Chapter title |
Gelatin Zymography Using Leupeptin for the Detection of Various Cathepsin L Forms
|
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Chapter number | 16 |
Book title |
Lysosomes
|
Published in |
Methods in molecular biology, April 2017
|
DOI | 10.1007/978-1-4939-6934-0_16 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6932-6, 978-1-4939-6934-0
|
Authors |
Yoko Hashimoto |
Editors |
Karin Öllinger, Hanna Appelqvist |
Abstract |
Zymography is a highly sensitive method to assess the activities as well as molecular weights of enzymes in crude biological fluids and tissue extracts. Cathepsin L is a lysosomal cysteine proteinase that is optimally active at slightly acidic pH and is highly unstable in alkaline solutions such as electrode buffer (pH 8.3). Large amounts of cathepsin L are secreted by various cancer cells, where it promotes invasion and metastasis. Leupeptin is a tight-binding inhibitor of cysteine proteinases, and its complex with cathepsin L is stable in alkaline solutions. Moreover, leupeptin can be easily removed from the complex because it is a reversibly binding inhibitor. In addition, leupeptin is too small to influence the electrode migration distance of the complex with cathepsin L on a sodium dodecyl sulfate-polyacrylamide gel. Here, a novel gelatin zymography technique that employs leupeptin to detect pro-, intermediate, and mature cathepsin L forms on the basis of their gelatinolytic activities is described. Further, the differences in the glycosylation, phosphorylation, and processing statuses of lysosomal and secreted cathepsin L forms isolated from cultured HT 1080 cells are demonstrated using this method. |
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