Synthesis of a new phosphatidylserine spin-label and calcium-induced lateral phase separation in phosphatidylserine-phosphatidylcholine membranes.

A new phosphatidylserine spin label with nitroxide stearate attached at the 2 position has been synthesized by the reaction of spin-labeled CDP-diglyceride with L-serine under the catalytic action of phosphatidylserine synthetase. Some structural properties of pure phosphatidylserine (PS) and binary PS-phosphatidylcholine (PC) membranes were studied with the spin label. PS membrane became solidified on lowering solution pH, 50% solidification being attained at pH 3.5. The membrane was also solidified by addition of Ca-2+. The effect of Ba-2+,Sr-2+, and Mg-2+ was smaller than that of Ca-2+. The calcium-induced lateral phase separation in the binary membrane was studied from the side of the calcium-receiving lipid. The results confirmed and extended our previous conclusion drawn with PC spin label. The phase diagram of the binary membrane in the presence of Ca-2+ was determined. Not all PS molecules were aggregated to form the solid patches but some remained dissolved in the fluid PC matrix. The fluid PS fraction was larger for the membranes containing more PC. The membrane with 10% PS still had a significant fraction of solid phase. The rate of calcium-induced aggregation was greatly dependent on the PS content. The aggregation was almost complete within 5 min in the membrane containing 67% PS, while it was still proceeding after several hours in the membrane with 20% PS. The rate-limiting step was suggested to be in the formation of "stable" nuclei consisting of larger aggregates. The possible biological significance of the ionotropic phase separation was discussed whereby a transient density fluctuation was emphasized.