A rho gene product in human blood platelets. I. Identification of the platelet substrate for botulinum C3 ADP-ribosyltransferase as rhoA protein

Y. Nemoto, T. Namba, T. Teru-uchi, F. Ushikubi, N. Morii, S. Narumiya

    Research output: Contribution to journalArticle

    50 Scopus citations

    Abstract

    A substrate protein for botulinum C3 ADP-ribosyl-transferase (C3 exoenzyme) in human platelets was purified to apparent homogeneity from the cytosol by ammonium sulfate fractionation and successive chromatography on columns of DEAE-Sepharose, hydroxylapatite, phenyl-Sepharose, and TSK phenyl-5PW. The purified protein yielded an amino acid sequence identical to that of rhoA protein. When platelet cytosol and membranes were incubated with C3 exoenzyme and [32P]NAD and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, they gave only one [32P]ADP-ribosylated band on each electrophoresis that showed an Mr of 22,000 and a pI of 6.0. The radioactive bands from the two fractions co-migrated with each other and with the [32P] ADP-ribosylated purified protein. When these radioactive products were partially digested with either α-chymotrypsin or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same digestion pattern was found in the three samples. These results suggest that the ADP-ribosylation substrate for C3 exoenzyme in the platelet cytosol and membrane is rhoA protein and that it is the sole substrate detectable in human platelets.

    Original languageEnglish (US)
    Pages (from-to)20916-20920
    Number of pages5
    JournalJournal of Biological Chemistry
    Volume267
    Issue number29
    StatePublished - Jan 1 1992

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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