2-Azido-( sup 32 P)NAD+, a photoactivatable probe for G-protein structure: Evidence for holotransducin oligomers in which the ADP-ribosylated carboxyl terminus of alpha interacts with both alpha and gamma subunits

Journal Article · Tue May 01 00:00:00 EDT 1990 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.87.10.3645· OSTI ID:6727646

Vaillancourt, R R; Dhanasekaran, N; Johnson, G L; Ruoho, A E ^[1]

---

1. Univ. of Wisconsin Medical School, Madison (USA)

A radioactive and photoactivatable derivative of NAD+, 2-azido-(adenylate-32P)NAD+, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys347 of the alpha subunit (alpha T) of GT, the retinal guanine nucleotide-binding protein. ADP-ribosylation of alpha T followed by light activation of the azide moiety of 2-azido-(adenylate-32P)ADP-ribose produced four crosslinked species involving the alpha and gamma subunits of the GT heterotrimer: an alpha trimer (alpha-alpha-alpha), and alpha-alpha-gamma crosslink, an alpha dimer (alpha-alpha), and an alpha-gamma crosslink. The alpha trimer, alpha-alpha-gamma complex, alpha dimer, and alpha-gamma complexes were immunoreactive with alpha T antibodies. The alpha-alpha-gamma and the alpha-gamma complexes were immunoreactive with antisera recognizing gamma subunits. No evidence was found for crosslinking of alpha T to beta T subunits. Hydrolysis of the thioglycosidic bond between Cys347 and 2-azido-(adenylate-32P)ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys347 of alpha T in the crosslinked oligomers to alpha monomers, indicative of intermolecular photocrosslinking, and to gamma monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that GT exists as an oligomer and that ADP-ribosylated Cys347, which is four residues from the alpha T-carboxyl terminus, is oriented toward and in close proximity to the gamma subunit.

• OSTI ID: 6727646
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Vol. 87:10; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
NAD
CHEMICAL PREPARATION
PROTEINS
METABOLISM
ADP
AFFINITY
CROSS-LINKING
PHOSPHORUS 32
PHOTOLYSIS
PROTEIN STRUCTURE
RETINA
RIBOSE
TOXINS
TRACER TECHNIQUES
ALDEHYDES
ANTIGENS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
BODY AREAS
CARBOHYDRATES
CHEMICAL REACTIONS
COENZYMES
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
EYES
FACE
HEAD
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MATERIALS
MONOSACCHARIDES
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PENTOSES
PHOSPHORUS ISOTOPES
PHOTOCHEMICAL REACTIONS
POLYMERIZATION
RADIOISOTOPES
SACCHARIDES
SENSE ORGANS
SYNTHESIS
TOXIC MATERIALS
550201* - Biochemistry- Tracer Techniques