Dalton D A, Langeberg L, Robbins M
Department of Biology, Reed College, Portland, Oregon 97202.
Arch Biochem Biophys. 1992 Jan;292(1):281-6. doi: 10.1016/0003-9861(92)90080-g.
Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silver or Coomassie blue staining. Purified MR contained 0.7 mol FAD/mol enzyme and had a specific activity of 288 mumol NADH oxidized.min-1.mg protein-1. The enzyme was a single subunit occurring as two isozymes (MR I and MR II) with Mr values of 39,000 and 40,000. Isoelectric focusing revealed that each isozyme consisted of two forms with pl values of 4.6 to 4.7. Ferricyanide and 2,6-dichlorophenol-indophenol were effective as electron acceptors. The purified enzyme did not possess leghemoglobin reductase activity. Inhibition by p-chloromercuribenzoate indicated the involvement of a thiol group in MR activity. The Km values were 5.6, 150, and 7 microM for NADH, NADPH, and monodehydroascorbate, respectively. The pH optimum was 8 to 9. The N-terminal sequence of 10 amino acids of MR II had little homology to known protein sequences.
大豆（Glycine max (L.) Merr.）根瘤中含有抗坏血酸-谷胱甘肽循环的酶，作为对抗活性氧形式的重要防御机制。该循环中的关键酶——单脱氢抗坏血酸还原酶（MR）——被纯化了646倍，在SDS-PAGE上经银染或考马斯亮蓝染色后呈现为一条单一的条带。纯化后的MR每摩尔酶含有0.7摩尔FAD，比活性为288 μmol NADH氧化·分钟⁻¹·毫克蛋白⁻¹。该酶是一个单亚基，以两种同工酶（MR I和MR II）的形式存在，分子量分别为39,000和40,000。等电聚焦显示，每种同工酶由两种形式组成，其等电点值为4.6至4.7。铁氰化物和2,6-二氯酚靛酚作为电子受体是有效的。纯化后的酶不具有豆血红蛋白还原酶活性。对氯汞苯甲酸的抑制作用表明硫醇基团参与了MR的活性。NADH、NADPH和单脱氢抗坏血酸的Km值分别为5.6、150和7 μM。最适pH为8至9。MR II的10个氨基酸的N端序列与已知蛋白质序列的同源性很低。
