Remarkable stability of solubilized and delipidated sarcoplasmic reticulum Ca2+-ATPase with tightly bound fluoride and magnesium against detergent-induced denaturation

Abstract

Conditions were developed in the absence of Ca2+ for purification, delipidation, and long term stabilization of octaethylene glycol monododecyl ether (C12E8)-solubilized sarcoplasmic reticulum Ca2+-ATPase with tightly bound Mg2+ and F-, an analog for the phosphoenzyme intermediate without bound Ca2+. The Ca2+-ATPase activity to monitor denaturation was assessed after treatment with 20 mM Ca2+ to release tightly bound Mg2+/F-. The purification and delipidation was successfully achieved with Reactive Red-agarose affinity chromatography. The solubilized Mg2+/F--bound Ca2+-ATPase was very rapidly denatured at pH 8, but was perfectly stabilized at pH 6 against denaturation for over 20 days at 4°C even without exogenously added phospholipid and at a high C12E8/enzyme weight ratio (10:1). The activity was not restored unless the enzyme was treated with 20 mM Ca2+, showing that tightly bound Mg2+/F- was not released during the long term incubation. The perfect stability was attained with or without 0.1 mM dithiothreitol, but inactivation occurred with a half-life of 10 days in the presence of 1 mM dithiothreitol, possibly due to reduction of a specific disulfide bond(s). The remarkable stability is likely conferred by intimate gathering of cytoplasmic domains of Ca2+-ATPase molecule induced by tight binding of Mg2+/F-. The present study thus reveals an essential property of the Mg2+/F-/Ca2+-ATPase complex, which will likely provide clues to understanding structure of the Ca2+-released form of phosphoenzyme intermediate at an atomic level.