Ca-ATPase Activity Research Articles

Histochemistry of sarcoplasmic reticulum Ca-ATPase using dysprosium as capturing reagent

This report describes the development of a histochemical method for the demonstration of sarcoplasmic reticulum Ca-ATPase activity in cross-sections of skeletal muscle. The demonstration of sarcoplasmic reticulum Ca-ATPase activity is complicated by the fact that capturing reagents for phosphate inhibit the enzyme. We present a minimal model for heavy-metal-phosphate precipitation reactions which gives a theoretical description of the effect of enzyme inhibition on the rate of phosphate precipitation in the section. The model indicates that the choice of capturing reagent is crucial: whether or not ATPase activity can be demonstrated depends mainly on the inhibition constant and the solubility product of the phosphate salt of the capturing reagent (but also on a fairly large number of other factors). All lanthanides tested can be used to demonstrate sarcoplasmic reticulum Ca-ATPase activity, but dysprosium results in the highest staining intensity. This suggests that dysprosium inhibits sarcoplasmic reticulum Ca-ATPase to a lesser degree than the other lanthanides and/or the solubility product of its phosphate salt is smaller. As an example, the method is used to investigate the effect of thyroid hormone on sarcoplasmic reticulum Ca-ATPase activity in individual fibres of the rat soleus muscle.

Quercitin-dependent ATPase activity in the hypodermal tissue of Callinectes sapidus during the moult cycle

Ultrahistochemical evidence for quercitin-sensitive ATPase activity, believed to represent Ca-ATPase activity, in the epidermal tissue underlying the exoskeleton (=hypodermis) of the marine crab, Callinectes sapidus , is presented for different stages of the moult cycle. At premoult stage D 2 , activity in the epidermis was localized along the basolateral membrane below the apical junctions, but at D 3 , activity was only apparent in tissue associated with the carapace suture lines. No evidence of Ca-ATPase activity was found immediately after the moult (A 1 ), but by A 2 , calcification was occurring in the cuticle, and activity was present along the apical membranes and microvilli. At B 2 similar activity was seen, and the apical cytoplasm contained small vacuoles showing activity on their bounding membranes. No activity was seen in intermoult (C 4 ). These data were interpreted to mean that calcium entered the hypodermal cells from the cuticle in D 2 and was pumped out into the intercellular spaces by the activity of Ca-ATPase. This movement decreased greatly as the moult approached (D 3 ) except in the vicinity of the suture lines where resorption of calcium is known to be more extensive. In postmoult stages, the enzyme activity, concentrated at the apical surface of the epidermal cells, was consistent with the net outward transport of calcium toward the cuticle.

The Effect of the S14A Mutation on the Conformation and Thermostability of Saccharomyces cerevisiae G-Actin and Its Interaction with Adenine Nucleotides

The actin Ser14 hydroxyl is one of a number of ligands that binds to the gamma-phosphate of ATP thereby stabilizing the actin.ATP complex. In yeast actin, conversion of Ser14 to Ala (S14A), causes a temperature-sensitive phenotype in vivo and temperature-sensitive polymerization defects in vitro (Chen, X., and Rubenstein, P. A. (1995) J. Biol. Chem. 270, 11406-11414). Here, using a new luciferase-based procedure, we show that the mutation results in a 40-60-fold decrease in actin's affinity for ATP. The mutation causes a decrease in the intrinsic ATPase activity of both Ca- and Mg-G-actin at 30 degrees C and alters the protease susceptibility of sites on subdomain 2. Ca-S14A-actin but not Mg-S14A-actin binds etheno-ATP at 37 degrees C. Intrinsic tryptophan fluorescence measurements show that at 37 degrees C, Mg-S14A-actin but not the calcium form unfolds. CD measurements show the mutation causes a decrease in the apparent denaturation temperature for Ca-actin from 57 to 45 degrees C and for the magnesium form a decrease from 52 to 40 degrees C. Based on a re-examination of actin's crystal structure coordinates, we propose that the Ser14 hydroxyl forms a polar bridge between the ATP gamma-phosphate and the amide nitrogen of Gly74, thus conferring additional stability on the actin small domain.

Functional Reconstitution of Recombinant Phospholamban with Rabbit Skeletal Ca2+-ATPase

Phospholamban (PLB) is a small, transmembrane protein that resides in the cardiac sarcoplasmic reticulum (SR) and regulates the activity of Ca(2+)-ATPase in response to beta-adrenergic stimulation. We have used the baculovirus expression system in Sf21 cells to express milligram quantities of wild-type PLB. After purification by antibody affinity chromatography, the function of this recombinant PLB was tested by reconstitution with Ca(2+)-ATPase purified from skeletal SR. The results obtained with recombinant PLB were indistinguishable from those obtained with purified, canine cardiac PLB. In particular, PLB reduced the apparent calcium affinity of Ca(2+)-ATPase but had no effect on Vmax. At pCa 6.8, PLB inhibited both calcium uptake and ATPase activity of Ca(2+)-ATPase by 50%. This inhibition was fully reversed by addition of a monoclonal antibody to PLB, which mimics the physiological effects of PLB phosphorylation. Maximal PLB regulatory effects occurred at a molar stoichiometry of approximately 3:1, PLB/Ca(2+)-ATPase. We also investigated peptides corresponding to the two main domains of PLB. The membrane-spanning domain, PLB26-52, appeared to uncouple ATPase hydrolysis from calcium transport, even though the permeability of the reconstituted vesicles was not altered. The cytoplasmic peptide, PLB1-31, had little effect, even at a 300:1 molar excess over Ca(2+)-ATPase.

Lithium-induced inhibition of Na-K ATPase and Ca ATPase activities in rat brain synaptosome.

To explore the action mechanism of lithium in the brain, the author investigated the effects of lithium on Na-K ATPase and Ca ATPase in rat brain synaptosomes prepared from forebrains by the method of Booth and Clark. The activities of Na-K ATPase and Ca ATPase were assayed by the level of inorganic phosphate liberated from the hydrolysis of ATP. Lithium at the optimum therapeutic concentration of 1 mM decreased the activity of Na-K ATPase from the control value of 19.08 +/- 0.29 to 18.27 +/- 0.10 micromoles Pi/mg protein/h and also reduced the activity of Ca ATPase from 6.38 +/- 0.12 to 5.64 +/- 0.12 micromoles Pi/mg protein/h. The decreased activity of Na-K ATPase will decrease the rate of Ca2+ efflux, probably via an Na-Ca exchange mechanism and will increase the rate of Ca2+ entry by the depolarization of nerve terminals. The reduced activity of Ca ATPase will result in the decreased efflux of Ca2+. As a Conclusion, it can be speculated that lithium elevates the intrasynaptosomal Ca2+ concentration via inhibition of the activities of Na-K ATPase and Ca ATPase, and this increased [Ca2+]i will cause the release of neurotransmitters and neurological effects of lithium.

スケトウダラ塩漬肉の乾燥中に起こる筋原繊維タンパク質の変性に及ぼすソルビトールの影響

Meat blocks from walleye pollack were soaked in solutions containing 0.5-1.5M NaCl, 0.5-1.0M sorbitol. or their combinations with varied ratios for 24 hours at 4°C. After soaking, the cured meats were subjected to dehydration at 15°C. Changes in weight, NaCl and sorbitol concentrations of the meat, and myofibrillar Ca-ATPase activity together with crosslinking of myosin heavy chain of the same meat were investigated as a function of dehydration time. The results were as follows: (1) The meats containing 0.40-0.70M NaCl, 0.37-1.02M sorbitol, or combinations of 0.38-1.68M NaCl and 0.34-0.85M sorbitol were subjected to the dehydration process. (2) The rates of reduction of the weight of cured meats agreed well with the increase in NaCl and sorbitol concentration of the same meats induced by the dehydration. (3) The rate of dehydration of the cured meats was not related to the rate of denaturation of myofibrillar protein of the same meats. It is thus likely that the water retention ability of the meat is controlled by the concentration of NaCl and sorbitol, and their relative proportion in the meat, which was prepared by curing.

スケトウダラ筋肉の塩漬中に起こる筋原繊維タンパク質の変性に及ぼすソルビトールの影響

Meat blocks from walleye pollack were soaked in solutions containing 0.5-1.0M NaCl (N), 0.5-1.5M sorbitol (S), or their combinations with varied ratios for 24 hours at 4°C. During soaking, the increase in N and S of the meat, and the denaturation of myofibrillar protein as measured by the loss of Ca-ATPase activity and the cross-linking of myosin heavy chain of the same meat were investigated. The results were as follows: (1) The permeation of N into the meat depended on the concentration of N but not on the copresence of S concentration. (2) The permeation of S depended on the concentration of S, although the rate of permeation varied with the copresence of N concentration. (3) With increasing the concentration of N in the meat, the denaturation of myofibrillar protein accelerated, whereas increasing the concentration of S suppressed the same change. These findings suggest that the quality of meat block soaked in NaCl-sorbitol solution can be controlled by selecting the concentration and relative proportion of N and S in the soaking solution.

Halothane and cyclopiazonic acid modulate Ca-ATPase oligomeric state and function in sarcoplasmic reticulum.

We have studied the effects of cyclopiazonic acid (CPA) and halothane on the enzymatic activity, oligomeric state, and conformational equilibrium of the Ca-ATPase in skeletal muscle sarcoplasmic reticulum (SR). CPA is a potent inhibitor of Ca-ATPase activity, and this inhibition is competitive with respect to ATP concentration. Time-resolved phosphorescence anisotropy was used to detect the fraction of Ca-ATPase monomers, dimers, and larger aggregates in the absence and presence of CPA. CPA increased the fraction of dimers and larger aggregates of the Ca-ATPase. Addition of halothane to SR, or detergent solubilization of the Ca-ATPase, increased the apparent KI of CPA inhibition, and increased the fraction of Ca-ATPase present as monomers. CPA stabilized the E2 conformational state of the Ca-ATPase relative to the E1 and E2-P states, as measured by fluorescein 5-isothiocyanate fluorescence and enzyme phosphorylation from inorganic phosphate. E2-P formation in the presence of CPA was partially restored by halothane and solubilization. We conclude that CPA inhibits the Ca-ATPase in part by overstabilizing dimers or small oligomers of the Ca-ATPase, which is correlated with stabilization of the E2 conformation of the enzyme.

Reversible inactivation of the sarcoplasmic reticulum Ca(2+)-ATPase coupled to rearrangement of cytoplasmic protein domains as revealed by changes in trypsinization pattern.

Repetitive homogenization of skeletal muscle sarcoplasmic reticulum (SR) membranes in the presence of chelating agents at low ionic strength leads to the loss of the Ca-ATPase activity. This inactive state of the enzyme is coupled to an extensive rearrangement of the cytosolic domains as visualized by a completely different trypsinization pattern of the enzyme. In addition to the primary cleavage site (Arg 505), a novel trypsinization site (Arg 334), just N-terminal of the phosphorylation domain and localized on the primary tryptic fragment A, becomes exposed. Cleavage at the latter site yields a soluble fragment of M(r) 20,117 and the membrane-bound N-terminal one-third of the ATPase of M(r) 35,279. Two additional trypsinization sites C-terminal of the nucleotide binding domain become exposed in the inactive Ca(2+)-ATPase conformation. Rapid cleavage at these sites yields two soluble fragments of about 15 and 10 kDa. All together, the three soluble fragments comprise most of the large cytosolic loop of the Ca(2+)-ATPase. The inactivation and the change in trypsinization pattern can be reversed by rehomogenization of the extracted membranes in the presence of divalent cations. The results suggest the presence of an occluded site for divalent cations which can be depleted or refilled during application of sheer forces. Occupation of this site is essential to confer to the enzyme an active conformation.

The hydrophilic domain of phospholamban inhibits the Ca2+ transport step of the Ca2+-ATPase

The peptide MEKVQYLTRSAIRRASTIEMPQQAR-Cys corresponding to residues 1-25 of phospholamban was found to inhibit the ATPase activity of skeletal muscle Ca(2+)-ATPase, but to have no effect on the Ca(2+)-dependence of its activity. The peptide was found to decrease the rate of the Ca2+ transport step (E1PCa2-->E2P) by a factor of 2.4. The rate of this same step was decreased by poly(L-Arg) by a factor of 2.2. The peptide shifted the E2-E1 equilibrium of the ATPase towards E1 by a factor of 4 due to stronger binding to the E1 than to the E2 conformation of the ATPase; dissociation constants for binding to E1 and E2 were estimated as 3 and 10 microM respectively. The peptide had no effect on the level of phosphorylation by Pi in the absence of Ca2+ or on the rate of phosphorylation by ATP in the presence of Ca2+.

Theoretical analysis of calcium wave propagation based on inositol (1,4,5)-trisphosphate (InsP 3) receptor functional properties

In the presence of inositol (1,4,5)-trisphosphate (InsP 3) repetitive waves of elevated cytosolic free Ca 2+ (Ca waves) that travel through cellular cytoplasm are observed. Investigation of this phenomenon stimulated the view of cellular cytoplasm as ‘an excitable medium composed of Ca release processes (InsP 3R), coupled by a common stimulatory signal (Ca) through diffusion’ [Lechleiter JD. Clapham DE. (1992) Molecular mechanisms of intracellular calcium excitability in Xenopus laevis oocytes. Cell, 69, 283–294]. Using a kinetic model of InsP 3R gating, an analytical expression for the amplitude of Ca wave propagating through this excitable medium has been obtained. The ampiitude of the Ca wave is determined by the combination of cell-specific parameters and the functional properties of a single InsP 3R. An analytical expression for Ca wave propagation velocity has been also obtained using the Luther equation for diffusion-driven autocatalytic reaction. Both equations provided reasonable estimations for Ca wave amplitude (1.3 μM free Ca) and the velocity of the wave propagation (21 μm/s) for Ca waves in Xenopus oocytes when numerical values of parameters were used. The duration of refractory period has been shown to be determined mainly by the activity of CaATPase. Obtained results provide an insight into the mechanisms underlying the process of Ca wave propagation and define the interrelationship between different factors involved in this process. Some experimentally testable predictions can be done based on the analytical expressions obtained for Ca wave amplitude, the velocity of Ca wave propagation and the duration of refractory period.

Comparison of calcium,magnesiunn-ATPase activity and membrane fluidity in patients with essential hypertension and in normotensive controls

To measure calcium,magnesium-ATPase (Ca-ATPase) activity and membrane fluidity in patients with essential hypertension compared with normotensive subjects; to investigate the interrelationship between membrane fluidity and the activity of the Ca-ATPase; and to assess the importance of circulating lipids on the Ca-ATPase and membrane fluidity. Ca-ATPase and membrane fluidity were measured in erythrocyte membranes. Kinetic parameters [maximal activity (Vmax), apparent dissociation constant and allosteric number] of the Ca-ATPase activity were measured, in the presence of saturating calmodulin, in 38 normotensives and 57 essential hypertensives. Fluorescent polarization anisotropy, as an index of membrane fluidity, was measured, using the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium DPH (TMA-DPH) in 37 normotensives and 44 hypertensives. Of these 22 were paired for age, sex and race. There was no significant difference in the Vmax and allosteric number of the Ca-ATPase between the normotensives and hypertensives, but there was a trend for the hypertensives to have a reduced calcium affinity. In contrast, hypertensive subjects had significantly lower membrane fluidity. Sex and serum triglycerides level were important determinants of membrane fluidity in both groups. Comparisons between normotensives and hypertensives demonstrated decreased fluidity in the hypertensives independent of sex and serum triglycerides level, although the differences, especially with TMA-DPH, were more pronounced in the females. In both groups there were negative correlations between Vmax and both DPH and TMA-DPH anisotropy. The present study demonstrates that essential hypertension is associated with a generalized alteration in the erythrocyte membrane physical and chemical properties. However, despite the positive correlation between Vmax and membrane fluidity, the present study also demonstrates that essential hypertension is not associated with a major abnormality in the activity of the erythrocyte Ca-ATPase in isolated membranes.

A membrane potential model with counterions for cytosolic calcium oscillations

An initial model has been proposed to describe a mechanism for cytosolic calcium oscillations [Jafri MS. Vajda S. Pasik P. Gillo B. (1992) A membrane model for cytosolic calcium oscillations: a study using Xenopus oocytes. Biophys. J., 63, 235–246]. In this paper we extend our original model to include the effects of counterion movement Into the ER in response to calcium release. This produces smoother oscillations over a wider parameter range. We have lowered the endoplasmic reticulum (ER) Intraluminal free calcium concentration and shown that the oscillations can occur at lower ER membrane potentials, consistent with physiological values. The improved model is then tested with two representative paradigms that are currently under investigation by many researchers. The model predicts that the reduction of the ER calcium pump (Ca-ATPase) rate can cause the termination of cytosolic calcium oscillations in an active cell, and induce oscillations in a resting cell. This result is consistent with experiments with thapsigargin, a Ca-ATPase activity Inhibitor. In addition, we simulate the latency period for the response to the application of agonist and offer a plausible explanation for it. Our mathematical model Is currently the only model that formulates the contributions of calcium binding proteins, ER membrane potential, ER counterion movements, and distinct calcium pump populations, and describes their effects on cytosolic calcium oscillations.

Comparison of the effects of fluoride on the calcium pumps of cardiac and fast skeletal muscle sarcoplasmic reticulum: evidence for tissue-specific qualitative difference in calcium-induced pump conformation

Comparison of the effects of fluoride (NaF, 1–10 mM) on the catalytic and ion transport functions of the Ca 2+-ATPase in sarcoplasmic reticulum (SR) vesicles isolated from rabbit cardiac and fast-twitch skeletal muscles revealed similarities as well as striking tissue-specific differences depending on the experimental conditions employed. Short preincubation (3 min at 37°C) of cardiac or fast muscle SR with fluoride in the absence of Ca 2+ and ATP prior to initiating enzyme turnover by simultaneous addition of Ca 2+ and ATP to the assay medium resulted in a strong inhibitory effect of fluoride on ATP-energized (oxalate-facilitated) Ca 2+ uptake and Ca 2+-ATPase activity. On the other hand, when turnover was initiated by the addition of ATP to SR preincubated with fluoride in the presence of Ca 2+ but in the absence of ATP, fluoride caused concentration-dependent stimulation of active Ca 2+ uptake by fast muscle SR with no appreciable change in Ca 2+-dependent phosphoenzyme (EP) formation (from ATP) or Ca 2+-ATPase activity but inhibition of active Ca 2+ uptake by cardiac SR with concomitant inhibition of EP formation and Ca 2+-ATPase activity. Exposure of cardiac or fast muscle SR to fluoride in the presence of both Ca 2+ and ATP resulted in concentration-dependent stimulatory effect of fluoride on Ca 2+ uptake with no change in EP formation or Ca 2+-ATPase activity; this effect diminished substantially at saturating oxalate concentration in the assay. Assessment of the effects of deferoxamine (1 mM) and exogenous aluminum (10 μM) did not indicate a requirement for aluminum in the inhibitory or stimulatory effect of fluoride. These results suggest that (a) the Ca 2+ and ATP-deprived (E 1/E 2) but not the Ca 2+ plus ATP-liganded (CaE 1ATP) conformation of the SR Ca 2+-ATPase is susceptible to inhibition by fluoride in both cardiac and fast muscle; (b) the Ca 2+-bound conformation (CaE 1) of the SR Ca 2+-ATPase is susceptible to inhibition in cardiac muscle but is refractory to fluoride in fast muscle; and (c) the stimulatory effect of fluoride is largely secondary to its ability to mimic the action of oxalate in intravesicular Ca 2+ trapping when the fluoride-resistant enzyme is turning over normally. Fluoride inhibited phosphorylation of the Ca 2+-free enzyme by P i in cardiac and fast muscle SR indicating that fluoride sensitivity of the phosphorylation site of the SR Ca 2+-ATPase is similar in cardiac and fast muscle. In cardiac SR, disruption of the functional interaction between Ca 2+-ATPase and its regulatory protein phospholamban, through phosphorylation of the latter (by cAMP kinase) did not alter the fluoride sensitivity of the Ca 2+-bound enzyme (CaE 1). These results, coupled with the refractoriness of CaE 1ATP to fluoride in cardiac and fast muscle SR, suggest that a tissue-specific difference in the accessibility (reactivity) of the nucleotide binding site to fluoride upon Ca 2+ binding to the enzyme may account for the observed difference in fluoride sensitivity of the cardiac versus fast muscle enzyme - i.e., when the ATPase is in CaE 1 conformation, its ATP binding site is ‘fluoride-reactive’ in the cardiac enzyme but is ‘fluoride-resistant’ in the fast muscle enzyme.

Effects of Membrane Thickness on the Molecular Dynamics and Enzymic Activity of Reconstituted Ca-ATPase

We have studied the effect of phospholipid chain length on the activity and molecular dynamics of reconstituted Ca-ATPase from skeletal sarcoplasmic reticulum (SR), using time-resolved phosphorescence anisotropy (TPA) and electron paramagnetic resonance (EPR). We used reconstituted Ca-ATPase in exogenous phosphatidylcholines with monounsaturated chains 14-24 carbons long, to determine their effects on the physical properties of the Ca-ATPase and to correlate these physical changes with changes in the ATPase activity. In agreement with previous studies, we found that the enzyme activity was maximal with a chain length of 18 and decreased substantially with longer or shorter chains. Our TPA results show that chain lengths longer or shorter than the optimal 18 result in a significantly decreased mobility of the Ca-ATPase, indicated by higher residual anisotropy and suggesting extensive protein aggregation. Saturation-transfer EPR data obtained with a spin label bound to a different site also indicates substantial immobilization of the enzyme, supporting the TPA results. There is good agreement between the fractional inhibition of the Ca-ATPase activity and the fraction of the enzyme in large aggregates. Solubilization in the nonionic detergent C12E8 demonstrated that inhibition of enzyme activity is reversible. In contrast to the large effects on protein mobility, these changes in chain length had little or no effect on hydrocarbon chain mobility as detected by conventional EPR at different depths in the membrane. We conclude that the Ca-ATPase has an optimum lipid bilayer thickness, presumably matching the thickness of the hydrophobic transmembrane surface of the enzyme, and that deviation from this optimum thickness produces a hydrophobic mismatch that induces protein aggregation and hence Ca-ATPase inhibition. This is consistent with our proposal that protein dynamics and protein-protein interactions are of primary importance to the Ca-ATPase mechanism.

Interdependence of H+ and K+ fluxes during the Ca(2+)-pumping activity of sarcoplasmic reticulum vesicles.

The release of H+ during the oxalate-supported Ca2+ uptake in sarcoplasmic reticulum vesicles is kinetically coincident with the initial phase of Ca2+ accumulation. The Ca2+ uptake is increased and the H+ release is decreased in the presence of KCl and other monovalent chloride salts as expected for a H(+)-monovalent cation exchange. The functioning of the Ca(2+)-pump is disturbed by the presence of potassium gluconate and to a lesser extent, of choline chloride. These salts do not inhibit the ATPase activity of Ca(2+)-permeable vesicles, suggesting a charge imbalance inhibition which is specially relevant in the case of gluconate. Therefore, K+, and also Cl-, appear to be involved in secondary fluxes during the active accumulation of Ca2+. The microsomal preparation seems homogeneous with respect to the K(+)-channel, showing an apparent rate constant for K+ release of approximately 25 s-1 measured with the aid of 86Rb+ tracer under equilibrium conditions. A Rb+ efflux, sensitive to Ca(2+)-ionophore, can be also detected during the active accumulation of Ca2+. The experimental data suggest that both monovalent cations and anions are involved in a charge compensation during the Ca2+ uptake and H+ release. Fluxes of these highly permeable ions would contribute to cancel the formation of a resting membrane potential through the sarcoplasmic reticulum membrane.

Distribution of calcium-ATPase in developing teeth of embryonic American alligators (Alligator mississippiensis).

Light microscopic and ultrastructural observations were carried out to evaluate the cell morphology and histochemistry (calcium-ATPase activity) of developing teeth in embryonic American alligators (Alligator mississippiensis). Ca-ATPase activity was observed in the distal and lateral cell membranes, rough endoplasmic reticulum (rER), mitochondria, vacuoles, and other organelles of the ameloblast, but only in the distal cell membrane and process of the odontoblast. Enzyme activity in the ameloblasts increased gradually during development. These sites of enzyme activity are related to mineralization of the enamel layer, similar to that in mammalian tooth development. Alligator teeth are heavily mineralized like mammalian teeth; however, alligator ameloblasts have undeveloped distal processes during mineralization in contrast to mammalian ameloblasts in which Tomes' processes are found near the distal portion of ameloblasts at maturation stage. The localization of intense enzyme activity in the distal and lateral ameloblast cell membrane suggests that these regions are the site of accumulation of calcium as enamel differentiates in the developing tooth. © 1993 Wiley-Liss, Inc.

Formation of a stable inactive complex of the sarcoplasmic reticulum calcium ATPase with magnesium, beryllium, and fluoride.

Incubation of leaky or nonionic detergent-solubilized sarcoplasmic reticulum vesicles in solutions containing magnesium, beryllium, and fluoride caused a time-dependent complete inhibition of calcium ATPase activity. The inhibited state persisted through dialysis for 2 days versus EGTA and was reversed within minutes by the presence of 0.5 mM calcium. Calcium-independent ATPase activity was unaffected. Omission of magnesium or fluoride resulted in retention of activity, while omission of beryllium produced slower inactivation, as described previously (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235). Incubation of nonleaky vesicles had a similar effect, although it occurred more than 10-fold more slowly, suggesting that a component, probably beryllium, must enter the vesicles for inhibition to occur. By contrast, inhibition of nonleaky vesicles by magnesium and fluoride developed less than 2-fold more slowly. Including calcium in the incubation mixtures resulted in partial protection, so that the time course of CaATPase activity leveled off at nonzero values (for example, at 0.1 mM calcium, the activity leveled off at 43% of control). This result is most simply accounted for by a model involving simultaneous binding of calcium and a form of fluoroberyllium to the CaATPase (e.g. Ca2EMgBeFn).

Recording maximal Ca-ATPase activity in intact human erythrocytes by means of a Ca-selective electrode

Recording maximal Ca-ATPase activity in intact human erythrocytes by means of a Ca-selective electrode

A structural and kinetic study on myofibrils prevented from shortening by chemical cross-linking.

In previous work, we studied the early steps of the Mg(2+)-ATPase activity of Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. The myofibrils were free to contract, and the results obtained refer to the ATPase cycle of myofibrils contracting with no external load. Here we studied the ATPase of myofibrils contracting isometrically. To prevent shortening, we cross-linked them with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). SDS-PAGE and Western blot analyses showed that the myosin rods were extensively cross-linked and that 8% of the myosin heads were cross-linked to the thin filament. The transient kinetics of the cross-linked myofibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4 degrees C, by the rapid-flow quench method. The ATP binding steps were studied by the cold ATP chase and the cleavage and release of products steps by the Pi burst method. In Pi burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concentrations (as determined by the cold ATP chase methods) for both cross-linked (isometric) and un-cross-linked (isotonic) myofibrils. This shows that in both cases the rate-limiting step is after the cleavage of ATP. When cross-linked, the kcat of Ca(2+)-activated myofibrils was reduced from 1.7 to 0.8 s-1. This is consistent with the observation that fibers shortening at moderate velocity have a higher ATPase activity than isometric fibers.(ABSTRACT TRUNCATED AT 250 WORDS)