Tanuma S, Endo H
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Japan.
Eur J Biochem. 1990 Jul 20;191(1):57-63. doi: 10.1111/j.1432-1033.1990.tb19093.x.
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.
从人红细胞中纯化出一种（ADP - 核糖）n糖水解酶，纯化倍数约为13000倍，并对其进行了特性鉴定。在十二烷基硫酸钠/聚丙烯酰胺凝胶上，纯化后的酶呈现均一性，估计相对分子质量（Mr）为59000。氨基酸分析表明，该酶酸性氨基酸残基含量相对较高，碱性氨基酸残基含量较低。等电聚焦显示该酶是一种酸性蛋白，pI值为5.9。此酶对（ADP - 核糖）n的水解方式为外切糖苷水解，最终产物为ADP - 核糖。（ADP - 核糖）n（平均链长，n = 15）的Km值为5.8 μM，其水解的最大速度为21 μmol·min-1·mg蛋白-1。酶活性的最适pH为7.4，KCl的抑制作用比NaCl更强。该酶活性受到ADP - 核糖和cAMP的抑制，但不受二丁酰衍生物（Bt2 - cAMP）、cGMP或AMP的抑制。这些物理和催化特性与胞质（ADP - 核糖）n糖水解酶II相似，但与从豚鼠肝脏纯化的核（ADP - 核糖）n糖水解酶I不同[田沼，S.，川岛，K. & 远藤，H.（1986年）《生物化学杂志》261，965 - 969]。因此，人红细胞中含有（ADP - 核糖）n糖水解酶II。纯化的红细胞（ADP - 核糖）n糖水解酶对与组蛋白H1结合的聚（ADP - 核糖）的降解动力学与相应的游离聚（ADP - 核糖）基本相同。相比之下，该糖水解酶对游离寡聚（ADP - 核糖）有明显活性，而对与组蛋白H1结合的相应寡聚（ADP - 核糖）活性则低得多。该酶对与线粒体和胞质游离mRNA核糖核蛋白颗粒（mRNP）蛋白结合的寡聚（ADP - 核糖）的活性比对与组蛋白H1结合的寡聚（ADP - 核糖）的活性更高。它不会降解分别用霍乱毒素和百日咳毒素制备的单（ADP - 核糖基） - 刺激性鸟嘌呤核苷酸结合蛋白（Gs）和 - 抑制性鸟嘌呤核苷酸结合蛋白（Gi）。这些结果表明，胞质（ADP - 核糖）n糖水解酶II可能参与核外去（ADP - 核糖基）化作用，但不参与膜去单（ADP - 核糖基）化作用。
