N_2和NH_4^+培养下粪产碱菌固氮酶铁蛋白的生物合成及特性比较

Biosynthesis and Properties of Fe Protein of Nitrogenase from N_2-Grown and NH_4^+-Grown Alcaligenes faecalis

应用DEAE-纤维素和凝胶柱层析,分别将N_2和NU_1^+(30 mmol/L)培养的粪产碱菌固氮酶铁蛋白(Af2和Af^+2)分离并提纯52倍,在SDS-PAGE上呈均一状态。Af2的比活性达1540 nmol C_2H_4mg^(-1)protein min^(-1),Af~*2无活性。Af2和Af~*2理化性质基本相同,分子量为64.5 kD;均由2个亚基组成,每个亚基分子量为32.5kD;氨基酸种类相同,总残基数分别为537和553,不含色氨酸;每分子Af2和Af^+2均含有4个Fe原子和4个酸不稳定S^(2-)原子;UV-vis光谱吸收特征相同;荧光探剂测定结果为:每分于Af2和Af~*2均络合2个分子MgATP或2个分子MgADP。

Alcaligenes faecalis strain A 1501 isolated from strain A15 was used in this experiment. Its main physiological and biochemical properties were the same as those of strain A15. Starin A1501, however,could use more sorts of carbohydrates as sole carbon source. A. faecalis A1501 was grown in N-free and combined N(NH_4^+30 mmol/L) culture medium, respectively. The Fe protein from N_2-grown cells (Af2) and NH_4^+grown cells (Af~* 2) were purified by treating the cell-free crude extract with DNase and RNase followed by 56℃ heating for 5 min. After centrifugation, the supernatant was twice passed through DEAE-cellulose (DE-52) columns(Fig. 1,2) followed by passing twice through Sephacryl S-200 column (Fig. 3) for gel filtration. Both Af2 and Af~* 2 were shown to be homogeneous by SDS-PAGE (Fig. 4). Af2 was purified 52-fold with specific activity of 1540 nmol C_2H_4 mg^(-1) protein min^(-1). No activity was detected from Af~* 2 (Table 1). The amino acid composition, metal contents, molecular weight, subunit composition, UV-visible spectrum and the properties of binding with MgATP and MgATP of Af2 and Af~* 2 were compared. The results showed that their physical and biochemical properties were almost the same. The molecular weight in both Af2 and Af~* 2 was 64.5 kD (Fig. 5) and the molecules were composed of two homogeneous subunits with the same molecular weight of 32.5 kD (Fig. 4). The number of amino acid resedues was 537 in Af2 and 553 in Af~* 2 and tryptophane was absent from both proteins(Table 2). Both Af2 and Af~* 2 contain 4 Fe and 4 acid-labile S^(2-). Their UV-visible spectrum features were the same,with a broad absorbance between 380 nm and 650 nm and with a small shoulder at 410 nm after being exposed to oxygen for 10 min(Fig. 6). Fluorescence probe FMA showed that both Af2 and Af~* 2 bound with 2 MgATP or 2 MgADP per molecule (Table 3).