Evidence that lipid hydroperoxides inhibit plasma lecithin:cholesterol acyltransferase activity.
The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. Recently, we found that polar lipids isolated from minimally oxidized LDL produced a dramatic inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, suggesting that HDL-cholesterol transport may be impaired during early atherogenesis. In this study, we have identified molecular species of oxidized lipids that are potent inhibitors of LCAT activity. Treatment of LDL with soybean lipoxygenase generated small quantities of lipid hydroperoxides (20 +/- 4 nmol/mg LDL protein, n = 3); but when lipoxygenase-treated LDL (1 mg protein/ml) was recombined with the d > 1.063 g/ml fraction of human plasma, LCAT activity was rapidly inhibited (25 +/- 4 and 65 +/- 16% reductions by 1 and 3 h, respectively). As phospholipid hydroperoxides (PL-OOH) are the principal oxidation products associated with lipoxygenase-treated LDL, we directly tested whether PL-OOH inhibited plasma LCAT activity. Detailed dose-response curves revealed that as little as 0.2 and 1.0 mole % enrichment of plasma with PL-OOH produced 20 and 50% reductions in LCAT activity by 2 h, respectively. To gain insight into the mechanism of LCAT impairment, the enzyme's free cysteines (Cys31 and Cys184) and active site residues were "capped" with the reversible sulfhydryl compound, DTNB, during exposure to either minimally oxidized LDL or PL-OOH. Reversal of the DTNB "cap" after such exposures revealed that the enzyme was completely protected from both sources of peroxidized phospholipids. We, therefore, conclude that PL-OOH inhibited plasma LCAT activity by modifying the enzyme's free cysteine and/or catalytic residues. These studies are the first to suggest that PL-OOH may accelerate the atherogenic process by impairing LCAT activity.
Bielicki
J K
JK
Lawrence Berkeley National Laboratory, Life Sciences Division 1-213, University of California at Berkeley, One Cyclotron Road, Berkeley, CA 94720, USA.
Forte
T M
TM
D001161:Arteriosclerosis / Q000097:blood / Q000209:etiology; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid; D004791:Enzyme Inhibitors / Q000097:blood*; D006801:Humans; D066298:In Vitro Techniques; D008054:Lipid Peroxides / Q000097:blood / Q000494:pharmacology*; D008077:Lipoproteins, LDL / Q000097:blood / Q000494:pharmacology; D008084:Lipoxygenase / Q000494:pharmacology; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000037:antagonists & inhibitors* / Q000097:blood* / Q000737:chemistry
D004791:Enzyme Inhibitors; D008054:Lipid Peroxides; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D004228:Dithionitrobenzoic Acid; D008084:Lipoxygenase; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D003545:Cysteine