Reconstitution of Ca2�-ATPase of Sarcoplasmic Reticulum with13C-Labelled Lipids13C-NMR Spectroscopic Studies

Abstract

Sarcoplasmic reticulum of rabbit muscle was isolated and delipidated. The enzymatically inactive apo-Ca2+-ATPase [EC 3.6.1.3] regained up to 50% of its original enzyme activity upon addition of 13C/14C-labeled oleic or linoleic acid and lysolecithin. There were 90-180 fatty acyl chains bound with reactivation of the Ca2+-ATPase, which obtained a density of 1.13-1.17 equal to that of native sarcoplasmic reticulum. No bilayer structure was formed. Only the firm binding of the fatty acid chains was required and no interactions with the polar head groups. This was expressed by a 60% reduction of the spin-lattice relaxation time (T1-time) of free [14-13C]linoleic acid and 2-[14-13C]linoleoyllysolecithin. The [N-13CH3]choline head group of lysolecithin was not altered in its mobility after binding to the apo-Ca2+-ATPase. A phospholipid exchange method is described, which allows an almost complete phospholipid exchange in the presence of sodium cholate. Cholate is completely removed by Sephadex G-25 chromatography and subsequent sucrose gradient centrifugation. The Ca2+-ATPase is enriched by this procedure. Proteins with MW below 100,000 are removed. The native phospholipids of sarcoplasmic reticulum were exchanged against lecithins specifically labelled in their fatty acyl chains and the choline polar head groups without loss of enzymatic activity. Ninety to 180 molecules were bound, which is twice the number of fatty acyl chains in the reactivation with free fatty acids and lysolecithins. It was concluded from the T1-time measurements that only half of the fatty acids of the phospholipids are bound to the apoprotein, the rest forming the bilayer and being freely mobile in it. The Ca2+-ATPase activity is 2 to 3 times as high as in native sarcoplasmic reticulum when highly unsaturated lecithin species (dilinoleoyl-phosphatidylcholine) are used in the exchange method. Ca2+ translocation of these sarcoplasmic reticulum vesicles with highly unsaturated lecithins but depleted of phosphatidylethanolamine is very low. Exchange of less unsaturated lecithin species and phosphatidylethanolamines enhanced partially the 45Ca2+ storage.