Detection of arginine-ADP-ribosylated protein using recombinant ADP-ribosylarginine hydrolase

Anal Biochem. 1995 Oct 10;231(1):115-22. doi: 10.1006/abio.1995.1510.

Abstract

We made use of ADP-ribosylarginine hydrolase to detect arginine-ADP- ribosylated proteins. The hydrolase was expressed in Escherichia coli as a protein fused with glutathione S-transferase (GST). The fusion protein GST-ADP-ribosylarginine hydrolase catalyzed the hydrolysis of alpha-ADP-ribosylarginine to produce ADP-ribose and arginine. Casein ADP-ribosylated with [32P]NAD and chicken heterophil arginine-specific ADP-ribosyltransferase served as a substrate for the recombinant ADP-ribosylarginine hydrolase and the released ADP-ribose was determined. Protein ADP-ribosylated by cholera toxin could serve as substrate of the hydrolase but protein ADP-ribosylated by pertussis toxin, diphtheria toxin, or C(3) enzyme of Clostridium botulinum could not. The hydrolase did not release the radioactivity incorporated into isolated rat liver nuclei incubated with [(32)P]NAD or in bovine brain cytosol incubated with [(32)P]ADP-ribose. In homogenate of mouse heart which contained arginine-specific ADP-ribosyltransferase, labeling of a 55-kDa protein by incubation with [(32)P]NAD was removed by ADP-ribosylarginine hydrolase treatment; hence, the specific hydrolysis of ADP-ribose-arginine bond by GST-ADP-ribosylarginine hydrolase can be used to detect the arginine-ADP-ribosylated proteins in crude preparations. Arginine--ADP-ribosylated proteins in crude preparations. Arginine-ADP-ribosylated proteins in mouse spleen lymphocytes were identified using this method.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate Ribose / metabolism*
  • Animals
  • Arginine / metabolism*
  • Base Sequence
  • Escherichia coli
  • Glycoside Hydrolases / genetics
  • Glycoside Hydrolases / metabolism*
  • Hydrolysis
  • Mice
  • Molecular Sequence Data
  • N-Glycosyl Hydrolases*
  • Organ Specificity
  • Poly(ADP-ribose) Polymerases / analysis
  • Proteins / analysis*
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Substrate Specificity

Substances

  • Proteins
  • Recombinant Fusion Proteins
  • Adenosine Diphosphate Ribose
  • Arginine
  • Poly(ADP-ribose) Polymerases
  • Glycoside Hydrolases
  • N-Glycosyl Hydrolases
  • ADP-ribosylarginine hydrolase