Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 86; no. 13; pp. 4948 - 4952
• Main Authors: Hicks, G.R. (Oregon State University, Corvallis, OR), Rayle, D.L, Jones, A.M, Lomax, T.L
• Format: Journal Article
• Language: English
• Published: Headquarters National Academy of Sciences of the United States of America 01-07-1989; National Acad Sciences
• Subjects: 550201 - Biochemistry- Tracer Techniques; ACIDE INDOLACETIQUE; ACIDO INDOLACETICO; Adenosine triphosphatases; Affinity Labels; Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; AUXINAS; AUXINE; AUXINS; Azides - metabolism; BASIC BIOLOGICAL SCIENCES; BIOCHEMICAL REACTION KINETICS; Biological and medical sciences; Biological Sciences: Botany; CELL CONSTITUENTS; Cell Membrane - chemistry; Cell Membrane - metabolism; Cell Membrane - ultrastructure; CELL MEMBRANES; CELL STRUCTURE; CUCURBITA PEPO; ELECTROMAGNETIC RADIATION; ELECTROPHORESIS; Enzymes; ESTRUCTURA CELULAR; ETAPAS DE DESARROLLO DE LA PLANTA; FAR ULTRAVIOLET RADIATION; Fundamental and applied biological sciences. Psychology; Gels; HYDROGEN COMPOUNDS; Hydrogen-Ion Concentration; Hypocotyl - chemistry; Hypocotyl - metabolism; Hypocotyl - ultrastructure; Hypocotyls; IAA; Indoleacetic Acids - analysis; Indoleacetic Acids - metabolism; KINETICS; Life Sciences (General); MEMBRANE PROTEINS; MEMBRANES; MOLECULAR STRUCTURE; ORGANIC COMPOUNDS; PEPTIDE; PEPTIDES; Peptides - analysis; Peptides - metabolism; PEPTIDOS; Photolysis; PLANT DEVELOPMENTAL STAGES; Plant growth; PLANT GROWTH REGULATORS; Plant Proteins; POLYPEPTIDES; Product labeling; PROTEINS; RADIATIONS; REACTION KINETICS; RECEPTORS; Receptors, Cell Surface - analysis; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; SEEDS; Space life sciences; STADE DE DEVELOPPEMENT VEGETAL; STRUCTURE CELLULAIRE; Temperature; Tritium; TRITIUM COMPOUNDS; ULTRAVIOLET RADIATION; Ultraviolet Rays; Vegetables - chemistry; Vegetables - metabolism; Vegetables - ultrastructure; Nasa Program Space Biology; Nasa Discipline Plant Biology; Nasa Discipline Number 29-20; Non-Nasa Center; Vegetals; Auxin; Radiolabelling; Peptides; Cucurbitaceae; Cucurbita pepo; Photoaffinity labelling; Specificity; Photolysis; Dicotyledones; Angiospermae; Plasma membrane; Spermatophyta; Biological receptor; NASA Program Space Biology; NASA Discipline Number 29-20; NASA Discipline Plant Biology; Non-NASA Center
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.13.4948

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.