Characterisation of boar sperm dynein heavy chains by UV-vanadate dependent photocleavage.
Intact and Triton X-100 demembranated boar spermatozoa possess two main heavy chains with molecular masses (M(r)) of 430 and 460 kDa. These heavy chains were photo-cleaved within the axoneme under V1 conditions and produced two main fragments at 245 kDa and 185 kDa. Two minor fragments 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 heavier chain was cleaved in the absence of ATP to give the 245 kDa fragment. The boar axonemal heavy chains cannot be directly extracted by high salt treatment of the demembranated sperm in presence of high level of protease inhibitors (HPI) but were extracted when the solution contained low protease inhibitor (LPI) concentrations. Electron microscopy showed that high salt treatment in presence of LPI extracted the outer arm mainly from the principal piece of the flagellum and less from the intermediate piece. Fractionation of the LPI high salt by chromatography 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 and 340 kDa. These heavy chains can be photo-cleaved 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 chains or proteolytic fragments of the heavy chains. A chain at 63 kDa was also associated with the 20S fraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Gatti
J L
JL
URA 1291 INRA-CNRS, Laboratoire de Physiologie de la Reproduction, INRA Nouzilly, Monnaie, France.
Nicolle
J C
JC
Dacheux
J L
JL
D000818:Animals; D005591:Chemical Fractionation; D004398:Dyneins / Q000737:chemistry* / Q000528:radiation effects; D008297:Male; D008854:Microscopy, Electron; D010782:Photolysis; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000378:metabolism; D013094:Spermatozoa / Q000737:chemistry*; D013552:Swine; D014466:Ultraviolet Rays; D014638:Vanadates / Q000494:pharmacology*
D017319:Photosensitizing Agents; D011480:Protease Inhibitors; D014638:Vanadates; D004398:Dyneins