Active unit of solubilized sarcoplasmic reticulum calcium adenosinetriphosphatase: an active enzyme centrifugation analysis.

Abstract

Sarcoplasmic reticulum calcium adenosinetriphosphatase (Ca2+-ATPase) was solubilized to monomeric form with the nonionic detergent n-dodecyl octaethylene glycol monoether (C12E8). Equilibrium ultracentrifugation analysis indicated that this preparation is initially greater than 75% monomer, the remainder being best described as a tetramer. In the presence of substrates, this preparation has ATPase activity comparable to that of leaky sarcoplasmic reticulum vesicles. The possibility of substrate-induced oligomerization of the monomer under ATPase activity assay conditions was tested. Active enzyme centrifugation analysis demonstrated that ATPase activity sedimented with a rate which can only be attributed to a monomeric particle. The sedimentation rate was invariant over a 6-fold concentration range comparable to that used in activity assays. The portion of the protein that sediments as an oligomer when measurements are based on the movement of protein (A280) is not seen when measurements are based on the movement of activity. The data demonstrate that the monomer represents the minimal ATPase active unit of Ca2+-ATPase.