|Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1991: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1990: ¥1,200,000 (Direct Cost: ¥1,200,000)
In the present study we intended to clarify the relation between structure and function of heme oxygenase and its insertion mechanism to microsomal membranes. As a result we obtained the following interesting findings.
1. A 28 kDa-tryptic peptide of heme oxygenase obtained after solubilization of rat liver microsomes by mild trypsin treatment was purified. The tryptic peptide, like the native heme oxygenase, readily bound with substrate heme forming a hemeprotein transiently. The absorption spectra of the ferric, ferrous, ferrous-CO and ferrous-O_2 forms of the resulting complex resembled those of the corresponding forms of the complex of heme and the native enzyme. Ferric heme bound to the tryptic peptide was quantitatively decomposed to biliverdin on incubation with a mixture of ascorbic acid and desferrioxamine, indicating that the tryptic peptide still retained catalytic activity. These observations suggest that heme oxygenase has two domains, a hydrophilic and a hydrophobic domain,
and that the two domains are folded almost independently of each other.
2. A plasmid, pKK-RHO, was constructed by incorporating the coding sequence of a cDNA for rat heme oxygenase into the expression vector pKK233-2. Escherichia coli strain XLl-blue transformed with pKK-RHO produced a catalytically active, full-length heme oxygenase. The 32-kDa native enzyme expressed, was localized in the bacterial membranes, possibly due to the spontaneous membrane-binding properties of a hydrophobic segment in its C-terminal region. During cultivation, a few degraded forms of heme oxygenase that had lost their membraneassociative properties appeared. A 30-kDa polypeptide, one of the degraded forms of heme oxygenase, retained ability to accept electrons from NADPH-cytochrome P450 reductase and also activity for catalyzing breakdown of heme to biliverdin.
3. A truncated, soluble, and enzymatically active rat heme oxygenase lacking its membraneassociative, C-terminal segment was expressed in E. coli strain JM109. The roles of its four histidine residues were examined by determining the enzymatic activities of mutant enzymes in which each of these residues in turn was replaced by alanine. Mutation of histidine residue 25 to alanine resulted in marked decrease in activity for heme breakdown, indicating that this histidine residue has an important role in the heme oxygenase reaction.
4. We established an expression system of rat heme oxygenase in COS cells. Expressed native and truncated enzymes were located in microsome and cytosol, respectively, suggesting that the C-terminal region is important for insertion to microsomal membranes. We are now trying to determine amino acid sequence essential for binding. Less