Robertson D L, Alberte R S
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637, USA.
Plant Physiol. 1996 Aug;111(4):1169-75. doi: 10.1104/pp.111.4.1169.
Two peaks of glutamine synthetase (GS) activity were resolved by anion-exchange chromatography from the marine diatom Skeletonema costatum Grev. The second peak of activity accounted for greater than 93% of total enzyme activity, and this isoform was purified over 200-fold. Results from denaturing gel electrophoresis and gel-filtration chromatography suggest that six 70-kD subunits constitute the 400-kD native enzyme. The structure of the diatom GS, therefore, appears more similar to that of a type found in bacteria than to the type common among other eukaryotes. Apparent Michaelis constant values were 0.7 mM for NH4(+), 5.7 mM for glutamic acid, and 0.5 mM for ATP. Enzyme activity was inhibited by serine, alanine, glycine, phosphinothricin, and methionine sulfoximine. Polyclonal antiserum raised against the purified enzyme localized a single polypeptide on western blots of S. costatum cell lysates and recognized the denatured, native enzyme. Western analysis of the two peak fractions derived from anion-exchange chromatography demonstrated that the 70-kD protein was present only in the later eluting peak of enzyme activity. This form of GS does not appear to be unique to S. costatum, since the antiserum recognized a similar-sized protein in cell lysates of other chromophytic algae.
通过阴离子交换色谱法从海洋硅藻中肋骨条藻（Skeletonema costatum Grev.）中分离出谷氨酰胺合成酶（GS）活性的两个峰。第二个活性峰占总酶活性的93%以上，该同工型被纯化了200多倍。变性凝胶电泳和凝胶过滤色谱的结果表明，六个70-kD亚基构成了400-kD的天然酶。因此，硅藻GS的结构似乎与细菌中发现的一种类型更相似，而不是其他真核生物中常见的类型。NH4(+)的表观米氏常数为0.7 mM，谷氨酸为5.7 mM，ATP为0.5 mM。酶活性受到丝氨酸、丙氨酸、甘氨酸、草丁膦和甲硫氨酸亚砜亚胺的抑制。针对纯化酶产生的多克隆抗血清在中肋骨条藻细胞裂解物的western印迹上定位了一条单一多肽，并识别变性的天然酶。对阴离子交换色谱得到的两个峰级分的western分析表明，70-kD蛋白仅存在于酶活性较晚洗脱的峰中。这种形式的GS似乎并非中肋骨条藻所特有，因为抗血清在其他色藻的细胞裂解物中识别出了类似大小的蛋白。
