ADP-ribosylation of transducin by pertussis toxin

Paul A Watkins, D. L. Burns, Y. Kanaho, T. Y. Liu, E. L. Hewlett, J. Moss

Research output: Contribution to journalArticle

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 languageEnglish (US)
Pages (from-to)13478-13482
Number of pages5
JournalJournal of Biological Chemistry
Volume260
Issue number25
StatePublished - 1985
Externally publishedYes

Fingerprint

Transducin
Pertussis Toxin
Adenosine Diphosphate
Guanylyl Imidodiphosphate
Rhodopsin
Labeling
Rod Cell Outer Segment
Guanosine
Guanosine Triphosphate
Proteins
Trypsin
Substrates
Nucleotides
Retinal Photoreceptor Cell Outer Segment
Proteolysis
Retinal Rod Photoreceptor Cells
Light
Radioactivity
Phosphoric Diester Hydrolases
Photons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Watkins, P. A., Burns, D. L., Kanaho, Y., Liu, T. Y., Hewlett, E. L., & Moss, J. (1985). ADP-ribosylation of transducin by pertussis toxin. Journal of Biological Chemistry, 260(25), 13478-13482.

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 journalArticle

Watkins, PA, Burns, DL, Kanaho, Y, Liu, TY, Hewlett, EL & Moss, J 1985, 'ADP-ribosylation of transducin by pertussis toxin', Journal of Biological Chemistry, vol. 260, no. 25, pp. 13478-13482.
Watkins PA, Burns DL, Kanaho Y, Liu TY, Hewlett EL, Moss J. ADP-ribosylation of transducin by pertussis toxin. Journal of Biological Chemistry. 1985;260(25):13478-13482.
Watkins, Paul A ; Burns, D. L. ; Kanaho, Y. ; Liu, T. Y. ; Hewlett, E. L. ; Moss, J. / ADP-ribosylation of transducin by pertussis toxin. In: Journal of Biological Chemistry. 1985 ; Vol. 260, No. 25. pp. 13478-13482.
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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.",
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AU - Watkins, Paul A

AU - Burns, D. L.

AU - Kanaho, Y.

AU - Liu, T. Y.

AU - Hewlett, E. L.

AU - Moss, J.

PY - 1985

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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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