Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum at Glu-439 with 5-(bromomethyl)fluorescein.

The (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum was labeled with 5-(bromomethyl)fluorescein. A stoichiometry of one label per ATPase molecule was found, which was unaffected by the presence of ATP. Labeling resulted in a 60% decrease in ATPase activity. Sequencing identified the labeled residue as Glu-439. The fluorescence emission spectrum of the labeled ATPase was unaffected by the addition of Ca2+ or vanadate or by phosphorylation with either Pi or ATP. Measurement of the pK of the bound fluorescein and observation of quenching by KI were consistent with a relatively exposed location for the fluorophore. Measurements of fluorescence energy transfer located the position of Glu-439 relative to Lys-515 and Cys-344 and relative to the membrane surface. None of these distances changed in binding Ca2+ or vanadate.

Localization of Cys-344 on the (Ca(2+)-Mg(2+)-ATPase of sarcoplasmic reticulum using resonance energy transfer.

4-Bromomethyl-6,7-dimethoxy-coumarin labels the (Ca(2+)-Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum at Cys-344. Resonance energy transfer has been used to measure the distance between this site and Lys-515 labelled with fluorescein isothiocyanate as about 37 A. The height of Cys-344 above the phospholipid/water interface has been measured by resonance energy transfer for the ATPase reconstituted into bilayers containing fluorescein-labelled phosphatidylethanolamine; the height was found to be about 45 A. None of these distances was found to alter on changing pH, or on addition of Mg2+, Ca2+ or vanadate. Quenching of the fluorescence of the coumarin-labelled ATPase with KI suggested that the fluorophore is not fully exposed on the ATPase.

Reactivity of lysyl residues on the (Ca(2+)-Mg2+)-ATPase to 7-amino-4-methylcoumarin-3-acetic acid succinimidyl ester.

The (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum was labeled with the succinimidyl ester of 7-amino-4-methylcoumarin-3-acetic (AMCA). Although a large number of residues were labeled, it was found that Lys-492 was labeled preferentially at pH values between 6 and 8, consistent with an unusual environment for this residue. Labeling was reduced in the presence of ATP, suggesting that Lys-492 is in or near the ATP binding site of the ATPase. Other identified residues labeled by AMCA were Lys-35, Lys-135, Lys-218, Lys-371, and Lys-605. It is suggested that these represent surface-exposed lysyl residues. Lys-515, labeled by fluorescein isothiocyanate (FITC), was not labeled by AMCA. Labeling with AMCA at pH 6.0 has no effect on ATPase activity, suggesting that Lys-492 is not essential for activity. The fluorescence of AMCA-labeled ATPase did not change on addition of either ATP in the presence of Ca2+ or Pi in the absence of Ca2+, suggesting that Lys-492 was not affected by any major conformational changes on the ATPase. The efficiency of fluorescence energy transfer between AMCA and FITC labels on the ATPase was unaffected by binding Ca2+ or vanadate, arguing against any large-scale movement of the cytoplasmic domains of the ATPase.

Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum with 4-(bromomethyl)-6,7-dimethoxycoumarin: detection of conformational changes.

The (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum was labeled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It was shown that a single cysteine residue (Cys-344) was labeled on the ATPase, with a 25% reduction in steady-state ATPase activity and no reduction in the steady-state rate of hydrolysis of p-nitrophenyl phosphate. The fluorescence intensity of the labeled ATPase was sensitive to pH, consistent with an effect of protonation of a residue of pK 6.8. Fluorescence changes were observed on binding Mg2+, consistent with binding to a single site of Kd 4 mM. Comparable changes in fluorescence intensity were observed on binding ADP in the presence of Ca2+. Binding of AMP-PCP produced larger fluorescence changes, comparable to those observed on phosphorylation with ATP or acetyl phosphate. Phosphorylation with P(i) also resulted in fluorescence changes; the effect of pH on the fluorescence changes was greater than that on the level of phosphorylation measured directly using [32P]P(i). It is suggested that different conformational states of the phosphorylated ATPase are obtained at steady state in the presence of Ca2+ and ATP and at equilibrium in the presence of P(i) and absence of Ca2+.

Effects of Mg2+ and ATP on the phosphate transporter of sarcoplasmic reticulum.

The extra uptake of Ca2+ by vesicles of sarcoplasmic reticulum (SR) observed in the presence of Pi, attributable to transport of Pi by the Pi-transporter, has been studied. It has been shown that the Pi transporter is stimulated by ATP. Single channel conductance measurements have shown that the Cl- channel in the SR membrane is impermeable to Pi. It is suggested that the transporter could be an ion antiporter system. Studies of uptake as a function of pH and Mg2+ concentration suggest that transport of MgHPO4 and H2PO-4 are faster than transport of HPO2-4. For oxalate and pyrophosphate, Mg2+ binding inhibits transport. It is suggested that protonation of lysine residue(s) at the anion binding site increase the rate of transport.

Covalent and non-covalent inhibitors of the phosphate transporter of sarcoplasmic reticulum.

The sarcoplasmic reticulum (SR) of skeletal muscle contains a Pi transporter which transports Pi into the lumen of the SR, increasing the level of accumulation of Ca2+ by SR by forming insoluble salts with Ca2+. Phosphonocarboxylic acids inhibit the transport of Pi by the transporter, phosphonoformic acid itself being transported into the SR increasing the level of accumulation of Ca2+. Phenylphosphonic acid also inhibits Pi transport, distinguishing the Pi transporter of SR from the Na+/Pi transporter of brush-border membranes. Oxalate transport is also inhibited by the phosphono-carboxylic acids, consistent with the suggestion that oxalate and phosphate are carried on the same transporter. The effects of maleate are, however, not inhibited, suggesting a separate carrier for the dicarboxylic acids. Acetic anhydride and phenylglyoxal inhibit the transporter, Pi providing protection against the effects of acetic anhydride, suggesting the presence of a lysine residue at the Pi binding site. ATP provides protection against the effects of acetic anhydride and phenylglyoxal, suggesting the presence of an ATP binding site on the transporter.

Effects of Mg2+, anions and cations on the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.

In a previous paper [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227] we presented a kinetic model for the activity of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum. Here we extend the model to account for the effects on ATPase activity of Mg2+, cations and anions. We find that Mg2+ concentrations in the millimolar range inhibit ATPase activity, which we attribute to competition between Mg2+ and MgATP for binding to the nucleotide-binding site on the E1 and E2 conformations of the ATPase and on the phosphorylated forms of the ATPase. Competition is also suggested between Mg2+ and MgADP for binding to the phosphorylated form of the ATPase. ATPase activity is increased by low concentrations of K+, Na+ and NH4+, but inhibited by higher concentrations. It is proposed that these effects follow from an increase in the rate of dephosphorylation but a decrease in the rate of the conformational transition E1'PCa2-E2'PCa2 with increasing cation concentration. Li+ and choline+ decrease ATPase activity. Anions also decrease ATPase activity, the effects of I- and SCN- being more marked than that of Cl-. These effects are attributed to binding at the nucleotide-binding site, with a decrease in binding affinity and an increase in 'off' rate constant for the nucleotide.

A kinetic model for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.

The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1'PCa2 and to E2 modulate the rates of the transport step E1'PCa-E2'PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.