Preparative isolation of Apo(Ca2+-ATPase) from sarcoplasmic reticulum and the reactivation by lysophosphatidylcholine of Ca2+-dependent ATP hydrolysis and partial-reaction steps of the enzyme.
1. A preparative method for the isolation of the lipid free apoprotein, the Ca2+-ATPase of sarcoplasmic reticulum, from the partially purified lipoprotein, Ca2+-ATPase vesicles, is presented. 2. By enzymatic hydrolysis of the phospholipids and removal of the splitting products and endogenous neutral lipids, the apoprotein was consistently delipidated to 0.02 mumol Pi/mg protein. 3. Reactivation of the splitting of ATP and the pseudo substrate, dinitrophenyl phosphate, was demonstrated with a variety of lipids and detergents. 4. A total reactivation of ATP splitting was achieved after a mild ultrasonication of the apoprotein with myristoylglycerophosphocholine which resulted in solubilization of the enzyme as an optically clear solution. 5. The stable resolubilized enzyme could be stored for several weeks maintaining full enzymatic activity. Gel chromatography suggested that under the assay conditions, the monomeric form of the enzyme predominated. 6. In comparison with the native enzyme, the resolubilized enzyme showed differences in the temperature dependence of the activation of ATP hydrolysis and a reduced apparent affinity for MgATP. 7. The phosphate-transferring activities of the resolubilized enzyme were only partially reactivated in the forward direction, and none of the reverse partial-reaction steps of the enzyme could be demonstrated.
D000818:Animals; D001051:Apoenzymes / Q000302:isolation & purification / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification* / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D008854:Microscopy, Electron; D009132:Muscles / Q000201:enzymology*; D010743:Phospholipids / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013329:Structure-Activity Relationship
D001051:Apoenzymes; D003902:Detergents; D008244:Lysophosphatidylcholines; D010743:Phospholipids; D000252:Calcium-Transporting ATPases