A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella.
Treatment of demembranated sea urchin sperm axonemes with an extraction solution containing 0.6 M NaCl, pH 7.0 for 10 min at 4 degrees C yields a solution of dynein 1 having a low, latent specific ATPase activity of about 0.25 mumol of Pi mg(-1) min(-1). Exposure of this dynein solution to 0.1% Triton-X-100 for 10 min at 25 degrees C causes an increase in its ATPase activity to about 3 mumol of Pi mg(-1) min(-1). A similar activation can be obtained by treating at 42 degrees C or by reacting with 60 mol of p-chloromercuribenzene sulfonate/10(6) g of protein. The effects of these activating procedures are not additive, suggesting that they lead to a common activated state. Purification of the latent activity dynein 1 by sucrose density gradient centrifugation yields a monodisperse preparation sedimenting at 21 S, and having a molecular weight of 1,250,000 as determined by sedimentation diffusion and sedimentation equilibrium. Activation of the latent dynein 1 with Triton X-100 converts it to a form sedimenting at 10 to 14 S. The 21 S dynein is also converted to a 10 S form by dialysis against 5 mM imidazole/NaOH buffer, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, pH 7, although in this case, the ATPase activity is increased only about 3-fold, with another 3-fold activation being obtainable upon subsequent treatment with Triton X-100. The 21 S latent form of dynein 1 may represent the intact dynein arms that form moving cross-bridges and generate active sliding between adjacent doublet tubules of the flagellar axoneme. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate suggests a model in which the 21 S dynein 1 particle is composed of three subunits of about 330,000 daltons and one of each of three medium weight subunits of 126,000, 95,000, and 77,000 daltons. When latent dynein 1 is added back to NaCl-extracted axonemes in the presence of 0.15 M NaCl, it recombines stoichiometrically and restores the arms on the doublet tubules with a 6-fold activation of its ATPase activity measured in the absence of KCl.
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004398:Dyneins / Q000378:metabolism*; D004789:Enzyme Activation; D005407:Flagella / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D009994:Osmolar Concentration; D011487:Protein Conformation; D012617:Sea Urchins; D013094:Spermatozoa / Q000201:enzymology*
D002731:4-Chloromercuribenzenesulfonate; D004228:Dithionitrobenzoic Acid; D000251:Adenosine Triphosphatases; D004398:Dyneins