Characterization of an Important Enzymatic Component in Collagenase That Is Essential for the Effective Digestion of the Human and Porcine Pancreas
Abstract:Recent clinical results from Edmonton have demonstrated the feasibility of achieving normoglycemia in type I diabetic patients by islet transplantation. One of the key issues in obtaining this success was transplanting sufficient numbers of islets by sequential transplants. Although the development of semipurified endotoxin-free Clostridium histolyticum-derived collagenase (Liberase) has improved islet yields from the human pancreas, batch-to-batch variation and loss of activity with time still hampers progress in obtaining consistent islet preparations. In order to define key components of crude collagenase, a panel of monoclonal antibodies (McAbs) was raised against crude collagenase. Monoclonal antibodies were generated by fusions between splenocytes of BALB/c mice immunized with Boheringer P collagenase and the myeloma cell line NS-0. These monoclonal antibodies were used as probes to study molecular differences between effective and ineffective collagenase batches using Western blotting. Two monoclonal antibodies (LDS71 and LDS81) were raised and characterized as recognizing separate epitopes on a 125-kDa component. Western blotting indicated that the 125-kDa band was rapidly broken down by storage or by dialysis in the presence of dithiothreitol. However, this breakdown could be prevented by the addition of leupeptin (a protease inhibitor) to the dialysis buffer. On testing fractions at 5-min intervals from the “Ricordi” digestion circuit during porcine and human pancreas digestion, the 125-kDa component was rapidly broken down in relatively ineffective collagenase batches but in effective batches was present throughout the digestion process. The correlation between the presence of the 125-kDa band and effectiveness of pancreas digestion suggests that this may be a key component in the formulation of C. histolyticum collagenase.
Document Type: Research Article
Affiliations: Department of Surgery, University of Leicester, RKB, Royal Infirmary, Leicester LE2 7LX, UK
Publication date: 2001-08-01
More about this publication?
- Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.