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 A novel socio-ecological approach helps identifying suitable wolf habitats
17.02.2017 | Universität Zürich

nachricht New, ultra-flexible probes form reliable, scar-free integration with the brain
16.02.2017 | University of Texas at Austin

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

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

Im Focus: Breakthrough with a chain of gold atoms

In the field of nanoscience, an international team of physicists with participants from Konstanz has achieved a breakthrough in understanding heat transport

In the field of nanoscience, an international team of physicists with participants from Konstanz has achieved a breakthrough in understanding heat transport

Im Focus: DNA repair: a new letter in the cell alphabet

Results reveal how discoveries may be hidden in scientific “blind spots”

Cells need to repair damaged DNA in our genes to prevent the development of cancer and other diseases. Our cells therefore activate and send “repair-proteins”...

Im Focus: Dresdner scientists print tomorrow’s world

The Fraunhofer IWS Dresden and Technische Universität Dresden inaugurated their jointly operated Center for Additive Manufacturing Dresden (AMCD) with a festive ceremony on February 7, 2017. Scientists from various disciplines perform research on materials, additive manufacturing processes and innovative technologies, which build up components in a layer by layer process. This technology opens up new horizons for component design and combinations of functions. For example during fabrication, electrical conductors and sensors are already able to be additively manufactured into components. They provide information about stress conditions of a product during operation.

The 3D-printing technology, or additive manufacturing as it is often called, has long made the step out of scientific research laboratories into industrial...

Im Focus: Mimicking nature's cellular architectures via 3-D printing

Research offers new level of control over the structure of 3-D printed materials

Nature does amazing things with limited design materials. Grass, for example, can support its own weight, resist strong wind loads, and recover after being...

Im Focus: Three Magnetic States for Each Hole

Nanometer-scale magnetic perforated grids could create new possibilities for computing. Together with international colleagues, scientists from the Helmholtz Zentrum Dresden-Rossendorf (HZDR) have shown how a cobalt grid can be reliably programmed at room temperature. In addition they discovered that for every hole ("antidot") three magnetic states can be configured. The results have been published in the journal "Scientific Reports".

Physicist Dr. Rantej Bali from the HZDR, together with scientists from Singapore and Australia, designed a special grid structure in a thin layer of cobalt in...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Booth and panel discussion – The Lindau Nobel Laureate Meetings at the AAAS 2017 Annual Meeting

13.02.2017 | Event News

Complex Loading versus Hidden Reserves

10.02.2017 | Event News

International Conference on Crystal Growth in Freiburg

09.02.2017 | Event News

 
Latest News

Switched-on DNA

20.02.2017 | Materials Sciences

Second cause of hidden hearing loss identified

20.02.2017 | Health and Medicine

Prospect for more effective treatment of nerve pain

20.02.2017 | Health and Medicine

VideoLinks
B2B-VideoLinks
More VideoLinks >>>