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Biochemical characterization of 14S dynein isolated from the cilia of Tetrahymena thermophila.

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posted on 2015-11-19, 09:07 authored by Hazel Ann. Tharia
Dynein is the ATPase responsible for cilia and flagella motility in eukaryotic organisms. At least two ATPases have been identified in Tetrahymena thermophila and are termed 22S and 14S on account of their differing sedimentation coefficients. 14S dynein was fractionated, using anion-exchange Fast Protein Liquid Chromatography, into four fractions (designated 1-4). Electron microscopy analysis revealed that the four fractions were structurally distinct. Fraction 1 comprised two globular heads interconnected via two stems; fraction 2 consisted of at least two clearly different globular structures; fraction 3 was a single globular head; and fraction 4 comprised three globular heads interconnected via three stems to a base. Further structural characterisation involved the use of hydrodynamic techniques in which the mass and sedimentation coefficient were determined for each fraction. In the presence of 40mM NaCl, fraction 1 had a mass of 654kDa and a sedimentation coefficient of 20.IS. Fraction 2 had a variable mass due to aggregation (616-966kDa), and a sedimentation coefficient of 16.6S, whereas fractions 3 and 4 had variable sedimentation coefficients but were of mass 701kDa and 527kDa respectively. These parameters were then utilised, in conjunction with electron microscopy data, in the construction of low-resolution bead models to represent the fractions. The four fractions had a unique polypeptide composition as shown by SDS polyacrylamide gel electrophoresis. At least four unique heavy chains were identified immunologically; two associated with fraction 1, one associated with fraction 2, and one associated with fractions 3 and 4. Fractions 1 and 2 were readily distinguished from fractions 3 and 4 using the technique of vanadate-dependent photolysis. The requirement for ATP in the photolysis reaction indicated differences in the structure of the heavy chain. The fractions were also distinguished with respect to the number and location of V2 sites, and the specific ATPase activity. These latter studies also showed that, for all four fractions, an increase in ionic strength resulted in a decrease in ATPase activity. This was coincident with an observed change in the structure and/or conformation of the fractions as determined by hydrodynamic analysis under equivalent conditions.


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University of Leicester

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  • Doctoral

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  • PhD



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