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-P-32]GTP. Two sets of proteins of M(r) = 32-36 kDa and 22-24 kDa were strongly recognized by the antisera. Binding of [alpha-P-32]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 GTPgammaS, GTP or GDP; binding was not affected by 10(-6)-10(-5) M ATPgammaS or ADP. The kinetics, specificity and the effects of phytohormones in a [S-35]GTPgammaS binding assay were also studied in isolated plasma membranes of barley aleurone protoplasts.