Assay of ornithine decarboxylase activity by reversed-phase high-performance liquid chromatography

Cynthia S. Beeman, Edward F. Rossomando

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Ornithine decarboxylase (l-ornithine carboxylase; EC 4.1.1.17; ODCase) is a key enzyme in the biosynthesis of polyamines. It catalyzes the decarboxylation of l-ornithine to putrescine. The high-performance liquid chromatographic (HPLC) method described here for determining ODCase activity combines the sensitivity of radiochemical detection with the separative capacity of HPLC without the necessity of generating a pre-column derivative. In this study, [1,2-3H]putrescine was separated from l-[2,3-3H]ornithine using revresed-phase HPLC eluted isocratically. This method was used to study ODCase from both prokaryotic and mammalian sources. With the ODCase from Escherichia coli we found the reaction rates to be linear for 5 min with an apparent Michaelis constant (KM) of 20 mM. After 1 h this activity had produced approximately four-fold more product at pH 5.0 than at pH 7.3. In contrast, the initial rate of ODCase from submandibular glands was linear for 60 min. Also, the rate of putrescine synthesis was ten-fold higher in the embryonic gland than in the adult which was 8-80 times lower than that of E. coli.

Original languageEnglish
Pages (from-to)101-110
Number of pages10
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume496
Issue numberC
DOIs
StatePublished - 1989

Bibliographical note

Funding Information:
Support for this research was derived in part from the National Institutes of Health No. DE05333 (C.&B. ). We wish to thank Mr. S.T. Ingalls for many useful conversations regarding the development of this assay, Dr. T. Gay for the use of his word processing equipment, and Dr. E.J. Kollar for editing the manuscript.

ASJC Scopus subject areas

  • Chemistry (all)

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