Effect of Human Plasma Apolipoproteins on the Activity of Purified Lecithin:Cholesterol Acyltransferase

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Abstract
A homogeneous preparation of active lecithin:cholesterol acyltransferase (LCAT EC 23.1.43) was isolated from human plasma by density ultracentrifugation, high density lipoprotein affinity chromatography, DEAE-Sepharose. and hydroxylapatite chromatography. 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 35 moles of glycosamine/105 g protein and no galactosamine. The purified enzyme, stored at 40 µg/ml at 4 °C, had a half life of 18-30 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 nM):cholesterol (10 µM) vesicles prepared in 1.25% ethanol in the absence or presence of 0.5% albumin. Significant transacylation occurred only with apoA-I or apoC-I, with maximum activation at 0.18-0.44 µM A-I (n = 11), and 0.71 µM C-I (n = 9). Addition of albumin to the incubation mixture enhanced the activation of LCAT by apoA-Iby approximately 100%, whereas it slightly inhibited the activation by apoC-I. Maximum activation by apoC-I yielded only 20 + 4 (n = 4. vesicles with albumin), or 42 ± 5% (n = 7. vesicles without albumin), of the LCAT activity obtained with apoA-I. The apoproteins A-II, C-I, C-II and C-III and D, partially inhibited the activation of LCAT by A-I and All, C-II, C-III and D inhibited the activation of LCAT by C-I. A-I or CT in excess of that needed for maximum activation also inhibited the enzyme reaction. Though apoA-I is the most effective activator of the LC AT reaction when egg lecithin is the acyl donor, apoC-I can also stimulate the reaction. The other apoproteins. All, C-II, C-III and D, inhibit the reaction and thereby potentially play a regulatory role in the LCAT reaction.