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Purification and characterization of an (ADP‐ribose) n glycohydrolase from human erythrocytes

1990; Wiley; Volume: 191; Issue: 1 Linguagem: Inglês

10.1111/j.1432-1033.1990.tb19093.x

1432-1033

Sei‐ichi Tanuma, Hiroyoshi Endo,

Neonatal Health and Biochemistry

An (ADP‐ribose) n , glycohydrolase from human erythrocytes was purified approximately 13000‐fold and characterized. On sodium dodecyl sulfate/polyacrylamide gel the purified enzyme appeared homogeneous and had an estimated relative molecular mass ( M r ) of 59000. 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 K m value for (ADP‐ribose) n , (average chain length, n = 15) was 5.8 μM. and the maximal velocity of its hydrolysis was 21 μmol · min −1 . mg protein −1 . The optimum pH for enzyme activity was 7.4. KCI was more inhibitory than NaCl. The enzyme activity was inhibited by ADP‐ribose and cAMP but not the dibutyryl‐derivative (Bt 2 ‐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 mitochon‐drial and cytosolic free mRNA ribonucleoprotein particle (mRNP) proteins than on oligo(ADP‐ribose) bound to Histone HI. 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.