A CONVENIENT SPECTROPHOTOMETRIC METHOD FOR MEASURING THE KINETIC-PARAMETERS OF GLYCERYL-ETHER MONOOXYGENASE (EC1.14.16.5)

Details of a direct spectrophotometric method for assaying glyceryl-ether monooxygenase activity are described. The assay has several advantages over previous methods including the convenient determination of the kinetic parameters of lipid substrates and tetrahydropterin cofactors with acceptable accuracy. The apparent K(m) and V(max) values have been measured for 6-methyl- and 6,7-dimethyl-5,6,7,8-tetrahydropterins and 6R-tetrahydrobiopterin as well as twelve lipid ethers including lyso-PAF (platelet activating factor), and the V/K values are a better index for comparing substrate efficiencies. The monooxygenase activities of a variety of assorted lipids are also compared with RS-batyl alcohol, some of which are weak inhibitors. The effects on monooxygenase activity by various concentrations of six detergents are compared and showed that Mega-10 is the most satisfactory for solubilising alkyl ether substrates at low concentrations (ca. 0.08%) of detergent. The syntheses of a variety of ether lipids used in this work, together with their H-1-NMR and IR spectra, are described.