Russell R, Scopes R K
Centre for Protein and Enzyme Technology, La Trobe University, Bundoora, V3083, Australia.
Bioseparation. 1994 Aug;4(4):279-84.
Choline dehydrogenase has been purified using hydrophobic chromatography 250-fold from a Pseudomonas strain. Although the enzyme is associated with the cell membrane and could be extracted from membrane preparations, it was best purified from a complete cell extract made with a non-ionic detergent. Only phenazine methosulfate was able to act as electron acceptor; there was no evidence of bound flavin, but there was evidence of pyrroloquinoline quinone cofactor. The purified enzyme had a specific activity of up to 67 units/mg, which is at least ten times higher than the values reported for mitochondrial choline dehydrogenases, and up to 100 times higher than previous reports for the Pseudomonas enzyme. The estimated subunit size of 66 kDa, which corresponds with the native size, is close to that deduced from the gene sequence of the Escherichia coli betA gene, and preliminary N-terminal sequencing shows homology with this deduced sequence. The next enzyme in the degradation pathway of choline, betaine aldehyde dehydrogenase, was also purified from the same extract.
胆碱脱氢酶已通过疏水色谱法从一种假单胞菌菌株中纯化了250倍。尽管该酶与细胞膜相关，并且可以从膜制剂中提取，但最好从用非离子洗涤剂制备的完整细胞提取物中进行纯化。只有硫酸吩嗪甲酯能够作为电子受体；没有结合黄素的证据，但有吡咯喹啉醌辅因子的证据。纯化后的酶比活性高达67单位/毫克，这至少比线粒体胆碱脱氢酶的报道值高十倍，比之前假单胞菌酶的报道值高多达一百倍。估计亚基大小为66 kDa，与天然大小相对应，与从大肠杆菌betA基因序列推导出来的大小相近，初步的N端测序显示与该推导序列具有同源性。胆碱降解途径中的下一种酶，即甜菜碱醛脱氢酶，也从同一提取物中纯化出来。
