Evidence of Endogenous Mono-Adp-Ribosylation of Cardiac Proteins Via Anti-Adp-Ribosylarginine Immunoreactivity (44506)

Abstract

Arginine-specific mono-ADP-ribosylatlon of proteins and arginine-specific mono-ADP-ribosyltransferase occur in heart. We developed a polyclonal antiserum, R-28, against ADP-ribosyipolyarginine that recognized mono-ADP-ribosylated proteins and identified the major mono-ADP-ribosylatlon products of quail heart. Treatment of Immobilon-bound ADP-ribosylated G₂ protein with hydroxylamlne under conditions that remove ADP-ribose from Its arginines eliminated R-28 Immunoreactivity to G₂ Also, R-28 Immunoreactivity to quail heart proteins was removed by NaOH and phosphodiesterase I treatments. Similar treatment with mercuric chloride did not remove the immunoreactivity but did remove exogenously (via in vitro pertussis toxin treatment) added ADP-ribose from cysteine of cardiac G₁/G_o proteins. The antiserum did not appear to react with ADP-ribosyiasparagine of Rho (formed by C₃ toxin), ADP-ribosyidiphthamide of elongation factor 2 (formed by diphtheria toxin) in quail heart preparations, or polyADP-ribosylated proteins of a neonate rat cardiac nuclear preparation. Thus, the R-28 antiserum appears to contain predominantly antibodies directed against ADP-ribosyiarginine. To test the usefulness of R-28, immunoblotting of subcellular fractions of quail heart was performed. R-28 showed the greatest Immunoreactivity in the sarcolemma with significant Immunoreactivity in denser membrane fractions. The cytosol also contained an Immunoreactlve band distinct from those found in the membranes. Hydroxylamine treatment eliminated immunoreactivity in the sarcolemma and denser membrane fractions but not the cytosol, suggesting the membranous Immunoreactlve bands contain ADP-ribosylarginlne. In conclusion, a polyclonal antiserum that recognizes ADP-ribosyiarginine proteins has been raised. The usefulness of the antiserum is demonstrated by the characterization of endogenous arginine mono-ADP-ribosylatlon products in quail heart. The quail heart has several sarcolemmal and denser membrane fraction proteins that appear to be mono-ADP-rlbosylated on arginines.