by altering both Vmax and Km of ATP as well as Ca2+-dependent enzyme activities, suggesting that after binding to a single independent site, Plictran inhibits enzyme catalysis by decreasing the affinity of enzyme for ATP as well as for Ca2+. Preincubation of enzyme with 15 microM cAMP or the addition of 2mM ATP to the reaction mixture resulted in slight activation of Plictran-inhibited enzyme. Pretreatment of SR with 5 X 10(-7) M propranolol and 5 X 10(-8) M Plictran resulted in inhibition of basal activity in addition to the loss of stimulated activity. Preincubation of heart SR preparation with 5 X 10(-5) M coenzyme A in combination with 5 X 10(-8) M Plictran partly restored the beta-adrenergic stimulation. These results suggest that some critical sites common to both basal- and beta-adrenergic-stimulated Ca2+-ATPase are sensitive to binding by Plictran, and the resultant conformational change may lead to inhibition of beta-adrenergic stimulation.     

Brain Ca2+/Mg(2+)-ATPase activity and seasonal adaptation of the Djungarian dwarf hamster Phodopus sungorus.

1. Of three sets of Djungarian dwarf hamster, two groups were raised during winter under greatly differing circumstances. One winter group was raised within a climate controlled cage in which the ambient temperature was maintained at 22 degrees C and whereby conditions of light vs darkness were maintained in a constant 12 hr cycle. The second winter group was raised out of doors whereby the hamsters were subjected to prevailing seasonal environmental conditions. A third group was studied under summer conditions, as well. Ca(2+)-, Mg(2+)- and (Ca2+/Mg2+)-ATPase activity was analysed in cellular (= total homogenate) and subcellular fractions (P1-, synaptosomal fraction, synaptic membranes) from cortex, cerebellum and basal brain. 2. The data obtained indicate similar ATPase activity in the cortical homogenates of the winter indoor and summer hamsters. 3. Winter outdoor animals experiencing normal torpidity, however, exhibited reduced ATPase activity by about 50%. 4. Cortical subcellular fractions yielded different results: both the winter and the summer groups showed high ATPase activity in the synaptosomal and synaptic membrane fractions. 5. In the total cerebellar homogenate, the hamsters raised under summer and winter conditions showed the greatest enzyme activity, although less activity was seen in the subcellular fractions. 6. The ATPase activity in the basal brain was found to be nearly identical in all three hamster groups.     

Ischemia-induced structural change in SR Ca2+-ATPase is associated with reduced enzyme activity in rat muscle

In this study, we employed an in vivo model of prolonged ischemia in rat skeletal muscle to investigate the hypothesis that structural modifications to the sarcoplasmic reticulum (SR) Ca2+-ATPase can explain the alterations in Ca2+-ATPase activity that occur with ischemia. To induce total ischemia, a tourniquet was placed around the upper hindlimb in 27 female Sprague-Dawley rats weighing 256 +/- 6.7 g (mean +/- SE) and was inflated to 350 mmHg for 4 h. The contralateral limb served as control (C) to the ischemic limb (I), and the limbs of animals killed immediately after anesthetization served as a double control (CC). Mixed gastrocnemius and tibialis anterior muscles were sampled and used for SR vesicle preparation. Maximal Ca2+-ATPase activity (micromol x g protein(-1) x min(-1)) of C (15,802 +/- 1,246) and I (11,609 +/- 1,029) was 90 and 73% (P < 0.05) of CC (17,562 +/- 1,682), respectively. No differences were found between groups in either the Hill coefficient or the free Ca2+ at half-maximal activity. The fluorescent probes, FITC and N-cyclohexyl-N'-(dimethylamino-alpha-naphthyl) carbodiimide, used to assess structural alterations in the regions of the ATP binding site and the Ca2+ binding sites of the Ca2+-ATPase, respectively, indicated a 26% reduction (P < 0.05) in FITC binding capacity (absolute units) in I (0.22 +/- 0.01) compared with CC (0.29 +/- 0.02) and C (0.29 +/- 0.03). Our results suggest that the reduction in maximal SR Ca2+-ATPase activity in SR vesicles with ischemia is related to structural modification in the region of the nucleotide binding domain by mechanisms that are as yet unclear.     

Cholesterol effect on enzyme activity of the sarcolemmal (Ca2+ + Mg2+)-ATPase from cardiac muscle

The effect of cholesterol incorporation and depletion of the cardiac sarcolemmal sacs on (Ca2+ + Mg2+)-ATPase activity was examined. Cholesterol incorporation to the sarcolemmal sacs was achieved utilizing an in vivo and an in vitro procedure. Cholesterol depleted membranes were obtained in vitro after incubation of the sarcolemmal sacs with inactivated plasma. Arrhenius plots of the (Ca2+ + Mg2+)-ATPase activity showed a triphasic curve when the assays were carried out using a temperature range between 0 and 40 degrees C. The sarcolemmal (Ca2+ + Mg2+)-ATPase activity was shown to be inversely proportional to the cholesterol concentration of the membranes, showing a low ATPase activity with a high cholesterol content and a high ATPase activity when the cholesterol concentration was low. Although the (Ca2+ + Mg2+)-ATPase activity was found to be inhibited in the cholesterol incorporated sarcolemmal sacs, the withdrawal of small amounts of cholesterol from the membranes produced an important stimulatory effect. Changes in (Ca2+ + Mg2+)-ATPase activity due to variation in the membrane cholesterol concentration were shown to be reversible. Our results indicate the possibility of a slow exchange of cholesterol between the tightly bound lipid surrounding the (Ca2+ + Mg2+)-ATPase and the bulk lipid of the sarcolemma.     

Monoclonal antibodies to dog heart sarcoplasmic reticulum. Antibodies that inhibit Ca2+-pump systems of cardiac and skeletal muscles

Purified sarcoplasmic reticulum (SR) vesicles from dog heart were used as an antigen to produce monoclonal antibodies (mAbs) to the Ca2+-ATPase. Nine of twelve clones of hybridoma cells produce mAbs which cross-react with seven SR preparation isolated from cardiac and skeletal muscles of various species. Three mAbs of IgM type interact with the 45-kDa tryptic fragment of rabbit skeletal muscle Ca2+-ATPase and markedly inhibit Ca2+ uptake (by 95%) and ATPase activity (by 80%) and decrease (by 30-50%) the steady-state level of the Ca2+-ATPase phosphoenzyme. The ATPase activity could be completely blocked by one of these mAbs if the incubation medium was supplemented with 2 microM orthovanadate. On the other hand, when SR vesicles were treated with increasing concentrations of a nonionic detergent C12E8, the inhibiting effect of mAb 4B4 is diminished. It is concluded that the mAbs inhibit the Ca2+-ATPase only if the enzyme exists in an oligomeric form. The inhibition of the SR activities is due to an effect of the mAbs on the whole active center of the enzyme, rather than on a single partial reaction.     

Proton pump-linked Mg2+-ATPase activity in isolated rat liver lysosomes

ATPase activity in highly purified rat liver lysosome preparations was evaluated in the presence of other membrane cellular ATPase inhibitors, and compared with lysosome ATP-driven proton translocating activity. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 50% inhibition in divalent cation-dependent ATPase activity, and an 80% inactivation of ATP-linked lysosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+, ATPase activity was similar to that seen in an Mg2+ medium. Mg2+-dependent ATPase activity was greatly inhibited (from 70 to 80%) by the platinum complexes; cis-didimethylsulfoxide dichloroplatinum(II) (CDDP) at approximately 90 microM and cis-diaminedichloroplatinum(II) at twofold higher concentrations. Less inhibition, about 30 and 45%, was obtained with N,N'-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. The concentration dependence of inhibition by the above drugs was determined for both proton pumping and ATPase activities, and half-maximal inhibition concentration of each activity was found at nearly similar values. A micromolar concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented ATP from setting up a pH gradient across the lysosomal membranes, but stimulated Mg2+-ATPase activity significantly. ATPase activity in Ca2+ medium was also inhibited by CDDP and stimulated by FCCP, but both effects were two- to threefold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in a CDDP-supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton pump, was anion dependent. The lowest activity was recorded in a F-medium, and increased in the order of F- less than SO2-4 less than Cl- approximately equal to Br-. The CDDP-sensitive ATPase activity observed, supported by Mg2+ and less so by Ca2+, may be related to lysosome proton pump activity.     

Postnatal development of Ca2+-sequestration by the sarcoplasmic reticulum of fast and slow muscles in normal and dystrophic mice

Ca2+-uptake activities of the sarcoplasmic reticulum (SR) were determined with a Ca2+-sensitive electrode in homogenates from fast- and slow-twitch muscles from both normal and dystrophic mice (C57BL/6J strain) of different ages. Immunochemical quantification of tissue Ca2+-ATPase content allowed determination of the specific Ca2+-transport activity of the enzyme. In 3-week-old mice of the dystrophic strain specific Ca2+ transport was already significantly lower than in the normal strain. It progressively decreased with maturation and reached only 40-50% and 30-50% of the normal values in fast- and slow-twitch muscles of adult dystrophic animals, respectively. Tissue contents of calsequestrin were reduced in both types of muscle leading to an increased Ca2+-ATPase to calsequestrin protein ratio. Equal amounts of the Ca2+-ATPase protein (detected by Coomassie blue staining of polyacrylamide gels) were present in SR vesicles isolated by Ca2+-oxalate loading from adult normal and dystrophic fast-twitch muscles. However, the specific ATP-hydrolysing activity of the enzyme was approximately 50% lower in dystrophic than in normal SR. The reduced ATP-hydrolysing activity was correlated with decreased Ca2+-transport activity, phosphoprotein formation and fluorescein isothiocyanate labeling as determined in total microsomal and heavy SR fractions. Although the Ca2+ and ATP affinities of the enzyme were unaltered, its ATPase activity was reduced at all levels of ATP in the dystrophic SR. Taken together, these findings point to a markedly impaired function of the SR and an increase in the population of inactive SR Ca2+-ATPase molecules in murine muscular dystrophy.     

Interaction of myotoxin a with the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum

Myotoxin a is a muscle-damaging toxin isolated from the venom of Crotalus viridis viridis. Its interaction with the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles purified from rabbit skeletal muscle was investigated. Myotoxin a inhibited Ca2+ loading and stimulated Ca2+-dependent ATPase without affecting unidirectional Ca2+ efflux. Its action was dose, time, and temperature dependent. Myotoxin a partially blocked the binding of specific anti-(rabbit SR Ca2+-ATPase) antibodies. It is concluded that myotoxin a attaches to the SR Ca2+-ATPase and uncouples Ca2+ uptake from Ca2+-dependent ATP hydrolysis. Myotoxin a also prevented the formation of decavanadate-induced two-dimensional crystalline arrays of the SR Ca2+-ATPase.