PARTIAL CHARACTERIZATION OF A PUTATIVE 110kDa MYOSIN FROM THE GREEN ALGA<i>CHARA CORALLINA</i>BY<i>IN VITRO</i>BINDING OF FLUORESCENT F‐ACTIN

Abstract

Using the binding of heterologous, rhodamine phalloidin-labelled F-actin in vitro, two F-actin binding proteins were identified in protein extracts from the green alga Chara corallina after fractionation by anion exchange chromatography. The first protein, a putative myosin, released laterally bound F-actin at ATP-concentrations as low as 1 microM; equivalent concentrations of ADP were not effective. Binding of F-actin was inhibited by the sulfhydryl-alkylating agent N-ethylmaleimide (NEM). Binding of F-actin was also abolished by a monoclonal anti-myosin (J14) previously used for immunodetection and immunolocalization in internodal cells (Grolig et al., 1988, Eur J Cell Biol 47: 22-31). Immunoblotting with J14 detected a 110 kDa polypeptide only in those protein fractions that had revealed ATP-sensitive binding of F-actin. The putative myosin bound with mediocre affinity to immobilized calmodulin and free Ca(2+)-concentration made no difference to this binding affinity. In contrast to the putative myosin, the second, less abundant protein revealed ATP-insensitive and end-wise binding to the microfilament and was not recognized by the anti-myosin antibody.