Evidence for functional differences between two flagellar dynein ATPases

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Abstract
Energy coupling in flagellar motility was investigated using demembranated, reactivated sea urchin spermatozoa (Arbacia punctulata). The ATP-dependence of ATPase activity was investigated for ATP concentrations ranging from 4 μM to 600 μM ATP. Using Eadie-Scatchard plot analysis, we identified two axonemal dynein ATPase activities. Their apparent Michaelis constants were calculated to be equal to 4 μM and 161 μM ATP, and they were referred to, respectively, as the high-affinity dynein ATPase (HADA) and the low-affinity dynein ATPase (LADA). Investigation of movement-coupled ATPase activity (difference between the ATPase activities of reactivated and broken, immotile spermatozoa) indicated that HADA and LADA were both 65% movement-coupled. The apparent Michaelis constants of movement-coupled HADA and LADA, 12 μM and 271 μM ATP, respectively, were two- to four-fold greater than the apparent Michaelis constants of movement-uncoupled HADA and LADA. The apparent Michaelis constants for force generation and beat frequency of reactivated spermatozoa were determined to be 24 μM and 290 μM ATP, respectively. These results raise the possibility that flagellar force generation is controlled primarily by movement-coupled HADA, and that flagellar beat frequency is controlled primarily by movement-coupled LADA. Thus, mechanochemical activity in flagellar motility may be divided between two enzymatically and functionally distinct classes of flagellar dyneins.