A heme-binding protein with a molecular mass of 22 kDa, termed p22 HBP, was purified from mouse liver cytosol, using blue Sepharose CL-6B. We identified a cDNA encoding p22 HBP, and sequence analysis revealed that p22 HBP comprises 190 amino acid residues (M r 21,063) and has no homology to any other known heme-binding protein.

To investigate the involvement of peripheral-type benzodiazepine receptors (PBR) in heme metabolism, we examined the interaction of [55Fe]heme with PBR. Transfection of the cloned mouse PBR-isoquinoline carboxamide-binding protein (PBR/IBP) cDNA into monkey kidney Cos-1 cells resulted in a 2.5-fold increase in [55Fe]hemin binding sites, concomitant with the increase in [3H]PK11195 binding sites, as compared with those seen in antisense PBR/IBP cDNA-transfected cells. The binding of hemin to the transfected receptors exhibited a relatively high affinity with a Kd of 12 nM, and was inhibited by several benzodiazepine ligands, including PK11195, Ro 5-4864, diazepam and protoporphyrin IX. When mouse liver mitochondria were incubated with [55Fe]hemin, the binding to PBR had a Kd of 15 +/- 1.8 nM. The Bmax of [55Fe]hemin binding to the mitochondria was 6.88 +/- 0.76 pmol/mg of protein, a value consistent with that of [3H]PK11195 binding, with a lower affinity. Coproporphyrinogen III, a precursor porphyrin produced in the cytosol, is translocated into mitochondria, then is converted to protoporphyrinogen IX; this conversion decreased in the presence of benzodiazepine ligands. To examine whether this decrease was related to a decrease in the binding of coproporphyrinogen to the mitochondria, the effects of benzodiazepines on the binding of coproporphyrinogen were examined. As the binding was dose-dependently inhibited by PK11195, Ro 5-4864, and diazepam, porphyrins are likely to be endogenous ligands for PBR. We propose that PBR play a role in the intracellular transport of porphyrins and heme.

The identification of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. Previous studies demonstrated that CD34− severe combined immunodeficiency (SCID)-repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34+CD38− SRCs. However, the purification level of rare CD34− SRCs in 18 lineage marker-negative (Lin−) CD34− cells (1/1000) is still very low compared with that of CD34+CD38− SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a high degree of purification of rare human CD34− SRCs. Using 18Lin−CD34− cells, we analyzed the expression of candidate positive markers by flow cytometric analysis. We finally identified CD133 as a reliable positive marker of human CB-derived CD34− SRCs and succeeded in highly purifying primitive human CD34− HSCs. The limiting dilution analysis demonstrated that the incidence of CD34− SRCs in 18Lin−CD34−CD133+ cells was 1/142, which is the highest level of purification of these unique CD34− HSCs to date. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin−CD34− cells, as well as 18Lin−CD34+ cells, in their positive fractions, indicating its functional significance as a common cell surface maker to isolate effectively both CD34+ and CD34− SRCs.

Exposure of HeLa and HL60 cells to sodium arsenite or cadmium chloride led to marked increases in cellular heme oxygenase activity. SDS-polyacrylamide gel electrophoresis of P%]methionine-labeled cellular proteins indicated that these treatments also resulted in the induction of a 32-kDa protein. Immunoblot analysis further showed that the 32-kDa protein reacted with anti-bovine heme oxygenase antibodies. Treatment of the cells with cobaltic chloride or heat induced neither the 32-kDa protein nor heme oxygenase activity. It is concluded that the 32-kDa stress protein induced by arsenite and cadmium ions in these human cells is heme oxygenase.

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To investigate the role of the iron-sulphur cluster in mammalian ferrochelatases, the terminal enzyme of the haem biosynthetic pathway, we examined the interaction of nitric oxide (NO) and ferrochelatase. When macrophage cell line RAW 264.7 cells were treated with interferon-gamma and lipopolysaccharide NO synthesis in the cells was stimulated, and a decrease in ferrochelatase activity was observed, with no change in the amount of ferrochelatase. The addition of NG-monomethyl-L-arginine, a selective inhibitor of NO synthesis, reduced the effect of interferon-gamma and lipopolysaccharide, while the effect of NG-monomethyl-L-arginine was suppressed by the addition of L-arginine, a substrate of NO synthase. When purified recombinant human ferrochelatase was treated with 3-morpholinosydnonimine, a NO-generating compound, ferrochelatase activity decreased with disappearance of characteristic absorbance spectra of the iron-sulphur cluster. S-Nitroso-N-acetylpenicillamine also reduced the activity, in a dose-dependent manner. These results indicate that ferrochelatase activity can be modulated by NO synthesis probably through disruption of the iron-sulphur cluster. We propose that inactivation of ferrochelatase mediated by NO (or NO-derived species) may play a role in the regulation of haem metabolism.

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