and TGF-␤2 in fetal bovine heart endothelial (FBHE) cell proliferation assays; FP4 was inactive in this assay. FP3 also increased NO synthesis by RAW 264.7 cells, mimicking an ␣ 2 M activity that has been attributed to the neutralization of endogenously synthesized TGF-␤. Thus, we have isolated a peptide corresponding to 13% of the ␣ 2 M sequence that binds TGF-␤ and neutralizes the activity of TGF-␤ in two separate biological assays.1 is a 718-kDa glycoprotein that was originally characterized as a broad spectrum proteinase inhibitor (1). The structure of ␣ 2 M consists of four identical subunits, each with 1451 amino acids (2). The subunits are linked into dimers by disulfide bonds and into intact homotetramers by noncovalent interactions (3, 4). Proteinases react with ␣ 2 M by cleaving any of a number of susceptible peptide bonds in the "bait region," which includes amino acids 666 -706 (1, 3, 5). Bait region cleavage causes ␣ 2 M to undergo a major conformational change, which effectively "traps" the attacking proteinase in a complex that is nondissociable, even when the proteinase and the inhibitor are not covalently linked (1, 6 -8).Conformational change also reveals binding sites for the ␣ 2 M receptor/low density lipoprotein receptor-related protein (LRP) (9). These binding sites have been localized to 18-kDa peptides at the C terminus of each ␣ 2 M subunit; Lys-1370 and Lys-1374 play particularly important roles (10 -13).Like the complement components, C3 and C4, each ␣ 2 M subunit contains a novel thiol ester bond, which is formed from the side chains of . The thiol esters may be instrumental in determining the conformational state of ␣ 2 M (17, 18). When ␣ 2 M reacts with a proteinase, the thiol esters emerge from within hydrophobic, solvent-restricted clefts and are cleaved by nucleophiles or H 2 O (14, 18). Small primary amines, such as methylamine, penetrate the hydrophobic clefts and react with ␣ 2 M thiol esters independently of proteinases, inducing an equivalent or nearly equivalent conformational change (6, 7).In addition to its activity as a proteinase inhibitor, ␣ 2 M functions as a major carrier and regulator of certain cytokines, including isoforms of the transforming growth factor-␤ (TGF-␤) family. O'Connor-McCourt and Wakefield (19) first identified ␣ 2 M as a physiologically significant carrier of TGF-␤ in human serum (19). Their studies demonstrated that nearly all of the TGF-␤1 in serum is associated with ␣ 2 M and that the bound TGF-␤1 is inactive. Huang et al. (20) More recent studies have demonstrated the function of ␣ 2 M as a TGF-␤-carrier in animal model systems. When radioiodinated TGF-␤1 is injected intravascularly in mice, the cytokine is cleared rapidly at first; however, this is followed by a slow clearance phase, during which time the TGF-␤ is almost entirely ␣ 2 M-associated (21-23). In cell culture systems, ␣ 2 M neutralizes both exogenously added and endogenously synthesized TGF-␤ (24 -28). Neutralization of endogenously synthesized TGF-␤ results in altered gene expr...

Human ␣ 2 M induced NO synthesis comparably to m␣ 2 M; however, MUG had no effect. These studies demonstrate that the ability to neutralize TGF-␤ is a property of m␣ 2 M, which is not redundant in the murine ␣-macroglobulin family or in murine plasma. M␣ 2 M is the only murine ␣-macroglobulin that promotes NO synthesis. The absence of m␣ 2 M, in ␣ 2 M(؊/؊) mice, may allow TGF-␤ to more efficiently suppress excessive iNOS expression following endotoxin challenge.