Histidine regulation in Salmonella typhimurium: XVI. A sensitive radiochemical assay for histidinol dehydrogenase |
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Authors: | Zygmunt Ciesla Francesco Salvatore James R Broach Stanley W Artz Bruce N Ames |
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Institution: | Department of Biochemistry, University of California, Berkeley, California 94720 USA |
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Abstract: | A sensitive radiochemical assay for measurement of histidinol dehydrogenase is presented. The method is based upon separation of the product of the reaction. 14C]histidine, from the substrate, 14C]histidinol, on small Dowex 50 columns. The assay can be performed on cell extracts or on toluenized cells and is approximately 100 times more sensitive than previously reported assays for this enzyme.14C]histidinol is obtained in high yields through conversion of uniformly labeled 14C-glucose by a strain of Salmonella typhimurium derepressed for the histidine operon and blocked at the histidinol dehydrogenase step. Accumulated 14C]histidinol is purified from the culture supernatant by ion-exchange chromatography.This sensitive assay has facilitated measurement of reduced levels of histidine operon expression in promoter mutants, and has been adapted for study of histidine operon regulation in a cell free protein synthesizing system. |
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Keywords: | To whom reprint requests should be addressed |
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