PARTIAL CHARACTERIZATION OF A PUTATIVE 110-KDA MYOSIN FROM THE GREEN-ALGA CHARA-CORALLINA BY IN-VITRO BINDING OF FLUORESCENT F-ACTIN

Using the binding of heterologous, rhodamine phalloidin-labelled F-act in in vitro, two F-actin binding proteins were identified in protein e xtracts from the green alga Chara corallina after fractionation by ani on exchange chromatography. The first protein, a putative myosin, rele ased laterally bound F-actin at ATP-concentrations as low as 1 mu M; e quivalent concentrations of ADP were not effective. Binding of F-actin was inhibited by the sulfhydryl-alkylating agent N-ethylmaleimide (NE M). Binding of F-actin was also abolished by a monoclonal anti-myosin (J14) previously used for immunodetection and immunolocalization in in ternodal cells (Grolig et al., 1988, Eur J Cell Biol 47: 22-31). Immun oblotting with J14 detected a 110 kDa polypeptide only in those protei n fractions that had revealed ATP-sensitive binding of F-actin. The pu tative myosin bound with mediocre affinity to immobilized calmodulin a nd free Ca2+-concentration made no difference to this binding affinity . In contrast to the putative myosin, the second, less abundant protei n revealed ATP-insensitive and end-wise binding to the microfilament a nd was not recognized by the anti-myosin antibody. (C) 1996 Academic P ress Limited