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Immunological determination of transducin content in retinas exhibiting inherited degeneration
Abstract:
The inherited disorders of rd mice and affected Irish setter dogs are characterized by the accumulation of cyclic GMP (cGMP). Since the cGMP level in normal retinal rods is regulated by a light-activated enzyme cascade involving rhodopsin, transducin, and phosphodiesterase, an abnormality associated with any of these three proteins would cause cGMP accumulation. In order to determine the relationship between different forms of retinal degeneration and the transducin content in the affected retinas, affinity-purified antibodies directed against the individual subunits of bovine transducin were prepared. These antibodies, which recognized transducin in many vertebrate species, were used to compare the retinal content of this protein at various stages of inherited photoreceptor degeneration. In each of the disorders studied (rd and rds mice, RCS rat, and affected Irish setter dog), retinas at early stages of degeneration displayed two characteristics similar to those of normal control retinas. First, all three subunits of transducin were detected and found to have normal electrophoretic mobility, suggesting that these disorders are unlikely to be due to changes in the composition of transducin subunits. Second, the amount of cross-reactive T beta always exceeded those of T alpha and T gamma. This disproportionately higher amount of T beta-like protein became more pronounced as the visual cells degenerated. In retinas which had undergone complete photoreceptor degeneration, cross-reactive T alpha and T gamma were undetectable. In contrast, anti-T beta gamma antibodies detected an amount of T beta-like polypeptide corresponding to 10-25% of the control. Since our anti-T beta gamma antibodies recognize the beta subunit of the GTP-binding N proteins of the adenylate cyclase system, this finding suggests that this residual T beta-like protein, which is not part of transducin, may be associated with other GTP-binding regulatory proteins.