Abstract
Quantitative aspects of the binding of free fatty acid to human erythrocytes were studied by measuring the distribution of various amounts of [1-(14)C]lauric acid between washed human erythrocytes and defatted human plasma albumin. Incubations were done at 37 degrees C in an isotonic phosphate-buffered salt solution. Laurate uptake approached a steady state value within 1 hr of incubation over the range of laurate-albumin molar ratios that were tested. Uptake was due primarily to a transfer of laurate from albumin to the cell, not to incorporation of the intact laurate-albumin complex. The fatty acid binding sites of the erythrocyte are located predominantly on or within the cell membrane. The binding model which best fitted the laurate uptake data consisted of two classes of erythrocyte binding sites. This model contains a small number of sites, 2.0 x 10(-13) moles/10(6) cells, that have an average apparent association constant of 1.8 x 10(6) m(-1) for laurate. Thus, the average strength of these sites is of the same order of magnitude as the stronger laurate binding sites of albumin. The binding model also contains a relatively large number of weaker fatty acid binding sites, 1.3 x 10(-11) moles/10(6) cells, that have an average apparent association constant of 1.3 x 10(4) m(-1) for laurate. These sites are too weak to bind appreciable amounts of laurate unless the fatty acid-albumin molar ratio is elevated.
Highlights
Quantitative aspects of the binding of free fatty acid to human erythrocytes were studied by measuring the distribution of various amounts of [l-'C]lauric acid between washed human erythrocytes and defatted human plasma albumin
THE FIRSTSTEP in the utilizationof free fatty acid bya mammalian cell is binding of the fattyacid in unesterified form to the cell membrane [1,2,3]
Goodman [2] analyzed the binding reaction quantitativelyu,sing washed human erythrocytes and palmitic acid. His analysis detected the presence of only a single class of erythrocyte bindingsites, and these sites were located on the cell membrane
Summary
Lauric acid was obtained from The Hormel Institute, Austin, Minn.[1J4C]Lauric acid was purchasedfrom. The experimental data for the binding of laurate to human erythrocytes consisted of measurements of the molar ratio of fatty acid to albumin after the steady state was achieved, 8, and of the amount of fatty acid associated with the cells, 9. For each value of 8, the corresponding value for the unbound fatty acid concentration, c, was calculated by inverting Equation 3 This was done by substituting into Equation 3 the equilibricuomnstants, K t , calculated from the laurate-albumin binding data, and using a Newton-Raphson iterative technique to determine c for each value of 8. This determinationis unique because Equation 3 is a strictly increasing function of c, and thenumerical results can be calculated to an accuracy within the experimental error of the data. The fitting was done on an IBM System/370 computer using a least-squares model fitting procedure in conjunction withan IBM 2250 graphic display unit
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