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 extracti on of newt lung axonemes selectively removes >95% of the outer arm dyn ein (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 stu dy was undertaken to ascertain basic information on the structure and molecular composition of newt OAD. Populations of demembranated axonem es were extracted with 0.375 M salt. Each lung released similar to 1.4 x 10(7) axonemes during isolation, yielding similar to 120 ng of salt extractable OAD. Electron microscopy of negatively stained samples re vealed that newt OAD consisted of two globular heads joined together b y a Y-shaped stem, similar to sea urchin and trout sperm OAD. Each hea d appeared to be roughly spherical in shape, measuring similar to 17 n m in diameter. Electrophoretic analysis of whole axonemes revealed mor e than six dynein heavy chains when resolved in silver stained 0-8 M u rea, 3-5% acrylamide gradients. Extracted OAD, either crude in high sa lt or purified by alloaffinity, was composed of two heavy chains. UV-i nduced (366 nm) photolytic cleavage at the V1 site, performed in the p resence of Mg2+, vanadate, and ATP, produced four new polypeptides (M( r) 234, 232, 197, and 189 kD). Photolysis was supported by Mg2+ and Ca 2+, but did not occur in the presence of Mn2+. The apparent M(r) of th e 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 microtubul e-alloaffinity. Based on its extraction characteristics, polypeptide c omposition in purified and crude samples, and structure, we conclude t hat this two-headed particle represents the entire newt respiratory ou ter arm dynein. (C) 1995 Wiley-Liss, Inc.