Glyceryl Etherase [EC 1.14.16.5] from Rat Liver: A Convenient Assay for Structure-Activity Relationship Studies

W. L. F. Armarego, B. Kosar-Hashemi
1991 Pteridines  
In 1964 a microsomal preparation from rat liver was shown by Teitz et al. (1) to cleave 1-glyceryl ethers of CI6 and CI8 fatty alcohols to glycerol and the respective aliphatic aldehyde. The enzyme was shown to be a hydroxylase and required a tetrahydropterin as an essential cofactor and oxygen. The enzymic reaction was followed by studying the cleavage of 1glyceryl ethers labeled with 14 C at the oc-carbon atom of the CI6 and CI8 side-chains and measuring the amounts of 14 C labeled aldehydes
more » ... labeled aldehydes after separation by chromatography. With this assay Snyder and coworkers (2) extended the study and obtained considerable information about substrate specificity and cofactor activity. Kötting et al. (3) devised a continuous assay, whereby the hydroxylase reaction was coupled with dihydropteridine reductase which reduced the quinonoid dihydropterin formed back to the tetrahydropterin and observed the oxidation of NADH to NAD by the rate of decreased absorption at 340 nm. We have found that the assay using radioactive labeled substrate was time consuming and could not be used to readily evaluate the kinetic parameters for various substrates and pteridine cofactors. Similarly, the kinetic parameters could not be obtained by the coupled assay with the desired confidence although they are useful for obtaining a rough indication of the relative initial rates of various lipid substrates. For comparing the activity of substrates it is necessary to measure the kinetic parameters [K m and V ma J. In order to overcome the disadvantages of previous methods and for measuring the kinetic parameters of substrates we have adapted a spectrophotometric procedure (4) based on the assay that Ayling et al. (5) developed for phenylalanine hydroxylase. The assay involves the enzymic hydroxylation of the α-methylene group of the alkyl chain of the glyceryl (or related) ethers with concomitant oxidation of a 5,6,7,8-tetrahydropterin cofactor to the respective quinonoid 7,8-dihydro(6//)pterin. The rate of hydroxylation is measured by the rate of conversion of the tetrahydropterin to the quinonoid dihydropterin at a convenient wavelength. The wavelength is chosen for each tetrahydropterin used and is at the isosbestic point of the spectra for the conversion of the quinonoid dihydropterin to the respective 7,8-dihydro(3//)pterin. The reaction extinction coefficient is the difference between the extinction coefficients of the tetrahydropterin and the quinonoid dihydropterin at the isosbestic point. A double beam spectrophotometer (e. g. Varian 118) in which the cuvette holders arc closc enough for simultaneous addition of tetrarhydropterin is used. The glyceryl ether and related substrates solubilised by sonication in 0.8% Mega-10 (100 μΐ), M Tris/HCl (100 μΐ, at the desired pH), 1 mg/ ml catalase (100 μΐ), microsomal fraction (containing ca 0.5 mg protein) and water to make 950 μΐ are placed in one cuvette. The blank cuvette contained the same ingredients except that the Mega-10 solution did not contain the ether substrate. When the absorption at the pre-determined wavelengths is steady, the pterin cofactor (50 μΐ) is added simultaneously to both cuvettes and the rate of change of absorption is measured. The kinetic parameters are computed from the initial rates measured at varying concentrations of the ether substrates at saturating, or near saturating, concentrations of pterin cofactor and vice-versa. The kinetic parameters of three pterin cofactors and several lipid ether substrates are in the Table 1. The Unauthenticated Download Date | 7/25/18 6:32 PM
doi:10.1515/pteridines.1991.3.12.95 fatcat:rz6sux3sava2vjpcphz2bn2c3q