Physical characterization and ATPase activity of 14S dynein fractions from Tetrahymena thermophila.
Using anion-exchange fast protein liquid chromatography, 14S dynein was separated into four fractions (designated 1-4). These fractions were distinguished with respect to polypeptide composition, and at least four unique heavy chains were identified. Each fraction was shown to exhibit ATPase activity. Fraction 2 has a specific activity 2-3 times greater than that of fractions 1, 3, and 4; the fractions showed a consistent 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 contrast to 22S dynein, which was previously shown to exhibit increased activity under identical conditions. Electron microscopy analysis revealed that the four fractions of 14S dynein were structurally distinct. Fraction 1 comprises two globular head domains interconnected via two stems; fraction 2 consists of at least two clearly different globular structures; fraction 3 is a single globular head; and fraction 4 comprises three globular head domains interconnected by three stems to a basal structure. Further structural characterization using hydrodynamic techniques enabled a determination of mass and sedimentation coefficient for each fraction. Fraction 1 had a mass of 654 kDa and a sedimentation coefficient of 20.1 S. Fraction 2 had a variable mass due to association (616-966 kDa), and a sedimentation coefficient of 16.6 S, whereas fractions 3 and 4 had variable sedimentation coefficients but were of mass 701 kDa and 527 kDa respectively. Where possible, hydrodynamic parameters were utilized, in conjunction with electron microscopy data, to construct low-resolution hydrodynamic bead models to represent the fractions. Optimal models, which were consistent with all the available data, were produced for fractions 1 and 4. Bead modelling was also carried out for 22S dynein, using previously published data, to validate the 14S dynein modelling.
Tharia
H A
HA
Department of Biochemistry, University of Leicester, UK.
Rowe
A J
AJ
Byron
O
O
Wells
C
C
D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004398:Dyneins / Q000737:chemistry / Q000378:metabolism*; D008854:Microscopy, Electron; D016808:Tetrahymena thermophila / Q000201:enzymology* / Q000648:ultrastructure*
D004398:Dyneins