MEMBRANE ASSOCIATION AND OLIGOMERIC ORGANIZATION OF THE ALPHA-SUBUNITAND BETA-SUBUNIT OF MOUSE MEPRIN-A

Meprins are oligomeric cell surface metalloproteinases of the ''astaci n family.'' They consist of two types of subunits (alpha and beta), wh ich are evolutionarily related and whose cDNA sequences predict a simi lar domain structure. The present work shows that reducing agents solu bilized meprin alpha subunits (approximately 90%), but not beta subuni ts, from mouse kidney brush border membranes. In addition, immunoblott ing of membranes or purified meprins with an antibody raised to the al pha subunit epidermal growth factor like domain, predicted to be near the COOH terminus from the cDNA-deduced amino acid sequence, indicated that this domain is not present in the mature alpha subunit. By contr ast, an epitope predicted to be near the COOH terminus of the beta sub unit was present in the mature form of beta. When meprins were solubil ized from brush border membranes by papain, the size of the alpha subu nit (approximately 90 kDa) did not change, while the beta subunit decr eased from 110 to 90 kDa with concomitant loss of the COOH-terminal ep itope. These data indicate that beta is a type I transmembrane protein , while alpha does not transverse the membrane and its association is dependent on disulfide bonds. The oligomeric organization of purified meprin A (EC 3.4.24.18), examined by sedimentation equilibrium analysi s and native gradient gel electrophoresis, is that of disulfide-bridge d dimers which aggregate noncovalently to form higher molecular weight complexes, predominantly tetramers. Western blotting of ICR kidney br ush border membrane proteins identified alpha(2) homodimers and alpha beta heterodimers. Treatment of mouse or rat kidney brush border membr anes with 7 M urea solubilized alpha(2), but not alpha beta dimers. Th us, the mature alpha subunit exists in alpha(2) and alpha beta disulfi de linked dimers which form tetramers, and the alpha(2) homodimers ass ociate with the membrane through noncovalent interactions with alpha b eta.