Development of a Rapid Assay for Prolyl Hydroxylase in Mouse Lung

Authors

Nathan Pearson, West Virginia University
Dale Porter, West Virginia University
Kenneth P. Blemmings, West Virginia University

Document Type

Article

Abstract

The National Institute for Occupational Safety and Health is conducting research on the effects of respiratory exposure to carbon nanotubes. Exposure to some kinds of carbon nanotubes has been associated with a fibrogenic response in lungs that has negative effects on lung physiology and human health. A thorough understanding of the molecular events leading to fibrosis could offer prophylactic or therapeutic approaches to avoid the fibrosis. Several different enzymes are associated with fiber formation in the lung ,and one of interest is prolyl hydroxylase (PH-4). Current protocols for measuring PH-4 activity are expensive, cumbersome, and time-consuming. A rapid assay protocol would aid in our understanding of the regulation on the enzyme’s activity. To prepare the tissue for assay, it was homogenized and then microsomes were prepared by differential centrifugation. Then, a surfactant was used to solubilize the protein, allowing substrate access. The incubation occurred in stoppered vials. The vials were placed in a 37 degree C water bath with shaking. A radioactive co-substrate for the reaction, 2-oxo[1-14C]-glutarate, was incubated in the presence and absence of a synthetic peptide containing proline and the liberated 14CO2 was captured. The reaction was terminated by adding pH 5 phosphate buffer to the reaction vial. Radioactivity was determined using liquid scintillation spectrometry. The peptide-dependent 14CO2 captured was used to estimate enzyme activity. This assay has been determined to be linear with respect to enzyme concentration as well as incubation time. This is a useful method as it can be completed in a matter of hours and requires no previous preparation of tissue or substrate. This rapid assay will be used to assess PH-4 in mouse lung from mice exposed or not exposed to carbon nanotubes. PH-4 regulation, or a lack thereof, after nanotube exposure suggests the molecular pathway by which the fibrogenic response associated with carbon nanotube exposure is elicited.

Recommended Citation

Pearson, Nathan; Porter, Dale; and Blemmings, Kenneth P. (2009) "Development of a Rapid Assay for Prolyl Hydroxylase in Mouse Lung," Mountaineer Undergraduate Research Review: Vol. 1, Article 7.
Available at: https://researchrepository.wvu.edu/murr/vol1/iss1/7