Abstract
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.
Highlights
We have investigatedthe self-association properties of this unassociated pool may subsequently reassociate with of human apolipoprotein A-IV using severaI comple- high density lipoproteins [14,15,16], verylittle apo-A-IV is found mentary physical techniques
In experiments ported that human apo-A-IV contains ahigh percentage o f awhere apolipoproteinA-IV was incubated withuman helical structure and contains at least one discrete, highly high density lipoproteins, fractionatedby gradient gel hydrophobic domain [20].It is highly probable that, electrophoresis, and localized by immunoblotting, di- like other apolipoproteins, apo-A-IV will undergo self-associmer formation occurred, but very little binding to lipoproteinswas observed
Studies from several laboratories have consistently demonstrated that theformation of oligomeric species is a char; acteristic feature of the human apolipoproteins [18, 19]
Summary
The plasma apolipoproteins are distinguished foremost by their ability to combine with lipid, and by their ability to undergo self-association in aqueous solution [18, 19] These protein concentrations, and that the extenotf dimeri- biophysical properties are determined largely by their ability zation is minimally influenced by pH. Immunoblots of human serum fractionated on acrylamide gradient gels and isopycnic density gradients demonstratedan apolipoprotein A-IV band of size and density consistent with a circulating dimeric formun,associated with lipid. The nitrocellulose membranes were incubated in ablocking solution of 1%bovine serum albumin (Sigma) in a 20 mM Tris, 500 mM sodium chloride, pH 7.5 (TBS) overnight, the corresponding gel was stained with Coomassie Blue to confirm protein elution. Analytical Procedures-The protein concentration of apolipoprok i n solutions was determined by the method of Lowry et al [28] using crystalline bovine serum albumin (Sigma) as a standard.Transmission densitometry of nitrocellulose membranes was done using a Zeineh SL-504-XL soft laser densitometer (Biomed Instruments)
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