ISOLATION AND CHARACTERIZATION OF FIBRONECTIN-ALPHA(1)-MICROGLOBULIN COMPLEX IN RAT PLASMA

Molecules containing the 28 kDa immunoregulatory protein alpha(1)-micr oglobulin (alpha(1)-m), also known as protein HC, were isolated from r at plasma or serum by immunoaffinity chromatography. Three molecular s pecies were distinguished on the basis of nondenaturing PAGE. Two of t hese have been described previously: uncomplexed alpha(1)-m, and the c omplex of alpha(1)-m with alpha(3)-inhibitor-3. The third species was analysed by denaturing PAGE, immuno-blotting, proteinase digestion and N-terminal-sequence analyses, and shown to consist of a complex betwe en alpha(1)-m and fibronectin. This complex, with a mass of about 560 kDa, was resistant to dissociation in the presence of denaturants, but not in the presence of reducing agents in combination with denaturant s, and we conclude that the two components are linked by disulphide bo nds. About 60% of the total detectable plasma alpha(1)-m exists as hig h-molecular-mass complexes distributed approximately evenly between fi bronectin and alpha(1)-inhibitor-3. Immunochemical analyses were used to determine the proportion of the total plasma pools of fibronectin a nd alpha(1)-inhibitor-3 that circulate in complex with alpha(1)-m. Abo ut 3-7% of the total plasma fibronectin from three different rat strai ns contained alpha(1)-m, whereas 0.3-0.8% of the total plasma alpha-in hibitor-3 contained alpha(1)-m. Complexes were found at similar levels in plasma and serum, indicating that coagulation is not responsible f or complex formation. Moreover, immunochemical analyses of human plasm a revealed small amounts of alpha(1)-m in complex with fibronectin and alpha(2)-macroglobulin (an alpha(1)-inhibitor-3 homologue). The exist ence of a complex between alpha(1)-m and fibronectin in rats and human s suggests a mechanism for the incorporation of the immunoregulatory m olecule alpha(1)-m into the extracellular matrix.