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Detection of GTP-binding proteins in barley aleurone protoplasts

Author: Wang, M. · Sedee, N.J.A. · Heidekamp, F. · Snaar-Jagalska, B.E.
Type:article
Date:1993
Source:FEBS Letters, 3, 329, 245-248
Identifier: 36713
doi: doi:10.1016/0014-5793(93)80230-R
Keywords: Barley aleurone · GTP-binding protein · Phytohormone · Guanine nucleotide binding protein · Phytohormone · Barley · Nonhuman · Protein purification · Protoplast · Binding Sites · Blotting, Western · Cell Membrane · Electrophoresis, Polyacrylamide Gel · GTP-Binding Proteins · Hordeum · Plant Growth Regulators · Protoplasts · Signal Transduction · Support, Non-U.S. Gov't · Temperature

Abstract

We report the existence of several families of GTP-binding proteins in barley aleurone protoplasts. Partial purified plasma membrane proteins were separated by SDS-PAGE, transferred to a nitrocellulose filter and incubated with either antisera raised against a highly conserved animal G protein α subunit peptide/or Ras protein, or with [α-32P]GTP. Two sets of proteins of M(r) = 32-36 kDa and 22-24 kDa were strongly recognized by the antisera. Binding of [α-32P]GTP was detected on Western blots with proteins of M(r) = 22-24 kDa and 16 kDa. Binding was inhibited by 10-7-10-6 M GTPγS, GTP or GDP; binding was not affected by 10-6-10-5 M ATPγS or ADP. The kinetics, specificity and the effects of phytohormones in a[35S]GTPγS binding assay were also studied in isolated plasma membranes of barley aleurone protoplasts. Chemicals/CAS: GTP-Binding Proteins, EC 3.6.1.-; Plant Growth Regulators