The changes in phospholipid composition sf the inner (cytoplasmic) surface of the human erythrocyte membrane resulting from the digestion of sealed inside-out vesicles with phospholipases A, and C were determined. Virtually all of the phosphatidylethanolamine and phosphatidylserine and 30-48%; of the phosphatidylcholine and sphingomyelin sf insideout vesicles were found to be accessible to enzyme hydrolysis. In contrast, all of the above phospholipids of unsealed ghosts were susceptible to phospholipolytic digestion. These results are a direct demoi~stration of an asymmetric distribution of phospholipids in the human erythrocyte membrane. KahHenberg, A., Walker, C. & Rohrlick, W. (1974) Evidence for an Asymmetric Distribution of PhosphoIipids in the Human Erythrocyte Membrane. Can. J. Biochem. 52, 803-886 Wous avons dCterminC les changements apportks dans la composition ghospholipidique B la surface intkrieure (cytoplasmiqile) de la membrane des Crytkrscytes humains suite k H a digestion par les phsspholipases As et C de vksicules morp-pkslogiquemerat inverskes et scellbes, Preque toutes les phssphatidylCthano1amines et les phosphatidylsCrines et 30-40CjI des phosphatidylcholiraes et des sphingomy6Enes des vCsisules inverskes sont touchCes par H'hydrolyse enzymatique. Au contraire, tous ces phospholigides sont susceptibles de digestion ghssgholipolytique dam les membranes strsmatiques non scelldes. Ces rCsuHtats dbmontrent direetement la distribution asymdtrique des phospholipides dans la membrane Crythrocytaire hurnaine. [Traduit par le jounral]lntrgeductim ghosts have provided evidence for an asym-~h~ action of group-specific labels and metrical distribution of membrane phospholipids phospholipases (4, 5 ) towards erythrocytes and (see Ref. 6). In contrast to cell ghosts9 the -

No abstract

The mechanism underlying the inactivation of the stereospecific uptake of D-glucose by isolated human-erythrocyte membranes following digestion with phospholipase A2 (Kahlenberg, A. &Banjo, B. (1972) J. Biol. Chem 247, 1156–1160) was investigated. This inactivation was not accompanied by any significant change in the uptake of L-glucose. The decrease in D-glucose uptake following limited (25–30%) cleavage of membrane phospholipids by phospholipase A2 was characterized by a twofold increase in the apparent dissociation constant of the D-glucose–membrane complex and a 34% decrease in the membrane's maximum capacity for D-glucose uptake. These effects of phospholipase A2 were completely reversed upon removal of the membrane-bound phospholipid byproducts (fatty acids and lysophospholipids) by washing the membranes with defatted bovine-serum albumin. Oleic acid and various lysophosphatides added to albumin-washed, phospholipase A2-treated membranes in amounts formed by the enzyme treatment produced negligible inhibition of D-glucose uptake. With more extensive phospholipase A2 digestion of membrane phospholipids, defatted bovine-serum albumin did not restore D-glucose uptake despite the removal of the phospholipid by-products formed.In addition to the inactivation of D-glucose uptake, limited enzyme treatment transforms the appearance of the membranes collected by centrifugation from opaque white to transparent and gelatinous. Both of these effects of phospholipase A2 are completely reversed upon incubation of the membranes at pH 5.5 for 2 h at 37 °C without loss of any of the membrane lysophosphatides and fatty acids formed by the enzyme treatment. It is suggested that this pH- and temperature-dependent restoration of D-glucose uptake is due to a conformational change resulting in the relocation of the membrane D-glucose-binding sites into a functional environment.These results indicate that the inactivation of D-glucose uptake by phospholipase A2, which was not accompanied by any change in L-glucose uptake, occurs by two different mechanisms. With limited hydrolysis of membrane phospholipids, one or both of the resulting phospholipid by-products reversibly inhibit the uptake of D-glucose by decreasing the affinity of the membrane for D-glucose and by masking a portion of the total available D-glucose-binding sites. However, upon extensive cleavage of phospholipids in the hydrophobic region of the membrane, there is an apparently irreversible disorganization of the membrane D-glucose-binding component. This might be due to destruction of vital phospholipids and/or a disturbance of the interactions between the lipid and protein components of the membrane.