Studies on the mechanism and reversal of the phospholipase-A2 inactivation of D-glucose uptake by isolated human erythrocyte membranes.

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.