Different Roles of Class I and Class II Clostridium Histolyticum Collagenase in Rat Pancreatic Islet Isolation

Abstract

Crude Clostridium histolyticum collagenase was purified by gel filtration and fractionated by anion exchange chromatography into class I with high collagen digestion activity (CDA) and low FALGPA (2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine) hydrolysis activity (FHA), class II with low CDA and high FHA, and a fraction called class WI with intermediate activities. The roles of these collagenase classes in rat pancreatic islet isolation were investigated. Dissociations were carried out with 360 mg of pancreatic tissue in 10 ml of buffer containing 10% (wt/vol) albumin to suppress endogenous proteolytic activity, 100 U of C. histolyticum neutral protease, and one or two purified collagenase(s). For purified nonfraction-ated (PNF) collagenase, 2.6 mg of enzyme containing 2.4 U CDA and 38.0 U FHA was used, and for the separate classes, comparable amounts of activity were added. PNF collagenase dissociated the tissue completely in 32 min and yielded 5.0 ± 0.4 μl islet tissue/g pancreas. Class I collagenase alone dissociated pancreatic tissue extremely slowly and incompletely; only a few islets were released (0.7 ± 0.2 μl/g pancreas). Class II collagenase alone dissociated the tissue adequately in 50 min, and a high islet yield of 5.7 ± 0.6 μl/g was obtained. With class I/II, a similar dissociation time (47 min) and islet yield (5.5 ± 0.3 μl/g) were obtained. Combining class I and class II collagenase resulted in a more rapid dissociation (32 min) and a higher islet yield (7.1 ± 0.8 μl/g) than that obtained with PNF collagenase (P < 0.05). Similar results were obtained when purified class I collagenase was replaced by recombinant class I collagenase (32 min, islet yield 6.1 ± 0.3 μl/g). We conclude that class U collagenase plays a major role in pancreas dissociation and islet isolation and that class I collagenase acts synergistically with class II collagenase.