Vessal M, Tabei S M
Department of Biochemistry, Shiraz University of Medical Sciences, Iran.
Comp Biochem Physiol B Biochem Mol Biol. 1996 Apr;113(4):757-63. doi: 10.1016/0305-0491(95)02099-3.
Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCI or barbital buffer. The Km values of oxaloacetate and NADH were 0.400 +/- 0.018 and 0.410 +/- 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65 degrees C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a Ki value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a Ki value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated.
通过QAE-和SP-葡聚糖凝胶色谱法，从绵羊肝脏细粒棘球蚴原头蚴中纯化出细胞质苹果酸脱氢酶，纯化了22倍。该酶在Tris-HCl或巴比妥缓冲液中的最适pH均为8.0。草酰乙酸和NADH的Km值分别为0.400±0.018和0.410±0.038 mM。该酶在65℃加热2分钟后活性丧失约90%。在焦碳酸二乙酯浓度为4 mM时，该酶受到61.4%的抑制。果糖1,6-二磷酸浓度为3 mM时，对该酶的抑制率为76.5%。这种抑制作用对NADH而言是非竞争性的，Ki值为0.85 mM。在甲苯达唑浓度为1 mM时，该酶受到75%的抑制，甲苯达唑通过与NADH竞争抑制该酶，Ki值为0.176 mM。柠檬酸浓度为2 mM时，该酶活性几乎增加了一倍。该酶在两种底物浓度较高时均受到抑制。该酶不受对羟基汞苯甲酸或富马酸的抑制。该酶对作为辅因子的NADH具有绝对特异性。将该酶的性质与来自宿主肝脏、囊液和其他一些动物来源的酶的性质进行了比较。根据宿主肝脏、囊液和原头蚴酶性质的差异对结果进行了讨论。还阐明了甲苯达唑用于治疗棘球蚴病的生化基础。
