TY - JOUR
T1 - Fractions from commercial collagenase preparations
T2 - Use in enzymic isolation of the islets of Langerhans from porcine pancreas
AU - Klöck, Gerd
AU - Kowalski, Matthias B.
AU - Hering, Bernhard J.
AU - Eiden, Martin E.
AU - Weidemann, Astrid
AU - Langer, Stefan
AU - Zimmermann, Ulrich
AU - Federlin, Konrad
AU - Bretzel, Reinhard G.
PY - 1996
Y1 - 1996
N2 - Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the liberation of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.
AB - Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the liberation of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.
KW - Collagenase
KW - FPLC
KW - Free flow electrophoresis
KW - Islet isolation
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U2 - 10.1016/0963-6897(96)00023-1
DO - 10.1016/0963-6897(96)00023-1
M3 - Article
C2 - 8889213
AN - SCOPUS:0029742474
SN - 0963-6897
VL - 5
SP - 543
EP - 551
JO - Cell transplantation
JF - Cell transplantation
IS - 5
ER -