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Two‐dimensional zymography for analysis of proteolytic enzymes in human pure pancreatic juice
Author(s) -
Kaino Seiji,
Furui Toshifumi,
Hatano Satoko,
Kaino Miyuki,
Okita Kiwamu,
Nakamura Kazuyuki
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150190530
Subject(s) - chemistry , isoelectric focusing , coomassie brilliant blue , isoelectric point , chromatography , gelatin , zymography , casein , biochemistry , proteolytic enzymes , enzyme , staining , sodium dodecyl sulfate , immobilized ph gradient , polyacrylamide gel electrophoresis , gel electrophoresis , microbiology and biotechnology , biology , genetics
Proteolytic enzymes in human pure pancreatic juice (PPJ), which was collected by cannulating the main pancreatic duct using endoscopy, were investigated by two‐dimensional zymography (2‐DZ). 2‐DZ was carried out by combining isoelectric focusing (IEF) in the first dimension with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in the second dimension, using gels containing casein or gelatin as a substrate for the proteolytic enzymes. After electrophoresis, the gels were incubated in Triton X‐100 followed by incubation at 37°C in Tris buffer (pH 8.5) containing CaCl 2 . By staining the gels with Coomassie Brilliant Blue (CBB R‐250), proteolytic enzymes were detected as clear spots and zones against a blue background. Proteinase inhibitors, such as a cysteine proteinase inhibitor (E‐64), a metalloproteinase inhibitor (EDTA), and a serine proteinase inhibitor (Pefabloc® SC), were added to PPJ in order to determine the types of proteinases. In patients with pancreatic cancer, spots of molecular weight ( M r ) 70 000 and isoelectric points (p I ) 5.3–5.5 were clearly detected on the gels containing casein and gelatin, while these spots were not detected in the PPJ from healthy subjects. The proteolytic activities of these spots were strongly inhibited by EDTA and Pefabloc SC but not E‐64. These results suggest that the spots of M r 70 000 and p I 5.3–5.5 in PPJ of pancreatic cancer might be matrix metalloproteinase 2, which is a candidate for tumor‐associated proteinase. 2‐DZ proved to be a tool for analysis of proteolytic enzymes in PPJ and for the clinical diagnosis of pancreatic cancer.