Huh W K, Kim S T, Yang K S, Seok Y J, Hah Y C, Kang S O
Department of Microbiology, College of Natural Sciences, Seoul National University, Republic of Korea.
Eur J Biochem. 1994 Nov 1;225(3):1073-9. doi: 10.1111/j.1432-1033.1994.1073b.x.
D-Erythroascorbic acid was detected from the cell extracts of a dimorphic fungus, Candida albicans. Its concentration in yeast cells grown at 25 degrees C was estimated to be about 0.45 mumol/ml cell water. D-Arabinono-1,4-lactone oxidase, which catalyses the final step in the biosynthesis of D-erythroascorbic acid, was purified 639-fold from the mitochondrial fraction of C. albicans to apparent homogeneity, with an overall yield of 21.2%, by a purification procedure consisting of Triton X-100 solubilisation, ammonium sulphate precipitation, anion-exchange, hydrophobic-interaction, gel-filtration and dye-ligand chromatographies. Gel-filtration chromatography and polyacrylamide-gradient gel electrophoresis in the presence of deoxycholate gave apparent molecular masses of 110 kDa and 84.4 kDa, respectively. SDS/PAGE showed only one protein band corresponding to a molecular mass of 66.7 kDa. Considering the binding of detergents, the enzyme is suggested to be a single polypeptide. The enzyme showed a typical fluorescence excitation spectrum of a flavin-containing enzyme. The flavin was not released by treatment with SDS, CCl3CO2H or boiling, indicating that it may be covalently bound to the enzyme protein. The enzyme was optimally active at 40 degrees C and at pH 6.1. The enzyme was stable in the range pH 7.5-10. An apparent Km value for D-arabinono-1,4-lactone was 44.1 mM. L-Galactono-1,4-lactone, L-gulono-1,4-lactone and L-xylono-1,4-lactone could also serve as substrates. Competitive inhibition was demonstrated with D-glucono-1,5-lactone, L-arabinono-1,4-lactone, D-galactono-1,4-lactone and D-gulono-1,4-lactone. p-Chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, iodoacetamide and divalent metal ions such as Cd2+, Hg2+, Mn2+ and Zn2+ exhibited inhibitory effects on the enzyme.
从二态真菌白色念珠菌的细胞提取物中检测到了D - 赤藓糖型抗坏血酸。其在25℃下生长的酵母细胞中的浓度估计约为0.45 μmol/ml细胞水。催化D - 赤藓糖型抗坏血酸生物合成最后一步的D - 阿拉伯糖 - 1,4 - 内酯氧化酶，通过由Triton X - 100增溶、硫酸铵沉淀、阴离子交换、疏水相互作用、凝胶过滤和染料配体色谱组成的纯化程序，从白色念珠菌的线粒体部分纯化了639倍，达到表观均一性，总产率为21.2%。在脱氧胆酸盐存在下进行的凝胶过滤色谱和聚丙烯酰胺梯度凝胶电泳分别给出了110 kDa和84.4 kDa的表观分子量。SDS/PAGE仅显示出一条对应于66.7 kDa分子量的蛋白带。考虑到去污剂的结合，该酶被认为是一条单一多肽。该酶显示出含黄素酶典型的荧光激发光谱。黄素不会因SDS、三氯乙酸或煮沸处理而释放，表明它可能与酶蛋白共价结合。该酶在40℃和pH 6.1时活性最佳。该酶在pH 7.5 - 10范围内稳定。D - 阿拉伯糖 - 1,4 - 内酯的表观Km值为44.1 mM。L - 半乳糖 - 1,4 - 内酯、L - 古洛糖 - 1,4 - 内酯和L - 木糖 - 1,4 - 内酯也可作为底物。用D - 葡萄糖酸 - 1,5 - 内酯、L - 阿拉伯糖 - 1,4 - 内酯、D - 半乳糖 - 1,4 - 内酯和D - 古洛糖 - 1,4 - 内酯证明了竞争性抑制作用。对氯汞苯甲酸、N - 乙基马来酰亚胺、碘乙酸、碘乙酰胺和二价金属离子如Cd2 +、Hg2 +、Mn2 +和Zn2 +对该酶表现出抑制作用。
