Characterization of proteoliposomes containing apoprotein A-I: a new substrate for the measurement of lecithin: cholesterol acyltransferase activity.

TLDR

The study introduces a cholate dialysis method that generates large, stable, homogeneous proteoliposomes for sensitive LCATase activity measurement and mechanistic investigation. Proteoliposomes were produced by cholate dialysis from apoA‑I, lecithin, and cholesterol at various molar ratios. The optimized 0.8:250:12.5 proteoliposome was stable for weeks, matched in size to controls, served as a superior LCATase substrate, and produced a linear, plasma‑enzyme‑dependent activity of about 95 nmol hr⁻¹ ml⁻¹.

Abstract

Proteoliposome vesicles containing apoA-I, lecithin, and cholesterol (including labeled cholesterol) were prepared from various molar ratios of the three components by the cholate dialysis technique. Comparative studies on the sensitivity and efficiency of these proteoliposomes to serve as substrate for lecithin:cholesterol acyltransferase (LCATase) indicated that the proteoliposome with apoA-I:lecithin:cholesterol molar ratio of 0.8:250:12.5 was ideal for assaying LCATase activity of both plasma and purified enzyme. This proteoliposome was shown to be comparable in size by gel filtration (radius, 131.9 +/- 4.8 A, n = 6) and by electron microscopy (radius, 123.4 +/- 5.1 A, n = 100). The proteoliposome preparation was stable as LCATase substrate for at least 3 and 5 weeks, respectively, when stored at 4 degrees C and -20 degrees C, and was a better substrate for the enzyme activity assay than were lecithin-cholesterol liposomes incubated with apoA-I. Under the standardized assay system LCATase activity was a linear function of plasma enzyme added and was independent of the amount of plasma cholesterol added to the proteoliposomes in the range of 3 to 20 microliters of plasma. The mean LCATase activity by this method was 95.1 +/- 14.0 (range 76.5-122.5) nmol/hr per ml of plasma from fifteen normal human subjects. This method of substrate formation using the cholate dialysis technique permits the preparation of large amounts of stable, efficient, homogeneous, and well-defined substrate that is suitable for measuring low levels of enzyme activity, comparative studies, and large scale investigations of plasma LCATase, as well as studies of the mechanism and regulation of LCATase reaction.