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.

Characterization of antibody to human phosphatidylcholine: cholesterol acyltransferase.

Purified preparations of phosphatidylcholine (lecithin): cholesterol acyltransferase (EC 2.3.1.43), were injected into goats to produce antisera reacting with this enzyme. The antisera and the gamma-globulin derived thereform were examined by the technics of immunodiffusion, immunoelectrophoresis and immunoinhibition of the enzyme. The antisera gave no precipitation lines with human high density lipoproteins (HDL) and human low density lipoproteins (LDL). A weak antibody titer towards human serum albumin was noted only after prolonged immunization. The enzymatically active band isolated from acrylamide gels gave a single arc in immunodiffusion and immunoelectrophoresis. The gamma-globulin derived from the antisera inhibited human phosphatidylcholine:cholesterol acyltransferase activity.

Relationship between high density lipoproteins and the rate of in vitro serum cholesterol esterification.

The rate of in vitro esterification of serum cholesterol and the concentrations of serum high density lipoprotein cholesterol (HDL-C) were measured in 18 women and 9 men, 74--95 years of age. Subjects in this age group can have HDL-C levels below accepted limits of normal. The subjects were divided into two groups, one with HDL-C less than 40 mg/dl and the other with HDL-C 40 mg/dl or higher. The rate of in vitro serum cholesterol esterification in the former group was 2.00 n. moles/ml/min, significantly higher (p less than 0.02) than 1.62 n. moles/ml/min present in the latter group. There was no correlation between the cholesterol esterification and a protein (s), which can produce an immune gamma globulin that completely inhibits in vitro serum cholesterol esterification. These findings are discussed with reference to the putative relationships between HDL-C and atherosclerosis.

Purification and characterization of lecithin:cholesterol acyltransferase.

The purification of lecithin:cholesterol acyltransferase (LCAT] from human plasma is reported. Hydroxylapatite fractions were approximately 16,000 fold purified over the starting plasma and were free of high-density lipoprotein (HDL) and albumin. The enzyme showed one band on polyacrylamide gel electrophoresis, SDS-urea polyacrylamide gel electrophoresis, isoelectric focusing, and one arc in immunodiffusion against a goat antiserum preparation. It was determined to be a glycoprotein with a molecular weight of approximately 70,000 and a pI of 3.7--4.0.

Immunological evaluation of LCAT deficiency.

Antibody towards a highly purified LCAT preparation was tested against LCAT-deficient sera by using the technic of immunodiffusion and immunoinhibition. Reaction of identity among normal serum, deficient sera, and purified LCAT was observed in immunodiffusion with two different antibodies obtained from two different goats. When the antibodies were mixed with deficient sera and tested for immunoinhibition of LCAT activity, suggesting that deficient sera contained an enzymatically inactive LCAT. The other antibody preparation showed inhibition of enzyme activity even in antigen excess. It appears that the precipitin lines observed in immunodiffusion do not represent LCAT in serum. In view of the higher titre of antibody in immunoinhibition experiments with this antibody, it remains to be determined whether at lower ratios of antibody to deficient serum, immunoinhibition by the antibody will be abolished in this case too.