The enzyme that catalyzes the decarboxylation of ornithine to putrescine is a key factor in the biosynthesis of polyamines; ornithine decarboxylase is involved in the control of cell regulation, differentiation, and growth.
The assay described by Haraguchi et al (1980) involves a preparation on CellexP, a conversion to the fluorescent derivative with fluorescamine, and separation on HPLC.
The column was LiChrosorb RP-18, and the separation was carried out by elution with a gradient of 45 to 80% methanol and 0.1 M ammonium chloride in an acetate buffer (pH. 4.0).
The incubation mixture contained ornithine, pyridoxyl phosphate, and the enzyme. After incubation for 1 hour, the reaction was terminated with perchloric acid. The precipitate was removed by centrifugation, the supernatant extracted with chloroform-methanol (2:1), and the aqueous layer applied to a CellexP column. The putrescine was eluted, reacted with fluorescamine, and quantitated by HPLC.
The enzyme was from rat intestinal mucosa and was partially purified by a 20 to 80% precipitation with ammonium sulfate.
In the assay described by Beeman and Rossomando (1989), a ¿¿Bondapak Cjg column (3.9 mm x 300 mm) was used to separate L-[2,3-3H]ornithine from [l,2-3H]putrescine. The mobile phase contained 0.05 M sodium phosphate (pH adjusted to 3.9 with phosphoric acid) containing 0.01 M SDS and 36% acetonitrile. Fractions were collected and the radioactivity determined by a liquid scintillation counter. The flow rate was 1.0 mL/min, and 0.5 mL fractions were collected.
The reaction mixture contained in a final volume of 1 mL 20 mM sodium phosphate buffer (pH 5.0), 2.5 mM dithiothreitol, 0.1 mM pyridoxal 5'-phosphate, 0.4 ^Ci L-[2,3-3H]ornithine, 100 mM ornithine hydrochloride, and 0.3 mL of either E. coli ornithine decarboxylase or tissue homogenate. The E. coli enzyme was assayed at pH 5.0 and 37°C; the pH values of reaction mixtures were adjusted to pH 7.3 before assaying homogenates of mammalian tissues. The reaction was stopped by injection onto the column.
The E. coli ornithine decarboxylase was purchased from Sigma. Supernates obtained by centrifugation of homogenates of submandibular glands of mice (homogenized in 20 mM sodium phosphate buffer, pH 7.3), 1.25 mM dithiothreitol, and 10 pM disodium EDTA.
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