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-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.
Grolig
F
F
Institut für Allgemeine Botanik und Pflanzenphysiologie, Justus Liebig-Universität, Giessen, Germany.
Schröder
J
J
Sawitzky
H
H
Lange
U
U
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002458:Cell Fractionation; D000460:Chlorophyta / Q000737:chemistry* / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D008970:Molecular Weight; D009218:Myosins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000199:Actins; D000911:Antibodies, Monoclonal; D002147:Calmodulin; D005456:Fluorescent Dyes; D010940:Plant Proteins; D013439:Sulfhydryl Reagents; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D009218:Myosins; D005033:Ethylmaleimide