Outer arm dynein from Newt lung respiratory cilia: purification and polypeptide composition

Dyneins are multimeric ATPases that comprise the inner and outer arms of cilia and flagella. It previously has been shown that salt extraction of newt lung axonemes selectively removes > 95% of the outer arm dynein (OAD), and that the beat frequency of OAD-depleted axonemes cannot be activated as compared to controls [Hard et al., 1992: Cell Motil. Cytoskeleton 21:199-209]. Therefore, expression of the activated state appears to require the presence of outer dynein arms. The present study was undertaken to ascertain basic information on the structure and molecular composition of newt OAD. Populations of demembranated axonemes were extracted with 0.375 M salt. Each lung released approximately 1.4 x 10(7) axonemes during isolation, yielding approximately 120 ng of salt extractable OAD. Electron microscopy of negatively stained samples revealed that newt OAD consisted of two globular heads joined together by a Y-shaped stem, similar to sea urchin and trout sperm OAD. Each head appeared to be roughly spherical in shape, measuring approximately 17 nm in diameter. Electrophoretic analysis of whole axonemes revealed more than six dynein heavy chains when resolved in silver stained 0-8 M urea, 3-5% acrylamide gradients. Extracted OAD, either crude in high salt or purified by alloaffinity, was composed of two heavy chains. UV-induced (366 nm) photolytic cleavage at the V1 site, performed in the presence of Mg2+, vanadate, and ATP, produced four new polypeptides (M(r) 234, 232, 197, and 189 kD). Photolysis was supported by Mg2+ and Ca2+, but did not occur in the presence of Mn2+. The apparent M(r) of the dynein heavy chains was determined to lie between 430-420 kD. Eight discrete polypeptides (putative intermediate chains, IC1-IC8, M(r), 175-56 kD) copurified with the alpha- and beta-heavy chains by microtubule-alloaffinity. Based on its extraction characteristics, polypeptide composition in purified and crude samples, and structure, we conclude that this two-headed particle represents the entire newt respiratory outer arm dynein.