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Measuring digestive protease activation in the mouse pancreas.
Pancreatology. 2020 Mar; 20(2):288-292.P

Abstract

Intrapancreatic activation of digestive proteases, trypsin and chymotrypsin in particular, is a hallmark of pancreatitis. In experimental rodent models, protease activation is routinely measured from pancreatic homogenates using fluorogenic peptide substrates. Here we investigated the optimal conditions for the determination of intrapancreatic trypsin and chymotrypsin activation elicited by a single intraperitoneal injection of cerulein in C57BL/6N mice. We found that these protease assays were significantly improved by using lower amounts of pancreatic homogenate and exclusion of bovine serum albumin from the assay buffer. Furthermore, pancreatic homogenates had to be freshly prepared and assayed; as freezing and thawing stimulated protease activation. Finally, replacement of the widely used Boc-Gln-Ala-Arg-AMC trypsin substrate with Z-Gly-Pro-Arg-AMC reduced the background activity in saline-treated control mice and thereby increased the extent of cerulein-induced trypsin activation. Using the optimized protocol, we reproducibly measured 20-fold and 200-fold increases in the intrapancreatic trypsin and chymotrypsin activity, respectively, in mice given cerulein.

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Authors+Show Affiliations

Mosztbacher D
Center for Exocrine Disorders, Department of Molecular and Cell Biology, Boston University, Henry M. Goldman School of Dental Medicine, Boston, MA, 02118, USA.
Demcsák A
Department of Surgery, University of California Los Angeles, Los Angeles, CA, 90095, USA.
Sahin-Tóth M
Center for Exocrine Disorders, Department of Molecular and Cell Biology, Boston University, Henry M. Goldman School of Dental Medicine, Boston, MA, 02118, USA; Department of Surgery, University of California Los Angeles, Los Angeles, CA, 90095, USA. Electronic address: msahintoth@mednet.ucla.edu.

MeSH

AnimalsCeruletideChymotrypsinEnzyme ActivationFemaleMaleMiceMice, Inbred C57BLPancreasPancreatitisPeptide HydrolasesSerum Albumin, BovineSodium ChlorideTrypsin

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31899136

Citation

Mosztbacher, Dóra, et al. "Measuring Digestive Protease Activation in the Mouse Pancreas." Pancreatology : Official Journal of the International Association of Pancreatology (IAP) ... [et Al.], vol. 20, no. 2, 2020, pp. 288-292.
Mosztbacher D, Demcsák A, Sahin-Tóth M. Measuring digestive protease activation in the mouse pancreas. Pancreatology. 2020;20(2):288-292.
Mosztbacher, D., Demcsák, A., & Sahin-Tóth, M. (2020). Measuring digestive protease activation in the mouse pancreas. Pancreatology : Official Journal of the International Association of Pancreatology (IAP) ... [et Al.], 20(2), 288-292. https://doi.org/10.1016/j.pan.2019.12.020
Mosztbacher D, Demcsák A, Sahin-Tóth M. Measuring Digestive Protease Activation in the Mouse Pancreas. Pancreatology. 2020;20(2):288-292. PubMed PMID: 31899136.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Measuring digestive protease activation in the mouse pancreas. AU - Mosztbacher,Dóra, AU - Demcsák,Alexandra, AU - Sahin-Tóth,Miklós, Y1 - 2019/12/26/ PY - 2019/10/07/received PY - 2019/12/21/revised PY - 2019/12/24/accepted PY - 2020/1/4/pubmed PY - 2021/2/27/medline PY - 2020/1/4/entrez KW - Chymotrypsin KW - Fluorescent substrate KW - Pancreatitis KW - Protease activation KW - Trypsin SP - 288 EP - 292 JF - Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.] JO - Pancreatology VL - 20 IS - 2 N2 - Intrapancreatic activation of digestive proteases, trypsin and chymotrypsin in particular, is a hallmark of pancreatitis. In experimental rodent models, protease activation is routinely measured from pancreatic homogenates using fluorogenic peptide substrates. Here we investigated the optimal conditions for the determination of intrapancreatic trypsin and chymotrypsin activation elicited by a single intraperitoneal injection of cerulein in C57BL/6N mice. We found that these protease assays were significantly improved by using lower amounts of pancreatic homogenate and exclusion of bovine serum albumin from the assay buffer. Furthermore, pancreatic homogenates had to be freshly prepared and assayed; as freezing and thawing stimulated protease activation. Finally, replacement of the widely used Boc-Gln-Ala-Arg-AMC trypsin substrate with Z-Gly-Pro-Arg-AMC reduced the background activity in saline-treated control mice and thereby increased the extent of cerulein-induced trypsin activation. Using the optimized protocol, we reproducibly measured 20-fold and 200-fold increases in the intrapancreatic trypsin and chymotrypsin activity, respectively, in mice given cerulein. SN - 1424-3911 UR - https://www.unboundmedicine.com/medline/citation/31899136/Measuring_digestive_protease_activation_in_the_mouse_pancreas_ DB - PRIME DP - Unbound Medicine ER -