Phosphoenzyme conformational states and nucleotide-binding site hydrophobicity following thiol modification of the Ca2+-ATPase of sarcoplasmic reticulum from skeletal muscle

Enhanced fluorescence of the ATP analogue 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)adenosine 5'-triphosphate (TNP-ATP), bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, is closely related to phosphoenzyme levels (Bishop, J. E., Johnson, J. D., and Berman, M. C. (1984) J. Biol. Chem. 259, 15163-15171) and has an emission maximum consistent with decreased polarity of the TNP-ATP-binding site. The phosphoenzyme conformation responsible for increased nucleotide-binding site hydrophobicity has been studied by redistribution of phosphoenzyme intermediates following specific thiol group modification. N-Ethylmaleimide, in the presence of 50 microM Ca2+, 1 mM adenyl-5'-yl imidodiphosphate, pH 7.0, at 25 degrees C for 30 min, selectively modified the SH group essential for phosphoenzyme decomposition, which resulted in decreased ATPase activity, Ca2+ uptake, and a decrease in ATP-induced TNP-ATP fluorescence. Phosphorylated (Ca2+, Mg2+)-ATPase levels from [gamma-32P] ATP remained relatively unaffected (3.1 nmol/mg), but the ADP-insensitive fraction decreased from 56 to 15%. Phosphoenzyme levels from 32Pi were also decreased to the same extent as turnover, with equivalent loss of Pi-induced TNP-ATP fluorescence. The E1 to E2 transition, as monitored by the change in intrinsic tryptophan fluorescence, was unaffected. Modification of thiol groups of unknown function did not modify turnover-induced TNP-ATP fluorescence. It is concluded that the ADP-insensitive phosphoenzyme, E2-P, is responsible for enhanced TNP-ATP fluorescence. This suggests that the conformational transition, 2Ca2+outE1 approximately P----2Ca2+inE2-P, is associated with altered properties of the noncatalytic, or regulatory, nucleotide-binding site.     

Mechanism of the stimulation of cardiac sarcoplasmic reticulum calcium pump by calmodulin

Calmodulin has been shown to stimulate the initial rates of Ca2+-uptake and Ca2+-ATPase in cardiac sarcoplasmic reticulum, when it is present in the reaction assay media for these activities. To determine whether the stimulatory effect of calmodulin is mediated directly through its interaction with the Ca2+-ATPase, or indirectly through phosphorylation of phospholamban by an endogenous protein kinase, two approaches were taken in the present study. In the first approach, the effects of calmodulin were studied on a Ca2+-ATPase preparation, isolated from cardiac sarcoplasmic reticulum, which was essentially free of phospholamban. The enzyme was preincubated with various concentrations of calmodulin at 0 degrees C and 37 degrees C, but there was no effect on the Ca2+-ATPase activity assayed over a wide range of [Ca2+] (0.1-10 microM). In the second approach, cardiac sarcoplasmic reticulum vesicles were prephosphorylated by an endogenous protein kinase in the presence of calmodulin. Phosphorylation occurred predominantly on phospholamban, an oligomeric proteolipid. The sarcoplasmic reticulum vesicles were washed prior to assaying for Ca2+ uptake and Ca2+-ATPase activity in order to remove the added calmodulin. Phosphorylation of phospholamban enhanced the initial rates of Ca2+-uptake and Ca2+-ATPase, and this stimulation was associated with an increase in the affinity of the Ca2+-pump for calcium. The EC50 values for calcium activation of Ca2+-uptake and Ca2+-ATPase were 0.96 +/- 0.03 microM and 0.96 +/- 0.1 microM calcium by control vesicles, respectively. Phosphorylation decreased these values to 0.64 +/- 0.12 microM calcium for Ca2+-uptake and 0.62 +/- 0.11 microM calcium for Ca2+-ATPase. The stimulatory effect was associated with increases in the apparent initial rates of formation and decomposition of the phosphorylated intermediate of the Ca2+-ATPase. These findings suggest that calmodulin regulates cardiac sarcoplasmic reticulum function by protein kinase-mediated phosphorylation of phospholamban.     

Evidence that platelet and skeletal sarcoplasmic reticulum Ca2+-ATPase are structurally distinct

Proteolytic digestion and indirect immunostaining were used to compare the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins. When the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins were digested in the native state with trypsin, the platelet Ca2+-ATPase, which had an apparent undigested molecular mass of 103 kDa, yielded 78-kDa and 25-kDa fragments. Calcium transport activity depended on the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic digestion of the sarcoplasmic reticulum pump protein, which also had an undigested molecular mass of 103 kDa, yielded products with apparent molecular masses of 55 kDa, 36 kDa, and 26 kDa. Distinct patterns were also observed when the platelet and sarcoplasmic reticulum calcium pump proteins were digested with chymotrypsin and Staphylococcus aureus protease in the presence of sodium dodecyl sulfate. Chymotrypsin digestion of the platelet protein resulted in the appearance of products with apparent molecular masses of 70 kDa, 39 kDa, and 31 kDa, while a similar digestion of the sarcoplasmic reticulum calcium pump protein yielded 54-kDa, 52.5-kDa, 46-kDa, 41-kDa, and 36-kDa fragments. Exposure of the sarcoplasmic reticulum and platelet Ca2+-ATPase proteins to S. aureus protease also yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPases from platelets and sarcoplasmic reticulum are distinct proteins.     

Interaction of valinomycin and monovalent cations with the (Ca2+,Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum

The interactions of monovalent cations and of the K+-specific ionophore, valinomycin, with the Ca2+-ATPase of skeletal muscle of sarcoplasmic reticulum have been studied in the absence of cation gradients by their effects on enzyme turnover and on the ATP plus Ca2+-dependent enhanced fluorescence of the ATP analogue, 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine 5'-triphosphate (TNP-ATP) (Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Monovalent cations decreased turnover-dependent TNP-ATP fluorescence in the series K+ greater than Rb+ approximately equal to Cs+ greater than Na+ greater than Li+ (K0.5 = 49, 73, 75, 94, and 246 mM, respectively), consistent with the known specificity of the monovalent cation binding site that stimulates turnover and E-P hydrolysis. Valinomycin (200 nmol/mg), in the absence of monovalent cations, decreased ATPase activity by 30% and abolished the stimulatory effects of 150 mM KCl or NaCl on turnover. The ionophore alone enhanced TNP-ATP fluorescence by 20% and altered the specificity and affinity of the site that inhibited TNP-ATP fluorescence to Cs+ greater than Rb+ greater than K+ approximately equal to Na+ greater than Li+ (K0.5 = 79, 111, 134, 136, and 270 mM, respectively), which follows the Hofmeister series for effectiveness of monovalent lyotropic cations. TNP-ATP binding was not affected by either monovalent cations or valinomycin. Inhibition of turnover-dependent TNP-ATP fluorescence appears to be a useful parameter for monitoring monovalent cation binding to the Ca2+-ATPase. It is concluded that the ionophore interacts directly with the Ca2+-ATPase, independent of its K+ conductance effects on the lipid bilayer, and modifies the affinity and specificity of the monovalent cation site, either by direct interaction or by the formation of a valinomycin-monovalent cation-enzyme complex.     

Characterization of the tryptophan fluorescence from sarcoplasmic reticulum adenosinetriphosphatase by frequency-domain fluorescence spectroscopy

We examined the tryptophan decay kinetics of sarcoplasmic reticulum Ca2+-ATPase using frequency-domain fluorescence. Consistent with earlier reports on steady-state fluorescence intensity, our intensity decays reveal a reproducible and statistically significant 2% increase in the mean decay time due to calcium binding to specific sites involved in enzyme activation. This Ca2+ effect could not be eliminated with acrylamide quenching, which suggests a global effect of calcium on the Ca2+-ATPase, as opposed to a specific effect on a single water-accessible tryptophan residue. The tryptophan anisotropy decays indicate substantial rapid loss of anisotropy, which can be the result of either intramolecular energy transfer or a change in segmental flexibility of the ATPase protein. Energy transfer from tryptophan to TNP-ATP in the nucleotide binding domain, or to IEADANS on Cys-670 and -674, indicates that most tryptophan residues are 30 A or further away from these sites and that this distance is not decreased by Ca2+. In light of known structural features of the Ca2+-ATPase, the tryptophan fluorescence changes are attributed to stabilization of clustered transmembrane helices resulting from calcium binding.     

Evidence of endoplasmic reticulum-related Ca2+ ATPase in human microvascular endothelial cells

We have demonstrated by immunological and molecular methods the presence of a reticulum endoplasmic-related Ca2+-ATPase in human omental microvascular endothelial cells (HOME cells). HOME cells reacted positively with a previously characterized sarcoplasmic reticulum Ca2+-ATPase antibody as demonstrated by indirect immunofluorescence. Western blotting revealed that the antibody recognized a 95-100 kDa protein. 35S-Metabolic labeling led to the detection of a similar protein with which the purified sarcoplasmic reticulum Ca2+-ATPase competed. Dot-blotting experiments indicated that a substantial amount of Ca2+-ATPase was present in HOME cell membranes. In addition, Northern blot analysis using a cDNA probe from cardiac sarcoplasmic reticulum showed the presence of mRNA species of 4-kb. As these experiments were conducted in comparison with cell types with well-defined Ca2+-ATPase in HOME cells.     

Influence of monovalent cations on the Ca2+-ATPase of sarcoplasmic reticulum isolated from rabbit skeletal and dog cardiac muscles. An interpretation of transient-state kinetic data

Transient-state kinetics of phosphorylation and dephosphorylation of the Ca2+-ATPase of sarcoplasmic reticulum vesicles from rabbit skeletal and dog cardiac muscles were studied in the presence of varying concentrations of monovalent and divalent cations. Monovalent cations affect the two types of sarcoplasmic reticulum differently. When the rabbit skeletal sarcoplasmic reticulum was Ca2+ deficient, preincubation with K+ (as compared with preincubation with choline chloride) did not affect initial phosphorylation at various concentrations of Ca2+, added with ATP to phosphorylate the enzyme. This is in contrast to preincubation with K+ of the Ca2+-deficient dog cardiac sarcoplasmic reticulum, which resulted in an increase in the phosphoenzyme level. When Ca2+ was bound to the rabbit skeletal sarcoplasmic reticulum, K+ inhibited E - P formation; but under the same conditions, E - P formation of dog cardiac sarcoplasmic reticulum was activated by K+ at 12 microM Ca2+ and inhibited at 0.33 and 1.3 microM Ca2+. Li+, Na+ and K+ also have different effects on E - P decomposition of skeletal and cardiac sarcoplasmic reticulum. The latter responded less to these cations than the former. Studies with ADP revealed differences between the two types of sarcoplasmic reticulum. For rabbit skeletal sarcoplasmic reticulum, 40% of the phosphoenzyme formed was 'ADP sensitive', and the decay of the remaining E - P was enhanced by K+ and ADP. Dog cardiac sarcoplasmic reticulum yielded about 40--48% ADP-sensitive E - P, but the decomposition rate of the remaining E - P was close to the rate measured in the absence of ADP. Thus, these studies showed certain qualitative differences in the transformation and decomposition of phosphoenzymes between skeletal and cardiac muscle which may have bearing on physiological differences between the two muscle types.     

(Ca2+ + Mg2+)-ATPase activity associated with the maintenance of a Ca2+ gradient by sarcoplasmic reticulum at submicromolar external [Ca2+]. The effect of hypothyroidism

The formation and maintenance of Ca2+-filling levels by sarcoplasmic reticulum vesicles from euthyroid (control) and hypothyroid skeletal muscle were investigated using the Ca2+-indicator quin-2, at [Ca2+] in the medium [( Cao2+]) of 0.05-0.3 microM. Rapid ATP-dependent Ca2+ uptake resulted in a steady-state Ca2+-filling level, Cai2+, within one minute. This Ca2+ gradient was maintained for at least three minutes, during which less than 20% of the ATP was consumed. Cai2+ was maximal (120 nmol/mg) for [Cao2+] greater than 0.3 microM and decreased to 40 nmol/mg at [Cao2+] of 0.05 microM. Preparations from both experimental groups showed qualitatively and quantitatively the same relationship between Cai2+ and [Cao2+] at steady state, despite a significantly lower Ca2+-pump content of hypothyroid sarcoplasmic reticulum, which resulted in a 25% lower maximal (Ca2+ + Mg2+)-ATPase activity. Maintenance of the steady state, at all levels of Cai2+, was associated with net ATP consumption by the Ca2+ pump and cycling of Ca2+, which processes were 30% slower in the hypothyroid group as compared to the control group. Determination of the passive efflux of Ca2+, as well as the fraction of leaky or unsealed sarcoplasmic reticulum fragments, excluded either of these possibilities as an explanation for the relatively high (Ca2+ + Mg2+)-ATPase rates at steady state. On the basis of these and previously reported results, it is concluded that the maintenance of a Ca2+ gradient by sarcoplasmic reticulum under physiological conditions with respect to external [Ca2+] and the concentrations of ATP, ADP and Pi, is associated with the cycling of Ca2+ coupled to net ATP hydrolysis. Using the obtained data it is calculated that the sarcoplasmic reticulum may account for 20% of the resting metabolic rate in skeletal muscle. Consequently, together with the previously reported lower sarcoplasmic reticulum content of skeletal muscle in hypothyroidism, we calculate that about one third of the decrease in basal metabolic rate in this thyroid state can be related to the alterations of the sarcoplasmic reticulum.     

An investigation of functional similarities between the sarcoplasmic reticulum and platelet calcium-dependent adenosinetriphosphatases with the inhibitors quercetin and calmidazolium

The platelet and skeletal sarcoplasmic reticulum calcium-dependent adenosinetriphosphatases (Ca2+-ATPases) were functionally compared with respect to substrate activation by steady-state kinetic methods using the inhibitors quercetin and calmidazolium. Quercetin inhibited platelet and sarcoplasmic reticulum Ca2+-ATPase activities in a dose-dependent manner with IC50 values of 25 and 10 microM, respectively. Calmidazolium also inhibited platelet and sarcoplasmic reticulum Ca2+-ATPase activities, with half-maximal inhibition measured at 5 and 4 microM, respectively. Both inhibitors also affected the calcium transport activity of intact platelet microsomes at concentrations similar to those which reduced Ca2+-ATPase activity. These inhibitors were then used to examine substrate ligation by the platelet and sarcoplasmic reticulum calcium pump proteins. For both Ca2+-ATPase proteins, quercetin has an affinity for the E-Ca2 (fully ligated with respect to calcium at the exterior high-affinity calcium binding sites, unligated with respect to ATP) conformational state of the protein that is approximately 10-fold greater than for other conformational states in the hydrolytic cycle. Quercetin can thus be considered a competitive inhibitor of the calcium pump proteins with respect to ATP. In contrast to the effect of quercetin, calmidazolium interacts with the platelet and sarcoplasmic reticulum Ca2+-ATPases in an uncompetitive manner. The dissociation constants for this inhibitor for the different conformational states of the calcium pump proteins were similar, indicating that calmidazolium has equal affinity for all of the reaction intermediates probed. These observations indicate that the substrate ligation processes are similar for the two pump proteins. This supports the concept that the hydrolytic cycles of the two proteins are comparable.     

Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.     

Inhibition of calcium-dependent ATPase from sarcoplasmic reticulum by a new class of indolizidine alkaloids, pumiliotoxins A, B, and 251D

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

Purification, calcium binding properties, and ultrastructural localization of the 53,000- and 160,000 (sarcalumenin)-dalton glycoproteins of the sarcoplasmic reticulum

The 53-kDa glycoprotein and sarcalumenin (160-kDa glycoprotein) were extracted from rabbit skeletal muscle sarcoplasmic reticulum with EGTA and purified by fractionation on DEAE-Sephadex A-25 and lentil lectin-Sepharose 4B. Sarcalumenin was shown to bind up to 400 nmol of Ca2+/mg of protein at pH 7.5, which is equivalent to binding of approximately 35 mol of Ca2+/mol of protein. The apparent dissociation constant was 300 microM in the presence of 20 mM KCl and 600 microM in 150 mM KCl. The 53-kDa glycoprotein did not bind any Ca2+ under the conditions examined. Immunoblot analysis of isolated sarcoplasmic reticulum subfractions demonstrated the presence of the two glycoproteins in both the longitudinal sarcoplasmic reticulum and the terminal cisternae. Their concentrations were higher, however, in the longitudinal sarcoplasmic reticulum vesicles. Comparative immunoelectron microscopic studies using monoclonal antibodies revealed a codistribution of the 53-kDa glycoprotein with the Ca2(+)-ATPase in all regions of the free sarcoplasmic reticulum. A similar distribution was found for sarcalumenin, although immunolabeling was much weaker. The colocalization of the 53-kDa glycoprotein and sarcalumenin with the Ca2(+)-ATPase and the Ca2+ binding properties of sarcalumenin suggest that the glycoproteins may be involved in the sequestration of Ca2+ in the nonjunctional regions of the sarcoplasmic reticulum.     

A monoclonal antibody to the Ca2+-ATPase of cardiac sarcoplasmic reticulum cross-reacts with slow type I but not with fast type II canine skeletal muscle fibers: an immunocytochemical and immunochemical study

Ca2+-ATPase of the sarcoplasmic reticulum was localized in cryostat sections from three different adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with monoclonal antibodies to the Ca2+-ATPase. Type I (slow) myofibers were strongly labeled for the Ca2+-ATPase with a monoclonal antibody (II D8) to the Ca2+-ATPase of canine cardiac sarcoplasmic reticulum; the type II (fast) myofibers were labeled at the level of the background with monoclonal antibody II D8. By contrast, type II (fast) myofibers were strongly labeled for Ca2+-ATPase of rabbit skeletal sarcoplasmic reticulum. The subcellular distribution of the immunolabeling in type I (slow) myofibers with monoclonal antibody II D8 corresponded to that of the sarcoplasmic reticulum as previously determined by electron microscopy. The structural similarity between the canine cardiac Ca2+-ATPase present in the sarcoplasmic reticulum of the canine slow skeletal muscle fibers was demonstrated by immunoblotting. Monoclonal antibody (II D8) to the cardiac Ca2+-ATPase binds to only one protein band present in the extract from either cardiac or type I (slow) skeletal muscle tissue. By contrast, monoclonal antibody (II H11) to the skeletal type II (fast) Ca2+-ATPase binds only one protein band in the extract from type II (fast) skeletal muscle tissue. These immunopositive proteins coelectrophoresed with the Ca2+-ATPase of the canine cardiac sarcoplasmic reticulum and showed an apparent Mr of 115,000. It is concluded that the Ca2+-ATPase of cardiac and type I (slow) skeletal sarcoplasmic reticulum have at least one epitope in common, which is not present on the Ca2+-ATPase of sarcoplasmic reticulum in type II (fast) skeletal myofibers. It is possible that this site is related to the assumed necessity of the Ca2+-ATPase of the sarcoplasmic reticulum in cardiac and type I (slow) skeletal myofibers to interact with phosphorylated phospholamban and thereby enhance the accumulation of Ca2+ in the lumen of the sarcoplasmic reticulum following beta-adrenergic stimulation.     

Stimulated human neutrophils damage cardiac sarcoplasmic reticulum function by generation of oxidants

An important aspect of myocardial injury is the role of neutrophils in post-ischemic damage to the heart. Stimulated neutrophils initiate a series of reactions that produce toxic oxidizing agents. Superoxide rapidly dismutases to H2O2 and neutrophils contain myeloperoxidase which catalyzes the oxidation of Cl- by H2O2 to yield hypochlorous acid (HOCl). The highly reactive HOCl combines non-enzymatically with nitrogenous compounds to generate long-lived, non-radical oxidants, monochloramine and taurine N-monochloramine. We investigated the role of oxygen radicals and long-lived oxidants on cardiac sarcoplasmic reticulum function, which plays a major role in the regulation of intracellular Ca2+ and thereby in the generation of force. Incubation of sarcoplasmic reticulum with phorbol myristate acetate (PMA)-stimulated neutrophils (4 x 10(6) cells/ml) significantly decreased calcium uptake rate (0.85 +/- 0.11 to 0.11 +/- 0.06 mumol/min per mg) and Ca2+-ATPase activity (1.67 +/- 0.08 to 0.46 +/- 0.10 mumol/min per mg). Inclusion of myeloperoxidase inhibitors (cyanide, sodium azide and 3-amino-1,2,4-triazole), catalase, superoxide dismutase plus catalase, and alpha-tocopherol significantly protected (P less than 0.01) calcium uptake rates and Ca2+-ATPase activity of sarcoplasmic reticulum. Superoxide dismutase (10 microgram/ml) alone or deferoxamine (1 mM) had no protective effect in this system. The maximum inhibition of sarcoplasmic reticulum function was observed with (3-4) x 10(6) cells/ml in 4-6 min. HOCl and NH2Cl inhibited calcium uptake rate and Ca2+-ATPase activity of sarcoplasmic reticulum in a dose-dependent manner (2-20 microM), whereas H2O2 damaged sarcoplasmic reticulum at concentrations ranging from 5 to 25 mM. HOCl (20 microM) inhibited 80-90% of Ca2+-uptake rate and Ca2+-ATPase activity and L-methionine (0.1-1 mM) provided complete protection. We conclude that stimulated neutrophils damage cardiac sarcoplasmic function by generation of myeloperoxidase-catalyzed oxidants.     

Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes.

Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar studies using mixed membranes revealed four other phosphoproteins (50, 40, 20 and 18 kDa) formed in addition to the 90 and 95 kDa components. The findings are discussed in the context of platelet Ca2+ mobilization for function and the mechanisms whereby Ca2+ homoeostasis is controlled in the unactivated cell.     

Enhanced Ca2+-induced calcium release by isolated sarcoplasmic reticulum vesicles from malignant hyperthermia susceptible pig muscle

To further define the possible involvement of sarcoplasmic reticulum calcium accumulation and release in the skeletal muscle disorder malignant hyperthermia (MH), we have examined various properties of sarcoplasmic reticulum fractions isolated from normal and MH-susceptible pig muscle. A sarcoplasmic reticulum preparation enriched in vesicles derived from the terminal cisternae, was further fractionated on discontinuous sucrose density gradients (Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374). The resultant MH-susceptible and normal sarcoplasmic reticulum fractions, designated F0-F4, did not differ in yield, cholesterol and phospholipid content, or nitrendipine binding capacity. Calcium accumulation (0.27 mumol Ca/mg per min at 22 degrees C), Ca2+-ATPase activity (0.98 mumol Pi/mg per min at 22 degrees C), and calsequestrin content were also similar for MH-susceptible and normal sarcoplasmic reticulum fraction F3. To examine sarcoplasmic reticulum calcium release, fraction F3 vesicles were passively loaded with 45Ca (approx. 40 nmol Ca/mg), and rapidly diluted into a medium of defined Ca2+ concentration. Upon dilution into 1 microM Ca2+, the extent of Ca2+-dependent calcium release measured after 5 s was significantly greater for MH-susceptible than for normal sarcoplasmic reticulum, 65.9 +/- 2.8% vs. 47.7 +/- 3.9% of the loaded calcium, respectively. The C1/2 for Ca2+ stimulation of this calcium release (5 s value) from MH-susceptible sarcoplasmic reticulum also appeared to be shifted towards a higher Ca2+-sensitivity when compared to normal sarcoplasmic reticulum. Dantrolene had no effect on calcium release from fraction F3, however, halothane (0.1-0.5 mM) increased the extent of calcium release (5 s) similarly in both MH-susceptible and normal sarcoplasmic reticulum. Furthermore, Mg2+ was less effective at inhibiting, while ATP and caffeine were more effective in stimulating, this Ca2+-dependent release of calcium from MH-susceptible, when compared to normal sarcoplasmic reticulum. Our results demonstrate that while sarcoplasmic reticulum calcium-accumulation appears unaffected in MH, aspect(s) of the sarcoplasmic reticulum Ca2+-induced calcium release mechanism are altered. Although the role of the Ca2+-induced calcium release mechanism of sarcoplasmic reticulum in situ is not yet clear, our results suggest that an abnormality in the regulation of sarcoplasmic reticulum calcium release may play an important role in the MH syndrome.     

Mechanism of the stimulation of Ca2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase

Sarcoplasmic reticulum isolated from moderately fast rabbit skeletal muscle contains intrinsic adenosine 3',5'-monophosphate (cAMP)-independent protein kinase activity and a substrate of 100 000 Mr. Phosphorylation of skeletal sarcoplasmic reticulum by either endogenous membrane bound or exogenous cAMP-dependent protein kinase results in stimulation of the initial rates of Ca2+ transport and Ca2+-ATPase activity. To determine the molecular mechanism by which protein kinase-dependent phosphorylation regulates the calcium pump in skeletal sarcoplasmic reticulum, we examined the effects of protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Skeletal sarcoplasmic reticulum vesicles were preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated sarcoplasmic reticulum) and absence (control sarcoplasmic reticulum) of adenosine 5'-triphosphate (ATP). Control and phosphorylated sarcoplasmic reticulum were subsequently assayed for formation (5-100 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase. Protein kinase mediated phosphorylation of skeletal sarcoplasmic reticulum resulted in pronounced stimulation of initial rates and levels of E approximately P in sarcoplasmic reticulum preincubated with either ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) prior to assay (Ca2+-free sarcoplasmic reticulum), or with calcium/EGTA buffer (Ca2+-bound sarcoplasmic reticulum). These effects were evident within a wide range of ionized Ca2+. Phosphorylation of skeletal sarcoplasmic reticulum by protein kinase also increased the initial rate of E approximately P decomposition. These findings suggest that protein kinase-dependent phosphorylation of skeletal sarcoplasmic reticulum regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the active calcium transport observed at steady state.     

Transient kinetic analysis of turnover-dependent fluorescence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP bound to Ca2+-ATPase of sarcoplasmic reticulum

The fluorescence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum is greatly enhanced during turnover induced by ATP plus Ca2+ (Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). We have studied the kinetics of induction of TNP-ATP fluorescence and of its decay and have found a close correlation with levels of phosphorylated intermediate of the enzyme, E-P. Steady-state kinetic studies suggested competitive binding of ATP and TNP-ATP to the catalytic site, with Km and Ki values of 2.4 and 1.0 microM, respectively. Rate constants for fluorescence enhancement and for E-P formation in the presteady state were 1.2 s-1 or 97-130 s-1 under conditions resulting in TNP-ATP or ATP saturation respectively, of the enzyme at inception of reaction. The slow process was concluded to be the koff for dissociation of TNP-ATP from the catalytic site. Following this dissociation, a second TNP-ATP site was detected, which both formed (97-130 s-1) and decayed (0.22 s-1) synchronously with E-P. TNP-ATP binding to this noncatalytic site was rapid (5 X 10(7) M-1 s-1) and resulted in high fluorescence during steady-state turnover. Fluorescence was found to be dissociated from E-P by KCl (100 mM). KCl had little effect on E-P levels, but decreased fluorescence by 68%. These studies provide independent kinetic evidence for the existence of both catalytic and noncatalytic, or "regulatory," nucleotide-binding sites, but cannot distinguish whether the two sites exist independently or whether the catalytic site is transformed into a regulatory site on phosphorylation. The latter site, which shows relatively high selectivity for TNP-ATP over ATP, and which is simultaneously hydrophobic and freely accessible to the medium, may play a role during energy transduction. The changes occurring at this site during catalysis are conveniently monitored with TNP-ATP fluorescence.     

Transmembranous organization of (Ca2(+)-Mg2+)-ATPase from sarcoplasmic reticulum. Evidence for lumenal location of residues 877-888

An antipeptide antibody was produced against a peptide corresponding to residues 877-888 of fast twitch rabbit sarcoplasmic reticulum ATPase. This antipeptide antibody bound strongly to the ATPase in sarcoplasmic reticulum vesicles only after the vesicles had been solubilized with the detergent C12E8 indicating that its epitope was located in the lumen of the sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum or purified (Ca2(+)-MG2+)-ATPase by proteinase K for up to 1 h resulted in a stable ATPase fragment of 30 kDa containing the epitope for the above antibody and the epitope for an antibody directed against the C terminus. Further proteolysis revealed smaller fragments (Mr 19,000 and 13,000) containing both epitopes. By contrast, small fragments of the ATPase (less than 29 kDa) containing the N-terminal epitope were not observed even after short exposures to proteinase K. These data support the view that the (Ca2(+)-MG2+)-ATPase has 10 transmembranous helices.     

Comparison of the (Ca2+ + Mg2+)-ATPase proteins from normal and dystrophic chicken sarcoplasmic reticulum.

The involvement of membrane protein in dystrophic chicken fragmented sarcoplasmic reticulum alterations has been examined. A purified preparation of the (Ca2+ + Mg2+)-ATPase protein from dystrophic fragmented sarcoplasmic reticulum was found to have a reduced calcium-sensitive ATPase activity and phosphoenzyme level, in agreement with alterations found in dystrophic chicken fragmented sarcoplasmic reticulum. An amino acid analysis of the ATPase preparations showed no difference in the normal and dystrophic (Ca2+ + Mg2+)-ATPase. The (Ca2+ + Mg2+)-ATPase was investigated further by isoelectric focusing and proteolytic digestion of the fragmented sarcoplasmic reticulum. Neither of these methods indicated any alteration in the composition of the dystrophic (Ca2+ + Mg2+)-ATPase. We have concluded that the alterations observed in dystrophic fragmented sarcoplasmic reticulum are not due to increased amounts of non-(Ca2+ + Mg2+)-ATPase protein, and that the normal and dystrophic (Ca2+ + Mg2+)-ATPase protein are not detectably different.