Saxena A K, Saxena P, Monnier V M
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 1996 Dec 20;271(51):32803-9. doi: 10.1074/jbc.271.51.32803.
Searching for novel approaches for uncoupling glycation from hyperglycemia as a cause of diabetic complications, a Pseudomonas sp. soil strain containing a membrane-bound enzyme that deglycates amino acids under release of free fructosamine was isolated (Gerhardinger, C., Marion, S. M., Rovner, A., Glomb, M., and Monnier, V. M. (1995) J. Biol. Chem. 270, 218-224). This enzymatic activity was found to be very sensitive to inactivation by most detergents. From the plasma membrane ( approximately 3 mg/ml protein concentration), the enzyme could be solubilized in active form using 10 mM 3-[(3-chlolamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulfonate aided by 2 M NaCl and 10% glycerol (27% optimal solubilization yield). The supernatant from a 55% saturation (NH4)2SO4 cut was fractionated onto a phenyl-Superose HR 5/5 column and enzymatic activity was eluted with a inverse gradient of (NH4)2SO4. Following removal of (NH4)2SO4 with PD-10 columns and fractionation with a Mono Q HR 5/5 column, a sharp peak of enzyme activity was eluted. Analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major band at 106 kDa and, on isoelectrofocusing gel, a pI of 5.1. The activity was completely inhibited by CN- and N3-, suggestive of copper as a likely cofactor. Identification of the protein was confirmed by affinity labeling with 14CN- and isoelectrofocusing. The "amadoriase" activity was also inhibited by Hg2+, Ag2+, Cu2+, and Zn2+ and had Km and Vmax values of 0.14 mM and 0.48 unit/ml (16 units/mg of protein), respectively, for epsilon-(1-deoxyfructosyl) aminocaproate. Significant activity was noted toward many glycated amino acids (highest with epsilon-fructosyl lysine) but not with glycated proteins. The sequence of the first 16 NH2-terminal amino acids and a search in various data bases revealed that this amadoriase enzyme is a novel protein. Based on its properties, this deglycating enzyme, which degrades Amadori products oxidatively into free fructosamine, is classified as fructosyl aminocaproate:oxygen oxidoreductase (EC 1.5.3).
为寻找将糖化反应与高血糖解偶联作为糖尿病并发症病因的新方法，分离出了一种假单胞菌属土壤菌株，该菌株含有一种膜结合酶，可在释放游离果糖胺的情况下使氨基酸脱糖基化（格哈德inger，C.，马里昂，S.M.，罗夫纳，A.，格洛姆，M.，和莫尼尔，V.M.（1995年）《生物化学杂志》270卷，218 - 224页）。发现这种酶活性对大多数去污剂的失活非常敏感。从质膜（蛋白质浓度约为3 mg/ml）中，该酶可以在10 mM 3 - [(3 - 氯氨丙基)二甲基铵基]-2 - 羟基 - 1 - 丙烷磺酸盐、2 M NaCl和10%甘油的辅助下以活性形式溶解（最佳溶解产率为27%）。55%饱和度硫酸铵沉淀的上清液在苯基 - 超级ose HR 5/5柱上进行分级分离，酶活性用硫酸铵的反向梯度洗脱。用PD - 10柱去除硫酸铵并在Mono Q HR 5/5柱上进行分级分离后，洗脱得到一个尖锐的酶活性峰。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析显示在106 kDa处有一条主要条带，等电聚焦凝胶分析显示其pI为5.1。该活性被CN - 和N3 - 完全抑制，提示铜可能是一种辅因子。通过用14CN - 进行亲和标记和等电聚焦确认了该蛋白质的鉴定。“阿玛多里酶”活性也被Hg2 +、Ag2 +、Cu2 +和Zn2 +抑制，对于ε - (1 - 脱氧果糖基)氨基己酸，其Km和Vmax值分别为0.14 mM和0.48单位/ml（16单位/mg蛋白质）。对许多糖化氨基酸有显著活性（对ε - 果糖基赖氨酸活性最高），但对糖化蛋白质无活性。前16个NH2 - 末端氨基酸的序列以及在各种数据库中的搜索表明，这种阿玛多里酶是一种新蛋白质。基于其特性，这种将阿玛多里产物氧化降解为游离果糖胺的脱糖基化酶被归类为果糖基氨基己酸：氧氧化还原酶（EC 1.5.3）。
