Purification of human plasma lecithin:cholesterol acyltransferase by covalent chromatography.
Human plasma lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) has been purified more than 20,000 fold from plasma in 10% yield. This new procedure is composed of only four steps, including ultracentrifugation of plasma to yield a 1.21-1.25 kg/l density fraction, covalent binding of LCAT in this fraction to thiopropyl-Sepharose followed by adsorption of the enzyme to wheat-germ lectin-Sepharose for elimination of albumin and finally batch-wise treatment of the desorbed LCAT with hydroxyapatite to remove residual impurities. The purified enzyme was free of apolipoprotein A-I, A-II, B, C-I, C-II, C-III and E as checked by double immunodiffusion and SDS-electrophoresis, which latter method also demonstrated the absence of hitherto characterized lipid transfer proteins. Only traces of apolipoprotein D were present in the preparation as detected by immunoblotting. The purified enzyme retained alpha- and beta-LCAT activities. Non-denaturing and denaturing polyacrylamide gel electrophoresis yielded apparent molecular masses of 69 and 66 kDa, respectively, for the enzyme which on isoelectric focusing produced one major and one minor isoform with pI values of 4.20 and 4.25, respectively. Apolipoprotein A-I was required to transform artificial lecithin-cholesterol liposomes into substrates for the purified LCAT.
Holmquist
L
L
King Gustaf V Research Institute, Karolinska Institute, Stockholm, Sweden.
D002845:Chromatography / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D017886:Durapatite; D006801:Humans; D006882:Hydroxyapatites; D007700:Kinetics; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000097:blood* / Q000302:isolation & purification
D006882:Hydroxyapatites; D017886:Durapatite; D007862:Phosphatidylcholine-Sterol O-Acyltransferase