Ultracentrifugal behavior of apolipoprotein A-I of rhesus monkey (Macaca mulatta)

Abstract

The sedimentation equilibrium and sedimentation velocity of apolipoprotein AI (apo-A-I) from high density lipoproteins of rhesus monkey plasma were studied in aqueous solutions of 0.02 M EDTA, pH 8.6, as a function of protein concentration. The sedimentation equilibrium results showed that, in the concentration range between 9 and 36 muM (0.25 to 1.0 g/liter), apo-A-I behaved as a single species of molecular weight 28,200. This molecular weight corresponds to that of the monomeric form as previously estimated from electrophoretic and chemical data. As the apo-A-I concentration was increased above 36 muM, the log c versus r2 plots from sedimentation equilibrium experiments became curvilinear, suggesting self-association. An analysis of these data generated nonlinear, nonoverlapping molecular weight versus concentration plots pointing at the existence of a heterogeneous population of apo-A-I. By sedimentation velocity studies apo-A-I at concentrations between 68 and 330 muM exhibited two distinct components, with S0/20,w = 1.9 and S0/20,w = 3.9. When separated by gel permeation chromatography, these two components had an identical amino acid composition and retained similar S20,w values as before column fractionation. The slow component had a diffusion coefficient of 8.27 X 10(-7) cm2/s and, assessed by the criteria of sedimentation equilibrium ultracentrifugation, remained monomeric even at concentrations of 125 muM. The fast component, when analyzed by low speed sedimentation equilibrium, behaved as a self-associating system which could best be fitted into a monomer-hexamer model in a rapid equilibrium. The equilibrium constant for this association was found to be 9.5 X 10(23) M(-5). Thus, rhesus apo-A-I, dissolved in 0.02 M EDTA, pH 8.6, consisted of two distinct species, one monomeric over a relatively wide range of concentrations, the other readily self-associating. Their structural relationship remains to be established. This ultracentrifugal behavior of rhesus apo-A-I differs markedly from that reported for human apo-AI (Vitello, L. B., and Scanu, A. M. (1976) J. Biol. Chem. 251, 1131-1136).