Interactions of bacterial lipopolysaccharide with acute-phase rabbit serum and isolation of two forms of rabbit serum amyloid A.

Abstract

Mixing lipopolysaccharide (LPS, Salmonella minnesota R595) with acute-phase rabbit serum (APRS) results in the formation of two types of complexes. The two complexes are separable from each other and from LPS by CsCl density gradient ultracentrifugation. LPS alone had density 1.38 g/cm3, complex 1.3 had density 1.3 +/- 0.02 g/cm3, and complex 1.2 had density less than 1.20 g/cm3. At 1 to 10 micrograms LPS/ml APRS, up to 23 micrograms of LPS could be found in complex 1.3, with the remainder of the LPS in complex 1.2. The capacity of complex 1.2 for LPS was 500 to 600 micrograms LPS/ml APRS. The ability of rabbit serum to form complex 1.3 rose to a maximum in 24 hr post-acute-phase induction. The two complexes were purified by equilibrium density gradient ultracentrifugation and immunoaffinity chromatography using antibodies to LPS and subjected to SDS-PAGE. Complex 1.3 had major peptides after reduction of 91, 64, 61 and 50 kilodaltons. The 61-kilodalton peptide is probably rabbit serum albumin; the others are unidentified. Complex 1.2 had major peptides after reduction whose mobility corresponded to apolipoprotein A-I and serum amyloid A. Complex 1.2 thus seems to be analogous to the LPS-high-density-lipoprotein complexes that form in normal serum except that the latter complexes do not contain serum amyloid A. Rabbit serum amyloid A (SAA) was purified by CsCl equilibrium density gradient ultracentrifugation, gel filtration, and ion-exchange chromatography into two forms, SAA-1 and SAA-2. The two forms of SAA have very similar amino acid compositions and m.w. (SAA-1, 11,526 daltons; SAA-2, 11,469 daltons). They differ primarily in their isoelectric points, 6.57 and 6.27 for SAA-1 and SAA-2, respectively. The complexes 1.2 from several individual rabbits after acute-phase induction were studied by isoelectric focusing. Seven of 10 rabbits showed both forms of SAA; three showed a single form. Two rabbits showing SAA-1 on the first acute-phase induction were reinduced; one rabbit again gave SAA-1 and the other gave SAA-2. These results suggest that SAA and perhaps other acute-phase reactants may modulate the biologic activity of LPS.