Two approaches to isolate cytoplasmic dynein ATPase from Neurospora crassa

Cytoplasmic dynein is a force-producing enzyme that, in association with dy nactin, conducts minus-end directed transport of various organelles along m icrotubules. Biochemical analyses of cytoplasmic dynein and dynactin have b een conducted primarily in vertebrate systems, whereas genetic analyses hav e been explored mainly in yeast and the filamentous fungi. To provide a com plementary biochemical approach for the study of fungal dynein, we isolated /partially purified cytoplasmic dynein ATPase from the filamentous fungus N eurospora crassa. N, crassa dynein was partially purified by slightly modif ying the existing procedures, described for mammalian cytoplasmic dynein th at uses dynein-microtubule binding, followed by release with ATP and sucros e gradient fractionation. A novel approach was also used to isolate dynein- specific ATPase by gel filtration (Sepharose CL-4B). The K-m, ATP obtained by isolating dynein ATPase using gel filtration was similar to that obtaine d by using conventional method, suggests that contaminant proteins do not i nterfere with the dynein ATPase activity. Like vertebrate dynein, N. crassa dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K-m, ATP for N. crassa c ytoplasmic dynein is 10- to 15-fold higher than that of the vertebrate enzy me. (C) 2000 Societe francaise de biochimie et biologie moleculaire / Editi ons scientifiques et medicales Elsevier SAS.