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Record 8 of 42
Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin
Author and Affiliation:
Hicks, G. R.(Oregon State University, Department of Botany and Plant Pathology, Corvallis 97331-2902)
Rayle, D. L.
Jones, A. M.
Lomax, T. L.
Abstract: 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.
Publication Date: Jul 01, 1989
Document ID:
20040112101
(Acquired Oct 05, 2004)
Subject Category: LIFE SCIENCES (GENERAL)
Document Type: Journal Article
Publication Information: Proceedings of the National Academy of Sciences of the United States of America (ISSN 0027-8424); Volume 86; 4948-52
Publisher Information: United States
Contract/Grant/Task Num: DCB-8718731; NAGW-1253; NAGW-230
Financial Sponsor: NASA; Washington DC United States
Description: In English
Distribution Limits: Unclassified; Publicly available; Unlimited
Rights: Copyright
NASA Terms: AUXINS; AZIDES (INORGANIC); AZIDES (ORGANIC); CELL MEMBRANES (BIOLOGY); HORMONES; INDOLEACETIC ACIDS; MEMBRANES; METABOLISM; PEPTIDES; PLANT GROWTH REGULATORS; PLANTS (BOTANY); PLASMAS (PHYSICS); POLYPEPTIDES; VEGETABLES; GENETICS; ION CONCENTRATION; PHOTOLYSIS; PROTEINS; STEMS; TEMPERATURE; TRITIUM; ULTRAVIOLET RADIATION
Other Descriptors: AUXINS/ANALYSIS/METABOLISM; AZIDES/METABOLISM; CELL MEMBRANE/CHEMISTRY/METABOLISM/ULTRASTRUCTURE; INDOLEACETIC ACIDS/ANALYSIS/METABOLISM; PEPTIDES/ANALYSIS/METABOLISM; PLANT GROWTH REGULATORS; VEGETABLES/CHEMISTRY/METABOLISM/ULTRASTRUCTURE; AFFINITY LABELS; HYDROGEN-ION CONCENTRATION; HYPOCOTYL/CHEMISTRY/METABOLISM/ULTRASTRUCTURE; PHOTOLYSIS; PLANT PROTEINS; RECEPTORS, CELL SURFACE/ANALYSIS/GENETICS/METABOLISM; SUPPORT, U.S. GOV'T, NON-P.H.S; SUPPORT, U.S. GOV'T, P.H.S; TEMPERATURE; TRITIUM; ULTRAVIOLET RAYS; NASA DISCIPLINE NUMBER 29-20; NASA DISCIPLINE PLANT BIOLOGY; NASA PROGRAM SPACE BIOLOGY; NON-NASA CENTER
Availability Source: Other Sources
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