Inhibin, activin and follistatin bind preferentially to the transformed species of  2 -macroglobulin D J Phillips, J R McFarlane, M T W Hearn 1 and D M de Kretser Institute of Reproduction and Development, and 1 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168, Australia (Requests for offprints should be addressed to D J Phillips, Institute of Reproduction and Development, Level 3, Block E, Monash Medical Centre, Clayton Road, Clayton, Victoria 3168, Australia) Abstract  2 -Macroglobulin ( 2 -M), a major serum glycoprotein, has been implicated as a low-affinity binding protein for inhibin and activin. In serum,  2 -M exists as two major species, a native form that is abundant and stable, and a transformed (‘fast’) species that is rapidly cleared from the circulation via  2 -M receptors. In this study inhibin, activin and their major binding protein follistatin were investigated for their ability to bind to the native or transformed species of purified human  2 -M. Using native PAGE and size exclusion chromatography, radiolabelled inhibin, activin and follistatin bound to the transformed  2 -M. Inhibin and follistatin did not bind significantly to native  2 -M, whereas activin was able to bind to the native species but with a lower capacity compared with that to transformed  2 -M. Under reducing conditions, binding of these hormones to  2 -M was abolished. These findings implicate  2 -M as a mechanism whereby inhibin, activin and follistatin may be removed from the circulation through  2 -M receptors, but also whereby activin can be maintained in the circulation through its ability to bind to native  2 -M. Journal of Endocrinology (1997) 155, 65–71 Introduction  2 -Macroglobulin ( 2 -M) is a major serum glycoprotein that has a multiplicity of functions including proteinase inhibition, immune regulation and cytokine binding (for review, see Chu & Pizzo 1994). This 725 kDa protein exists in at least two major forms, a native or so-called ‘slow’ species, which is present in high concentrations in serum and can entrap proteinases, and the transformed or electrophoretically ‘fast’ species, which appears after trapping of a proteinase or reaction with primary amines (Van Leuven et al. 1981). A number of growth factors have been shown to bind to  2 -M at a site distinct from that where proteinases are entrapped, including members of the transforming growth factor- (TGF) superfamily (Chu & Pizzo 1994). Importantly, some polypeptides have been shown to bind significantly only to the native form of  2 -M (e.g. carboxypeptidase A (Valnickova et al. 1996)), to both forms (e.g. TGF2 (Crookston et al. 1994)), and some to only the transformed species (e.g. growth hormone (Kratzsch et al. 1995)). The form of  2 -M to which factors can bind has significant implications for their physiology, as complexes of cytokines with native  2 -M remain stable in the circulation (Philip & O’Connor-McCourt 1991, Crookston et al. 1993), whereas factors binding to the transformed  2 -M are rapidly removed from the circulation via  2 -M receptors (Kristensen et al. 1990, Strickland et al. 1990, Misra et al. 1994). Inhibin and activin are members of the TGF super- family with diverse physiological roles within the body (dePaolo et al. 1991, Woodruff & Mather 1995). Follistatin is a polypeptide that is structurally distinct from inhibin or activin and is able to bind these cysteine knot proteins with high affinity (Nakamura et al. 1990, Kogawa et al. 1991, Sugino et al. 1993, Schneyer et al. 1994). Investi- gations into high molecular mass binding proteins of inhibin and activin have implicated  2 -M (Schneyer et al. 1992, Krummen et al. 1993, Vaughan & Vale 1993), but further characterizations of these interactions were not achieved in these earlier studies. More recently, Niemuller et al. (1995) demonstrated that activin A is able to bind to both the native and transformed  2 -M with a reported affinity of 510 60 and 190 30 n respectively. These data suggest that  2 -M has a dual function of both maintaining activin A in the circulation or within tissues and facilitating the clearance of activin A depending on the form of  2 -M present. Whether follistatin binds to  2 -M, and the different species of  2 -M that bind inhibin, have not been previously documented. In this study, we have investigated the interactions of inhibin, activin and follistatin with the native and trans- formed conformations of  2 -M. The results, showing that inhibin and follistatin bind significantly only to the 65 Journal of Endocrinology (1997) 155, 65–71  1997 Journal of Endocrinology Ltd Printed in Great Britain 0022–0795/97/0155–0065 $08.00/0