Kempner E S, Miller J H, Schlegel W, Hearon J Z
J Biol Chem. 1980 Jul 25;255(14):6826-31.
Recently, target analysis has been re-evaluated as a technique for the determination of molecular sizes (Kempner, E. S. & Schlegel, W. (1979) Anal. Biochem. 92, 2-10). The technique yields the size of the functional unit, i.e. the minimal assembly of structures necessary for a given function such as an enzymatic activity. Using this method, we have not determined the sizes of the functional units for different enzymatic activities on the "arom" conjugate from Euglena, a polyenzyme catalyzing five sequential reactions in the shikimic acid pathway, and on two conjugates from Escherichia coli carrying both aspartokinase and homoserine dehydrogenase activities. In each conjugate, the size for different enzymatic activities was measured and found to be the same. When compared to the molecular weight obtained with other techniques, the target size matched either the entire conjugate (aspartokinase-homoserine dehydrogenase conjugates I and II) or half the unit ("arom" conjugate). The information was obtained with minimal perturbation of the complexes and sparing laborious purification and reconstitution experiments. Tryptophan synthase was irradiated both as an intact conjugate and also as isolated subunits. In both structural forms, beta 2 was identified as the functional unit for the conversion of indole and serine to tryptophan. The results of this study give insight into the structural assembly of these polyenzymes.
最近，目标分析作为一种测定分子大小的技术已被重新评估（肯普纳，E.S. & 施莱格尔，W.（1979年）《分析生物化学》92卷，2 - 10页）。该技术可得出功能单元的大小，即实现特定功能（如酶活性）所需的最小结构组合。利用这种方法，我们尚未确定来自眼虫的“芳香”共轭物（一种在莽草酸途径中催化五个连续反应的多酶）上不同酶活性的功能单元大小，以及来自大肠杆菌的两种同时具有天冬氨酸激酶和高丝氨酸脱氢酶活性的共轭物上不同酶活性的功能单元大小。在每种共轭物中，对不同酶活性的大小进行了测量，发现它们是相同的。与用其他技术获得的分子量相比，目标大小要么与整个共轭物（天冬氨酸激酶 - 高丝氨酸脱氢酶共轭物I和II）匹配，要么与单元的一半（“芳香”共轭物）匹配。获得这些信息时对复合物的扰动最小，且无需费力的纯化和重组实验。色氨酸合成酶作为完整的共轭物以及分离的亚基均受到辐照。在这两种结构形式中，β2均被确定为将吲哚和丝氨酸转化为色氨酸的功能单元。这项研究的结果有助于深入了解这些多酶的结构组装。
