A proteolytic method for distinguishing between lipid-free and lipid-boundapolipoprotein A-I

Recent studies indicate that certain lipid-poor forms of apolipoprotein (ap o)A-I may be particularly important in promoting cholesterol release from o verburdened cells in the periphery. However, a detailed understanding of th e physiological relevance of these species has been hampered by the difficu lty in measuring them. As part of a search for a rapid assay for these form s of apoA-I, we have observed that the protease enteropeptidase can specifi cally cleave human lipid-free apoA-I but not its lipid-bound form. Enterope ptidase cleaved lipid-free apoA-I at a single site at amino acid 188, resul ting in an N-terminal fragment of 22 kDa, However, apoA-I was not susceptib le to enteropeptidase when present in reconstituted high-density lipoprotei n (rHDL) particles as small as 6 nm in diameter or in human HDL3 particles, even at extremely high enzyme-to-protein ratios and extended reaction time s. We capitalized on this observation to develop an assay for the measureme nt of lipid-poor apoA-I in in vitro systems, Densitometry was used to gener ate a standard curve from sodium dodecyl sulfate polyacrylamide gels to det ermine the amounts of the N-terminal proteolytic fragment in unknown sample s treated with enteropeptidase. This system could accurately quantify apoA- I that had been displaced from rHDL particles and human HDL3 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 meth od distinguishes forms of apoA-I bp a conformational parameter without prev ious separation of the species. This simple and inexpensive method will be useful for understanding the characteristics of plasma HDL that are favorab le for the dissociation of apoA-I. - Safi, W., J. N. Maiorano, and W. S. Da vidson. A proteolytic method for distinguishing between Lipid-free and Lipi d-bound apolipoprotein A-I.