[Effects of phospholipid layer on the dynamic microstructure of phosphorylation domain of Ca(2+)-ATPase from sarcoplasmic reticulum prepared from rabbit skeletal muscle].

The effects of phospholipids bilayers imposed on the intramolecular dynamic microstructure of Ca(2+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum were studied with a nanosecond time-resolved fluorometer. Ca(2+)-ATPase was purified and reconstituted into vesicle membranes. The phosphorylation domain of Ca(2+)-ATPase was labeled with a fluorophore, N-(1-anilinonaphthyl-4) maleimide (ANM). The phospholipids surrounding the hydrophobic segment of Ca(2+)-ATPase were exchanged with phosphatidylcholines of shorter acyl chain length by lipid titration. The membrane viscosity was measured by fluorometry using 1, 6-diphenyl-1, 3, 5-hexatriene (DPH). The membrane viscosity decreased when the intrinsic phospholipids were titrated with phosphatidylcholine having shorter acyl chains, and accompanied with a concurrent decrease in Ca(2+)-ATPase activity. The replacement of native lipids caused an increase in the fluorescence wavelength of ANM-labeled Ca(2+)-ATPase vesicles (red shift). This result suggests a conformational change in which the phosphorylation domain becomes more hydrophilic. The anisotropy decay time was was analyzed as two components, the slower being attributed to the intramolecular oscillation of the phosphorylation domain. The half-decay time of ANM fluorescence anisotropy was 72 +/- 4 nsec in the control vesicles, 69 +/- 3 nsec in di (18: 1) PC, 61 +/- 4 nsec in di (16: 1) PC, 54 +/- 3 nsec in di (14: 1) PC, and 49 +/- 2 nsec in di (12: 0) PC-titrated vesicles. This result suggests that the submolecular oscillation of the phosphorylation domain of Ca(2+)-ATPase is limited by the physical properties of boundary phospholipids, and that changes in the phospholipids cause alterations in the molecular motion of this domain, destabilize Ca(2+)-ATPase and reduce its activity.