Peterson J E, Larew J S, Graves D J
Biochemistry and Biophysics Department, Iowa State University, Ames 50011.
J Biol Chem. 1990 Oct 5;265(28):17062-9.
Integral membrane-associated arginine-specific mono-ADP-ribosyltransferase was purified from rabbit skeletal muscle microsomes. The ADP-ribosyltransferase was solubilized from the 100,000 x g pellet with 0.3% sodium deoxycholate and purified to greater than or equal to 95% homogeneity by successive DE52, concanavalin A-agarose, 3-aminobenzamide-agarose, and size-exclusion high-performance liquid chromatography (HPLC) steps in the presence of detergents. Two molecular weight forms of the enzyme were isolated and partially characterized. The apparent Mr of the alpha-form of the enzyme purified to greater than or equal to 95% homogeneity was approximately 39,000 +/- 500 as estimated by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr of the beta-form purified to greater than or equal to 80% homogeneity was 38,500 +/- 500. The rapid procedure resulted in a 200-fold purification for the alpha-form and a 645-fold purification for the beta-form, relative to the microsomal fraction. Positive identification of the enzyme was confirmed by utilizing a zymographic in situ gel assay and by HPLC assay of polyacrylamide gel slice incubations with an NAD and guanylhydrazone substrate. The specificity of the mono-ADP-ribosyltransferase zymographic assay was characterized by time course incubations, hydroxylamine sensitivity, 3-aminobenzamide inhibition, and histone dependence. The ADP-ribosyltransferase is inactivated by reducing agents.
整合膜相关精氨酸特异性单 ADP - 核糖基转移酶是从兔骨骼肌微粒体中纯化得到的。该 ADP - 核糖基转移酶用 0.3%脱氧胆酸钠从 100,000×g 沉淀中溶解出来，并在去污剂存在的情况下，通过连续的 DE52、伴刀豆球蛋白 A - 琼脂糖、3 - 氨基苯甲酰胺 - 琼脂糖和尺寸排阻高效液相色谱（HPLC）步骤纯化至均一性大于或等于 95%。分离出了该酶的两种分子量形式并对其进行了部分表征。通过银染十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳估计，纯化至均一性大于或等于 95%的酶的α形式的表观 Mr 约为 39,000±500。纯化至均一性大于或等于 80%的β形式的 Mr 为 38,500±500。相对于微粒体部分，该快速方法使α形式的酶纯化了 200 倍，β形式的酶纯化了 645 倍。通过使用原位凝胶酶谱法以及对用 NAD 和胍基腙底物孵育的聚丙烯酰胺凝胶切片进行 HPLC 分析，证实了该酶的阳性鉴定。通过时间进程孵育、羟胺敏感性、3 - 氨基苯甲酰胺抑制和组蛋白依赖性对单 ADP - 核糖基转移酶酶谱分析的特异性进行了表征。该 ADP - 核糖基转移酶被还原剂灭活。
