Abstract
Dolichol is one of the mevalonate pathway end products, present in all tissues and cellular membranes. The lipid is a primary alcohol consisting of 16-23 isoprenoid residues and occurs as a free alcohol, monophosphate or fatty acyl ester. The phosphorylated form functions as an obligatory intermediate in the biosynthesis of N-linked oligosaccharides of glycoproteins. Studies in model membranes indicate that dolichol has a destabilizing effect on phospholipid bilayers by increasing membrane fluidity and permeability. The aim of the present investigation was to identify and characterize the enzymes involved in dolichol esterification and thereby gain insight into the possible function of this lipid.
When phosphatidylcholine : phosphatidylethanolamine liposomes containing 2% dolichol were injected into rats, the polyisoprenoid lipid was esterified in the circulation. Pre-heparinization of the animal greatly increased the esterification and it was concluded that an enzyme was liberated into the plasma by heparin. It was further demonstrated that LCAT was not responsible for this activity and that human postheparin plasma also contained a dolichol acylating activity.
The enzyme was partially purified from heparinized liver perfusates and characterized with respect to its substrate specificity. It was shown that the enzyme was a transacylase and that phosphatidyl-ethanolamine and -serine were by far the preferred acyl donors. Only fatty acids in sn-1 position were utilized and cholesterol and retinol were not esterified.
The use of Brij-35 instead of Triton X-100 for delipidization of the enzyme made it possible to purify the enzyme to homogeneity without loosing the dolichol transacylase activity. The protein had a molecular mass of 61 kDa and was shown to be identical with hepatic lipase. The hepatic lipase-catalyzed dolichol esterification was strongly stimulated by a plasma cofactor that also shifted the pH optimum from 8.5 to 7.5. Gel filtration of whole plasma indicated that the cofactor might be associated with HDL.
The cofactor was purified from the lipoprotein-free high density fraction of plasma and the identity with apolipoprotein (apo) A-IV was demonstrated. The role of apoA-IV in hepatic lipase-catalyzed hydrolysis of human HDL-2 and VLDL was investigated. The cofactor strongly stimulated phospholipid hydrolysis and completely inhibited triglyceride hydrolysis in both lipoproteins. Thus, apo A-IV is an important cofactor to hepatic lipase, and it is suggested that apo A-IV rich HDL is a preferred lipoprotein substrate for hepatic lipase.
