A rapid large-scale procedure for purification of lecithin-cholesterol acyltransferase from human and animal plasma.
A rapid and convenient procedure was developed for large-scale purification of lecithin-cholesterol acyltransferase from the plasma of humans, pigs, dogs, goats and rabbits. The procedure included dextran sulfate-Mg2+ precipitation, Phenyl-Sepharose column, Affi-gel blue column, DEAE-Sepharose column, DEAE-Affi-gel blue column and hydroxyapatite column. Lecithin-cholesterol acyltransferase was purified approx. 20 000-fold with about 14% yield from human plasma. A similar result of purification was obtained from pigs, dogs, goats and rabbits. The final enzyme preparation from all five mammalian species was homogeneous as judged by SDS-polyacrylamide gel electrophoresis and high-performance gel filtration in the presence of SDS. The apparent molecular weight of the purified enzymes from humans, pigs, dogs, goats and rabbits were 65 000, 66 000, 65 000, 65 000 and 66 000, respectively, as determined by SDS-polyacrylamide gel electrophoresis and were 67 000, 67 000, 66 000, 66 000 and 67 000, respectively, by high-performance gel filtration. Studies with DEAE-Sepharose columns demonstrated that lecithin-cholesterol acyltransferase from these mammalian species was similar in molecular charge. The pH optimum for activity of purified enzyme ranged from 7.4 to 8.0 among these species. All purified lecithin-cholesterol acyltransferase species were activated by human apolipoprotein A-I and were similarly inhibited by p-hydroxymercuribenzoate and phenylmethylsulfonyl fluoride. We concluded that this purification scheme is suitable for rapid isolation of lecithin-cholesterol acyltransferase from the plasma of humans, pigs, dogs, goats and rabbits and, by inference, from other mammalian species. The availability of purified active lecithin-cholesterol acyltransferase from various animal species should facilitate phylogenetic and comparative studies of the enzyme.
D000818:Animals; D001053:Apolipoproteins / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006041:Goats; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007201:Indicator Dilution Techniques; D008970:Molecular Weight; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000037:antagonists & inhibitors / Q000097:blood*; D011817:Rabbits; D013552:Swine
D001053:Apolipoproteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase