International Journal ofPancreatologv, vol. 19,no. 1, 55~60, February1996 9 Copyright1996 by Humana PressInc. All rightsofany naturewhatsoever reserved. 0169-4197/96/I 9:55- 60/$6.50 Aprotinin Inhibits Unspecific Degradation of Collagen in Rat and Human Pancreas Jos . Manuel Lbpez,' Rodrigo Valderrama,' Salvador Navarro, .,1 and Santiago ImperiaF ~Gastroenterology Service, Hospital Clinic i Provincial, Barcelona, Spain; and :Department o.f Biochemistry and Physiology, University of Barcelona, Spain Summary Conclusion. Addition of aprotinin in human and rat pancreatic extracts inactiviates nonspecific proteases that completely degrade collagen. Background. We sought to clarify the relative roles of collagenase and nonspecific proteases in the breakdown of collagen by the pancreas. Methods. The degradation of [3H] collagen fibrils by pancreatic extracts to small fragments of low molecular weight was determined by SDS-electrophoresis and autoradiography. Aprotinin (0.14 mg/mL) was added to inhibit nonspecific protease activity. Results. Rat and human pancreas extracts contained a high collagenolytic activity that was demon- strated to be the result of the combined action ofcollagenase and other pancreatic proteases. Seventy percent of the total collagenolytic activity in rat pancreas extracts was inhibited by aprotinin. The same aprotinin concentration had no effect on two commercially available collagenases. The electrophoretic pattern obtained from [3H] collagen treated with rat and human pancreatic extracts containing aprotinin confirmed the presence of a true specific collagenase in the pancreas. Key Words: Collagen; collagenase; aprotinin; pancreas; rat; human. Introduction The breakdown of interstitial collagens in all higher organisms is believed to be initiated by spe- cific collagenases (1,2). The key feature of these enzymes is their ability to hydrolyze the triple-heli- cal collagen monomers of collagen fibrils at a spe- cific locus to produce characteristic 3/4 and 1/4 fragments at a physiological pH and temperature. After the first cleavage of collagen ~t chains by col- Received March 22, 1995; RevisedJune 12, 1995; Accepted August 14, 1995. *Author to whom all correspondence and reprint requests should be addressed: GastroenterologyService, Hospital Clinic i Provincial, c/Villarroel 170, 08036 Barcelona, Spain. lagenase other enzymes may act on these products and completely degrade the protein into small-mol- wt fragments. Neutral proteases, collagenolytic cathepsins, gelatinases, and collagen peptidases can act on these fragments (3). Trypsin, chymotrypsin, pepsin, pronase, and other proteases carry out a lim- ited digestion of the terminal ends of collagen, but do not degrade its main structure. For this reason, those enzymes are not considered as true collagenases, but as proteases showing collagenolytic activity (3,4). Most of the work on vertebrate collagenases has been performed on enzymes isolated from the culture media of an extended number of cell types: fibro- blasts (5), macrophages (6), keratinocytes (7), poly- morphonuclear leukocytes (8), and tumor cells (9). Few studies have concerned activities of collagen- 55