Rapid breakdown of diphosphoinositide and triphosphoinositide in erythrocyte membranes

Incubation of rabbit erythrocytes with 32Pi resulted in labeling of membrane diphosphoinositide, triphosphoinositide, and phosphatidic acid. Hypotonic lysis at 37°C resulted in an extremely rapid breakdown of the labeled polyphosphoinositides. This breakdown could be retarded by lysis in the presence of EDTA and by lowering the temperature to 0° thus allowing preparation of membranes with minimum breakdown of the labeled lipids. Rapid breakdown of di‐ and triphosphoinositide in isolated membranes could be initiated by Ca++ or to a lesser extent by Mg++ and prevented by detergents and by heating to 75°C. Assay of radiolabeled lipid was carried out by a method which bypassed prior lipid extraction and which enabled sequential sampling of reactions at 10‐second intervals. This method was more convenient than standard procedures and gave yields of di‐ and triphosphoinositide equivalent to that obtained by the method of Folch.