EFFECT OF HUMAN-PLASMA APOLIPOPROTEINS ON THE ACTIVITY OF PURIFIED LECITHIN - CHOLESTEROL ACYLTRANSFERASE

Abstract

An active preparation of lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) was isolated from human plasma by density ultracentrifugation, high-density lipoprotein affinity chromatography, DEAE-Sepharose and hydroxylapatite chromatography. This enzyme preparation gave a single band on polyacrylamide gel electrophoresis in 8 M urea and on sodium dodecyl sulfate gel electrophoresis. On analytical isoelectric focusing, the enzyme separated into at least 5 isoforms with isoelectric points of 5.1-5.5. The enzyme with an apparent MW of 66,000 .+-. 2000 was characterized by a high content of glutamic acid, aspartic acid, leucine and glycine and contained approximately 31 mol of glucosamine/103 mol of protein and no galactosamine. The purified enzyme, stored at 20-40 .mu.g/ml at 4.degree. C, had a half-life of 26 .+-. 4 days. The effect of purified human plasma apolipoproteins A-I, A-II, C-I, C-II, C-III and D on the activity of purified LCAT was studied, using egg yolk lecithin (40 .mu.M):cholesterol (10 .mu.M) vesicles prepared in 1.25% ethanol in the absence or presence of 0.5% albumin. Addition of albumin to the incubation mixture nearly doubled the esterification rate of LCAT with A-I was activator (n = 4), but it inhibited esterification by approximately 35% (n = 3) if C-I was the activator. Maximum activation by C-I yielded only 13 .+-. 6% (vesicles with albumin) or 42 .+-. 5% (vesicles without albumin) of the LCAT activity obtained with A-I. Each of the apoproteins A-II, C-II, C-III and D inhibited the LCAT reaction in the presence of A-I or C-I at concentrations needed for maximal activation. Contrary to previous work, apolipoprotein D apparently is not an activator of LCAT. LCAT activity is significantly affected by albumin and the apolipoproteins A-II, C-II, C-III and D.