The Use of Phospholipases in the Determination of Asymmetric Phospholipid Distribution in Membranes

Abstract

Specific information on the localization of components in the membrane is a necessary prerequisite to understanding the molecular organization and function of biological membranes. In the last decade, variety of techniques have been developed that provide information on the organization of membrane constituents. For example, various investigators have shown that several proteins may occupy different locations in the red cell membrane (for recent reviews, see Wallach, 1972 and Juliano, 1973), and a nonrandom distribution of phospholipids between the exterior and interior sides of the erythrocyte membrane was proposed by Bretscher (1972, 1973) [based on the observation that the relatively nonpermeant reagent formylmethionyl-sulfone methylphosphate (FMMP) failed to react with phosphatidylethanolamine and phosphatidylserine of intact cells]. These observations of Bretscher were essentially confirmed by Gordeski and Marinetti (1973). They used the nonpenetrating probe, 2,4,6-trinitrobenzene-sulfonate, although they could label some of the phosphatidylethanolamine of intact cells. Since both these reagents are intrinsically unable to react with cholinecontaining phospholipids, these results should be taken only as indirect evidence that lecithin and sphingomyelin form the outer monolayer of the erythrocyte membrane.