Identification and discrimination of extracellularly active cathepsins B and L in high-invasive melanoma cells

Analytical Biochemistry
Anke KloseNicolas Hunzelmann

Abstract

We established a novel protocol for lithium dodecyl sulfate (LDS) gelatin zymography, which operates under reducing conditions and at a slightly acidic pH value (6.5). This zymographic assay is based on polyacrylamide gel electrophoresis and facilitates the electrophoretic separation of human cathepsins in an active state. By this technique, activity of purified human liver cathepsin B was detected at a concentration as low as 50 ng and was blocked only in the presence of the cysteine protease inhibitor E-64 and the specific cathepsin B inhibitor CA-074 but not by aspartate, serine, or matrix metalloprotease inhibitors. The method was applied to analyze cathepsin activities in cell culture supernatants of the high-invasive melanoma cell line MV3. Interestingly, LDS zymography of MV3 cell supernatants in combination with specific inhibitors of cathepsins B and L identified three forms of extracellularly active cathepsin B and two forms of proteolytically active cathepsin L. We herein describe the generation and biochemical significance of acidic LDS zymography. This novel method permits not only the enzymatic analysis of purified cysteine proteases but also the identification and discrimination of different cathepsin activities ...Continue Reading

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Citations

May 18, 2011·Journal of Proteome Research·Catherine A FormoloYetrib Hathout
Jan 16, 2010·BMC Cancer·Jon J BriggsRigmor Solberg
Apr 24, 2015·The Journal of Reproduction and Development·Seon-Hyang KimNam-Hyung Kim
May 23, 2007·Pigment Cell Research·Cedric Gaggioli, Erik Sahai
Dec 17, 2009·Experimental Dermatology·Anke KloseNicolas Hunzelmann
Mar 9, 2010·Analytical Biochemistry·Weiwei A LiManu O Platt
Jan 9, 2007·Biochemical and Biophysical Research Communications·Nicola J NasserEviatar Nevo
Dec 19, 2007·Biochemical and Biophysical Research Communications·Michael FairheadChristopher F van der Walle
Aug 29, 2014·Environmental Science and Pollution Research International·Mateusz KędziorGrzegorz Terlecki
Aug 24, 2013·Nature·Pierrick UzureauEtienne Pays
Sep 12, 2019·Biochemistry. Biokhimii︠a︡·A I PetushkovaA A Zamyatnin
Dec 12, 2018·Analytical Biochemistry·Elena N ElpidinaIrina Y Filippova
May 16, 2021·Journal of Hematology & Oncology·Ziqi ZhangXiawei Wei

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