Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Medical Center Boulevard, Winston-Salem, North Carolina 27157-1016, and The Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute, Department of Pathology, University of California, San Francisco, California 94140-0608 Received October 3, 1991; Revised Manuscript Received December 3, 1991 abstract: We reported earlier that hepatic lipase (HL)-catalyzed hydrolysis of phospholipid monolayers is activated by apolipoprotein (apo) E [Thuren et al.(1991b) J. Biol. Chem. 266, 4853-4861]. On the basis of these studies, it was postulated that apoE-rich high-density lipoproteins (HDL) were preferred substrates for HL. In the present study, we tested this hypothesis, as well as further characterizing the activation of HL hydrolysis of phospholipid by apoE. The apoE-rich HDL, referred to as HDL-I, were isolated by heparin-Sepharose chromatography, and the phospholipid hydrolysis by HL was compared to an apoE-poor

HDL, designated HDL-II. The hydrolysis of HDL-I phosphatidylcholine was approximately 3-fold higher than HDL-II, supporting the hypothesis that HL preferably hydrolyzes the phospholipids in apoE-rich HDL. In order to gain additional insight into the nature of the activation, we used phospholipid monolayers as model systems. Comparison of the ability of the two thrombolytic fragments of apoE (22 kDa, residues 1-191; 12 kDa, residues 192-299) revealed that only the 12-kDa fragment was capable of activating the hydrolysis of phospholipid by HL (1.75-fold). However, activation was less than with the intact protein (2.8-fold for apoE3), suggesting that the intact protein was required for full activation. The fact that the 12-kDa fragment, which represents a major lipid region of the protein, did activate HL suggests that activation occurs at the lipid-water interface. The three common isoforms of apoE differed significantly in their abilities to activate HL; apoE3, apoE2, and apoE4 activated HL 2.8-, 2.3-, and 2.0-fold, respectively, at a surface pressure of 12.5 mN/m. The order ofaffinity of the isoforms for PC monolayers (E4> E2> E3) was not the same as activation, suggesting that lipid binding per se was not the major factor distinguishing the abilities of the three isoforms to activate hydrolysis. Determination of the ratio of HL and apoE at a phospholipid interface revealed that the stoichiometry was approximately 1: 1, suggesting that the activation