Polymerization of ADP-ribose pyrophosphatase: conversion mechanism of Mg(2+)-dependent ADP-ribose pyrophosphatase into Mg(2+)-independent form.
ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose (ADPR) into AMP and ribose-5'-phosphate. It is classified into two groups, Mg(2+)-dependent and Mg(2+)-independent ADPRase, depending on its Mg2+ requirement. Here, we purified Mg(2+)-dependent ADPRase from rabbit liver and examined what factors affect Mg2+ requirement. 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 ADPRase is a dimer made up of two identical subunits. Mg(2+)-dependent ADPRase with the highest ADPR affinity had a Km of 160 +/- 10 microM and a pH optimum of around pH 9.5. Treatment of the purified ADPRase with heated cytosol fractions at 37 degrees C for 3 h caused some changes in the chemical properties of the enzyme, including an increase in molecular weight, a decrease in solubility, and a loss of Mg(2+)-dependency. The molecular weight of the cytosol-treated ADPRase measured by gel filtration was over 420 kDa, suggesting, for the first time, that ADPRase could be polymerized by undefined cytoplasmic factors, and that polymerization is accompanied by changes in the solubility and metal ion dependency of the enzyme.
Kim
Dae-Ki
DK
Department of Biochemistry, Institute of Medical Science, Chonbuk National University Medical School, Chonju 561-756, Korea.
Kim
Jong-Hyun
JH
Song
Eun-Kyung
EK
Han
Myung-Kwan
MK
Kim
Jong-Suk
JS
D000246:Adenosine Diphosphate Ribose / Q000737:chemistry / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006358:Hot Temperature; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D008274:Magnesium / Q000737:chemistry / Q000502:physiology*; D015636:Magnesium Chloride / Q000737:chemistry / Q000494:pharmacology; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000145:classification; D011108:Polymers / Q000737:chemistry; D011755:Pyrophosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012267:Ribosemonophosphates / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013997:Time Factors
D011108:Polymers; D012267:Ribosemonophosphates; D015636:Magnesium Chloride; D000246:Adenosine Diphosphate Ribose; D000249:Adenosine Monophosphate; C031626:ribose-5-phosphate; D010744:Phosphoric Monoester Hydrolases; C058796:ADP-ribose pyrophosphatase I; D011755:Pyrophosphatases; D008274:Magnesium