Effect of human plasma apolipoproteins on the activity of purified lecithin: cholesterol acyltransferase.
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. Upon analytical isoelectric focusing the enzyme separated into at least five isoforms with isoelectric points ranging from 5.1 to 5.5. The enzyme with an apparent molecular weight of 66,000 +/- 2,000 was characterized by a high content of glutamic acid, aspartic acid, leucine and glycine and contained approximately 31 moles of glucosamine/10(3) moles of protein and no galactosamine. The purified enzyme, stored at 20-40 microgram/ml at 4 degrees 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 microM): cholesterol (10 microM) 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 as activator (n=4), whereas 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 does not appear to be an activator of LCAT. LCAT activity is significantly affected by albumin and the apolipoproteins A-II, C-II, C-III, and D.
D000596:Amino Acids / Q000032:analysis; D001053:Apolipoproteins / Q000097:blood / Q000494:pharmacology*; D002784:Cholesterol / Q000378:metabolism; D004530:Egg Yolk; D004789:Enzyme Activation; D005260:Female; D006595:Hexosamines / Q000032:analysis; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism
D000596:Amino Acids; D001053:Apolipoproteins; D006595:Hexosamines; D010713:Phosphatidylcholines; D002784:Cholesterol; D007862:Phosphatidylcholine-Sterol O-Acyltransferase