Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.     

The effect of cardiotoxin on (Ca2+ + Mg2+)-ATPase of the erythrocyte and sarcoplasmic reticulum

The effects of cardiotoxin on the ATPase activity and Ca2+-transport of guinea pig erythrocyte and rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (E.C.3.6.1.3) were investigated. Erythrocyte (Ca2+ + Mg2+)-ATPase was inhibited by cardiotoxin in a time- and dose-dependent fashion and inhibition appears to be irreversible. Micromolar calcium prevented this inhibitory effect. Specificity for (Ca2+ + Mg2+)-ATPase inhibition by cardiotoxin was indicated since a homologous neurotoxin had no effect. Cardiotoxin did not affect (Ca2+ + Mg2+)-ATPase activity from sarcoplasmic reticulum, but Ca2+-transport was 50% inhibited. This inhibition was not due to an increased Ca2+-efflux and could be the result of an intramolecular uncoupling of ATPase activity from Ca2+-transport. Inhibition of Ca2+-transport by cardiotoxin could not be prevented by millimolar concentrations of Ca2+. It is suggested that the biological effects of cardiotoxin could be a consequence of inhibition of plasma membrane (Ca2+ + Mg2+)-ATPases.     

Long-term stabilization and crystallization of (Ca2+ + Mg2+)-ATPase of detergent-solubilized erythrocyte plasma membrane.

Conditions which were optimal for the stabilization of Ca2(+)-transporting ATPase in solubilized sarcoplasmic reticulum membranes (Piku?la, S., Mullner, N., Dux, L. and Martonosi, A. (1988) J. Biol. Chem. 263, 5277-5286) were also found conducive for preservation of (Ca2+ + Mg2+)-ATPase activity in detergent-solubilized erythrocyte plasma membrane for up to 60 days. Of particular importance for the stabilization of calmodulin-stimulated Ca2(+)-dependent activity of (Ca2+ + Mg2+)-ATPase of solubilized erythrocyte plasma membrane was the presence of Ca2+ (10-20 mM), glycerol, anti-oxidants, proteinase inhibitors and appropriate detergents. Among eight detergents tested octaethylene glycol dodecyl ether, polyoxyethylene glycol(10) lauryl alcohol and polydocanol were found to be promotive in long-term preservation of the enzyme activity. Under these conditions (Ca2+ + Mg2+)-ATPase of erythrocyte ghosts became highly stable and developed microcrystalline arrays after storage for 35 days. Electron micrographs of the negatively stained and thin sectioned material indicated that crystals of purified, detergent-solubilized, lipid-stabilized erythrocyte (Ca2+ + Mg2+)-ATPase differ from those of Ca2(+)-ATPase of detergent-solubilized sarcoplasmic reticulum microsomes.     

Quercetin interaction with the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin.     

Silver ions trigger Ca2+ release by interaction with the (Ca2+-Mg2+)-ATPase in reconstituted systems

It has been suggested that vesicles derived from the sarcoplasmic reticulum of skeletal muscle contain Ca2+ channels which can be opened by interaction with sulfhydryl reagents such as Ag+ or Hg2+. We show that, in reconstituted vesicles containing the (Ca2+-Mg2+)-ATPase purified from sarcoplasmic reticulum as the only protein, the ATPase can act as a pathway for Ca2+ efflux and that Ag+ induces a rapid release of Ca2+ from such reconstituted vesicles. We also show that Ag+ has a marked inhibitory effect on the ATPase activity of the purified ATPase. We suggest that the (Ca2+-Mg2+)-ATPase can act as a pathway for rapid Ca2+ release from sarcoplasmic reticulum.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

(Ca2+ + Mg2+)-dependent ATPase mRNA from smooth muscle sarcoplasmic reticulum differs from that in cardiac and fast skeletal muscles

We have investigated some characteristics of the sarcoplasmic reticulum (Ca2+ + Mg2+)-dependent ATPase (Ca2+-ATPase) mRNA from smooth muscle using specific cDNA probes isolated from a rat heart cDNA library. RNA blot analysis has shown that the Ca2+-ATPase mRNA expressed in smooth muscle is identical in size to the cardiac mRNA but differs from that of fast skeletal muscle. S1 nuclease mapping has moreover shown that the cardiac and smooth muscle isoforms possess different 3'-end sequences. These results indicate that a distinct sarcoplasmic reticulum Ca2+-ATPase mRNA is present in smooth muscle.     

Ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections

The ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat gracilis muscle was determined by indirect immunoferritin labeling of ultrathin frozen sections. Simultaneous visualization of ferritin particles and of adsorption- stained cellular membranes showed that the Ca2+ + Mg2+-ATPase was concentrated in the longitudinal sarcoplasmic reticulum and in the nonjunctional regions of the terminal cisternae membrane but was virtually absent from mitochondria, plasma membranes, transverse tubules, and junctional sarcoplasmic reticulum. Ferritin particles were found preponderantly on the cytoplasmic surface of the membrane, in agreement with published data showing an asymmetry of the Ca2+ + Mg2+- ATPase within the sarcoplasmic reticulum membrane. Comparison of the density of ferritin particles in fast and slow myofibers suggested that the density of the Ca2+ + Mg2+-ATPase in the sarcoplasmic reticulum membrane in a fast myofiber is approximately two times higher than in a slow myofiber.     

A fast passive Ca2+ efflux mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles

The (Ca2+ + Mg2+)-ATPase from skeletal muscle sarcoplasmic reticulum was reconstituted into phospholipid bilayers. The permeability of lipid bilayers to Co2+ and glucose was increased slightly by incorporation of the ATPase, and the permeability of mixed bilayers of phosphatidylethanolamine and phosphatidylcholine increased with increasing content of phosphatidylethanolamine both in the presence and absence of the ATPase. The presence of the ATPase, however, resulted in a marked increase in permeability to Ca2+, the permeability decreasing with increasing phosphatidylethanolamine content. Permeability to Ca2+ was found to be dependent on pH and the external concentrations of Mg2+ and Ca2+, was stimulated by adenine nucleotides but was unaffected by inositol trisphosphate. A kinetic model is presented for Ca2+ efflux mediated by the ATPase. It is shown that the kinetic parameters that describe Ca2+ efflux from vesicles of sarcoplasmic reticulum also describe efflux from the vesicles reconstituted from the purified ATPase and phosphatidylcholine. It is shown that the effects of phosphatidylethanolamine on efflux can be simulated in terms of changes in the rates of the transitions linking conformations of the ATPase with inward- and outward-facing Ca2+-binding sites, and that effects of phosphatidylethanolamine on the ATPase activity of the ATPase can also be simulated in terms of effects on the corresponding conformational transitions. We conclude that the ATPase can act as a specific pathway for Ca2+ efflux from sarcoplasmic reticulum.     

State of aggregation of the (Ca2+ + Mg2+)-ATPase studied using chemical cross-linking

We have studied cross-linking of the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum and in reconstituted systems, using glutaraldehyde, cupric-1,10-phenanthroline and 3,3'-dithiobis (sulphosuccinimidylpropionate). All reagents produce extensive cross-linking, forming aggregates too large to enter polyacrylamide gels. Only traces of cross-linked dimeric ATPase species are formed. Saturation transfer electron spin resonance spectra of spin-labelled sarcoplasmic reticulum cross-linked with glutaraldehyde are also consistent with the formation of extensively cross-linked aggregates in the membrane. The results are interpreted in terms of dynamic clusters of ATPase molecules in the membrane, probably in the form of rows of ATPase molecules.     

Rapid kinetics of calcium ion transport and ATPase activity in the sarcoplasmic reticulum of dystrophic muscle.

Vesicular fragments of sarcoplasmic reticulum were isolated from pectoralis muscle of normal and dystrophic chicken. Purification of both preparations was equally satisfactory, as shown by a prominent ATPase band in electrophoresis gels. Measurements of ATPase phosphorylation, Ca2+ transport and Pi cleavage by rapid quench methods revealed a lower specific activity of the dystrophic vesicles with respect to all of these functions. On the other hand, Ca2+-independent ATPase activity was found to be increased in dystrophic vesicles. It is suggested that a fraction of ATPase units of dystrophic sarcoplasmic reticulum is not activated by Ca2+, owing to an altered protein assembly within the membrane bilayer. In fact, when the membrane structure is perturbed by detergents normal and dystropic preparations acquire an equally high Ca2+-dependent ATPase.     

The use of a model water-lipid system for the study of the electric function of Ca2+, Mg2+-ATPase of the sarcoplasmic reticulum

The method of dynamic capacity in the model organic phase-water system was used to investigate a possibility of studying the electrical function of Ca2+,Mg2+-ATPase from sarcoplasmic reticulum of the rabbit hind limb skeletal muscles. Decane and decane solution of azolectin were used as an organic phase. It is stated that in the model systems the sarcoplasmic reticulum Ca2+,Mg2+-ATPase did not cause ATP-dependent changes in the boundary Volta potential (delta phi) irrespective of the presence of polyvalent cation chelates in the organic phase. The fragmented sarcoplasmic reticulum is able of realizing Mg-ATP, Ca2+-dependent generation of delta phi only with phospholipids present in the organic phase. It is supposed that generation of delta phi of the fragmented sarcoplasmic reticulum is due to the active transport of calcium ions by the reticulum Ca2+,Mg2+-ATPase.     

A fluorescence quenching study of tryptophanyl residues of (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum

The tryptophan intrinsic fluorescence of the (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum was quenched by acrylamide at different temperatures. Sharp increases in the quenching constants were found in samples of ATPase reconstituted with dimyristoyl-phosphatidylcholine and dipalmitoylphosphatidylcholine at temperatures slightly below the Tc transition temperature of the pure phospholipid. It is suggested that acrylamide may diffuse more easily through proteins surrounded by a fluid phospholipid matrix than if they are in a rigid matrix, due to different states of protein fluidity.     

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.     

Interactions of hexachlorocyclohexanes with the (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum

Hexachlorocyclohexanes have been shown to inhibit the (Ca2+ + Mg2+)-ATPase of muscle sarcoplasmic reticulum reconstituted into bilayers of dioleoylphosphatidylcholine. However, for the ATPase reconstituted into bilayers of dimyristoleoylphosphatidylcholine, a pattern of activation at low concentration followed by inhibition at higher concentration is seen for hexachlorocyclohexanes and alkanes such as decane and hexadecane. The ATPase in sarcoplasmic reticulum vesicles is also inhibited by the hexachlorocyclohexanes. The effects of hexachlorocyclohexanes on activity are largely independent of concentrations of Ca2+ and ATP. Inhibition is more marked at lower temperatures. The hexachlorocyclohexanes quench the tryptophan fluorescence of the ATPase, and the quenching can be used to obtain partition coefficients into the membrane system. As for simple lipid bilayers, partition exhibits a negative temperature coefficient. Binding is related to effects on ATPase activity.     

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.     

Effect of the purified (Mg2+ + Ca2+)-activated ATPase of sarcoplasmic reticulum upon the passive Ca2+ permeability and ultrastructure of phospholipid vesicles.

The passive Ca2+ permeability of fragmented sarcoplasmic reticulum membranes is 10(4) to 10(61 times greater than that of liposomes prepared from natural or synthetic phospholipids. The contribution of membrane proteins to the Ca2+ permeability was studied by incorporating the purified [Ca2+ + Mg2+]-activated ATPase into bilayer membranes prepared from different phospholipids. The incorporation of the Ca2+ transport ATPase into the lipid phase increased its Ca2+ permeability to levels approaching that of sarcoplasmic reticulum membranes. The permeability change may arise from a reordering of the structure of the lipid phase in the environment of the protein or could represent a specific property of the protein itself. The calcium-binding protein of sarcoplasmic reticulum did not produce a similar effect. The increased rate of Ca2+ release from reconstituted ATPase vesicles is not a carrier-mediated process as indicated by the linear dependence of the Ca2+ efflux upon the gradient of Ca2+ concentration and by the absence of competition and countertransport between Ca2+ and other divalent metal ions. The increased Ca2+ permeability upon incorporation of the transport ATPase into the lipid phase is accompanied by similar increase in the permeability of the vesicles for sucrose, Na+, choline, and SO42- indicating that the transport ATPase does not act as a specific Ca2+ channel. Native sarcoplasmic reticulum membranes are asymmetric structures and the 75-A particles seen by freeze-etch electron microscopy are located primarily in the outer fracture face. In reconstituted ATPase vesicles the distribution of the particles between the two fracture faces is even, indicating that complete structural reconstitution was not achieved. The Ca2+ transport activity of reconstituted ATPase vesicles is also much less than that of fragmented sarcoplasmic reticulum. The density of the 40-A surface particles visible after negative staining of native or reconstituted vesicles is greater than that of the intramembranous particles and the relationship between these two structures remains to be established.     

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.     

Localization of Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle

Localization of the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in rat papillary muscle was determined by indirect immunofluorescence and immunoferritin labeling of cryostat and ultracryotomy sections, respectively. The Ca2+ + Mg2+-ATPase was found to be rather uniformly distributed in the free sarcoplasmic reticulum membrane but to be absent from both peripheral and interior junctional sarcoplasmic reticulum membrane, transverse tubules, sarcolemma, and mitochondria. This suggests that the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum is antigenically unrelated to the Ca2+ + Mg2+-ATPase of the sarcolemma. These results are in agreement with the idea that the sites of interior and peripheral coupling between sarcoplasmic reticulum membrane and transverse tubules and between sarcoplasmic reticulum and sarcolemmal membranes play the same functional role in the excitation-contraction coupling in cardiac muscle.     

Distances between functional sites of the Ca2+ + Mg2(+)-ATPase from sarcoplasmic reticulum using Co2+ as a spectroscopic ruler

Cobalt ion inhibits the Ca2+ + Mg2(+)-ATPase activity of sealed sarcoplasmic reticulum vesicles, of solubilized membranes and of the purified enzyme. To use Co2+ appropriately as a spectroscopic ruler to map functional sites of the Ca2+ + Mg2(+)-ATPase, we have carried out studies to obtain the kinetic parameters needed to define the experimental conditions to conduct the fluorimetric studies. 1. The apparent K0.5 values of inhibition of this ATPase are 1.4 mM, 4.8 mM and 9.5 mM total Co2+ at pH 8.0, 7.0 and 6.0, respectively. The inhibition by Co2+ is likely to be due to free Co2+ binding to the enzyme. Millimolar Ca2+ can fully reverse this inhibition, and also reverses the quenching of the fluorescence of fluorescein-labeled sarcoplasmic reticulum membranes due to Co2+ binding to the Ca2+ + Mg2(+)-ATPase. Therefore, we conclude that Co2+ interacts with Ca2+ binding sites. 2. Co2+.ATP can be used as a substrate by this enzyme with Vmax of 2.4 +/- 0.2 mumol ATP hydrolyzed min-1 (mg protein)-1 at 20-22 degrees C and pH 8.0, and with a K0.5 of 0.4-0.5 mM. 3. Co2+ partially quenches, about 10 +/- 2%, the fluorescence of fluorescein-labeled sarcoplasmic reticulum Ca2+ + Mg2(+)-ATPase upon binding to this enzyme at pH 8.0. From the fluorescence data we have estimated an average distance between Co2+ and fluorescein in the ATPase of 1.1-1.8 nm or 1.3-2.1 nm for one or two equidistant Co2+ binding sites, respectively. 4. Co2+.ATP quenches about 20-25% of the fluorescence of fluorescein-labeled Ca2+ + Mg2(+)-ATPase, from which we obtain a distance of 1.1-1.9 nm between Co2+ and fluorescein located at neighbouring catalytic sites.