The self-association of human apolipoprotein A-IV. Evidence for an in vivo circulating dimeric form.
We have investigated the self-association properties of human apolipoprotein A-IV using several complementary physical techniques. Sedimentation equilibrium analysis demonstrated that human apolipoprotein A-IV formed oligomeric species in aqueous solution at physiologic pH. Computer analysis established that the best model of self-association is a monomer-dimer-tetramer scheme, with an unusually large monomer-dimer association constant of 2.9 X 10(5) liters/mol. Fluorescence spectroscopy and electrophoretic analysis demonstrated that the rate of monomer-oligomer interconversion is sufficiently slow that a stable population of dimeric protein exists in solution, even at low total protein concentrations, and that the extent of dimerization is minimally influenced by pH. Moreover, these techniques established that the dissociation of oligomeric forms and the unfolding of the monomeric form are discrete and sequential events. In experiments where apolipoprotein A-IV was incubated with human high density lipoproteins, fractionated by gradient gel electrophoresis, and localized by immunoblotting, dimer formation occurred, but very little binding to lipoproteins was observed. Immunoblots of human serum fractionated on acrylamide gradient gels and isopycnic density gradients demonstrated an apolipoprotein A-IV band of size and density consistent with a circulating dimeric form, unassociated with lipid. We conclude that human apolipoprotein A-IV undergoes high affinity self-association in aqueous solutions, and that such self-association likely occurs in vivo. Self-association may thus be important in determining the biologic behavior of human apolipoprotein A-IV by influencing both the kinetics and distribution of its association with plasma lipoproteins.
D001054:Apolipoproteins A / Q000097:blood*; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007158:Immunologic Techniques; D008075:Lipoproteins, HDL / Q000097:blood; D046911:Macromolecular Substances; D008970:Molecular Weight; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation; D014508:Urea
D001054:Apolipoproteins A; D008075:Lipoproteins, HDL; D046911:Macromolecular Substances; D012996:Solutions; C022205:apolipoprotein A-IV; D014508:Urea