Cloning and expression of cDNA for soluble form of rat heme oxygenase-1.
Heme oxygenase catalyzes the oxidation of heme to biliverdin and carbon monoxide. The gene encoding the truncated soluble rat heme oxygenase-1 (Metl-Pro267) was cloned. The enzyme protein was expressed in E. coli JM109 and purified to homogeneity. The molecular weight of the recombinant enzyme was 30 kDa as assessed by SDS-polyacrylamide gel electrophoresis. From a 3-L culture, about 90 mg of the purified enzyme was routinely obtained. The dependency of the heme oxygenase reaction catalyzed by the soluble enzyme on the NADPH-cytochrome P-450 reductase concentrations and the effect of catalase on the reaction were examined to compare with the purified membrane-bound form of heme oxygenase-1 (Yoshida and Kikuchi, 1978b). The activity of the soluble enzyme was inhibited at high concentrations of NADPH-cytochrome P-450 reductase and the inhibition was not alleviated by addition of catalase unlike the membrane-bound form. The ferric iron of the heme-heme oxygenase complex was in a typical high spin state at acidic to neutral pH (pH 6.5-7.0) but conversion to low spin state was observed at basic pH (pH 9-10). The heme bound to heme oxygenase was converted to biliverdin at a stoichiometric ratio of unity in the presence of NADPH-cytochrome P-450 reductase system. During the heme degradation of the heme-heme oxygenase complex under atmospheric oxygen, several intermediates, that is, oxygenated heme and verdoheme, were spectrally discriminated.
Hidaka
T
T
Department of Medical Biochemistry, Kurume University School of Medicine, Japan.
Omata
Y
Y
Noguchi
M
M
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics*; D051547:Heme Oxygenase-1; D051381:Rats; D012995:Solubility
D018076:DNA, Complementary; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1