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.