Author/Authors :
Dagmar Kratky، نويسنده , , Achim Lass، نويسنده , , Peter M. Abuja، نويسنده , , Hermann Esterbauer PhD، نويسنده , , Hartmut Kühn، نويسنده ,
Latin Abstract :
Oxidative modification of low-density lipoprotein (LDL) has been implicated as a patho-physiological process in early atherogenesis and 15-lipoxygenases (15-LOX) may be involved. While studying the in vitro kinetics of the 15-LOX/LDL interaction, we found that the conventional spectrophotometric assays failed in the range of substrate saturation owing to the high optical density of concentrated LDL solutions. Therefore, we developed a much more sensitive assay system which was based on peroxide induced isoluminol enhanced chemiluminescence. With this method reliable kinetic data were obtained at LDL concentrations of up to 1 mg/ml. To validate this luminometric method the kinetic parameters of 15-LOX catalyzed oxygenation of linoleic acid (Km=3.7 μM, kcat=17 s−1) were determined and we observed a good agreement with previously published data obtained with a spectrophotometric assay. Moreover, we found that the kinetic constants of 15-LOX catalyzed LDL oxidation (Km=0.64 μM, kcat=0.15 s−1) are quite different from those of free fatty acid oxygenation and that the cholesterol esters are preferentially oxidized during 15-LOX/LDL interaction. Vitamin E depletion does not reduce the rate of LDL oxidation and analysis of the structure of the oxygenation products suggests that the majority of the products were formed via direct LOX catalyzed oxidation of LDL ester lipids. The luminometric method described here is not restricted to the measurement of LOX catalyzed LDL oxidation, but may also be used to determine kinetic constants for the oxidation of other complex substrates such as biomembranes or liposomes
NaturalLanguageKeyword :
CL , rabbit reticulocyte 15-lipoxygenase , cholesteryl linoleate hydroperoxides , 15-LOX , CE-OOH , hydroperoxy lipids , chemiluminescence , PbS , butylated hydroxytoluene , LOOH , USA.2 Deceased on 7 January 1997.Biochimica et Biophysica Acta 1437 (1999) 13^22 , AAPH , 2-azobis(2-amidinopropane) hydrochloride , LDL , Dallas , counts per second , PUFA , BHT , low-density lipoprotein , TX 75275 , Hydroperoxy lipid , 13-HPODE , tocopherol mediated lipid peroxidation* Corresponding author. Fax: +43 (316) 3809016 , absorbance at 234 nm , 2 , 11E-octadecadienoic acid , Southern Methodist University , EDTA , polyunsaturatedfatty acid , Lipid peroxidation , ethylenedinitrilotetraacetic acid , SDS-PAGE , 15-Lipoxygenase , HPLC , sodium dodecyl sulfate-polyacrylamide gel electrophoresis , low-density lipoprotein , High-performance liquid chromatography , TMP , Chemiluminescence1388-1981 / 99 / $ ^ see front matter ? 1999 Elsevier Science B.V. All rights reserved.PII: S 0 0 0 5 - 2 7 6 0 ( 9 8 ) 0 0 1 7 6 - 3Abbreviations: A234 , 13-(S)-hydroperoxy-9Z , E-mail: dagmar.kratky@kfunigraz.ac.at1 Present address: Department of Biological Sciences , CPS , phosphate-bu?ered saline
