Abstract
Lecithin: cholesterol acyltransferase (LCAT) is produced in liver and secreted into circulation. It catalyzes esterification of plasma cholesterol and formation of lysolecithin from lecithin, and is the major source of human plasma esterified cholesterol.
In the present study, LCAT was purified 1478 folds from human plasma by means of affinity chromatography, and was subjected to the study of enzyme-substrate complex formation. Purified LCAT was shown to bind delipidized protein of high density lipoproteins (apo HDL) which was coupled to Sepharose beads. However, LCAT had a higher affinity to apo HDL-Sepharose complex which had been relipidized by plasma lecithin.
When human plasma proteins were fractionated by Sephadex G 200 column, three main peaks were eluted. The LCAT present in D> 1.21 fraction appeared between the second and the third peaks. However, when D> 1.21 fraction was first incubated with VLDL, LDL and inactivated HDL and the incubation mixture was put to Sephadex G 200 gel filtration, the main peak of LCAT activity shifted to the area between the first and the second peaks, and it corresponded to HDL zone. On the other hand, almost no enzyme activity was found at the fractions where VLDL and LDL were eluted. The results are compatible with our earlier findings which showed substrate specificity among the three major lipoproteins.
LDL was coupled to cyanogen-activated Sepharose, and these fixed LDL were incubated with LCAT and HDL for 18 hr. at 37°C When LCAT reaction proceeded, the total content of cholesterol in LDL-Sepharose decreased significantly. LDL-Sepha rose is an artificial model of fixed lipoproteins, however the experimental design will be extended to the study of LDL bound to arterial wall.
