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用于解析参与谷氨酰胺代谢调节的酶的ω-氨基烷基琼脂糖。

Omega-aminoalkyl agaroses in the resolution of enzymes involved in regulation of glutamine metabolism.

作者信息

Shaltiel S, Adler S P, Purich D, Caban C, Senior P, Stadtman E R

出版信息

Proc Natl Acad Sci U S A. 1975 Sep;72(9):3397-401. doi: 10.1073/pnas.72.9.3397.

DOI:10.1073/pnas.72.9.3397

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC433000/

Abstract

Systematic examination of a homologous series of omega-aminoalkyl agaroses showed that the pentyl derivative (Seph-C5-NH2) was best suited for the retention and subsequent separation of several proteins involved in the regulation of glutamine metabolism in Escherichia coli, including: glutamine synthetase [EC 6.3.1.2; L-glutamate:ammonia ligase (ADP-forming)], ATP:glutamine synthetase adenylyltransferase (EC 2.7.7.42), the PII regulatory protein which regulates the adenylylation and deadenylylation activities of the adenylyltransferase, the UTP:PII protein uridylyltransferase, and the uridylyl removing enzyme which catalyzes the removal of uridylyl groups from uridylylated PII protein. Resolution of these proteins was achieved by gradually increasing the concentration of KCl in the eluant, which resulted in consecutive detachment of the proteins from the column. Proteins that co-elute from a DEAE-cellulose column can be resolved and further purified on epsilon-aminopentyl agarose, probably due to the fact that with the homologous series it is possible to adjust the contribution of hydrophobic interactions for optimal resolution.

摘要

对一系列ω-氨基烷基琼脂糖进行系统研究表明，戊基衍生物（Seph-C5-NH2）最适合保留并随后分离大肠杆菌中参与谷氨酰胺代谢调节的几种蛋白质，包括：谷氨酰胺合成酶[EC 6.3.1.2；L-谷氨酸：氨连接酶（ADP形成）]、ATP：谷氨酰胺合成酶腺苷酰转移酶（EC 2.7.7.42）、调节腺苷酰转移酶腺苷酰化和去腺苷酰化活性的PII调节蛋白、UTP：PII蛋白尿苷酰转移酶以及催化从尿苷酰化PII蛋白上去除尿苷酰基的尿苷酰去除酶。通过逐渐增加洗脱液中KCl的浓度实现了这些蛋白质的分离，这导致蛋白质依次从柱上脱离。从DEAE-纤维素柱上共洗脱的蛋白质可以在ε-氨基戊基琼脂糖上进行分离和进一步纯化，这可能是因为对于同系物系列，可以调节疏水相互作用的贡献以实现最佳分离效果。