A method for isolating human plasma lecithin:cholesterol acyltransferase without using anti-apolipoprotein D, and its characterization.
A method for isolating human plasma lecithin:cholesterol acyltransferase (EC 2.3.1.43) purified more than 50 000-fold is described. The crude enzyme obtained by initial ammonium sulfate and citric acid treatment of 21 of human plasma is subjected to repeated DEAE-cellulose chromatography to yield a preparation purified more than 600-fold. Hydroxyapatite chromatography of concentrates from this fraction using 0.5 mM phosphate buffer, pH 6.8, yields enzyme preparations purified more than 50 000-fold. The enzyme isolated by this procedure was free of apolipoprotein D, as shown by the absence of an arc in immunodiffusion with anti-apolipoprotein D. The enzyme showed a single band by polyacrylamide gel electrophoresis in the presence and absence of SDS. Upon analytical isoelectrofocusing the enzyme separated into three iso forms with isoelectric points below that of egg albumin (pI 4.6). The enzyme was characterized by a high content of glutamic acid, leucine and glycine, and a lower content of tyrosine. The enzyme possessed both transferase and phospholipase A2 activities and both activities show absolute requirement for apolipoprotein A-I. The purified enzyme was injected into Balb/c mice and the antiserum reacted both with the purified enzyme and normal human serum in immunodiffusion, giving lines of complete identity. The antiserum gave no precipitation lines with albumin or apolipoprotein D, providing additional evidence for the absence of apolipoprotein D in the purified enzyme. The gamma-globulin isolated from the antiserum inhibited human lecithin:cholesterol acyltransferase activity.
D000818:Animals; D000906:Antibodies / Q000032:analysis; D001053:Apolipoproteins / Q000276:immunology; D053399:Apolipoproteins D; D002848:Chromatography, DEAE-Cellulose; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000032:analysis; D007120:Immunochemistry; D005779:Immunodiffusion; D051379:Mice; D008807:Mice, Inbred BALB C; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000097:blood / Q000302:isolation & purification*
D000906:Antibodies; D001053:Apolipoproteins; D053399:Apolipoproteins D; D007106:Immune Sera; D007862:Phosphatidylcholine-Sterol O-Acyltransferase