Association of glyceraldehyde 3-phosphate dehydrogenase with the human erythrocyte membrane. Effect of detergents, trypsin, and adenosine triphosphate.

Glyceraldehyde 3-phosphate dehydrogenase is one of the major protein components associated with the erythrocyte membrane. This has been shown by activity studies of specific membrane extracts and by the specificity of iodoacetate inactivation and labeling of the enzyme. Studies on the association of the enzyme with the membrane are complicated by the tendency of the ghosts to seal under certain conditions, thus rendering the enzyme inaccessible to its substrates. This crypticity of the enzyme activity can be eliminated by detergent treatment. Sodium dodecyl sulfate is preferred, since at low concentrations it causes only minor decreases in the enzyme activity, while Triton X-100 gives more extensive inactivation at all concentrations tested. Trypsin digestion can release glyceraldehyde d-phosphate dehydrogenase from the membrane, but it also causes a fraction of the ghosts to seal. The fraction which reseals is somewhat variable between different membrane preparations and is related to the prior treatments of the membranes. Under standard hypotonic hemolysis conditions (10 mu Tris), 60 to 80% of the glyceraldehyde d-phosphate dehydrogenase remains associated with the membrane. Aldolase exhibits similar behavior, while lactic dehydrogenase and hemoglobin are almost completely released. In the presence of ATP (1 mM) the amount of glyceraldehyde 3-phosphate dehydrogenase associated with the membrane is reduced to 5 to 20 %. EDTA, Ca++, Mg++, and 2,3-diphosphoglycerate show little effect at concentrations of 1 mM, although release of the enzyme is promoted by these agents at higher concentrations. At O’, solubilized glyceraldehyde 3-phosphate dehydrogenase is inactivated by ATP, a process that can be prevented if the enzyme is preincubated with NAD or if the incubation with ATP is performed at 25”. The ATP-promoted release of glyceraldehyde 3-phosphate dehydrogenase during hemolysis is not altered by preincubation with NAD