Affordable Access

deepdyve-link
Publisher Website

Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system.

Authors
  • Huber, Warren J 3rd
  • Marohnic, Christopher C
  • Peters, Michelle
  • Alam, Jawed
  • Reed, James R
  • Masters, Bettie Sue Siler
  • Backes, Wayne L
Type
Published Article
Journal
Drug metabolism and disposition: the biological fate of chemicals
Publication Date
Apr 01, 2009
Volume
37
Issue
4
Pages
857–864
Identifiers
DOI: 10.1124/dmd.108.025023
PMID: 19131520
Source
Medline
License
Unknown

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 K(m) 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 microM), 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.

Report this publication

Statistics

Seen <100 times