Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum

The interaction between Ca2+-ATPase molecules in the native sarcoplasmic reticulum membrane and in detergent solutions was analyzed by chemical crosslinking, high performance liquid chromatography (HPLC), and by the polarization of fluorescence of fluorescein 5'-isothiocyanate (FITC) covalently attached to the Ca2+-ATPase. Reaction of sarcoplasmic reticulum vesicles with glutaraldehyde causes the crosslinking of Ca2+-ATPase molecules with the formation of dimers, tetramers and higher oligomers. At moderate concentrations of glutaraldehyde solubilization of sarcoplasmic reticulum by C12 E8 or Brij 36T (approximately equal to 4 mg/mg protein) decreased the formation of higher oligomers without significant interference with the appearance of crosslinked ATPase dimers. These observations are consistent with the existence of Ca2+-ATPase dimers in detergent-solubilized sarcoplasmic reticulum. Ca2+ (2-20 mM) and glycerol (10-20%) increased the degree of crosslinking at pH 6.0 both in vesicular and in solubilized sarcoplasmic reticulum, presumably by promoting interactions between ATPase molecules; at pH 7.5 the effect of Ca2+ was less pronounced. In agreement with these observations, high performance liquid chromatography of sarcoplasmic reticulum proteins solubilized by Brij 36T or C12 E10 revealed the presence of components with the expected elution characteristics of Ca2+-ATPase oligomers. The polarization of fluorescence of FITC covalently attached to the Ca2+-ATPase is low in the native sarcoplasmic reticulum due to energy transfer, consistent with the existence of ATPase oligomers (Highsmith, S. and Cohen, J.A. (1987) Biochemistry 26, 154-161); upon solubilization of the sarcoplasmic reticulum by detergents, the polarization of fluorescence increased due to dissociation of ATPase oligomers. Based on its effects on the fluorescence of FITC-ATPase, Ca2+ promoted the interaction between ATPase molecules, both in the native membrane and in detergent solutions.     

Emerging views on the structure and dynamics of the Ca2(+)-ATPase in sarcoplasmic reticulum

The ATP-dependent Ca2+ transport in sarcoplasmic reticulum involves transitions between several structural states of the Ca2(+)-ATPase, that occur without major changes in the secondary structure. The rates of these transitions are modulated by the lipid environment and by interactions between ATPase molecules. Although the Ca2(+)-ATPase restricts the rotational mobility of a population of lipids, there is no evidence for specific interaction of the Ca2(+)-ATPase with phospholipids. Fluorescence polarization and energy transfer (FET) studies, using site specific fluorescent indicators, combined with crystallographic, immunological and chemical modification data, yielded a structural model of Ca2(+)-ATPase in which the binding sites of Ca2+ and ATP are tentatively identified. The temperature dependence of FET between fluorophores attached to different regions of the ATPase indicates the existence of 'rigid' and 'flexible' regions within the molecule characterized, by different degrees of thermally induced structural fluctuations.     

The binding of monoclonal and polyclonal antibodies to the Ca2(+)-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules.

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.     

Selective inhibition by lasalocid of hydrolysis of the ADP-insensitive phosphoenzyme in the catalytic cycle of sarcoplasmic reticulum Ca2(+)-ATPase

The effect of a carboxylic ionophore (lasalocid) on the sarcoplasmic reticulum Ca2(+)-ATPase was investigated. The purified enzyme was preincubated with lasalocid in the presence of Ca2+ and the absence of K+ at pH 7.0 and 0 degrees C for 2 h. The Ca2(+)-dependent ATPase activity was strongly inhibited by this preincubation, whereas the activity of the contaminant Mg2(+)-ATPase was unaffected. The steady-state level of the phosphoenzyme (EP) intermediate remained constant over the wide range of lasalocid concentrations. The Ca2(+)-induced enzyme activation was unaffected. The kinetics of phosphorylation of the Ca2(+)-activated enzyme by ATP as well as the rate of conversion of ADP-sensitive EP to ADP-insensitive EP were also unaffected. Accumulation of ADP-insensitive EP was greatly enhanced, and almost all of the EP accumulating at steady state was ADP-insensitive. Hydrolysis of ADP-insensitive EP was strongly inhibited. A similar strong inhibition of the Ca2(+)-dependent ATPase activity by lasalocid was found with sarcoplasmic reticulum vesicles. To examine the effect of lasalocid on the conformational change in each reaction step, the Ca2(+)-ATPase of sarcoplasmic reticulum vesicles was labeled with a fluorescent probe (N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine) without a loss of catalytic activity and then preincubated with lasalocid as described above. The conformational changes involved in hydrolysis of ADP-insensitive EP and in the reversal of this hydrolysis were appreciably retarded by lasalocid. The conformational changes involved in other reaction steps were unaffected. These results demonstrate that hydrolysis of ADP-insensitive EP in the catalytic cycle of this enzyme is selectively inhibited by lasalocid.     

Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2(+)-ATPase with phospholamban in phospholipid vesicles

The Ca2(+)-ATPase in cardiac sarcoplasmic reticulum (SR) is under regulation by phospholamban, an oligomeric proteolipid. To determine the molecular mechanism by which phospholamban regulates the Ca2(+)-ATPase, a reconstitution system was developed, using a freeze-thaw sonication procedure. The best rates of Ca2+ uptake (700 nmol/min/mg reconstituted vesicles compared with 800 nmol/min/mg SR vesicles) were observed when cholate and phosphatidylcholine were used at a ratio of cholate/phosphatidylcholine/Ca2(+)-ATPase of 2:80:1. The EC50 values for Ca2+ were 0.05 microM for both Ca2+ uptake and Ca2(+)-ATPase activity in the reconstituted vesicles compared with 0.63 microM Ca2+ in native SR vesicles. Inclusion of phospholamban in the reconstituted vesicles was associated with a significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0. However, phosphorylation of phospholamban by the catalytic subunit of the cAMP-dependent protein kinase reversed the inhibitory effect on the Ca2+ pump. Similar findings were observed when a peptide, corresponding to amino acids 1-25 of phospholamban, was used. These findings indicate that phospholamban is an inhibitor of the Ca2(+)-ATPase in cardiac SR and phosphorylation of phospholamban relieves this inhibition. The mechanism by which phospholamban inhibits the Ca2+ pump is unknown, but our findings with the synthetic peptide suggest that a direct interaction between the Ca2(+)-ATPase and the hydrophilic portion of phospholamban may be one of the mechanisms for regulation.     

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.     

Transient kinetics of a Ca2+-induced fluorescence change from membrane-associated and solubilized sarcoplasmic reticulum Ca2+-ATPase

The kinetics and extent of the fluorescence change induced by Ca2+ interaction with the Ca2+-ATPase from sarcoplasmic reticulum have been compared by stopped flow fluorimetry for three preparations: sarcoplasmic reticulum; purified ATPase in membrane vesicles; and solubilized, delipidated ATPase. The kinetics of Ca2+ release and binding for both purified preparations could be described by a single exponential as has been observed for sarcoplasmic reticulum. The rate and extent of the fluorescence change for the solubilized and membrane-associated preparations are shown to be quite similar to those of the sarcoplasmic reticulum. From these results, it is concluded that all of the Ca2+-induced fluoescence change in sarcoplasmic reticulum originates from the Ca2+-ATPase. In addition, since the change in fluorescence is probably result of a conformational change in the ATPase during the Ca2+ pumping cycle, the results provide additional evidence that monomeric Ca2+-ATPase may be capable of Ca2+ transport since the delipidated preparation is monomeric under the conditions used for these experiments. Finally, it is concluded that phospholipid bilayer is not essential for this conformational change.     

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.     

Structural dynamics of the Ca2(+)-ATPase of sarcoplasmic reticulum. Temperature profiles of fluorescence polarization and intramolecular energy transfer

The temperature dependence of fluorescence polarization and F?rster-type resonance energy transfer (FRET) was analyzed in the Ca2(+)-ATPase of sarcoplasmic reticulum using protein tryptophan and site-specific fluorescence indicators such as 5-[2-[iodoacetyl)amino)ethyl]aminonaphthalene-1-sulfonic acid (IAEDANS), fluorescein 5'-isothiocyanate (FITC), 2',3'-O-(2,4,3-trinitrophenyl)adenosine monophosphate (TNP-AMP) or lanthanides (Pr3+, Nd3+) as probes. The normalized energy transfer efficiency between AEDANS bound at cysteine-670 and -674 and FITC bound at lysine-515 increases with increasing temperature in the range of 10-37 degrees C, indicating the existence of a relatively flexible structure in the region of the ATPase molecule that links the AEDANS to the FITC site. These observations are consistent with the theory of Somogyi, Matko, Papp, Hevessy, Welch and Damjanovich (Biochemistry 23 (1984) 3403-3411) that thermally induced structural fluctuations increase the energy transfer. Structural fluctuations were also evident in the energy transfer between FITC linked to the nucleotide-binding domain and Nd3+ bound at the putative Ca2+ sites. By contrast the normalized energy transfer efficiency between AEDANS and Pr3+ was relatively insensitive to temperature, suggesting that the region between cysteine-670 and the putative Ca2+ site monitored by the AEDANS-Pr3+ pair is relatively rigid. A combination of the energy transfer data with the structural information derived from analysis of Ca2(+)-ATPase crystals yields a structural model, in which the location of the AEDANS-, FITC- and Ca2+ sites are tentatively identified.     

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.     

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.     

Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum.

Ca2+-ATPase molecules were labeled in intact sarcoplasmic reticulum (SR) vesicles, sequentially with a donor fluorophore, fluorescein-5'-isothiocyanate (FITC), and with an acceptor fluorophore, eosin-5'-isothiocyanate (EITC), each at a mole ratio of 0.25-0.5 mol/mol of ATPase. The resonance energy transfer was determined from the effect of acceptor on the intensity and lifetime of donor fluorescence. Due to structural similarities, the two dyes compete for the same site(s) on the Ca2+-ATPase, and under optimal conditions each ATPase molecule is labeled either with donor or acceptor fluorophore, but not with both. There is only slight labeling of phospholipids and other proteins in SR, even at concentrations of FITC or EITC higher than those used in the reported experiments. Efficient energy transfer was observed from the covalently bound FITC to EITC that is assumed to reflect interaction between ATPase molecules. Protein denaturing agents (8 M urea and 4 M guanidine) or nonsolubilizing concentrations of detergents (C12E8 or lysolecithin) abolish the energy transfer. These results are consistent with earlier observations that a large portion of the Ca2+-ATPase is present in oligomeric form in the native membrane. The technique is suitable for kinetic analysis of the effect of various treatments on the monomer-oligomer equilibrium of Ca2+-ATPase. A drawback of the method is that the labeled ATPase, although it retains conformational responses, is enzymatically inactive.     

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.     

Effects of adenyl-5'-imidodiphosphate and vanadate Ion on the intermolecular cross-linking of Ca2(+)-ATPase in the sarcoplasmic reticulum membrane with N,N'-(1,4-phenylene)bismaleimide

The functional significance of the molecular interaction of Ca2(+)-ATPase in the sarcoplasmic reticulum (SR) membrane was examined using intermolecular cross-linking of Ca2(+)-ATPase with N,N'-(1,4-phenylene)bismaleimide (PBM). When SR vesicles were allowed to react with 1 mM PBM at pH 7 and 23 degrees C for various intervals and subjected to SDS-PAGE, the amount of the major band of monomeric ATPase decreased with a half life of about 20 min. Higher orders of oligomers were concurrently formed without accumulation of any particular species of oligomer. When SR vesicles were allowed to react with 1 mM PBM in the presence of 1 mM adenyl-5'-imidodiphosphate (AMP-PNP), the rate of oligomerization was markedly reduced and the amount of dimeric Ca2(+)-ATPase increased with time. After 1 h, more than 40% of the Ca2(+)-ATPase had accumulated in the dimeric form. When 1 mol of fluorescein isothiocyanate (FITC) was bound per mol of ATPase, the effects of AMP-PNP on the cross-linking with PBM were completely abolished. When SR vesicles were treated with PBM in the presence of 0.1 mM vanadate in Ca2+ free medium, the oligomerization of the Ca2(+)-ATPase by PBM was strongly inhibited. The vanadate effect on the cross-link formation was completely removed by the presence of Ca2+ and AMP-PNP in the reaction medium. When SR vesicles were pretreated with PBM in the presence of AMP-PNP and digested with trypsin for a short time, the dimeric ATPase was degraded to a peptide with an apparent molecular mass of about 170 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

N-terminal chimeric constructs improve the expression of sarcoplasmic reticulum Ca(2+)-ATPase in yeast.

Wild-type and chimeric constructs comprising rabbit sarcoplasmic reticulum (SR) Ca(2+)-ATPase and the N-terminal cytoplasmic portion of yeast plasma membrane H(+)-ATPase were expressed in yeast under control of a heat-shock regulated promoter. The wild-type ATPase was found predominantly in endoplasmic reticulum (ER) membranes. Addition of the first 88 residues of H(+)-ATPase to the Ca(2+)-ATPase N-terminal end promoted a marked shift in the localization of chimeric H(+)/Ca(2+)-ATPase which accumulated in a light membrane fraction associated with yeast smooth ER. Furthermore, there was a three-fold increase in the overall level of expression of chimeric H(+)/Ca(2+)-ATPase. Similar results were obtained for a chimeric Ca(2+)-ATPase containing a hexahistidine sequence added to its N-terminal end. Both H(+)/Ca(2+)-ATPase and 6xHis-Ca(2+)-ATPase were functional as demonstrated by their ability to form a phosphorylated intermediate and undergo fast turnover. Conversely, a replacement chimera in which the N-terminal end of SR Ca(2+)-ATPase was replaced by the corresponding segment of H(+)-ATPase was not stably expressed in yeast membranes. These results indicate that the N-terminal segment of Ca(2+)-ATPase plays an important role in enzyme assembly and contains structural determinants necessary for ER retention of the ATPase.     

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

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

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.     

Vanadate-catalyzed, conformationally specific photocleavage of the Ca2(+)-ATPase of sarcoplasmic reticulum

Vanadate-sensitized photocleavage of the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum was observed upon illumination of sarcoplasmic reticulum vesicles or the purified Ca2(+)-ATPase by ultraviolet light in the presence of 1 mM monovanadate or decavanadate. The site of the photocleavage is influenced by the Ca2+ concentration of the medium. When the [Ca2+] is maintained below 10 nM by EGTA, the vanadate-catalyzed photocleavage yields fragments of approximately equal to 87 and approximately equal to 22 kDa, while in the presence of 2-20 mM Ca, polypeptides of 71 and 38 kDa are obtained as the principal cleavage products. These observations indicate that the site of the vanadate-catalyzed photocleavage is altered by changes in the conformation of Ca2(+)-ATPase. Selective tryptic proteolysis, at Arg-505-Ala-506, combined with covalent labeling of Lys-515 by fluorescein 5'-isothiocyanate and with the use of anti-ATPase antibodies of defined specificity, permitted the tentative allocation of the sites of photocleavage to the A fragment near the T2 cleavage site in the absence of Ca2+, and to the B fragment between Lys-515 and Asp-659 in the presence of 2-20 mM Ca2+. The loss of ATPase activity during illumination is accelerated by calcium in the presence of vanadate. The vanadate-catalyzed photocleavage in the presence of Ca2+ is consistent with the existence of an ATPase-Ca2(+)-vanadate complex (Markus et al. (1989) Biochemistry 28, 793-799).     

Cloning, sequencing and functional expression in Escherichia coli of the gene for a P-type Na(+)-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum

Cloning and sequencing of the gene encoding a P-type Na(+)-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, were conducted. The structural gene was composed of 2628 nucleotides. The deduced amino acid sequence (876 amino acid residues; Mr, 96,664) suggested that the enzyme possesses 10 membrane-spanning regions. When the amino acid sequences of the four putative membrane regions, M4, M5, M6 and M8, of BL77/1 ATPase were aligned with those of fungal Na(+)-ATPase, Na(+)/K(+)-ATPase, H(+)-ATPases and sarcoplasmic reticulum Ca(2+)-ATPase, it exhibited the highest homology with Ca(2+)-ATPase except M5 region. By the transformation of Escherichia coli with the expression vector (pQE30) containing the ATPase gene, the enzyme was functionally expressed in E. coli membranes.