Abstract
Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the Km for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025–0.05 μM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment.
Footnotes
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This work was supported in part by the National Institutes of Health National Institute of Environmental Health Sciences [Grant ES004344]; the National Institutes of Health National Institute of General Medical Sciences [GM081568]; and the Robert A. Welch Foundation [AQ-0012] (to B.S.S.M.).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.025023.
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ABBREVIATIONS: HO-1, heme oxygenase-1, full-length; CO, carbon monoxide; Fe2+, ferrous iron; BVR, biliverdin reductase; P450, cytochrome P450; CPR, NADPH-cytochrome P450 reductase; ER, endoplasmic reticulum; hBVR, human biliverdin reductase; DLPC, dilauroylphosphatidylcholine; SOD, superoxide dismutase; H2O2, hydrogen peroxide; BSA, bovine serum albumin; RCS, reconstituted system; sHO-1, 30-kDa soluble human heme oxygenase-1.
- Received October 7, 2008.
- Accepted January 7, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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