Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T(α) and T(βγ). T(α) (39 kDa), which is [32P]ADP-ribosylated by pertussis toxin and [32P]NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T(βγ) (36 kDa and ~ 10 kDa); neither component of T(βγ) was a pertussis toxin substrate. Labeling of T(α) was enhanced by T(βγ) and was maximal at ~ 1:1 molar ratio of T(α):T(βγ). Limited proteolysis by trypsin of T(α) in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and 32 kDa. The amino terminus of both 38- and 32-kDa proteins was leucine, whereas that of T(α) could not be identified and was assumed to be blocked. The 32-kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T(βγ). Trypsin treatment of [32P]ADP-ribosyl-T(α) produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T(α) (in the presence of T(βγ) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTPγS), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDPβS) but was increased by ATP. When photolyzed rhodopsin and T(βγ) were present, Gpp(NH)p and GTP(γ)S decreased [32P]ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed [32P]ADP ribosylation of T(α) was affected by nucleotides, rhodopsin and light in addition to T(βγ). The amino terminus of T(α), while it does not contain the pertussis toxin ADP-ribosylation site, appeared critical to its reactivity.
|Original language||English (US)|
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|State||Published - 1985|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology