A proteolytic method for distinguishing between lipid-free and lipid-bound apolipoprotein A-I.
Recent studies indicate that certain lipid-poor forms of apolipoprotein (apo)A-I may be particularly important in promoting cholesterol release from overburdened cells in the periphery. However, a detailed understanding of the physiological relevance of these species has been hampered by the difficulty in measuring them. As part of a search for a rapid assay for these forms of apoA-I, we have observed that the protease enteropeptidase can specifically cleave human lipid-free apoA-I but not its lipid-bound form. Enteropeptidase cleaved lipid-free apoA-I at a single site at amino acid 188, resulting in an N-terminal fragment of 22 kDa. However, apoA-I was not susceptible to enteropeptidase when present in reconstituted high-density lipoprotein (rHDL) particles as small as 6 nm in diameter or in human HDL(3) particles, even at extremely high enzyme-to-protein ratios and extended reaction times. We capitalized on this observation to develop an assay for the measurement of lipid-poor apoA-I in in vitro systems. Densitometry was used to generate a standard curve from sodium dodecyl sulfate polyacrylamide gels to determine the amounts of the N-terminal proteolytic fragment in unknown samples treated with enteropeptidase. This system could accurately quantify apoA-I that had been displaced from rHDL particles and human HDL(3) with purified apoA-II. On the basis of the results, a system of nomenclature is proposed for "lipid-free," "lipid-poor," and "lipid bound" apoA-I. The reported method distinguishes forms of apoA-I by a conformational parameter without previous separation of the species. This simple and inexpensive method will be useful for understanding the characteristics of plasma HDL that are favorable for the dissociation of apoA-I.
Safi
W
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Department of Pathology and Laboratory Medicine, University of Cincinnati, 231 Bethesda Avenue, Cincinnati, OH 45267-0529, USA.
Maiorano
J N
JN
Davidson
W S
WS
D016632:Apolipoprotein A-I / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000378:metabolism*; D006801:Humans; D050356:Lipid Metabolism; D011485:Protein Binding; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D016632:Apolipoprotein A-I; D004765:Enteropeptidase