HISTOCHEMICAL ANALYSIS OF THE ROLE OF CLASS-I AND CLASS-II CLOSTRIDIUM-HISTOLYTICUM COLLAGENASE IN THE DEGRADATION OF RAT PANCREATIC EXTRACELLULAR-MATRIX FOR ISLET ISOLATION

To understand why class II Clostridium histolyticum collagenase is muc h more effective than class I in the isolation of rat pancreatic islet s, we analyzed the role of these collagenases in pancreatic tissue dis sociation. Crude collagenase was purified and then fractionated into c lass I and II with different enzyme activities and protein composition s, Pancreatic tissue was incubated,vith either class I, class II, or c lass I + II, with or without added protease, under conditions that eli minated endogenous proteolytic activity, The degradation of pancreatic extracellular matrix was monitored by selective histochemical stainin g of tissue samples, Class I and LI showed similar capacities to degra de glycoproteins and degraded about one-third of the glycoproteins dur ing 120 min of incubation, The degradation of collagens by class I and II was relatively more effective, 80 to 95% of the collagens being re moved in 120 min, and also class dependent. Bath in the presence and a bsence of protease, class II was more effective at degrading collagens than class I, but this difference in efficacy was less apparent than with islet isolation, Class I + II degraded collagens faster and more complete than did the individual classes, indicating a synergistic eff ect of class I and LI. Evaluation of collagen degradation at various p ancreatic locations did not show a selective degradation of collagens by any of the collagenase classes, The present data offer a partial ex planation for the major role of class II in islet isolation. (C) 1997 Elsevier Science Inc.