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.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology