COMPARISON OF PHOSPHOENOLPYRUVATE-CARBOXYKINASE FROM AUTOTROPHICALLY AND HETEROTROPHICALLY GROWN EUGLENA AND ITS ROLE DURING DARK ANAEROBIOSIS
COMPARISON OF PHOSPHOENOLPYRUVATE-CARBOXYKINASE FROM AUTOTROPHICALLY AND HETEROTROPHICALLY GROWN EUGLENA AND ITS ROLE DURING DARK ANAEROBIOSIS
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DOI：
10.1104/pp.86.2.457
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发表时间：
1988-02-01
期刊：
影响因子：
7.4
通讯作者：
BETZ, A
中科院分区：
文献类型：
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作者：
PONSGENSCHMIDT, E;SCHNEIDER, T;BETZ, A
在CO2光照下自养生长时，绿叶菊（1224-5/9）含有磷酸烯醇丙酮酸羧激酶。当添加葡萄糖等额外碳源时，其产率会更高。单独提供二氧化碳并保持在黑暗中的细胞缺乏这种酶，而如果在黑暗中同时提供葡萄糖和二氧化碳，则产量最高。经硫酸铵沉淀、Sephacryl S-300凝胶过滤和GMP-Sepharose亲和层析纯化该酶。后一个步骤是最有效的保护酶免于失活。用电泳和免疫学方法检测其同质性。自养和异养细胞产生的酶具有相同的最佳pH值和相似的等电点。凝胶过滤测定，自养酶分子量为761,000，异养酶分子量为550,000。两种酶的亚基分子量几乎相同。酶的动力学数据略有不同。糖酵解和三羧酸循环中间体在非生理性高浓度下对酶活性和抑制的影响有限。双免疫扩散和酶联免疫吸附试验表明，该酶定位于细胞质中。通过后一种定量试验发现，异养培养的细胞中磷酸烯醇丙酮酸羧激酶蛋白含量比自养培养的细胞高10倍。
Euglena gracilis (1224-5/9) contains phosphoenolpyruvate carboxykinase when grown autotrophic with CO2 in the light. Its yield is higher when an additional carbon source like glucose has been added. The enzyme is lacking in cells provided with CO2 alone and kept in the dark, whereas highest yields result if both glucose and CO2 are provided together in the dark. The enzyme was purified by ammonium sulfate precipitation, gel filtration on Sephacryl S-300 and affinity chromatography on GMP-Sepharose. The latter step was most effective to protect the enzyme from inactivation. Its homogeneity was tested electrophoretically and immunologically. Enzymes from autotrophic and heterotrophically grown cells have identical pH optima and similar isoelectric points. The molecular weight was different: 761,000 for the enzyme from autotrophic and 550,000 for that from heterotrophic cells as determined by gel filtration. The subunit molecular weight of both enzymes is nearly the same. The kinetic data of the enzymes are slightly different. Glycolytic and tricarboxylic acid cycle intermediates are of limited influence on enzyme activity and inhibitory in unphysiological high concentrations. From Ouchterlony double immunodiffusion and enzyme-linked immunosorbent assay, it is evident that the enzyme is localized in the cytosol. With the latter quantification test the phosphoenolpyruvate carboxykinase protein content was found 10 times higher in heterotrophically grown cells than when cultivated under autotrophic conditions.