Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system.
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.
Huber
Warren J
WJ
Department of Pharmacology and The Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, LA 70112, USA .
Marohnic
Christopher C
CC
Peters
Michelle
M
Alam
Jawed
J
Reed
James R
JR
Masters
Bettie Sue Siler
BS
Backes
Wayne L
WL
D000818:Animals; D001483:Base Sequence; D002374:Catalase / Q000378:metabolism; D017931:DNA Primers; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism*; D006801:Humans; D008081:Liposomes; D008565:Membrane Proteins / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism
D017931:DNA Primers; D008081:Liposomes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D002374:Catalase; D006419:Heme Oxygenase (Decyclizing); D013482:Superoxide Dismutase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase