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Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxinPlasma 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.
Document ID
20040112101
Acquisition Source
Headquarters
Document Type
Reprint (Version printed in journal)
Authors
Hicks, G. R.
(Oregon State University Corvallis 97331-2902)
Rayle, D. L.
Jones, A. M.
Lomax, T. L.
Date Acquired
August 21, 2013
Publication Date
July 1, 1989
Publication Information
Publication: Proceedings of the National Academy of Sciences of the United States of America
Volume: 86
ISSN: 0027-8424
Subject Category
Life Sciences (General)
Funding Number(s)
CONTRACT_GRANT: NAGW-230
CONTRACT_GRANT: DCB-8718731
CONTRACT_GRANT: NAGW-1253
Distribution Limits
Public
Copyright
Other
Keywords
NASA Discipline Number 29-20
NASA Program Space Biology
NASA Discipline Plant Biology
Non-NASA Center

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