Dr. Stacy Seeley has learned never to say "never."
Seeley, an assistant professor of Environmental Chemistry at Kettering University, swore she would never again work out the purification scheme for a protein after completing the process while earning her graduate degree.
"I essentially spent two years of my life purifying a protein during graduate school," she said. "My project was to determine the three-dimensional structure of this protein, so I needed it to be greater than 95 percent pure."
Then she got involved in a post-doctorate project at the New England Medical Center in Boston, requiring her to work out the purification scheme for a different protein related to blood coagulation.
"When very little is known about a protein, developing the purification scheme for it becomes largely trial and error," Seeley said. "For every thing that worked, there were five things I tried that didn't work. It taught me a lot about patience and persistence."
Seeley got through it a second time, more determined than ever to make this her last protein purification project.
But fate had other plans for Seeley. Shortly after arriving at Kettering, she met Dr. Arthur Bull of the Chemistry Department at Oakland University in Rochester, Michigan. In 1991, Bull had discovered a unique enzyme called13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase. This enzyme appeared to be involved in the regulation of tumor formation, particularly for colon cancer cells.
Since Bull's source of external funds was directed in other areas, he was unable to pursue investigations of 13-HODEdehydrogenase with the intensity the problem required. It was Seeley's experience in the protein purification process that prompted Bull to resurrect his 13-HODE project. The two entered into a collaborative research effort that focused on clarifying the role of 13-HODE dehydrogenase in the regulation of cellular activity.
For this research project, Seeley involved three Kettering Chemistry students and one Mechanical Engineering student. The research team traveled to Oakland University once a week to work in Bull's research lab.
Their starting point was the already established epidemiological and animal studies showing that dietary fat is known to enhance intestinal cancer formation, and that the type of fat consumed influences the development of particular types of cancer. In general, polyunsaturated fatty acids (PUFAs) seem to contribute more to the development of cancer than saturated fatty acids. Specifically, it is the oxidized metabolites of polyunsaturated fatty acids that encourage cancer cells to proliferate.
Dietary PUFAs are found in "just about everything," Seeley said. Most commonly they are found in all types of vegetable oils. The major dietary PUFA is linoleic acid and one of the major oxygen-containing metabolites of linoleic acid is 13-HODE. The human colon contains high levels of 13-HODE dehydrogenase, the enzyme that converts 13-HODE into a compound called13-OXO. 13-OXO has also proven to be significant in the effects of linoleic acid on cancer development.
"It is our working hypothesis that the formation and removal of 13-OXO is the mechanism by which linoleate oxygenation products regulate cellular activity," Seeley said. "Since 13-HODE dehydrogenase is the enzyme that produces 13-OXO from 13-HODE, it was essential that we fully purify and characterize this enzyme."
The isolated enzyme could then be used to unequivocally establish the role of this metabolic pathway in cellular regulation.
In June, Seeley successfully purified 13-HODE dehydrogenase from rat liver.
"Mass spectrometry and microsequencing results indicate that this is a novel protein," she said. "That means it is a unique protein and not just a previously known protein with a new function. So, we are pretty excited."
Currently, Seeley and Bull are in the process of cloning the 13-HODE dehydrogenase gene for future research. The long-term goal of this work is to define the role of 13-HODE dehydrogenase in cellular differentiation with the ultimate aim of evaluating the enzyme as a diagnostic and therapeutic target.
Seeley said the information furnished by these studies will provide insight in the role that specific dietary fatty acids play in colon cancer and eventually help formulate dietary recommendations to reduce colon cancer risks.