A method for isolating human plasma lecithin:cholesterol acyltransferase without using anti-apolipoprotein D, and its
Biochimica Et Biophysica Acta
|June 16, 1983
Summary
This study details a new method to isolate human plasma lecithin:cholesterol acyltransferase (LCAT), achieving over 50,000-fold purification. The highly purified LCAT enzyme demonstrates essential activity and is free of apolipoprotein D.
Area of Science:
- Biochemistry
- Enzymology
- Protein Purification
Background:
- Human plasma lecithin:cholesterol acyltransferase (LCAT) is crucial for high-density lipoprotein (HDL) maturation.
- Previous methods for LCAT isolation yielded preparations with lower purity and potential contaminants.
Purpose of the Study:
- To develop and describe a robust method for the high-fold purification of active human plasma LCAT.
- To characterize the purified LCAT enzyme and confirm its purity and enzymatic properties.
Main Methods:
- Sequential purification involving ammonium sulfate precipitation, DEAE-cellulose chromatography, and hydroxyapatite chromatography.
- Analysis of enzyme purity using immunodiffusion, SDS-PAGE, and analytical isoelectric focusing.
- Characterization of enzymatic activities and cofactor requirements.
Main Results:
- Achieved over 50,000-fold purification of LCAT from human plasma.
- The purified enzyme exhibited single bands on SDS-PAGE and separated into three isoforms upon isoelectric focusing.
- LCAT demonstrated both transferase and phospholipase A2 activities, both requiring apolipoprotein A-I.
- The purified enzyme was free of apolipoprotein D, confirmed by immunodiffusion.
Conclusions:
- A highly effective purification protocol for human plasma LCAT has been established.
- The purified LCAT is enzymatically active, requires apolipoprotein A-I, and is demonstrably free of apolipoprotein D.
- The developed method provides a valuable tool for further research into LCAT function and its role in lipid metabolism.
