[43] Preparation and purification of dynein

Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility

“…The vanadate sensitivity of the translocatorinduced microtubule movement, as well as the fact that ATP is a more effective substrate than GTP by an order of magnitude, suggests the presence of an ATPase. However, the fractions that induce the most movement have very little associated solution ATPase activity (ATP turnover rate of 0.1 sec −1 ) compared to the ATPase activity of dynein (1.5-30 sec −1 ; Bell et al, 1982). To obtain maximal ATPase hydrolysis of the translocator, a ternary complex consisting of translocator; microtubule; and bead, organelle, or glass surface may be necessary, though conditions for producing such complexes in high concentrations have not yet been found.…”

Isolation of dynein heavy chain cDNAs from trout testis which predict an extensive carboxyl-terminal alpha-helical coiled-coil domain.

“…(Bell et al, 1982; The DNA coding sequence for the carboxyl-termini of DYHCs is one of the most unusual ever reported for a cDNA. The largest cDNA we have sequenced to date contains 68 2 1-bp direct repeats.…”

“…The material was filtered through four layers of cheesecloth and the debris removed by centrifugation at 2500 g for 10 minutes at 4°C. The axonemes were then harvested from the supernatant and the dynein ATPase extracted in 0.6 M KCI according to Bell et al (1982). All manipulations were carried out on ice in solutions containing 10 mM benzamidine (Ensinck et al, 1972), 1 mM phenylmethylsulphonylfluoride (PMSF), 0.5 lAg/ml leupeptin and 0.1 U/ml soybean trypsin inhibitor.…”

“…Sucrose density gradient centrifugation of the 0.6 M KCI extract was performed as described (Bell et al, 1982) and sedimentation values determined using catalase and thyroglobulin as standards (Martin and Ames, 1961). ATPase activity and protein concentrations were determined by standard methods (King et al, 1986; Bio-Rad Protein Assay kit).…”

“…Extensive biochemical and electron microscopic analyses have provided insight into dynein polypeptide composition, enzymatic properties and fine structural features (Goodenough and Heuser, 1982;Johnson and Wall, 1983;Johnson et al, 1986;Gibbons, , 1988. Dynein ATPases consist of at least two heavy chains (300-500 kd), and a number of intermediate (76-122 kd) and low (14-24 kd) relative molecular mass polypeptides (Bell et al, 1982;Johnson, 1985). At present, little is known of the primary amino acid or nucleic acid sequence of any dynein molecule.…”