Myocardial collagenase: purification and structural characterization.

ABSTRACT

Interstitial collagenase (matrix metalloproteinase-1 [MMP-1]) plays an important role in extracellular matrix turnover. Myocardial MMP-1 may contribute to tissue remodelling in the heart. Little is known about collagenase and its regulation in the myocardium. To understand better the nature of this neutral proteinase in the rat myocardium, myocardial collagenase was purified to homogeneity. The purification procedure included a gel-filtration step on Sephacryl S-200 columns and substrate affinity chromatography on type I collagen-Sepharose. Under reducing conditions, collagenase was shown by SDS-PAGE to consist of a single polypeptide chain with a molecular mass of 54 kDa. Purified interstitial collagenase demonstrated a single lytic band on zymography. This band was inhibited by 1,10-phenanthroline (a metal chelator), which indicates that the 54 kDa protein is an MMP. Using a polyclonal antibody to proMMP-1, purified collagenase was characterized by immunoblot analysis. A single band of purified interstitial collagenase was observed on Western blot analysis. This indicated that the purified proenzyme was collagenase. Sequence analysis on cyanogen bromide-digested fragments of latent MMP-1 suggested that the active site sequence of rat myocardial MMP-1 is similar to that of the rat osteoblast collagenase, human skin fibroblast collagenase and Serratia proteinase. The substrate specificity of the purified collagenase was measured against fluorescent-labelled type I collagen. It was observed that after activation, purified collagenase was capable of degrading type I collagen in a time-dependent manner. The half-time for collagen degradation was estimated to be less than 30 s. These results suggest that collagenase is present in the normal adult rat myocardium and that collagen turnover may be regulated by this neutral metalloproteinase. A simple two-step purification protocol is demonstrated for interstitial collagenase. This procedure can be used for routine MMP-1 preparation from tissue sources.