Detection of GTP-binding proteins in barley aleurone protoplasts.
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 alpha subunit peptide/or Ras protein, or with [alpha-32P]GTP. Two sets of proteins of M(r) = 32-36 kDa and 22-24 kDa were strongly recognized by the antisera. Binding of [alpha-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 GTP gamma S, GTP or GDP; binding was not affected by 10(-6)-10(-5) M ATP gamma S or ADP. The kinetics, specificity and the effects of phytohormones in a [35S]GTP gamma S binding assay were also studied in isolated plasma membranes of barley aleurone protoplasts.
Wang
M
M
Center for Phytotechnology RUL/TNO, Department of Plant Molecular Biotechnology, Leiden, The Netherlands.
Sedee
N J
NJ
Heidekamp
F
F
Snaar-Jagalska
B E
BE
D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D001467:Hordeum / Q000737:chemistry*; D010937:Plant Growth Regulators / Q000494:pharmacology; D011523:Protoplasts / Q000737:chemistry*; D015398:Signal Transduction; D013696:Temperature
D010937:Plant Growth Regulators; D019204:GTP-Binding Proteins