Shikata S, Ozaki K, Kawai S, Ito S, Okamoto K
Tochigi Research Laboratories, Kao Corporation, Japan.
Biochim Biophys Acta. 1988 Feb 10;952(3):282-9. doi: 10.1016/0167-4838(88)90128-8.
We have succeeded in purifying to homogeneity a very labile NADP+-linked isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) from a strain of alkalophilic Bacillus, by a simple method, with an overall yield over 76% of the original activity. The molecular weight on Sephadex G-200 was around 90,000; and that by electrophoresis on SDS-polyacrylamide gels was about 44,000. The sedimentation coefficient (s020,w) and isoelectric point of the enzyme were determined to be 3.22 S and pH 4.7, respectively. The enzyme required Mn2+ for the reaction and for stability. The optimum pH for the reaction was in the range 7.8-8.4 at 30 degrees C; the optimum temperature at pH 8.0 was 75 degrees C; the activation energy of the reaction was 6.2 kcal/mol. The Km values for threo-Ds-isocitrate, DL-isocitrate, and NADP+ were 5.4 microM, 9.9 microM, and 7.3 microM, respectively. This enzyme was inhibited by NADPH, glyceraldehyde 3-phosphate, 3-phosphoglycerate, phosphoenol pyruvate, cis-aconitate, alpha-ketoglutarate, and oxaloacetate. In addition, it was subject to a concerted inhibition by a combination of glyoxylate and oxaloacetate, and also to a cumulative inhibition by nucleoside triphosphates.
我们通过一种简单的方法成功地从嗜碱芽孢杆菌菌株中纯化出了一种极不稳定的NADP⁺连接的异柠檬酸脱氢酶（异柠檬酸：NADP⁺氧化还原酶（脱羧），EC 1.1.1.42），总产率超过原始活性的76%。在Sephadex G - 200上测得的分子量约为90,000；在SDS - 聚丙烯酰胺凝胶上电泳测得的分子量约为44,000。该酶的沉降系数（s₀₂₀,w）和等电点分别测定为3.22 S和pH 4.7。该酶反应和保持稳定都需要Mn²⁺。30℃时反应的最适pH范围是7.8 - 8.4；pH 8.0时的最适温度是75℃；反应的活化能是6.2千卡/摩尔。对苏糖-Ds-异柠檬酸、DL-异柠檬酸和NADP⁺的Km值分别为5.4微摩尔、9.9微摩尔和7.3微摩尔。该酶受到NADPH、3-磷酸甘油醛、3-磷酸甘油酸、磷酸烯醇丙酮酸、顺乌头酸、α-酮戊二酸和草酰乙酸的抑制。此外，它还受到乙醛酸和草酰乙酸组合的协同抑制，以及核苷三磷酸的累积抑制。
