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 177% 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 μM 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 μM for Sn-, Co-, Zn- and Mg- protoporphyrins, respectively. Similarly, the sulfhydryl inactivating reagents, HgCl_2, 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, intei tine 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 deduced human heme oxygenase is also composed of 288 amino acids with a molecular mass of 32,800 Da. Following hemin treatment of human leukemic cell line K562 there was an increase in the amount of heme oxygenase protein and mRNA.