The role of endogenous serine proteinase on disintegration of collagen fibers from grass carp (Ctenopharyngodon idellus)

The dimethylmethylene blue assay for sulphated glycosaminoglycans has found wide acceptance as a quick and simple method of measuring the sulphated glycosaminoglycan content of tissues and fluids. The available assay methods have lacked specificity for sulphated glycosaminoglycans in the presence of other polyanions, however, and have not discriminated between the different sulphated glycosaminoglycans. We now describe a modified form of the dimethylmethylene blue assay that has improved specificity for sulphated glycosaminoglycans, and we show that in conjunction with specific polysaccharidases, the dimethylmethylene blue assay can be used to quantitate individual sulphated glycosaminoglycans.

Most abundant in the extracellular matrix are collagens, joined by elastin that confers elastic recoil to the lung, aorta, and skin. These fibrils are highly resistant to proteolysis but can succumb to a minority of the matrix metalloproteinases (MMPs). Considerable inroads to understanding how such MMPs move to the susceptible sites in collagen and then unwind the triple helix of collagen monomers have been gained. The essential role in unwinding of the hemopexin-like domain of interstitial collagenases or the collagen binding domain of gelatinases is highlighted. Elastolysis is also facilitated by the collagen binding domain in the cases of MMP-2 and MMP-9, and remote exosites of the catalytic domain in the case of MMP-12.

Gelatin zymography is a simple yet powerful method to detect proteolytic enzymes capable of degrading gelatin from various biological sources. It is particularly useful for the assessment of two key members of the matrix metalloproteinase family, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), due to their potent gelatin-degrading activity. This polyacrylamide gel electrophoresis-based method can provide a reliable assessment of the type of gelatinase, relative amount, and activation status (latent, compared with active enzyme forms) in cultured cells, tissues, and biological fluids. The method can be used to investigate factors that regulate gelatinase expression and modulate zymogen activation in experimental systems. The system provides information on the pattern of gelatinase expression and activation in human cancer tissues and how this relates to cancer progression. Interpretation of the data obtained in gelatin zymography requires a thorough understanding of the principles and pitfalls of the technique; this is particularly important when evaluating enzyme levels and the presence of active gelatinase species. If properly used, gelatin zymography is an excellent tool for the study of gelatinases in biological systems.