A Simple Purification of Apoliproteins A-I and B and Their Application to Cholestery Ester Transfer Assay

  • Cho, Kyung-Hyun (Dept. of Genetic Engineering, Kyungpook national University, Taegu 702-701) ;
  • Park, Myung-Sook (Dept. of Food Science and Nutrition) ;
  • Bok, Song-Hae (Bioproducts Research Group, Genetic Engineering Research Institute, KIST, Taejon 305-606) ;
  • Park, Young-Bok (Dept. of Genetic Engineering, Kyungpook national University, Taegu 702-701)
  • Published : 1996.06.01

Abstract

This study describes a stable and simple method for the measurement of cholesteryl ester transfer protein(CETP) activities using reconstituted HDL and LDL as substrates. Apolipoproteins (apo) A-I and -B were purified from hog plasma by a new strategy without ultracentrifugation and delipidation. a simple two-step column chromatography was administered. In the first step of phenyl-sepharose CL-4B column chro-matography, hydrophobic plasma proteins were isolated. The most hydrophobic proteins bound to the column appeared to be A-I and apo-B. Contaminat proteins were efficiently eliminated from the sample by washing the column with 0.3M NaCI containing buffer after loading the plasma on the column. Two pure proteins showing each single band on SDS-PSGE of apo A-I and apo-B were individually obtained by a subsequent gel filtration column chromatography(Sephadex G-200). This two-step purification was simple and inexpensive compared to the ultracentrifugation and/or delipidation method that are most commonly used. Reconstituted hight-density lipoproteins(HDL) and low-density lipoproteins(LDL) were prepared using the purified apo A-I and-B, respectively. When these artificially prepared HDL and LDL were used in the assays for CETP as the cholesteryl ester(CE) donor and acceptor respectively, the specific transfer of CE increased up to two fold compared to that used the native HSL and LDL.

Keywords

LDL;HDL;CETP;CE

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