Hussein A S, Walter R D
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Mol Biochem Parasitol. 1995 Jun;72(1-2):57-64. doi: 10.1016/0166-6851(94)00064-t.
We have purified and characterized the Ascaris suum gamma-glutamylcysteine synthetase, the rate-limiting step in the glutathione biosynthesis. The purified enzyme exhibited a specific activity of 18 U (mg protein)-1. Estimation of the molecular mass of the native enzyme by FPLC on Superdex S-200 revealed the presence of two enzyme activity peaks corresponding to molecular masses of 100 and 70 kDa. The higher-molecular-mass component could be dissociated by repeated gel filtration into the 70-kDa protein which is the enzymatically active subunit. The apparent Km values of the A. suum enzyme for L-aminobutyrate, L-cysteine and L-glutamate were 0.31, 0.41 and 0.94 mM, respectively. D,L-Buthionine-S,R-sulfoximine and cystamine showed time-dependent irreversible inhibitory effects on the A. suum enzyme activity with Ki values of 0.05 and 1.11 microM, respectively. The Ki values for the corresponding enzyme from rat kidney with D,L-buthionine-S,R-sulfoximine and cystamine were 7.19 and 22.2 microM, respectively. The time of half-inactivation of the enzyme at infinite concentration of D,L-buthionine-S,R-sulfoximine, tau 50, was determined to be 3.1 and 1.34 min, for the parasite and mammalian enzymes respectively. For cystamine, a tau 50 value of 3.32 min for the A. suum gamma-glutamylcysteine synthetase was determined, while a value of 2 min in case of rat kidney enzyme was found. The A. suum enzyme activity was competitively inhibited by glutathione with a Ki value of 0.11 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
我们已经纯化并鉴定了猪蛔虫γ-谷氨酰半胱氨酸合成酶，它是谷胱甘肽生物合成中的限速步骤。纯化后的酶比活性为18 U（mg蛋白）-1。通过在Superdex S-200上进行快速蛋白质液相色谱（FPLC）对天然酶的分子量进行估计，发现存在两个酶活性峰，分别对应于100 kDa和70 kDa的分子量。高分子量组分可通过反复凝胶过滤解离为具有酶活性的70 kDa蛋白质亚基。猪蛔虫酶对L-氨基丁酸、L-半胱氨酸和L-谷氨酸的表观Km值分别为0.31、0.41和0.94 mM。D，L-丁硫氨酸-S，R-亚砜亚胺和胱胺对猪蛔虫酶活性表现出时间依赖性的不可逆抑制作用，Ki值分别为0.05和1.11 μM。大鼠肾脏相应酶对D，L-丁硫氨酸-S，R-亚砜亚胺和胱胺的Ki值分别为7.19和22.2 μM。在D，L-丁硫氨酸-S，R-亚砜亚胺无限浓度下，寄生虫和哺乳动物酶的酶半失活时间tau 50分别确定为3.1和1.34分钟。对于胱胺，猪蛔虫γ-谷氨酰半胱氨酸合成酶的tau 50值为3.32分钟，而大鼠肾脏酶的该值为2分钟。猪蛔虫酶活性受到谷胱甘肽的竞争性抑制，Ki值为0.11 mM。（摘要截断于250字）
