Effects of phospholipids on the function of the calcium-magnesium ATPase

Abstract

The ATPase activity for the (Ca2(+)-Mg2+)-ATPase purified from rabbit skeletal muscle sarcoplasmic reticulum is lower when reconstituted into bilayers of dimyristoleoylphosphatidylcholine [(C14:1)PC] than when it is reconstituted into dioleoylphosphatidylcholine [(C18:1)PC]. The rate of formation of phosphoenzyme on addition of ATP is slower for (C14:1)PC-ATPase than for the native ATPase or (C18:1)PC-ATPase. The reduction in rate of phosphoenzyme formation is attributed to a reduction in the rate of a conformational change on the ATPase following binding of ATP but before phosphorylation. The level of phosphoenzyme formed from Pi is also less for (C14:1)PC-ATPase than for (C18:1)PC-ATPase. At steady state at pH 6.0 in the presence of ATP Ca2+ is released from (C18:1)PC-ATPase into the medium, but not from (C14:1)PC-ATPase. These effects of (C14:1)PC on the ATPase are reversed by addition of androstenol to a 1:1 molar ratio with (C14:1)PC. The results are interpreted in terms of a kinetic model for the ATPase.

Oxidative injury induced by hypochlorous acid to Ca-ATPase from sarcoplasmic reticulum of skeletal muscle and protective effect of trolox

“…6). Kinetic parameters concerning Ca 2+ dependence were calculated from the Hill equation (Gould et al 1986) and the dependence of Ca-ATPase activity on ATP concentration was described by a modified form of the bi-Michaelis-Menten equation according to Kosk-Kosicka et al (1983) and Michelangeli et al (1991). These authors suggest that ATP interacts at two sites: a high-affinity (catalytic) site and a low-affinity (regulatory) site.…”

“…The dependence of ATPase activity on ATP concentration was fitted to the bi-Michaelis-Menten equation according to Kosk-Kosicka (1983) and Michelangeli et al (1991):…”

The inhibitors thapsigargin and 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone favour the E2 form of the Ca2+, Mg2+‐ATPase

“…respectively, Ca:+,MB:+-ATPas¢ was purified from rabbit skeletal muscle SR and ATPase activities were determined using a coupled enzyme assay as described in Michelang¢li ¢t al. [12], The ATPase was labell~xt with NBD using a slight modification of the method of Wakabayashi et aL [t 1] as described previously 16]. The fluorescence of HBD-labelled ATPase was recorded using an SLM 8000 spectrofluorimeter with excitation and emission wavelengths of 430 and 510 nm.…”

“…I0 ml of Optiphase HiSafe 3 scintillant was added and the filters counted for both ~H and ~SCa' * in a liquid scintillation counter. The amount of [~Hlglucose trapped on the filter was used to calculate the filter wetting volume and thus the proportion of the total ~sCa:* on the filter that was bound to the ATPase, Free Ca ~* concentrations were calculated using the binding constants given in [14].…”

“…As described elsewhere [12,18] it is not possible to change the equilibrium constant for just one step alone in the reaction cycle of the ATPase, since the product of the equilibrium constants for all the steps around the cycle is fixed, equal to that for the hydrolysis of ATP. Table II shows that BHQ reduces the level of phosphorylation of the ATPase by Pi, consistent with a reduction in the equilibrium constant for phosphorylation.…”