PHYSICAL CHARACTERIZATION AND ATPASE ACTIVITY OF 14S DYNEIN FRACTIONSFROM TETRAHYMENA-THERMOPHILA

Using anion-exchange fast protein liquid chromatography, 14S dynein wa s separated into four fractions (designated 1-4). These fractions were distinguished with respect to polypeptide composition, and at least f our unique heavy chains were identified. Each fraction was shown to ex hibit ATPase activity. Fraction 2 has a specific activity 2-3 times gr eater than that of fractions 1, 3, and 4; the fractions showed a consi stent trend of decreasing activity in the order 2>3>1>4. In all cases, the specific ATPase activity was reduced by high ionic strength, in c ontrast to 22S dynein, which was previously shown to exhibit increased activity under identical conditions. Electron microscopy analysis rev ealed that the four fractions of 14S dynein were structurally distinct . Fraction 1 comprises two globular head domains interconnected via tw o stems; fraction 2 consists of at least two clearly different globula r structures; fraction 3 is a single globular head; and fraction 4 com prises three globular head domains interconnected by three stems to a basal structure. Further structural characterization using hydrodynami c techniques enabled a determination of mass and sedimentation coeffic ient for each fraction. Fraction 1 had a mass of 654 kDa and a sedimen tation coefficient of 20.1 S. Fraction 2 had a variable mass due to as sociation (616-966 kDa), and a sedimentation coefficient of 16.6 S, wh ereas fractions 3 and 4 had variable sedimentation coefficients but we re of mass 701 kDa and 527 kDa respectively. Where possible, hydrodyna mic parameters were utilized, in conjunction with electron microscopy data, to construct low-resolution hydrodynamic bead models to represen t the fractions. Optimal models, which were consistent with all the av ailable data, were produced for fractions 1 and 4. Bead modelling was also carried out for 22S dynein, using previously published data, to v alidate the 14S dynein modelling.