An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation
| Autor: | J. H. M. van Deijnen, Ghj Wolters, R van Schilfgaarde, G. H. Vos-Scheperkeuter |
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| Rok vydání: | 1992 |
| Předmět: |
Male
endocrine system PROTEASE Endocrinology Diabetes and Metabolism medicine.medical_treatment GRADIENTS EXTRACELLULAR MATRIX Cell Separation Biology Extracellular matrix ISLET ISOLATION Islets of Langerhans Clostridium histolyticum DIGESTION Endopeptidases Internal Medicine medicine Animals Pancreas chemistry.chemical_classification geography Protease geography.geographical_feature_category PURIFICATION TRANSPLANTATION CLOSTRIDIUM-HISTOLYTICUM Rats Inbred Strains Islet biology.organism_classification Rats Transplantation Kinetics Enzyme Microbial Collagenase chemistry Biochemistry Microbial collagenase Collagenase Chromatography Gel Electrophoresis Polyacrylamide Gel Indicators and Reagents COLLAGENASE medicine.drug |
| Zdroj: | Diabetologia, 35(8), 735-742. SPRINGER |
| ISSN: | 0012-186X |
| Popis: | Crude Clostridium histolyticum collagenase is widely used for the enzymatic degradation of pancreatic extracellular matrix in order to isolate the islets of Langerhans. The variable enzymatic composition of crude collagenases is a critical issue which contributes to the poor reproducibility of islet isolation procedures. In this study, the separate contributions of collagenase and protease to the islet isolation process were analysed by testing various combinations of purified collagenase and purified protease in rat pancreas dissociations under conditions which eliminated all other proteolytic activity. Under these conditions, complete tissue dissociation by purified collagenase required 99 +/- 10 min, whereas increasing amounts of protease progressively reduced this time to a minimum of 36 +/- 1 min. Histochemical analysis of the dissociation process showed that protease enhanced the degradation of all four major components of the extracellular matrix: collagen was degraded more completely, while proteoglycans, glycoproteins and elastin were degraded at a higher rate. Pancreas dissociation under the present, strictly controlled conditions resulted in a high yield of viable islets: 4.2-5.0 microliters islet tissue volume (3,300-3,800 islets) were isolated per g pancreas in the presence of a high or low protease concentration, respectively. Prolonged dissociation in the presence of protease resulted in a dramatic decrease in islet yield which correlated with the observation that the enzyme accelerated islet disintegration. It is concluded that the collagenase-induced dissociation of the extracellular matrix is facilitated by protease. Our study shows that high yields of viable islets can be obtained under controlled enzymatic conditions, provided that the exposure of islets to protease is limited. |
| Databáze: | OpenAIRE |
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