Purification and characterization of hamster serum amyloid A protein (SAA) by cholesteryl hemisuccinate affinity chromatography.
High-density lipoprotein (HDL) apolipoprotein was separated from hamster serum by cholesteryl hemisuccinate affinity chromatography (CHAC) in comparison with the density-gradient ultracentrifugation (DGUC). The apolipoprotein recovery from serum by CHAC was 70% and by DGUC 80%. This disadvantage is compensated for by the ease of purification by CHAC, a method particularly suited for the processing of large amounts of serum. From the acute-phase HDL CHAC fraction, apo SAA was isolated by gel filtration. Using isoelectrofocusing, two-dimensional gel electrophoresis, and titration curve, four isotypes of hamster apo SAA were identified and characterized. In the acute-phase serum, one of the isotypes was predominant (apo SAA1). In serum of amyloidotic animals, the relative contribution of apo SAA1 was considerably lower, suggesting selective removal of the latter during amyloidogenesis and possibly its deposition in hamster AA amyloid. Furthermore, the affinity chromatography method was modified with gradient elution of affinity-bound material. By this method HDL apolipoprotein was separated into three subclasses. Apo SAA was shown to associate with two different subclasses. In acute-phase serum most of the apo SAA1 was found in the subclass with the lowest affinity for the cholesteryl beads, whereas the latter was depressed in amyloidotic serum, suggesting that the amyloidogenicity of a particular apo SAA isotype is determined by its cholesteryl-binding properties.
Niewold
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TA
Department of Veterinary Pathology, University of Utrecht, The Netherlands.
Tooten
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PC
D000818:Animals; D001053:Apolipoproteins / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient; D002788:Cholesterol Esters; D002846:Chromatography, Affinity; D006224:Cricetinae; D007525:Isoelectric Focusing; D008075:Lipoproteins, HDL / Q000032:analysis; D008297:Male; D008647:Mesocricetus; D008970:Molecular Weight; D000685:Serum Amyloid A Protein / Q000302:isolation & purification*
D001053:Apolipoproteins; D002788:Cholesterol Esters; D008075:Lipoproteins, HDL; D000685:Serum Amyloid A Protein; C013440:cholesteryl succinate