家蚕5龄幼虫体内共轭酶和二氢叶酸还原酶的基本性质

共轭酶 (EC 3 4 2 2 12 )和二氢叶酸还原酶 (EC 1 5 1 3)是生物体内重要的叶酸代谢酶。以家蚕幼虫为材料分析了这两种酶的基本性质。 5龄幼虫体液共轭酶反应的最适pH为 7 8,最适温度为 5 0℃ ;反应体系中添加巯基乙醇、K+ 或Mg2 + 可显著提高酶活性 ,而对羟高汞苯磺酸、Co2 + 、Zn2 + 、Fe2 + 等对酶活性具有明显的抑制作用 ;以叶酸五谷氨酸为基质的水解产物为叶酸 ;另以叶酸三谷氨酸为底物 ,采用 33mmol/LHepse Ches缓冲液 (pH 7 8)在 37℃的反应条件下 ,求得Km 为 9 6 μmol/L。幼虫脂肪体等组织中二氢叶酸还原酶以NADPH为供氢体 ;在pH 4 5～7 5的范围内具有较强活性 ,最适反应温度 35℃ ;巯基乙醇、胍 ,以及高浓度的 1价金属离子 (K+ 、Na+ 等 )对酶具有活性化作用 ,对氯高汞苯甲酸、甲酰胺、Co2 + 等具有强烈的抑制作用 ;采用 35mmol/L柠檬酸 磷酸钠缓冲液 (pH 5 .0 ,10g/L抗坏血酸 )在 30℃的反应条件下 ,求得Km 值为 2 7μmol/L。家蚕体液可作为共轭酶的新酶源而加以利用。

Properties of Folic Acid γ-Glutamyl Hydrolase (Conjugase) and Dihydrofolate Reductase in 5th Instar Larvae Body of the Silkworm, Bombyx mori

Folic acid γ glutamyl hydrolase (conjugase) (EC 3 4 22 12) and dihydrofolate reductase (EC 1 5 1 3) are important folate metabolic enzymes. Some properties of conjugase and dihydrofolate reductase in silkworm larvae were investigated in this paper. The conjugase in the silkworm hemolymph had an optimum pH of 7 8, and optimum temperature of 50 ℃. The enzymatic activity was stimulated in the presence of 2 mercaptoehanol, K + and Mg 2+ , but inhibited by ρ hydroxymercuriphenylsulfonate, Co 2+ ,Zn 2+ and Fe 2+ . This conjugase converted pteroylpenta γ L glutamic acid to pteroylmonoglutamic acid exclusively, and the K m value was about 9 6 μmol/L when measured at pH 7 8, 37 ℃ with pteroyltri-γ-L glutamic acid as the substrate. The dihydrofolate reductase in the silkworm fat body required NADPH, had an optimum pH of 4 5～7 5, and an optimum temperature of 35 ℃. Its activity was stimulated in the presence of 2 mercaptoethanol, guanidine, Mg 2+ and high concentration of K + and Na +, but inhibited by ρ chloromercuribenzoate, formamide and Co 2+ . The K m value measured at pH 5 0, 30 ℃, was about 2 7 μmol/L when dihydropteroyl glutamic acid was used as the substrate. It was thought that the silkworm hemolymph can be used as a new conjugase source