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 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