High-pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner-arm dynein subunits |
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| Authors: | U W Goodenough B Gebhart V Mermall D R Mitchell J E Heuser |
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| Affiliation: | 1. Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia;;2. Department of Pediatrics, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia;;3. Department of Ophthalmology, Specialized Medical Center Hospital, Riyadh, Saudi Arabia;;4. Department of Obstetrics and Gynecology, Security Forces Hospital, Riyadh, Saudi Arabia;;5. Deparment of Obstetrics and Gynecology, College of Medicine, King Saud University, Riyadh, Saudi Arabia;;6. Human Genetics Department, Medical Research Institute, Alexandria University, Alexandria, Egypt;;7. Department of Pediatrics, Security Forces Hospital, Riyadh, Saudi Arabia;;8. Department of Pediatrics, College of Medicine, AlBaha University, AlBaha, Saudi Arabia;;9. Endocrinology Section, Department of Pediatrics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia;;10. Department of Pediatrics, Prince Sultan Military Medical City, Riyadh, Saudi Arabia;;11. Nephrology Section, Department of Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia;;12. Department of Neuroscience, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia;;13. Organ Transplant Center, King Faisal Specialist Hospital and Research Center, and College of Medicine, Alfaisal University, Riyadh, Saudi Arabia;;14. Nephrology Section, Department of Pediatrics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia;;15. Division of Pediatric Neurology, College of Medicine, King Saud University, Riyadh, Saudi Arabia;;16. Depatment of Obstetrics and Gynecology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia;;17. Department of Medical Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia;;18. Divison of Genetics, Department of General Pediatrics, King Fahad Central Hospital, Jazan, Saudi Arabia;;19. Vitreoretinal Division, King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia;;20. Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia;;21. Department of Pediatrics, King Fahad General Hospital, Jeddah, Saudi Arabia. |
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| Abstract: | 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. |
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