Sok D E
College of Pharmacy, Chungnam National University, Yuseong-Ku, Taejon, Korea.
Neurochem Res. 1996 Oct;21(10):1193-9. doi: 10.1007/BF02532395.
A Zn(2+)-glycerophosphocholine cholinephosphodiesterase was purified with a specific activity of 4.6 mumole/min.mg protein from bovine brain membranes by procedures involving PI-PLC solubilization, concanavalin A affinity chromatography, CM-sephadex chromatography and Sephadex G-150 chromatography. Based on molecular weight determination gel chromatography and SDS polyacrylamide gel electrophoresis, the phosphodiesterase activity appears to be a dimeric protein (110 kDa) composed of two subunits with a molecular weight of approximately 54 kDa. The K(m) value for p-nitrophenylphosphocholine and the optimum pH were found to be 16 microM and pH 10.5, respectively. The phosphodiesterase was inhibited by Cu2+, but not the other divalent metal ions. The activity of the apoenzyme was remarkably activated by Co2+ or Zn2+, but not Mn2+ or Mg2+. In addition, the inactivation of the enzyme in glycine buffer was prevented by Mn2+ or Zn2+, but not Co2+ or Mg2. In a separate experiment, comparing properties of the purified and membrane-bound phosphodiesterases, the forms of two enzymes were quite similar except in stability. Both enzymes were more stable at pH 7.4 than pH 5 or 10. However, the membrane-bound enzyme was more stable than the soluble enzyme at all three pHs. These data suggest that the activity of the phosphodiesterase may be stabilized in-vivo.
通过涉及磷脂酰肌醇特异性磷脂酶C（PI-PLC）溶解、伴刀豆球蛋白A亲和层析、CM-葡聚糖凝胶层析和葡聚糖凝胶G-150层析的方法，从牛脑膜中纯化出一种锌离子甘油磷酸胆碱胆碱磷酸二酯酶，其比活性为4.6微摩尔/分钟·毫克蛋白质。基于分子量测定凝胶层析和十二烷基硫酸钠聚丙烯酰胺凝胶电泳，磷酸二酯酶活性似乎是一种由两个亚基组成的二聚体蛋白（110 kDa），每个亚基的分子量约为54 kDa。对硝基苯基磷酸胆碱的米氏常数（K(m)）和最适pH值分别为16微摩尔和pH 10.5。该磷酸二酯酶受到铜离子抑制，但不受其他二价金属离子抑制。脱辅基酶的活性被钴离子或锌离子显著激活，但不被锰离子或镁离子激活。此外，锰离子或锌离子可防止该酶在甘氨酸缓冲液中失活，但钴离子或镁离子不能。在另一项实验中，比较纯化的和膜结合的磷酸二酯酶的特性，除稳定性外，两种酶的形式非常相似。两种酶在pH 7.4时比在pH 5或10时更稳定。然而，在所有三个pH值下，膜结合酶比可溶性酶更稳定。这些数据表明，磷酸二酯酶的活性在体内可能得到稳定。
