Detection of GTP‐binding proteins in barley aleurone protoplasts

Autor: Wang, Mei, Sedee, Norbert J.A., Heidekamp, Freek, Snaar-Jagalska, B.Ewa
Zdroj: FEBS Letters; August 1993, Vol. 329 Issue: 3 p245-248, 4p
Abstrakt: 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 Rasprotein, or with [α‐32P]GTP. Two sets of proteins of Mr= 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 Mr= 22–24 kDa and 16 kDa. Binding was inhibited by 10−7–10−6M GTPγS, GTP or GDP; binding was not affected by 10−6‐10−5M 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.
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