ALLOAFFINITY PURIFICATION OF DYNEIN (CILIA, MT-ASSOCIATED ATPASE)

Abstract

The bending movements of eukaryotic flagella and cilia have been shown to originate in a sliding motion between adjacent microtubule doublets. This sliding force can be attributed to the ciliary ATPase, dynein. Dynein can be extracted from the axonemes of Tetrahymena thermophila by low ionic strength dialysis. The soluble dynein will rebind to microtubules in an ATP sensitive way if the appropriate ionic conditions are reestablished. This phenomenon forms the basis of a rapid and highly specific purification procedure based on the ATP-regulated binding and release cycle.;Tetrahymena ciliary doublet microtubules, extracted free of dynein and other axonemal components, can be retained on the surface of a GVWP filter (Millipore). Buffer (+OR-)ATP passed through the microtubule-loaded filter yields neither microtubules nor ATPase in the filtrate. Crude dynein-containing extract (in the absence of microtubules) is able to pass through the filter unaffected, but when the dynein is allowed to bind to the microtubules before application to the filter, the dynein is retained. If buffer + ATP is now passed through the filter, the dynein is released into the filtrate. Since the technique demands the dynein to recognise two ligands, the procedure has been named alloaffinity chromatography.;Alloaffinity purified dynein from Tetrahymena has been extensively characterised. It is a monodisperse oligomer consisting of about 11 polypeptides, ATPase activity highly specific for ATP and, at physiological pH, the purified dynein displays non-linear saturation kinetics suggestive of negative cooperativity. The activity is stimulated 40% by the presence of microtubules. An adenylate kinase activity copurifies with the dynein in significant and reproducible amounts. Alloaffinity purified dynein is capable of rebinding to microtubules with a morphology appropriate for the in situ dynein arm. Thus, alloaffinity chromatography yields an authentic dynein molecule, suitable for further study. Alloaffinity chromatography has been applied to a crude flagellar extract of Chlamydomonas. Using Tetrahymena microtubules, the alloaffinity procedure yields a group of polypeptides which correspond well to the reported subunit composition of Chlamydomonas outer arm dynein. Alloaffinity chromatography is therefore potentially applicable to the study of axonemal dyneins from many species. In addition, the technique is a promising tool to search for dynein like molecules in non ciliated cells.