Abstract
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) |
|---|---|
| Pages (from-to) | 13478-13482 |
| Number of pages | 5 |
| Journal | Journal of Biological Chemistry |
| Volume | 260 |
| Issue number | 25 |
| State | Published - 1985 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
ADP-ribosylation of transducin by pertussis toxin. / Watkins, Paul A; Burns, D. L.; Kanaho, Y.; Liu, T. Y.; Hewlett, E. L.; Moss, J.
In: Journal of Biological Chemistry, Vol. 260, No. 25, 1985, p. 13478-13482.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - ADP-ribosylation of transducin by pertussis toxin
AU - Watkins, Paul A
AU - Burns, D. L.
AU - Kanaho, Y.
AU - Liu, T. Y.
AU - Hewlett, E. L.
AU - Moss, J.
PY - 1985
Y1 - 1985
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=0022398989&partnerID=8YFLogxK
M3 - Article
C2 - 3863817
AN - SCOPUS:0022398989
VL - 260
SP - 13478
EP - 13482
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 25
ER -