PRESERVATION OF THE NATIVE STRUCTURE AND FUNCTION OF CA2-ATPASE FROM SARCOPLASMIC-RETICULUM - SOLUBILIZATION AND RECONSTITUTION BY NEW SHORT-CHAIN PHOSPHOLIPID DETERGENT 1,2-DIHEPTANOYL-SN-PHOSPHATIDYLCHOLINE()

The properties of Ca2+-ATPase purified and reconstituted from rabbit s keletal sarcoplasmic reticulum (SR) has been studied in comparison wit h the preparations obtained by the commonly used detergent poly(oxyeth ylene)8-lauryl ether (C12E8) and the bile salt detergents cholate and deoxycholate. 1,2-Diheptanoyl-sn-phosphatidylcholine (DHPC) has been s hown to be excellent for solubilizing a wide variety of membrane prote ins [Kessi, Poiree, Wehrli, Bachofen, Semenza and Hauser (1994) Bioche mistry 33, 10825-10836]. The DHPC method consistently gave higher yiel ds of purified Ca2+-ATPase with a greater specific activity than the m ethods with C12E8, cholate, or deoxycholate. DHPC and C12E8 were super ior to cholate and deoxycholate in active enzyme yields and specific a ctivity. DHPC-solubilized Ca2+-ATPase purified on a density gradient r etained the E1Ca-E1Ca conformational transition, whereas the enzyme f rom the C12E8 purification did not retain this transition. The couplin g of Ca2+ transported to ATP hydrolysed in the DHPC-purified enzyme wa s maximal and matched the values obtained with native SR, whereas the coupling was much lower for the C12E8-purified enzyme. The specific ac tivity of Ca2+-ATPase reconstituted into dioleoylphosphatidylcholine v esicles with DHPC was up to 2-fold greater than that achieved with C12 E8, and is comparable to that measured in the native SR. Finally, the dissociation of Ca2+-ATPase into monomers by DHPC preserved the ATPase activity, whereas similar dissociation by C12E8 gave only one-sixth t he activity of that obtained with DHPC. These studies show that the Ca 2+-ATPase solubilized, purified and reconstituted with DHPC is superio r to that obtained with C12E8 in significant ways, making it a prepara tion suitable for detailed studies on the mechanism of ion transport a nd the role of protein-lipid interactions in the function of membrane proteins.