Commercial Collagenase* prepared from Clostridium histolyticum is widely used in isolation of pancreatic islets. It is known that the enzyme is very impure and that there are substantial variations in effectiveness between batches. Our studies suggest that one of the impurities of importance in islet isolation is a protease that has not been very well characterized. Comparison of two batches of enzyme, one of which was known to give good yields of islets and the other poor yields, showed that they had very similar activity against collagen (measured by digestion of insoluble collagen followed by assay of soluble products with ninhydrin) but substantially different activities against azocasein as measured by optical density increase (measured by release of dye). Eighteen batches of Collagenase were examined for efficiency in islet isolation, and the yields obtained correlated with manufacturer's data of activity against casein. The data show that low caseinase activity is associated with performance in islet isolation (r = .5 after adjusting for collagenase activity). The effect of supplementing a batch of collagenase, known to be poor in isolating islets, with proteolytic enzymes was investigated. Trypsin and papain had apparently no effect, but dispase significantly increased yield. Dispase alone failed to digest pancreas. Size-exclusion high-performance liquid chromatography identified a peak associated with high protease activity and efficiency in islet isolation, having an Mr of ∼30,000, compared to 78,000 for collagenase. The protease, like collagenase, is inhibited by EDTA. Increased Ca2+ and Mg2+ (up to 10 mM) did not affect activity. Both the protease and collagenase are stable under normal use but are inactivated by heating at 56°C. Other properties, e.g., effect of temperature, pH, and dithiothreitol, were also investigated.
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Techniques for Islet Preparation| January 01 1989
Protease Activity in Pancreatic Islet Isolation by Enzymatic Digestion
Derek WR Gray;
Address correspondence and reprint requests to Dr. P. McShane, Nuffield Department of Surgery, John Radcliffe Hospital, Headington, Oxford 0X3 OEX, England, UK
Philip McShane, Robert Sutton, Derek WR Gray, Peter J Morris; Protease Activity in Pancreatic Islet Isolation by Enzymatic Digestion. Diabetes 1 January 1989; 38 (Supplement_1): 126–128. https://doi.org/10.2337/diab.38.1.S126
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