Isolation of cilia from porcine tracheal epithelium and extraction of dynein arms.
Milligram amounts of mammalian ciliary axonemes were isolated from porcine tracheas. These were reactivated upon addition of ATP, indicating intact functional capability with a mean beat frequency at 37 degrees C of 8.2 Hz. Electron microscopy showed typical ultrastructure of the isolated demembranated axonemes. Electrophoresis into polyacrylamide gradient gels containing sodium dodecyl sulfate revealed reproducible protein profiles from ten different tracheal preparations. Four major protein bands were observed in the 300-330 K molecular weight region, as well as tubulin at 51-54K. Extraction of the isolated tracheal axonemes with 0.6M KCl removed the outer dynein arms seen in electron microscopic cross-section of axonemes, preferentially solubilized two of the high molecular weight proteins at 320 and 330 K, and resulted in a three- to four-fold increase in ATPase specific activity. Sedimentation of the dialyzed salt extract on a 5-30% sucrose density gradient and subsequent fractionation yielded two peaks of ATPase activity. The faster migrating, 19S major ATPase peak correlated with the 320 and 330 K proteins, and two other proteins at 81 and 67 K. The slower sedimenting, 12S minor ATPase peak corresponded to a 308 K protein and two smaller proteins at 33 and 48 K. Thus, the outer dynein arm of tracheal cilia appeared to be associated with at least two high molecular weight proteins. These results demonstrate that adequate quantities of functionally intact axonemes can be reproducibly isolated from porcine tracheas, allowing further fractionation and analysis of mammalian cilia.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002923:Cilia / Q000032:analysis* / Q000502:physiology / Q000648:ultrastructure; D004398:Dyneins / Q000302:isolation & purification* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000032:analysis / Q000648:ultrastructure; D008854:Microscopy, Electron; D008970:Molecular Weight; D009068:Movement; D013552:Swine; D014132:Trachea / Q000166:cytology*
D000251:Adenosine Triphosphatases; D004398:Dyneins