Calmodulin confers calcium sensitivity on ciliary dynein ATPase.

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Abstract
Extraction of demembranated cilia of Tetrahymena [T. pyriformis] by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca2+. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yields a 14S-K dynein with a low basal ATPase activity in the presence of Ca2+. Subsequent addition of calmodulin causes marked activation (up to 10-fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca2+-dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, .apprx. 0.1 .mu.M. 30S-K and 30S-E dyneins require .apprx. 0.7 .mu.M bovine brain calmodulin to reach half-maximal activation of their Ca2+-dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein, but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca2+-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in < 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca2+, but at much higher concentrations than required for prevention by ATP. .beta.,.gamma.-methyl-ATP appears not to prevent this activation. Ca2+-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca2+-dependent retention of the dyneins on a calmodulin-Sepharose 4B column. Gel electrophoresis of 14S dynein, purified by the affinity-chromatography, showed 2 major and 1 minor high MW components. Similar analysis of 30S dynein purified by this procedure also revealed 1 major and 1 minor high MW components that were different from the major conponents of 14S dynein. Ca2+-dependent binding sites for calmodulin were on axonemes that were extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. 14S and 30S dyneins of Tetrahymena contain Ca2+-dependent binding sites for calmodulin and that calmodulin mediates the Ca2+-regulation of the dynein ATPases of Tetrahymena cilia.