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.
Ota
Hiroko
H
Asahi Kasei Pharma Corporation, Shizuoka, Japan.
Tamezane
Hideto
H
Sasano
Yoshie
Y
Hokazono
Eisaku
E
Yasuda
Yuko
Y
Sakasegawa
Shin-ichi
S
Imamura
Shigeyuki
S
Tamura
Tomohiro
T
Osawa
Susumu
S
D006631:Amine Oxidase (Copper-Containing) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D017124:Conserved Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D010714:Phosphatidylethanolamines; C483858:phosphatidylethanolamine; D006631:Amine Oxidase (Copper-Containing)