Dynein inner arm heavy chain identification in cAMP-activated flagella using class-specific polyclonal antibodies.
While studying cAMP-dependent dynein alpha-heavy chain phosphorylation, we found previously [Stephens and Prior, 1992: J. Cell Sci. 103:999-1012] that high salt extraction of sperm flagella from the mussel Mytilus edulis or the clam Spisula solidissima removed most visible dynein arms, accompanied by an amount of Mg+2-ATPase that correlated with the mass of dynein alpha- and beta-heavy chains removed. However, although almost devoid of ATPase activity, such extracted axonemes retained one third of the heavy chain mass as two sets of electrophoretically-distinct, vanadate-cleavable, non-phosphorylated proteins. To explore the nature of these dynein-like proteins, antibodies to the alpha- and beta-heavy chains were blot affinity-purified from a rabbit antiserum raised against gradient-purified Spisula 18-20S flagellar outer arm dynein. Although able to recognize common epitopes of the opposite chain type, neither the alpha- nor the beta-heavy chain antibody recognized the tightly-bound proteins in either species, proving that they are immunologically distinct. While the beta-antibody recognized its heavy chain homolog in gill cilia, the alpha-antibody did not, demonstrating immunological distinction between flagellar and ciliary dynein alpha-heavy chains. Immunization of a mouse with nitrocellulose strips containing one of the two tightly-bound Spisula flagellar proteins produced an antiserum that cross-reacted with each tightly-bound protein in both species and also recognized alpha- and beta-heavy chains. The anti-molluscan serum cross-reacted strongly with sea urchin sperm flagellar dynein B-, C-, and D-bands, considered to be inner arm components, but not with sea urchin outer arm alpha- or beta-heavy chains. These data indicate that the electrophoretically and immunologically distinct, tightly-bound proteins of molluscan flagella are inner arm dynein heavy chains.
Stephens
R E
RE
Department of Physiology, Boston University School of Medicine, Massachusetts 02118, USA.
Prior
G
G
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D049872:Bivalvia / Q000378:metabolism*; D003429:Cross Reactions; D000242:Cyclic AMP / Q000378:metabolism; D004398:Dyneins / Q000032:analysis* / Q000276:immunology; D008297:Male; D013082:Sperm Tail / Q000201:enzymology
D000906:Antibodies; D000242:Cyclic AMP; D004398:Dyneins