EFFECT OF THE CHOLESTEROL CONTENT OF RECONSTITUTED LPA-I ON LECITHIN-CHOLESTEROL ACYLTRANSFERASE ACTIVITY

The production of cholesteryl ester (CE) by lecithin: cholesterol acyl transferase (LCAT) is elevated significantly in hyperlipidemic subjec ts at high risk for coronary artery disease. To elucidate the molecula r events involved, the relationship between LCAT activation and apolip oprotein (apo) A-I charge and structure in high density lipoproteins ( HDL) has been studied in both native HDL and homogeneous recombinant H DL (Lp2A-I) particles containing apoA-I, palmitoyloleoyl phosphatidylc holine and cholesterol. Increasing the cholesterol content of discoida l Lp2A-I from 4 to 26 molecules/particle raises the maximum rate of ch olesterol esterification by LCAT (V-max) from 3.1 to 9.2 nmol CE/h/uni t of LCAT and increases the apparent K-m from 0.5 to 3.5 mu M choleste rol. Similarly, increasing the cholesterol content in triolein core-co ntaining Lp2A-I (4-18 molecules/particle) and in native HDL(3) (12-21 molecules/particle) also significantly increases the V-max for LCAT (2 .8-7.7 and 0.5-3.6 nmol CE/h, respectively) and raises the K-m values (7.6-36.9 and 7.3-8.5 mu M cholesterol, respectively). In contrast, ch anges in the cholesterol content of native and recombinant HDL have no significant effect on the apparent K-m values when expressed in terms of the concentration of either apoA-I or palmitoyloleoyl phosphatidyl choline. This appears to indicate that interfacial cholesterol content has no effect on the binding affinity of LCAT to different LpA-I part icles but directly affects catalysis by modulating the interaction of cholesterol molecules with the active site of LCAT. Increasing the cho lesterol content of the different HDL particles progressively increase s the particle net negative charge, and these changes in apoA-I charge are strongly correlated with both the V-max and apparent K-m values f or LCAT, This suggests that the conformation and charge of apoA-I play a central role in LCAT activation and that these parameters are influ enced by the amount of cholesterol in the surface of HDL particles.