A research team has developed an experiment that reliably detects and quantifies mature cathepsin K – an enzyme implicated in osteoporosis, arthritis, atherosclerosis and cancer metastasis.
Researchers at the Georgia Institute of Technology have detected cathepsin K using a technique called gelatin zymography.
“This assay is important because researchers and pharmaceutical companies need a dependable method for sensitively detecting a small amount of cathepsin K and quantifying its activity to develop inhibitors to the enzyme that can fight the diseases while minimizing side effects,” said Manu Platt, an assistant professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University.
Cathepsin K is required to maintain adequate calcium levels in the body, but it can be highly destructive because it has the ability to break down bone by degrading collagen and elastin.
According to Platt, the major advantage of this protocol is the definitive knowledge that mature cathepsin K is being detected in cells and tissues – and not its immature form or one of the other 10 cathepsin varieties: B, H, L, S, C, O, F, V, X or W.
Another advantage is the reduced cost but more sensitive. The new assay allows cathepsin K to be detected in quantities as small as a few femtomoles and does not require antibodies, which can be expensive and cannot be used across different species.
In addition, zymography allowed the researchers to measure the activity of the enzyme, whereas Western blotting just measured its presence.
“Cathepsins are implicated in many different diseases and the value of this assay is that it enables the measurement of previously undeterminable cathepsin activity in normal and diseased cells and tissues,” noted Platt.
“This research should provide new information on a number of existing pathophysiological conditions where cathepsin K activity had been previously undetectable,” added Platt.
The study was published in the June issue of the journal Analytical Biochemistry