INSULIN-LIKE-GROWTH-FACTOR BINDING-PROTEIN (IGFBP) SUBSTRATE ZYMOGRAPHY - A NEW TOOL TO IDENTIFY AND CHARACTERIZE IGFBP-DEGRADING PROTEINASES

Insulin-like growth factor binding protein (IGFBP) degrading proteinas e activities have been described in biological fluids and conditioned media from numerous cell lines. To identify and characterize IGFBP-deg rading proteinases, our laboratory has developed IGFBP substrate zymog raphy. Herein, we illustrate how IGFBP substrate zymography can be use d both to identify candidate IGFBP-degrading proteinases and character ize their degradative capabilities. For this purpose, human matrix met alloproteinase-3 (MMP-3), a proteinase that degrades IGFBP-3 in human fibroblast cultures, was first electrophoresed through a polyacrylamid e gel containing IGFBP-3 as substrate and then analyzed for its abilit y to degrade the substrate into immunoreactive fragments that were abs orbed onto a polyvinylidene difluoride membrane. IGFBP-3 substrate zym ography was capable of detecting as little as 20 ng of human MMP-3, de monstrating a sensitivity similar to casein substrate zymography. Usin g the zymogram as a template, MMP-3 was identified in a standard SDS-p olyacrylamide gel run in parallel with the zymogram, and the correspon ding area of the gel was excised. Electroelution of the gel slice yiel ded active MMP-3 when examined by casein substrate zymography. Further more, digestion of IGFBP-3 in solution by the electroeluted MMP-3 reve aled the same fragmentation pattern of the binding protein as that pro duced by MMP-3, which had not been electroeluted. Together, these stud ies demonstrate that IGFBP substrate zymography can be a useful tool f or both the identification and the characterization of IGFBP-degrading proteinases.