Kinetic properties of microtubule-activated 13 S and 21 S dynein ATPases. Evidence for allosteric behaviour associated with the inner row and outer row dynein arms.

The 13 S and 21 S dynein ATPases from Tetrahymena cilia rebind to extracted doublet microtubules as inner row and outer row arms. Rebinding is accompanied by four- to ninefold activation of the ATPase activity. The soluble (microtubule-free) forms of the two dyneins exhibit simple saturation kinetics (h = 1.0) with Vmax much less than mumol Pi mg-1 min-1 and Km = 20-40 microM-ATP. Mixing a fixed quantity of free dynein with increasing concentrations of extracted doublets results in systematic increases in all three kinetic parameters for each dynein. At infinite concentrations of doublets and ATP, each enzyme undergoes a significant shift to sigmoid saturation kinetics (h = 2-3), Vmax increases to a turnover rate of about 90 mol ATP per mol Es-1 and the Michaelis constant increases to much greater than 100 microM-ATP. These data suggest that both enzymes are allosteric and can be interpreted in terms of positive cooperativity relative to a minimum of two or three interacting sites. It is less clear whether this cooperativity is related to subunit interactions within the 21 S or 13 S particles, or to subunit interactions between adjacent particles (arms) on the microtubule lattice.

Rebinding of Tetrahymena 13 S and 21 S dynein ATPases to extracted doublet microtubules. The inner row and outer row dynein arms.

Ciliary axonemes from Tetrahymena contain a second salt-extractable ATPase distinguishable from outer arm 21 S dynein by sedimentation velocity (congruent to 13 S), electrophoretic mobility and substrate specificity. As characterized by turbidimetric assay, gel electrophoresis in the presence of sodium dodecyl sulphate, ATPase activity and electron microscopy, the 13 S dynein ATPase rebinds to extracted doublet microtubules. Compared to structural-side (ATP-insensitive) 21 S dynein binding, which is moderately specific for the 24 nm outer row arm position, rebinding of 13 S dynein is highly specific but for the inner row arm position. However, 13 S dynein rebinds to the A subfibre with a spacing that coincides with the triplet spacing of the radial spokes (24-32-40 nm periods; 96 nm repeat). All of the major protein components present in the 13 S or 21 S fractions rebind to extracted doublets under conditions that both restore and activate dynein ATPase activity. Unlike active-side (ATP-sensitive) rebound 21 S dynein, rebound 13 S dynein is completely insensitive to dissociation by ATP-vanadate and does not independently decorate the B subfibre. The saturation profile for rebinding of 13 S dynein exhibits a lack of cooperativity between binding events (h = 1.0) similar to structural-side rebinding of 21 S dynein. At low 21 S/doublet stoichiometry there is no measureable competition between the 13 S and 21 S dyneins for binding sites on the A subfibre lattice, although at saturating concentrations of 21 S dynein, rebinding of 13 S dynein is blocked completely.

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.