title: Expression of heme oxygenase in hemopoiesis.
abstract: Heme oxygenase has been purified to electrophoretic homogeneity from detergent solubilized adult human liver microsomes. Treatment of microsomes with Triton X-100, sodium cholate and subsequent batchwise DEAE-cellulose, 2', 5' ADP-sepharose 4B, Sepharose CLB and hydroxylapatite column resulted in 17% yield of the purified heme oxygenase. The reconsituted system of heme oxygenase, composed of heme oxygenase, NADPH cytochrome c (P450) reductase and biliverdin reductase was equiactive with 1 mM NADPH and 4 nM NADH and showed complete dependence on added heme for catalytic activity. The Km values for NADPH and NADH were .046 and .526 mM, respectively. While NADPH concentration was held constant, the Km value for heme was 1.01 microM with a specific activity of 583 unit/mg protein. The activity of the reconstituted heme oxygenase system was not affected by preincubation with heavy metals despite their inhibitory effect of NADPH cytochrome c (P450) reductase and biliverdin reductase. However, the metalloporphyrins of these heavy metals were found to be strong inhibitors of the reconsituted system with Ki values of 0.015, 0.6, 2.3 and 5 microM for Sn-, Co-, Zn- and Mg- protoporphyrins, respectively. Similarly, the sulfhydryl inactivating reagents, HgCl2, iodoacetamide and p-chloromercurylbenzoate, inhibited the reconstituted heme oxygenase activity. Rabbits were immunized with purified human liver heme oxygenase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Microsomal protein with a molecular weight of 32,000 from rat and human liver as well as HepG2 cells were identified on dot and Western blots by their reaction with the anti-heme oxygenase similar to the purified enzyme protein. Anti-heme oxygenase precipitated quantitatively, the entire heme oxygenase of rat liver microsomes obtained from animals maintained on standard diet. The human bone marrow microsomal heme oxygenase activity was also quantitatively precipitated by this antibody. Antibody inhibition of rat and human heme xoygenase demonstrated a degree of conservation of both enzyme proteins between the species. As judged by Western blotting, the anti-heme oxygenase recognized only a single protein in spleen, liver, kidney, brain, heart, bone marrow, integtine and corneal epithelium. The human heme oxygenase cDNA was isolated by screening a cDNA library in the Okayama-Berg vector with a rat liver cDNA and was subjected to nucleotide sequence analysis. The deducted human heme oxygenase is also composed of 288 amino acids with a molecular mass of 32,800 Da.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Medicine, New York Medical College Valhalla 10595.
authors.forename: N G
authors.initials: NG
authors.lastname: Abraham
authors.forename: S M
authors.initials: SM
authors.lastname: Mitrione
authors.forename: W J
authors.initials: WJ
authors.lastname: Hodgson
authors.forename: R D
authors.initials: RD
authors.lastname: Levere
authors.forename: S
authors.initials: S
authors.lastname: Shibahara
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003429:Cross Reactions; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004247:DNA / Q000235:genetics; D006410:Hematopoiesis; D006419:Heme Oxygenase (Decyclizing) / Q000302:isolation & purification / Q000378:metabolism*; D008665:Metalloporphyrins / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D011233:Precipitin Tests; D051381:Rats; D015183:Restriction Mapping; D013045:Species Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014018:Tissue Distribution
chemical_list: D008665:Metalloporphyrins; D008670:Metals; D013439:Sulfhydryl Reagents; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D006419:Heme Oxygenase (Decyclizing); D009251:NADPH-Ferrihemoprotein Reductase