Abstract
Abstract A method for measuring the stereospecific uptake of d-glucose by isolated human erythrocyte membranes has recently been developed in our laboratory (Kahlenberg, A., Urman, B., and Dolansky, D. (1971) Biochemistry, 10,3154). Data obtained with this method were interpreted to suggest that the uptake activity measured represented the binding of d-glucose to a specific site on the erythrocyte membrane. To investigate the possible role of phospholipids in this d-glucose uptake activity, the phospholipid structure of erythrocyte membranes was modified by treatment with phospholipase A2, C, or D, and the effect of each modification on their d-glucose uptake activity and phospholipid composition was determined. Phospholipase A2 treatment produced a maximum decrease in uptake activity of 75% in association with the hydrolysis of approximately 74% of the total available β-fatty acid ester linkages of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; the maximum hydrolysis of these phospholipids obtainable was 91%. In contrast, hydrolysis of approximately 64% and 46% of the membrane phospholipid phosphodiester linkages by phospholipase C and D, respectively, resulted in only a 25% decrease in d-glucose uptake activity. Likewise a greater decrease in the activity of the erythrocyte membrane (sodium plus potassium)-activated adenosine triphosphatase was observed after phospholipase A2 than after phospholipase C or D treatment. Acetylcholinesterase activity of the membrane was not affected by any of these phospholipolytic enzymes. The above results suggest that perturbation of phospholipid hydrophobic groups in the interior of the membrane, proximal or hydrophobically bound to membrane proteins, causes a decrease in the d-glucose uptake activity of these proteins.
Highlights
Likewise a greater decrease in the activity of the erythrocyte membrane-activated adenosine triphosphatase was observed after phospholipase Az than after phospholipase
Erythrocyte Membrane Phospholipid Composition and D-Glucose Uptake Activity c Effects of Phospholipases C a& D-The concomitant changes in membrane phospholipid composition and D-glucose uptake activity resulting from the action of increasing amounts of phospholipases C and D are shown in Figs. 1 and 2, respectively
The results of the present study indicate that removal of membrane phospholipid hydrophilic groups by phospholipase C or D had no marked effect on the functional activity of the membrane protein components responsible for the stereospecific uptake of D-glucose
Summary
D-glucose by isolated human erythrocyte membranes has recently been developed in our laboratory (KAHLENBERG, A., URMAN, B., and DOLANSKY, D. (1971) Biochemistry, 10,3154). Data obtained with this method were interpreted to suggest that the uptake activity measured represented the binding of D-glucose to a specific site on the erythrocyte membrane. D-glucose uptake activity, the phospholipid structure of erythrocyte membranes was modified by treatment with phospholipase Az, C, or D, and the effect of each modification on their D-glucose uptake activity and phospholipid composition was determined. In order to gain some understanding of the role various phospholipid molecules might play in the u-glucose up take activity of isolated human erythrocyte membranes [19], these membranes were subjected to digestion with phospholipases AZ, C, and I>, and the resulting changes in their D-glucose uptake activity and phospholipid composition were measured. Preparation of Iiemoglobin-free Human Erythrocyte A//embranes --Erythrocyte membranes were prepared from recently outdated whole human blood by a modification of the method of Dodge, ;\litchell, and Hanahan [20] as previously described
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