Detection of GTP‐binding proteins in barley aleurone protoplasts

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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.