STUDY OF HUMAN ERYTHROCYTE-MEMBRANE PROTEIN INTERACTIONS BY SELECTIVESOLUBILIZATION OF TRITON-SKELETONS

The membrane skeleton, responsible for shape and mechanical properties of the red cell, was purified by the Triton extraction procedure in p resence of 5 mM, 150 mM or 600 mM NaCl. The proportion of spectrin, pr otein 4.1 and actin present in erythrocyte skeletons does not depend o n the molarity of NaCl used. In contrast ankyrin, protein band 3 and p rotein 4.2 are removed from skeletons as the ionic strength increased. Solubilization assays of membrane skeletons were used to study protei n interactions inside the skeleton. Solubilization was performed by Tr is, a non-selective disruptive reagent, or by p-mercuribenzene sulfoni c acid (PMBS), which principally release spectrin and actin. Tris acti on was assessed by calculation of the percentage of solubilized protei ns, which increased proportionally with Tris molarity. PMBS action was kinetically determined as the decrease in skeleton turbidity. With th ese two reagents, we observed a lower dissociation of skeletons prepar ed with high ionic strength buffer. Erythrocyte pretreatment with okad aic acid, an inhibitor of serine-threonine phosphatases, revealed a ph osphorylation-induced skeleton gelation and a better resistance to Tri s-solubilization.