Separation of free and apolipoprotein D-associated human plasma lecithin: cholesterol acyltransferase.
A high performance gel filtration method for the rapid and reproducible separation of free and apolipoprotein D-associated lecithin: cholesterol acyltransferase (LCAT) originating from human plasma has been developed. Starting from step 3 of a previously invented covalent chromatography procedure, free LCAT was obtained as a well separated fraction in a yield of 55% of that injected into the column. The free LCAT had a specific activity of over 34,000 units/mg and did not contain apolipoprotein D or any other contaminant in the injected sample. Further 28% of LCAT with fully retained activity was recovered in a second fraction, demonstrating a 66,000 u LCAT associated with all apolipoprotein D occurring as a mean 33,000 u and a minor 66,000 u species and with at least two unidentified proteins with apparent molecular masses of 76,000 u and 43,000 u, respectively. Both free and apolipoprotein D-associated LCAT accepted the free cholesterol of heat-inactivated plasma selectively depleted of VLDL and LDL (alpha-LCAT activity) and of HDL (beta-LCAT activity) as substrate.
Holmquist
L
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King Gustaf V Research Institute, Karolinska Insitute, Stockholm, Sweden.
D001053:Apolipoproteins / Q000097:blood*; D053399:Apolipoproteins D; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007700:Kinetics; D008722:Methods; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000097:blood* / Q000302:isolation & purification; D011485:Protein Binding; D012016:Reference Values
D001053:Apolipoproteins; D053399:Apolipoproteins D; D007862:Phosphatidylcholine-Sterol O-Acyltransferase