A method for the purification of milligram quantities of stable human phosphatidylcholine-cholesterol acyltransferase.
A method for processing 3 litres of human plasma for the purification of phosphatidylcholine-cholesterol acyltransferase is described. The method involves (NH4)2SO4 fractionation, citric acid treatment, and DEAE-cellulose and hydroxyapatite chromatography. At this stage the enzyme preparation is purified approx. 8000-fold. This preparation appears to be free of lipoproteins as determined by immunoelectrophoresis against anti-human serum and is minimally contaminated with albumin (less than 30 mug/mg of enzyme protein) as determined by immunodiffusion. The activity of the enzyme was stable for 4 days, but most of its activity was lost after 20 days On electrophoresis on 5% polyacrylamide gel, a fast-moving band with enzyme activity and a slow-moving band with no enzyme activity was observed. A faint band of albumin was also present. Extracts of enzymically active bands cut from ten gels and then pooled and extracted with 0.15 M-NaC1/4mM-sodium phosphate, pH 7.0, showed a single band on re-electrophoresis on 5% polyacrylamide gel.
D000217:Acyltransferases / Q000302:isolation & purification*; D000645:Ammonium Sulfate; D002848:Chromatography, DEAE-Cellulose; D002951:Citrates; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006882:Hydroxyapatites; D008722:Methods; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000302:isolation & purification*
D002951:Citrates; D006882:Hydroxyapatites; D000217:Acyltransferases; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D000645:Ammonium Sulfate