Some properties of bound and soluble dynein from sea urchin sperm flagella.
Axonemes were isolated from sperm of Colobocentrotus by a procedure involving two extractions with 1% Triton X-100 and washing The isolated axonemes contained 7 x 10(15) g protein per microm of their length. Treatment of the axonemes with 0 5 M KCl for 30 min extracted 50-70% of the flagellar ATPase protein, dynein, and removed preferentially the outer arms from the doublet tubules. Almost all of the dynein (85-95%) could be extracted from the axonemes by dialysis at low ionic strength. In both cases the extracted dynein sedimented through sucrose gradients at 12-14S, and no 30S form was observed The enzymic properties of dynein changed when it was extracted from the axonemes into solution. Solubilization had a particularly marked effect on the KCl- and pH-dependence of the ATPase activity. The pH-dependence of soluble dynein was fairly simple with a single peak extending from about pH 6 to pH 10. The pH-dependence of bound dynein was more complex. In 0.1 M KCl, the bound activity appeared to peak at about pH 9, and dropped off rapidly with decreasing pH, reaching almost zero at pH 7; an additional peak at pH 10 0 resulted from the breakdown of the axonemal structure and solubilization of dynein that occurred at about this pH. A similar curve was obtained in the absence of KCl, except for the presence of a further large peak at pH 8 Measurement of the kinetic parameters of soluble dynein showed that both K(m) and V(max) increased with increasing concentrations of KCl up to 0.5 M When bound dynein was assayed under conditions that would induce motility in reactivated sperm (0 15 M KCl with Mg(++) activation), it did not obey Michaelis-Menten kinetics, although it did when assayed under other conditions. The complex enzyme-kinetic behavior of bound dynein, and the differences between its enzymic properties and those of soluble dynein, may result from its interactions with tubulin and other axonemal proteins
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate; D000818:Animals; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D005407:Flagella / Q000032:analysis* / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008854:Microscopy, Electron; D011189:Potassium Chloride; D011506:Proteins / Q000032:analysis*; D012617:Sea Urchins / Q000378:metabolism*; D012995:Solubility; D013094:Spermatozoa / Q000166:cytology*
D011506:Proteins; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases