CHARACTERIZATION OF BOAR SPERM DYNEIN HEAVY-CHAINS BY UV-VANADATE DEPENDENT PHOTOCLEAVAGE

Intact and Triton X-100 demembranated boar spermatozoa possess two mai n heavy chains with molecular masses (M(r)) of 430 and 460 kDa. These heavy chains were photocleaved within the axoneme under V1 conditions and produced two main fragments at 245 kDa and 185 kDa. Two minor frag ments at 170 and 90 kDa were also obtained. In the presence of low Mg2 + (1 mM) a supplementary fragment of 200 kDa was also observed. The he avier chain was cleaved in the absence of ATP to give the 245 kDa frag ment. The boar axonemal heavy chains cannot be directly extracted by h igh salt treatment of the demembranated sperm in presence of high leve l of protease inhibitors (HPI) but were extracted when the solution co ntained low protease inhibitor (LPI) concentrations. Electron microsco py showed that high salt treatment in presence of LPI extracted the ou ter arm mainly from the principal piece of the flagellum and less from the intermediate piece. Fractionation of the LPI. high salt by chroma tography or sucrose gradient allowed the obtention of a particle with ATPase activity, a size of 1.2 MDa and a sedimentation coefficient of about 20S. The particle was composed of two heavy chains of M(r) 320 a nd 340 kDa. These heavy chains can be photocleaved under V1 conditions and in absence of Mg2+. The sucrose gradient 20S fractions contained also two chains at 110 and 87 kDa which could be either intermediate c hains or proteolytic fragments of the heavy chains. A chain at 63 kDa was also associated with the 20S fraction. These results show that boa r sperm dynein heavy chains are similar in molecular mass and photolyt ic properties compared to invertebrate or lower vertebrate sperm dynei ns, and suggest that the outer arm dyneins are maintained differently within the axoneme.