Nguyen Lieu Thi, Nguyen Kien Trung, Spízek Jaroslav, Behal Vladislav
Institute of Microbiology, Academy of Sciences of the Czech Republic, Víde:ntskà 1083, 142 20 Prague 4,Czech Republic.
Microbiology (Reading). 1995 May;141 ( Pt 5):1139-1145. doi: 10.1099/13500872-141-5-1139.
A second NAD-dependent valine dehydrogenase (VDH) of Streptomyces fradiae was detected and purified to homogeneity by affinity chromatography on Reactive-Blue 2 Sepharose followed by gel filtration and Mono Q fast protein liquid chromatography. The relative molecular masses of the native enzyme and its subunits were determined to be 80,000 and 41,000, respectively, indicating that the enzyme is a homodimer. The enzyme was the only active VDH in S. fradiae; its activity was significantly induced by L-valine, but was repressed by ammonia. Among branched- and straight-chain amino acids that serve as enzyme substrates, L-2-aminobutyrate and L-valine are preferred. Significant activities were found with deamino-NAD+ and 3-pyridinealdehyde-NAD+. The molecular and catalytic properties of the enzyme distinguish it from the enzyme previously purified, and thus indirectly indicate the existence of two VDHs in S. fradiae.
通过在活性蓝2琼脂糖凝胶上进行亲和层析，随后进行凝胶过滤和Mono Q快速蛋白质液相色谱，检测并纯化了弗氏链霉菌的第二种依赖NAD的缬氨酸脱氢酶（VDH），使其达到同质。天然酶及其亚基的相对分子质量分别测定为80,000和41,000，表明该酶是一种同型二聚体。该酶是弗氏链霉菌中唯一具有活性的VDH；其活性受到L-缬氨酸的显著诱导，但受到氨的抑制。在作为酶底物的支链和直链氨基酸中，L-2-氨基丁酸酯和L-缬氨酸是首选。发现该酶对脱氨基-NAD+和3-吡啶醛-NAD+具有显著活性。该酶的分子和催化特性使其与先前纯化的酶有所不同，从而间接表明弗氏链霉菌中存在两种VDH。
