Okabayashi K, Nakano E
J Biochem. 1984 Jun;95(6):1625-32. doi: 10.1093/oxfordjournals.jbchem.a134775.
Purification and characterization of mitochondrial malate dehydrogenase [EC 1.1.1.37] from unfertilized eggs of the sea urchin, Anthocidaris crassispina, are described. The purification method consisted of dextran sulfate fractionation, Blue Dextran Sepharose chromatography, Phenyl-Sepharose hydrophobic chromatography and DEAE-cellulose chromatography. The enzyme was purified 771-fold with a 7% yield from the crude extract. The purified enzyme appeared homogeneous on polyacrylamide gel electrophoresis under both native and denatured conditions. After incubation at 45 degrees C for 50 min, the enzyme lost about 90% of its activity. In the presence of NADH, however, the enzyme was protected against the heat denaturation. The native enzyme had a molecular weight of about 65,000 and probably consisted of two identical subunits. In the reduction of oxaloacetate with NADH, a broad optimum pH ranging from 8.2 to 9.4 was found with 50 mM Tris-HCl and glycine-NaOH buffers. Sodium phosphate buffer apparently activated the enzyme. The apparent Km values for oxaloacetate and NADH were 19 microM and 30 microM, respectively. The optimum pH for malate oxidation with NAD+ was 10.2 in 50 mM NaHCO3-Na2CO3 buffer. The apparent Km values for malate and NAD+ were 7.0 mM and 0.6 mM, respectively. Zinc ion, sulfite ion, p-chloromercuriphenylsulfonate and adenine nucleotides strongly inhibited the enzyme.
本文描述了从厚刺海胆未受精卵中纯化和鉴定线粒体苹果酸脱氢酶[EC 1.1.1.37]的过程。纯化方法包括硫酸葡聚糖分级分离、蓝色葡聚糖琼脂糖层析、苯基琼脂糖疏水层析和DEAE-纤维素层析。该酶从粗提物中纯化了771倍，产率为7%。在天然和变性条件下，纯化后的酶在聚丙烯酰胺凝胶电泳上均呈现单一条带。在45℃孵育50分钟后，该酶失去了约90%的活性。然而，在NADH存在的情况下，该酶受到热变性的保护。天然酶的分子量约为65,000，可能由两个相同的亚基组成。在用NADH还原草酰乙酸的过程中，发现50 mM Tris-HCl和甘氨酸-NaOH缓冲液的最适pH范围为8.2至9.4。磷酸钠缓冲液明显激活了该酶。草酰乙酸和NADH的表观Km值分别为19 microM和30 microM。在50 mM NaHCO3-Na2CO3缓冲液中，用NAD+氧化苹果酸的最适pH为10.2。苹果酸和NAD+的表观Km值分别为7.0 mM和0.6 mM。锌离子、亚硫酸根离子、对氯汞苯磺酸盐和腺嘌呤核苷酸强烈抑制该酶。
