Biochemistry 1996 Jul 16;3528:9204-11. doi: 10.1021/bi960662u.

Phase partition analysis of nucleotide binding to axonemal dynein.

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The binding of nucleoside triphosphates and related ligands to dynein ATPase from sea urchin sperm flagella has been studied by equilibrium partition analysis in an aqueous biphasic system containing dextran and poly(ethylene glycol). The stoichiometry of binding and the corresponding stepwise binding constants are obtained from direct binding isotherms fitted to the primary data. The results suggest that dynein possesses four different binding sites for nucleoside triphosphates per mole of heavy chain. The stepwise binding constants for MgATP range from approximately 10(4) M-1 to approximately 10(5) M-1. The isolated alpha and beta heavy chains have binding parameters similar to intact dynein. The amount of ADP bound normally is approximately 75% that of ATP, both for the intact dynein and for the separated heavy chains, although full saturation is achieved at high nucleotide concentrations. In the presence of the ATPase inhibitor vanadate, ADP binds with affinities similar to those of ATP, with binding constants close to those of ATP in the absence of vanadate. No appreciable binding of AMP or EDTA/ATP is observed. The substitution of Ca2+ or Fe3+ for Mg2+ does not significantly alter the amount of ATP bound; however, CaATP is bound with a somewhat lower affinity. Scatchard and Hill plots of the binding data and the calculated site-binding constants suggest that ATP and ADP bind in a weakly cooperative manner. These results suggest that the multiple binding of nucleotide to dynein heavy chains occurs at physiological concentrations, putatively at the four binding sites predicted earlier on the basis of their amino acid sequences. The data are consistent with a model in which, in addition to a single catalytic site, nucleotide binding occurs at additional noncatalytic sites that represent an as yet unknown functional aspect of dynein.

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Genes referenced: dnah3 LOC100887844 LOC581395 LOC592542