Antiserum against the a subunit of bovine rod-outer-segment transducin was used in an immunocytochemical study that identified the protein in retina (human, baboon, owl monkey, cow, rat, quail, newt, frog, salmon, eel, and lamprey), pineal organ (quail, newt, frog, salmon, eel, and lamprey), and parapineal organ (salmon and lamprey). No reaction was observed in the cow or rat pineal organ or the eel parapineal organ. The immunoreaction was very strong in outer segments but weak in perikarya. Immunoblots of crude tissue extracts of bovine rod-outer-segment membranes and frog and fish retina revealed a 39-kDa immunopositive band. The fish retina also contained two additional bands of mass 43 kDa and 25 kDa. Only the 43-kDa band was present in the fish pineal organ, which is photosensitive. This raises the possibility that the 43-kDa a transducin-immunopositive molecule present in the fish pineal organ and retina may be involved in phototransduction.Transducin (TD) is a member of a family of heterotrimeric GTP-binding (G) An important development in knowledge of G proteins has been a better understanding of their subunits. It appears that the ,B subunits (35 kDa) of all G proteins are similar but that the a subunit (39-42 kDa) of each is unique (4, 5).The unique nature of the a subunit of TD (TDa) makes it possible to specifically identify TD immunohistochemically. We were interested in the precise localization of TD in vertebrate retinae and also in determining whether it was present in vertebrate pineal complexes. The latter issue is of interest because it will provide some insight into the evolution of the vertebrate pineal gland from a photosensitive organ in lower forms (6), which contains the retinal proteins opsin and S antigen (7,8), to a nonphotosensitive organ in mammals, which contains only some retinal proteins including rhodopsin kinase and S antigen (9-11). TDa in the mammalian pineal gland could play an important role in neural control mechanisms. To investigate these issues, we used a highly specific antiserum against TDa, which does not recognize purified a subunits of other G proteins (12).
MATERIALS AND METHODSImmunochemistry. TD was purified from bovine rod-outersegment membranes (2); TDc. was purified by blue Sepharose chromatography (12). Rabbits were immunized with purified (>95%) TDa as described (4, 12); the antiserum used in this study is GI-2/10-12-84.The details of NaDodSO4/PAGE and immunoblotting techniques and the sources of all materials have been given (4, 12). Crude plasma membrane preparations were made by either sonication or teflon/glass homogenization of tissues, followed by 10,000 x g centrifugation. The crude membrane pellet was resuspended in water and NaDodSO4/PAGE sample buffer (4), boiled for 2 to 3 min, and cooled before loading on gels.Immunohistochemistry. Light-adapted retinae and pineal organs of adult human, baboon (Papio), owl monkey (Aotes), cattle (Bos taurus), rat (Wistar rat), quail (Coturnix coturnix japonica), newt (Triturus vulgaris), frog (Rana es...