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 Myti lus edulis or the clam Spisula solidissima removed most visible dynein arms, accompanied by an amount of Mg+2-ATPase that correlated with th e mass of dynein alpha- and beta-heavy chains removed. However, althou gh almost devoid of ATPase activity, such extracted axonemes retained one third of the heavy chain mass as two sets of electrophoretically-d istinct, vanadate-cleavable, non-phosphorylated proteins. To explore t he 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 dy nein. 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 i mmunologically 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 conta ining one of the two tightly-bound Spisula flagellar proteins produced an antiserum that cross-reacted with each tightly-bound protein in bo th 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, bu t not with sea urchin outer arm alpha- or beta-heavy chains. These dat a indicate that the electrophoretically and immunologically distinct, tightly-bound proteins of molluscan flagella are inner arm dynein heav y chains. (C) 1995 Wiley-Liss, Inc.