Analysis of mammalian dynein using antibodies against A polypeptides of sea urchin sperm flagellar dynein

Abstract

Two different affinity-purified polyclonal antibodies were prepared against A polypeptides of dynein 1 extracted from sea urchin sperm. These antibodies, named AD1 and AD2, reacted exclusively with the alpha and beta heavy chains of dynein 1. Using these antibodies, we analyzed their cross-reactivity with dynein of mammalian cells. Immunohistochemically, both AD1 and AD2 stained dynein-related structures such as cilia of rabbit tracheal epithelia and flagella of rat spermatozoa. Immunoblots of the proteins extracted from mammalian cilia and flagella revealed the presence of A polypeptide-like proteins which cross-reacted with AD1 and AD2. Immunoblot analysis showed that the cross-reactive proteins were localized to the 370-kDa band of rabbit cilia and the 390- and 350-kDa bands of rat sperms. The reaction patterns showed that there were some differences between the two antibodies. On ciliary protein immunoblots, AD1 recognized about half of the broad band region which reacted with AD2, and AD1 also recognized only the 350-kDa band of the flagella extract, suggesting that the antibody reveals only a beta-like polypeptide. Immunoprecipitation studies using the ciliary proteins and AD2 confirmed that the immunoreactive protein had ATPase activity. Given these results, we have characterized mammalian dyneins previously reported by other laboratories.