Gibberellin-photoaffinity labeling of wild oat (Avena fatua L.) aleurone protoplasts |
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Authors: | Robert P Walker Wanda M Waterworth Michael H Beale Richard Hooley |
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Institution: | (1) Department of Agricultural Sciences, University of Bristol, 1 ACR Long Ashton, Long Ashton Research Station, BS18 9AF Bristol, UK;(2) Present address: Department of Plant and Animal Sciences, Robert Hill Institute, P.O. Box 601, S10 2UQ Sheffield, UK;(3) Present address: School of Biological Sciences, Department of Biochemistry and Molecular Biology, University of Manchester, Stopford Building, Oxford Road, M13 9PT Manchester, UK |
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Abstract: | Aleurone protoplasts of wild oat (Avena fatua L.), and subcellular fractions isolated from them, were photoaffinity labeled using the synthetic gibberellin (GA) derivative GA4-17-yl-1-(1-thia)propan-3-ol-4-azido-5-125I]iodosalicylate. Labeled polypeptides were identified by electrophoresis under denaturing conditions followed by autoradiography. GA-photoaffinity labeling of both intact protoplasts and isolated subcellular fractions led to the covalent attachment of the reagent to many polypeptides. A 50 kD polypeptide in the soluble fraction of homogenates of aleurone protoplasts GA-photoaffinity labeled in vivo showed specific binding. The biologically active GA1, GA4 and GA4-17-yl-1(1-thia)propan-3-ol-4-azidosalicylate completed for binding whereas the biologically inactive GA8 and GA34 did not. The GA-photoaffinity labeling characteristics of this polypeptide suggested that it might interact specifically with biologically active GAs in vivo. Attempts to detect specific GA-binding in in vitro GA-photoaffinity labeling experiments met with only limited success perhaps indicating the labile nature of specific binding observed in vivo. The potential of GA-photoaffinity labeling for identifying GA-binding proteins in aleurone and other GA-responsive tissues is discussed.Abbreviations azido IAA =
5-azido-7-3H]indole-3-acetic acid
- azido NPA =
5-azido-3,6-3H]1-N-napthylpthalamic acid
- BTP =
1,3-bis(Tris(hydroxymethyl)methylamino)-propane
- GA4-O-ASA =
GA4-17-yl-1-(1-thia)propane-3-ol-4-azidosalicylate
- 125I]GA4-O-ASA =
GA4-17-yl-1-(1-thia)propan-3-ol-4-azido-5-125I]iodosalicylate
- NPA =
1-Naphthylphthalmic acid
- PAGE =
Polyacrylamide gel electrophoresis
- PMSF =
phenylmethylsulfonyl fluoride
- SDS =
Sodium dodecyl sulphate
- TLCK =
L-1-Chloro-3-(4-tosylamido)-7-amino-2-heptanone-HCl |
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Keywords: | -amylase" target="_blank">gif" alt="agr" align="BASELINE" BORDER="0">-amylase Avena fatua gibberellin-binding proteins gibberellins Gramineae photoaffinity wild oat |
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