Studies on Plasma Lecithin: Cholesterol Acyltransferase Reaction

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When whole plasma is incubated at 37°, a net increase of cholesterol esters occurs. The enzyme which catalyzes this reaction is present in high concentration in plasma, and mainly transfers fatty acids from the 2-position of lecithin to cholesterol. The enzyme is calledlecithin: cholesterol acyltransferase (LCAT). The LCAT reaction appears to be a physiologically important source of human plasma cholesterol esters.<BR>In our previous studies on plasma cholesterol esterification, we obtained an evidence indicating that the rate of cholesterol esterification of HDL was much higher than that of VLDL and LDL. In the present communication, further studies on the mechanism of this enzyme reaction are presented.<BR>The method involves formation of HDL-agarose, and subsequent utilization of this insoluble but fully hydrated product in the studies on enzyme-substrate complex formation. HDL-agarose was relatively stable and no changes in lipid occurred during a 24 hours' incubation at 37°. In the presence of the enzyme, esterification of cholesterol in HDL-agarose occurred, indicating that a reactive enzyme-substrate complex had been formed (TABLE I). In the experiment shown in Fig. 1, equal amounts of partially purified LCAT were applied to a column of HDL-agarose and 2 control columns. The fact that the effluent from the HDL-agarose column was of much less LCAT activity is consistent with the spetific binding of LCAT to HDL. The HDL-agarose complex was used for investigation of relative affinity of LCAT to various lipoproteins or their derivatives. Tubes containing HDL-agarose and LCAT together with different kinds of free substrates were incubated at 4° and LCAT activity bound to HDL-agarose was determined. The results shown in TABLE II demonstrate a higher affinity of LCAT to HDL than to LDL or VLDL. This fact suggests involvement of some kind of structural specificity of HDL.<BR>To investigate further the specificity of this enzyme-substrate complex formation, lipid composition of HDL was modified by LCAT reaction and these HDL were subjected to a competitive binding experiment. However, the binding of LCAT to HDL was not affected by any change of lipid composition of HDL (TABLE. III).<BR>Finally, apoprotein of HDL was coupled to agarose, and the experiment was performed to study binding of LCAT to this complex. The results shown in TABLE IV clearly demonstrate the binding of LCAT to HDL apoprotein.
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Studies on Plasma Lecithin: Cholesterol Acyltransferase Reaction

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