PURIFICATION OF GUANOSINE TRIPHOSPHATE CYCLOHYDROLASE-I AND DIHYDROFOLATE-REDUCTASE ON A DIHYDROFOLATE-SEPHAROSE AFFINITY COLUMN

被引:12
作者
THEN, RL
机构
[1] Pharmaceutical Research Department, F. Hoffmann-La Roche and Co., Ltd., 4002 Basle
关键词
D O I
10.1016/0003-2697(79)90120-9
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Folate was coupled to AH-Sepharose 4B, the gel poured into small columns, and the Sepharose-bound folate reduced in situ to dihydrofolate by dithionite/ascorbate at pH 6 to 7. The dihydrofolate-Sepharose column was used to purify guanosine triphosphate cyclohydrolase I (EC 3.5.4.16) and dihydrofolate reductase (EC 1.5.1.3). All steps were carried out in the cold and in the presence of 20 mm mercaptoethanol. GTP cyclohydrolase I bound strongly to the dihydrofolate-Sepharose column and was purified several-hundred-fold in a single step. It did not bind to folate-Sepharose. Binding to dihydrofolate-Sepharose is assumed to reflect a physiological role of dihydrofolate. GTP cyclohydrolase II did not bind to either folate- or dihydrofolate-Sepharose. Dihydrofolate reductase from Escherichia coli B and from rat liver did not bind to folate-Sepharose under the test conditions, but could be well purified on the dihydrofolate-Sepharose column. This column is judged to beuseful for the purification of other folate-converting enzymes. © 1979.
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页码:122 / 128
页数:7
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