Polyamine oxidase is a flavin-dependent peroxisomal enzyme involved in the degradation of polyamines to putrescine. This assay measures the conversion of N1-ace ty Spermidine to putrescine.
Following derivatization with o-phthalaldehyde, N'-acetylspermidine, and putrescine are separated on a Beckman Ultrasphere ion-pair column (4.6 mm x 250 mm, 5 /urn). Solvent A was composed of 0.1 M sodium acetate and 10 mAf octanesulfonic acid adjusted to pH 4.5 with acetic acid. Solvent B contained 0.2 M sodium acetate (pH 4.5)-acetonitrile (10:3, v/v) with 10 mAf octanesulfonic acid. A linear gradient from 35% A and 65% B to 100% B was achieved in 10 minutes, followed by continued flow of solvent B for 15 minutes. The flow rate was 1 mL/min. Postminutes, column derivatization of polyamines with o-phthalaldehyde was accomplished with a pump. Fluorescence detection was used, with excitation and emission wavelengths of 340 and 455 nm.
The standard reaction mixture contained 0.1 M glycine (pH 9.5), 5 mAf dithiothreitol, 250 (jlM iV'-acetylspermidine, and 250 to 400 ju.g of protein in a final volume of 250 fiL. The reaction mixtures also contained 0.56 mM aminoguanidine and 0.04 mAf pargyline to inhibit diamine oxidase and monoamine oxidase, respectively. The reaction was initiated by adding enzyme and stopped by the addition of 50 fiL of 50% trichloroacetic acid. After filtration, 25 to 100 fiL aliquots were injected. The reaction was linear with time and protein up to 60 to 500 /xg of protein.
Enzyme activities were measured in cytosolic fractions of small intestinal or colonic mucosa from rats.
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