High-pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner-arm dynein subunits.
A rapid procedure for fractionating salt-stable dynein subunits from high-salt extracts of Chlamydomonas axonemes has been developed using a high-pressure liquid chromatography system with an anion exchange column and gradient salt elution. Five distinct fractions are shown to be highly enriched for five distinct subunits or subunit complexes by SDS/polyacrylamide gel electrophoresis. ATPase activity and electron microscopy. Peaks 1 and 4 contain, respectively, the single-headed gamma-subunit and the two-headed alpha/beta-heteropolymer that form the outer arm in situ and are dissociated by salt exposure; both peaks are absent from the outer arm-less mutant pf-28. Peaks 2, 3 and 5 contain, respectively, two distinct single-headed species and a double-headed species that derive from inner arms; all three peaks are missing from the inner arm-less mutant pf-23. Sucrose-gradient sedimentation analysis confirms these assignments and provides additional information on the intermediate-chain and light-chain composition of the inner-arm species. Electron microscopy of the purified inner-arm species visualized by the quick-freeze deep-etch technique complements a previous analysis of outer-arm species. Each protein is shown to have a unique morphology, and both the inner- and outer-arm proteins clearly belong to a common family whose structural divergence presumably reflects functional specialization.
D000251:Adenosine Triphosphatases / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D002696:Chlamydomonas / Q000032:analysis* / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D004398:Dyneins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008854:Microscopy, Electron
D046911:Macromolecular Substances; D000251:Adenosine Triphosphatases; D004398:Dyneins