Enzymatic assay of phosphatidylethanolamine in serum using amine oxidase from Arthrobacter sp.

BACKGROUND: In human serum, as for phospholipids not containing choline, phosphatidylethanolamine (PE) exists approximately 5% in a whole phospholipid. PE is well known as one of the main components of biological membranes, and also plays important roles that contribute to apoptosis and cell signaling. However, it could not measure PE with other phospholipids due to a lack of choline in them. METHODS: Using an amine oxidase (EC 1.4.3.6), from Arthrobacter species, a simple and rapid enzymatic assay for measurements of PE in serum was established. That assay used the Hitachi 7170 analyzer to evaluate the analytical performance. RESULTS: The average within-run CVs were 0.38-1.27% (n=20) at 69-160 µmol/l. The correlation between values obtained with the present method (y) and the high-performance liquid chromatography (HPLC) method (x) was: y=0.944x+9.441 (r=0.977, S(y|x)=5.82, n=34). In addition, the reference interval of healthy subjects was 115±45 µmol/l. CONCLUSIONS: This new enzymatic method shows a high specificity for serum PE and can be easily applied to an automated analyzer. The present method is available as a novel marker of changes in the clinical condition of serum phospholipids.

Enzymatic characterization of an amine oxidase from Arthrobacter sp. used to measure phosphatidylethanolamine.

Ethanolamine oxidase was screened with the aim of using it to establish a novel enzymatic phosphatidylethanolamine assay. Ethanolamine oxidase activity was detected in the crude extract of Arthrobacter sp., and the enzyme was purified more than 15-fold in three steps with a 54% yield. SDS-PAGE revealed the presence of only one band, which migrated, with an apparent molecular mass of 70 kDa. Biochemical characterization of the enzyme showed phenylethylamine to be the preferred substrate, with the highest kcat/Km value. The primary structure, determined by sequencing the cloned gene, showed a high degree of identity to Cu-containing phenylethylamine oxidase (64%). When heterologously overexpressed in Escherichia coli, the enzyme exhibited only a trace of amine oxidase activity, but high levels of activity emerged after exposure to Cu2+, as is typical of recombinant copper amine oxidases. Preliminary application of this enzyme coupled with phospholipase D for determination of phosphatidylethanolamine is also described. This is the first enzymatic method for the measurement of phosphatidylethanolamine.