Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

New technique reliably detects enzyme implicated in cancer and atherosclerosis

02.06.2010
An enzyme implicated in osteoporosis, arthritis, atherosclerosis and cancer metastasis – cathepsin K -- eluded reliable detection in laboratory experiments in the past. Now, a research team at the Georgia Institute of Technology has developed an assay that reliably detects and quantifies mature 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.

Platt described the cathepsin K detection protocol in the June issue of the journal Analytical Biochemistry. This research was funded by new faculty support from Georgia Tech, and the Facilitating Academic Careers in Engineering and Science Scholars (FACES) and Summer Undergraduate Research in Engineering (SURE) programs at Georgia Tech.

The benefits of this assay over existing techniques are numerous, according to Platt. The major advantage of this protocol, he said, 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 of this technique is that it is more sensitive and less expensive than current, less reliable techniques. 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 our experiments we were able to detect mature cathepsin K activity in quantities as small as 3.45 femtomoles with zymography, which was 10 to 50 times more sensitive at detecting the enzyme than conventional Western blotting," noted Platt, who is also a Georgia Cancer Coalition Distinguished Cancer Scholar.

In addition, zymography allowed the researchers to measure the activity of the enzyme, whereas Western blotting just measured its presence.

To detect mature cathepsin K with gelatin zymography, Platt and Georgia Tech undergraduate student Weiwei Li first separated the enzymes present in cells by their molecular weights. This allowed them to distinguish the mature form of cathepsin K from the immature form and other cathepsin varieties.

Then, to verify the identity and presence of mature cathepsin K, the team activated the enzymes in the gel. They created the perfect acidic environment for cathepsin K to thrive and added inhibitors to block the activity of all enzymes except mature cathepsin K.

To validate the cathepsin K activity detected in the laboratory experiments, Platt and Georgia Tech undergraduate student Zachary Barry developed a computational kinetic model of the enzyme's activity. By solving a system of differential equations, they were able to calculate the concentrations of immature, mature and inactive cathepsin K present at all times during the experimental procedure.

"It is more challenging to work with enzymes than proteins because enzymes have to be functional, which means they have to be folded correctly to be active," explained Platt. "The model suggested that even after the slight denaturation and refolding required by our assay, the cathepsin K activity determined by zymography reflected what happens in nature and was not an artifact of the experimental procedure."

The model also predicted something unexpected -- the inactive form of cathepsin K commonly purchased from supply houses contained 20 percent mature enzyme.

"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.

With this assay, Platt's team is currently investigating whether cathepsin K activity is different in the cells of individuals with metastatic and non-metastatic breast and prostate cancers, and the role of cathepsin K in cardiovascular diseases, such as stroke, in children with sickle cell anemia. They are also examining whether cathepsin K plays a role in the inflammation associated with HIV.

"This research should provide new information on a number of existing pathophysiological conditions where cathepsin K activity had been previously undetectable," added Platt.

Additional contributors to this work included Georgia Tech research technologists Catera Wilder and Philip Keegan; former graduate student Rebecca Deeds; and Joshua Cohen, a summer researcher at Georgia Tech and currently an undergraduate at the Massachusetts Institute of Technology.

John Toon | EurekAlert!
Further information:
http://www.gatech.edu

More articles from Life Sciences:

nachricht Topologische Quantenchemie
21.07.2017 | Max-Planck-Institut für Chemische Physik fester Stoffe

nachricht Topological Quantum Chemistry
21.07.2017 | Max-Planck-Institut für Chemische Physik fester Stoffe

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Manipulating Electron Spins Without Loss of Information

Physicists have developed a new technique that uses electrical voltages to control the electron spin on a chip. The newly-developed method provides protection from spin decay, meaning that the contained information can be maintained and transmitted over comparatively large distances, as has been demonstrated by a team from the University of Basel’s Department of Physics and the Swiss Nanoscience Institute. The results have been published in Physical Review X.

For several years, researchers have been trying to use the spin of an electron to store and transmit information. The spin of each electron is always coupled...

Im Focus: The proton precisely weighted

What is the mass of a proton? Scientists from Germany and Japan successfully did an important step towards the most exact knowledge of this fundamental constant. By means of precision measurements on a single proton, they could improve the precision by a factor of three and also correct the existing value.

To determine the mass of a single proton still more accurate – a group of physicists led by Klaus Blaum and Sven Sturm of the Max Planck Institute for Nuclear...

Im Focus: On the way to a biological alternative

A bacterial enzyme enables reactions that open up alternatives to key industrial chemical processes

The research team of Prof. Dr. Oliver Einsle at the University of Freiburg's Institute of Biochemistry has long been exploring the functioning of nitrogenase....

Im Focus: The 1 trillion tonne iceberg

Larsen C Ice Shelf rift finally breaks through

A one trillion tonne iceberg - one of the biggest ever recorded -- has calved away from the Larsen C Ice Shelf in Antarctica, after a rift in the ice,...

Im Focus: Laser-cooled ions contribute to better understanding of friction

Physics supports biology: Researchers from PTB have developed a model system to investigate friction phenomena with atomic precision

Friction: what you want from car brakes, otherwise rather a nuisance. In any case, it is useful to know as precisely as possible how friction phenomena arise –...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Closing the Sustainability Circle: Protection of Food with Biobased Materials

21.07.2017 | Event News

»We are bringing Additive Manufacturing to SMEs«

19.07.2017 | Event News

The technology with a feel for feelings

12.07.2017 | Event News

 
Latest News

NASA looks to solar eclipse to help understand Earth's energy system

21.07.2017 | Earth Sciences

Stanford researchers develop a new type of soft, growing robot

21.07.2017 | Power and Electrical Engineering

Vortex photons from electrons in circular motion

21.07.2017 | Physics and Astronomy

VideoLinks
B2B-VideoLinks
More VideoLinks >>>