Purification of biologically active apolipoproteins by chromatofocussing.
Chromatofocussing has been used to isolate homogeneous apolipoproteins (apo) from human very-low-density lipoproteins and high-density lipoproteins with protein recovery of 70%. The inclusion of sulfhydryl-reducing agent (dithiothreitol) was required during solubilization of the lipoproteins (following delipidation) to achieve reproducible elution profiles. Removal of polyvalent buffers from apoproteins was rapidly accomplished on small columns of hydroxylapatite. The biological activity of purified apo AI and apo CII was confirmed by assessment of their ability to activate lecithin:cholesterol acyltransferase or lipoprotein lipase, respectively. Functional properties of isolated apo E were assessed by in vitro interaction with the low-density lipoprotein receptor expressed by cultured fibroblasts. Apolipoproteins purified by this rapid procedure exhibit identical physical, chemical and biological properties to those purified by other, more tedious techniques.
D000818:Animals; D001053:Apolipoproteins / Q000302:isolation & purification* / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D002021:Buffers; D002417:Cattle; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007525:Isoelectric Focusing; D008049:Lipase / Q000378:metabolism; D008075:Lipoproteins, HDL / Q000302:isolation & purification; D008079:Lipoproteins, VLDL / Q000302:isolation & purification; D008892:Milk / Q000201:enzymology; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism
D001053:Apolipoproteins; D002021:Buffers; D008075:Lipoproteins, HDL; D008079:Lipoproteins, VLDL; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D008049:Lipase