کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1173877 | 1491405 | 2012 | 8 صفحه PDF | دانلود رایگان |
There is strong experimental evidence associating cathepsin S with the pathogenesis of atherosclerosis, with emerging data to support its role in diseases such as abdominal aortic aneurysm, obesity, and type 2 diabetes. To further our understanding of cathepsin S, we have developed a novel sandwich immunoassay to measure the mature form of cathepsin S in plasma (mean values from 12 healthy donors of 53 ± 17 ng/ml, range = 39–102). We also developed a targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay to measure in vitro cathepsin S activity to compare activity levels with the protein mass levels determined by enzyme-linked immunosorbent assay (ELISA). Interestingly, we observed that only 0.4 to 1.1% of circulating cathepsin S was enzymatically active. We subsequently demonstrated that the attenuated activity we observed resulted from binding between cathepsin S and its endogenous inhibitor cystatin C in plasma. These data were obtained through immunoprecipitation coupled with either Western blotting analysis or in-gel tryptic digestion and LC–MS/MS characterization of Coomassie-stained gel bands. Although many laboratories have explored the relationship between cathepsin S and cystatin C, this is the first study to demonstrate their association in human circulation, a finding that could prove to be important in furthering our understanding of cathepsin S biology.
Journal: Analytical Biochemistry - Volume 430, Issue 2, 15 November 2012, Pages 130–137