Purification and characterization of adenosine diphosphate ribose pyrophosphatase from human erythrocytes.
Free ADP-ribose is a turnover product of NAD+, protein-bound polymeric and monomeric ADP-ribose, and cyclic ADP-ribose. But little is known about the specific cellular roles or metabolism of free ADP-ribose. ADP-ribose pyrophosphatase (EC 3.6.1.13), which hydrolyzes ADP-ribose into AMP and ribose-5'-phosphate, was purified from human erythrocytes. Purification was achieved to homogeneity by successive chromatographic steps, resulting in a final purification of 75,790-fold from the hemolysate. The purified enzyme showed a single band with the molecular weight of 34 kDa on SDS-PAGE both in the presence and absence of 2-mercaptoethanol. The molecular weight of the native enzyme calculated by gel filtration was 68 kDa, indicating that the active enzyme is a dimer of identical subunits. The enzyme requiring Mg2+ showed highest activity toward ADP-ribose, and about 40-70% activities with IDP-ribose, ADP-mannose and GDP-mannose. The enzyme showed a Km of 169 +/- 11 microM for ADP-ribose, broad pH optimum around pH 9.5, and pI of 5.1. ADP was a potent noncompetitive inhibitor with a Ki of 16 +/- 1.2 microM. These results suggest that our enzyme is unique, and different from the other ADP-ribose pyrophosphatases reported. ADP-ribose pyrophosphatase may play an important role in the regulation of intracellular steady-state of free ADP-ribose.
Kim
J S
JS
Department of Biochemistry, Medical School, Chonbuk National University, Chonju, South Korea.
Kim
W Y
WY
Rho
H W
HW
Park
J W
JW
Park
B H
BH
Han
M K
MK
Kim
U H
UH
Kim
H R
HR
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000645:Ammonium Sulfate; D005591:Chemical Fractionation; D002847:Chromatography, Agarose; D002848:Chromatography, DEAE-Cellulose; D003636:DEAE-Cellulose; D003911:Dextrans; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011755:Pyrophosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives; D013379:Substrate Specificity
D003911:Dextrans; D000246:Adenosine Diphosphate Ribose; C027176:octyl-sepharose CL-4B; D012685:Sepharose; D003636:DEAE-Cellulose; C025614:sephadex; D011755:Pyrophosphatases; D000645:Ammonium Sulfate