Purification and characterization of an (ADP-ribose)n glycohydrolase from human erythrocytes.
An (ADP-ribose)n glycohydrolase from human erythrocytes was purified approximately 13,000-fold and characterized. On sodium dodecyl sulfate/polyacrylamide gel the purified enzyme appeared homogeneous and had an estimated relative molecular mass (Mr) of 59,000. Amino acid analysis showed that the enzyme had a relatively high content of acidic amino acid residues and low content of basic amino acid residues. Isoelectrofocusing showed that the enzyme was an acidic protein with pI value of 5.9. The mode of hydrolysis of (ADP-ribose)n by this enzyme was exoglycosidic, yielding ADP-ribose as the final product. The Km value for (ADP-ribose)n (average chain length, n = 15) was 5.8 microM and the maximal velocity of its hydrolysis was 21 mumol.min-1.mg protein-1. The optimum pH for enzyme activity was 7.4 KCl was more inhibitory than NaCl. The enzyme activity was inhibited by ADP-ribose and cAMP but not the dibutyryl-derivative (Bt2-cAMP), cGMP or AMP. These physical and catalytic properties are similar to those of cytosolic (ADP-ribose)n glycohydrolase II, but not to those of nuclear (ADP-ribose)n glycohydrolase I purified from guinea pig liver [Tanuma, S., Kawashima, K. & Endo, H. (1986) J. Biol. Chem. 261, 965-969]. Thus, human erythrocytes contain (ADP-ribose)n glycohydrolase II. The kinetics of degradation of poly(ADP-ribose) bound to histone H1 by purified erythrocyte (ADP-ribose)n glycohydrolase was essentially the same as that of the corresponding free poly(ADP-ribose). In contrast, the glycohydrolase showed appreciable activity of free oligo(ADP-ribose), much less activity on the corresponding oligo(ADP-ribose) bound to histone H1. The enzyme had more activity on oligo(ADP-ribose) bound to mitochondrial and cytosolic free mRNA ribonucleoprotein particle (mRNP) proteins than on oligo(ADP-ribose) bound to histone H1. It did not degrade mono(ADP-ribosyl)-stimulatory guanine-nucleotide-binding protein (Gs) and -inhibitory guanine-nucleotide-binding protein (Gi) prepared with cholera and pertussis toxins, respectively. These results suggest that cytosolic (ADP-ribose)n glycohydrolase II may be involved in extranuclear de(ADP-ribosyl)n-ation, but not in membrane de-mono(ADP-ribosyl)ation.
Tanuma
S
S
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Japan.
Endo
H
H
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006168:Guinea Pigs; D006657:Histones / Q000378:metabolism; D006801:Humans; D008930:Mitochondria, Liver / Q000201:enzymology; D009711:Nucleotides; D011189:Potassium Chloride; D014176:Protein Biosynthesis; D013379:Substrate Specificity
D000596:Amino Acids; D006657:Histones; D009711:Nucleotides; D000246:Adenosine Diphosphate Ribose; D011189:Potassium Chloride; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase