Histidine regulation in Salmonella typhimurium. XVI. A sensitive radiochemical assay for histidinol dehydrogenase

Zygmunt Ciesla, Francesco Salvatore, James Broach, Stanley W. Artz, Bruce N. Ames

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)44-55
Number of pages12
JournalAnalytical Biochemistry
Volume63
Issue number1
DOIs
StatePublished - Jan 1 1975

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Histidinol
Salmonella
Salmonella typhimurium
Histidine
Assays
Operon
Ion Exchange Chromatography
Enzyme Assays
Chromatography
Cell Extracts
Ion exchange
Glucose
histidinol dehydrogenase
Substrates
Enzymes
Proteins

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Ciesla, Zygmunt ; Salvatore, Francesco ; Broach, James ; Artz, Stanley W. ; Ames, Bruce N. / Histidine regulation in Salmonella typhimurium. XVI. A sensitive radiochemical assay for histidinol dehydrogenase. In: Analytical Biochemistry. 1975 ; Vol. 63, No. 1. pp. 44-55.
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Histidine regulation in Salmonella typhimurium. XVI. A sensitive radiochemical assay for histidinol dehydrogenase. / Ciesla, Zygmunt; Salvatore, Francesco; Broach, James; Artz, Stanley W.; Ames, Bruce N.

In: Analytical Biochemistry, Vol. 63, No. 1, 01.01.1975, p. 44-55.

Research output: Contribution to journalArticle

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AB - 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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