Kim J S, Kim W Y, Rho H W, Park J W, Park B H, Han M K, Kim U H, Kim H R
Department of Biochemistry, Medical School, Chonbuk National University, Chonju, South Korea.
Int J Biochem Cell Biol. 1998 May;30(5):629-38. doi: 10.1016/s1357-2725(97)00142-8.
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.
游离ADP - 核糖是NAD⁺、蛋白质结合的聚合和单体ADP - 核糖以及环ADP - 核糖的周转产物。但关于游离ADP - 核糖的具体细胞作用或代谢情况却知之甚少。从人红细胞中纯化出了将ADP - 核糖水解为AMP和5'-磷酸核糖的ADP - 核糖焦磷酸酶（EC 3.6.1.13）。通过连续的色谱步骤实现了纯化至同质，最终从溶血产物中纯化了75790倍。纯化后的酶在SDS - PAGE上，无论有无2 - 巯基乙醇，均显示出一条分子量为34 kDa的条带。通过凝胶过滤计算出的天然酶分子量为68 kDa，表明活性酶是由相同亚基组成的二聚体。该酶需要Mg²⁺，对ADP - 核糖的活性最高，对IDP - 核糖、ADP - 甘露糖和GDP - 甘露糖的活性约为40 - 70%。该酶对ADP - 核糖的Km为169±11 μM，最适pH约为9.5，pI为5.1。ADP是一种有效的非竞争性抑制剂，Ki为16±1.2 μM。这些结果表明我们的酶是独特的，与已报道的其他ADP - 核糖焦磷酸酶不同。ADP - 核糖焦磷酸酶可能在调节细胞内游离ADP - 核糖的稳态中起重要作用。
