The effect of delipidation on the adenosine triphosphatase of sarcoplasmic reticulum. Electron microscopy and physical properties.

Abstract

Electron micrographs of negatively stained vesicles regenerated from the purified ATPase of sarcoplasmic reticulum show projections from the membrane which are similar to those previously observed on the intact parent membrane. They were also seen on sarcoplasmic vesicles from which all proteins other than the ATPase had been extracted with low concentrations of deoxycholate. These projections consist of a knob joined to the membrane by a narrow stalk, which together account for about for about half of the ATPase molecule. The overall length of the molecule is probably 10–12 nm. Inactivation of the enzyme in 1% deoxycholate followed by removal of the detergent gave non-vesicular aggregates which lacked projections and remained inactive. If lipid was removed from the protein before the detergent, optically clear solutions were obtained which contained much smaller particles, including single molecules of ATPase protein. These were approximately equidimensional (about 6.0 nm across) and could not be clearly related to the projections of the parent membrane. The cireular dichroism spectrum showed only a small decrease in content of α-helix relative to the active enzyme (35% to 28%), but no more than 5% of the original activity could be regenerated by recombination with phospholipids. The disappearance of the projections and the limited aggregation of the lipid-free molecule suggested an irreversible loss of hydrophobic surface following the replacement of lipid by deoxycholate. A structural model which would account for this behaviour is discussed.