PURIFICATION AND CHARACTERIZATION OF ERYTHROCYTE CALDESMON - HYPOTHESIS FOR AN ACTIN-LINKED REGULATION OF A CONTRACTILE ACTIVITY IN THE RED-BLOOD-CELL MEMBRANE

We have previously shown that in human or pig whole erythrocytes, only a single 71-kDa polypeptide cross-reacts with the affinity purified a ntibody to pig platelet caldesmon (der Terrossian et al., 1989). In th e present paper, we demonstrate that this polypeptide represents a gen uine caldesmon which remains attached to the membrane prepared in the presence of an excess of free Mg2+ but not in its absence. Immunoreact ivity of this peptide is specific towards the antibody to pig platelet caldesmon since it is not labelled with antibodies to other component s of the red cell membrane. Erythrocyte caldesmon was purified to 95% homogeneity and displays well known characteristics of caldesmons from other sources. Together with tropomyosin, it has the ability to regul ate platelet actin-activated rabbit skeletal muscle myosin ATPase acti vity. The stoichiometry of I caldesmon/1 tropomyosin/7-9 actin molecul es indicates that the amount of caldesmon, in the red cell membrane, c orresponds precisely to the amount of tropomyosin. Immunofluorescent l abelling of whole erythrocytes gave similar punctate patterns with pur ified antibodies to myosin, to caldesmon, to tropomyosin and to actin (but not to spectrin), suggesting colocalization of these proteins. To gether, and for the first time, our results give strong evidence that caldesmon, bound on the actin protofilament, might represent the inhib itory component, so far uncharacterized, of a thin-filament-like syste m in erythrocyte.