Binding stoichiometry of 21S dynein to A and B subfiber microtubules.
The binding properties of Tetrahymena 21S dynein to doublet A and B subfiber microtubules were analyzed by both a turbidimetric assay (delta A350 nm) and electron microscopy. KCl-extracted, sucrose-gradient, purified 21S dynein binds to each of the two kinds of axonemal microtubules in both ATP-insensitive and ATP-sensitive modes, even though only a single type of binding occurs to each of the subfibers in situ. Total dynein bound to axonemal microtubules is a composite of binding that is sensitive to dissociation by ATP and binding that is insensitive to ATP. Each exhibits a different binding profile. Total binding exhibits a sigmoid profile (h = 1.93) and saturates at 1.49 mg D/mg T. ATP-sensitive binding likewise exhibits a sigmoid profile (h = 2.66) but saturates at 1.06 mg D/mg T. Binding occurs with a similar affinity for both A and B subfibers. The Hill coefficient (h) for ATP-sensitive binding implies positive cooperativity between binding events. ATP-insensitive binding was studied independently in 20 microM ATP, 10 microM vanadate, which blocks ATP-sensitive binding. ATP-insensitive binding exhibits a hyperbolic profile (h = 1.0) and likewise occurs along each of the two kinds of axonemal tubules. Binding saturates at 0.87 mg D/mg T. The binding data suggest that the tubulin dimer has conserved both ATP-sensitive and ATP-insensitive binding sites for 21S dynein, even though the sites may not be expressed in vivo.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D004398:Dyneins / Q000378:metabolism*; D008870:Microtubules / Q000201:enzymology* / Q000648:ultrastructure; D013768:Tetrahymena / Q000648:ultrastructure; D013816:Thermodynamics; D014404:Tubulin / Q000378:metabolism*; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D014404:Tubulin; D014639:Vanadium; D014638:Vanadates; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004398:Dyneins