Michihiko Sato

Crystal structure of rat biliverdin reductase.

Biliverdin reductase (BVR) is a soluble cytoplasmic enzyme that catalyzes the conversion of biliverdin to bilirubin using NADH or NADPH as electron donor. Bilirubin is a significant biological antioxidant, but it is also neurotoxic and the cause of kernicterus. In this study, we have determined the crystal structure of rat BVR at 1.4 Å resolution. The structure contains two domains: an N-terminal domain characteristic of a dinucleotide binding fold (Rossmann fold) and a C-terminal domain that is predominantly an antiparallel six-stranded β-sheet. Based on this structure, we propose modes of binding for NAD(P)H and biliverdin, and a possible mechanism for the enzyme.

Posttranslational and direct integration of heme oxygenase into microsomes

Rat liver heme oxygenase has a large cytoplasmically exposed domain containing the N-terminus that can be cleaved from the membranes by a low concentration of trypsin, indicating that heme oxygenase is embedded in membranes with an insertion sequence near its C-terminal portion. Heme oxygenase synthesized in a cell-free system or purified from microsomes after detergent-solubilization was integrated into microsomal membranes posttranslationally and directly, like cytochrome b5.

Functional expression of putative H+-K+-ATPase from guinea pig distal colon

A guinea pig cDNA encoding the putative colonic H+-K+-ATPase α-subunit (T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854 ) was functionally expressed in HEK-293, a human kidney cell line. The cDNA for the putative colonic H+-K+-ATPase was cotransfected with cDNA for either rabbit gastric H+-K+-ATPase or TorpedoNa+-K+-ATPase β-subunit. In both expressions, Na+-independent, K+-dependent ATPase (K+-ATPase) activity was detected in the membrane fraction of the cells, with a Michaelis-Menten constant for K+ of 0.68 mM. The expressed K+-ATPase activity was inhibited by ouabain, with its IC50 value being 52 μM. However, the activity was resistant to Sch-28080, an inhibitor specific for gastric H+-K+-ATPase. The ATPase was not functionally expressed in the absence of the β-subunits. Therefore, it is concluded that the cDNA encodes the catalytic subunit (α-subunit) of the colonic H+-K+-ATPase. Although the β-subunit of the colonic H+-K+-ATPase has not been identified yet, both gastric H+-K+-ATPase and Na+-K+-ATPase β-subunits were found to act as a surrogate for the colonic β-subunit for the functional expression of the ATPase. The present colonic H+-K+-ATPase first expressed in mammalian cells showed the highest ouabain sensitivity in expressed colonic H+-K+-ATPases so far reported (rat colonic in Xenopus oocytes had an IC 50 = 0.4-1 mM; rat colonic in Sf9 cells had no ouabain sensitivity).

Neonatal hyperbilirubinemia in Japanese neonates: Analysis of the heme oxygenase-1 gene and fetal hemoglobin composition in cord blood

Neonatal hyperbilirubinemia is frequent and severe in Japanese infants. Although the G71R mutation of the bilirubin uridine diphosphate-glucuronosyltransferase gene is associated with severe neonatal hyperbilirubinemia in this population, it accounts for only half of the neonates with severe hyperbilirubinemia. It was suggested that increased bilirubin production would also be associated with severe neonatal hyperbilirubinemia in Japanese infants. To elucidate the genetic factors causing severe hyperbilirubinemia in these patients, we studied two notable factors associated with bilirubin production: heme oxygenase-1, a key enzyme of heme metabolism, and fetal Hb composition, a factor possibly associated with heme load in neonates. We first determined the sequences of promoter and all coding regions of the heme oxygenase-1 gene in Japanese neonates who had undergone phototherapy, but found no mutation except for the polymorphic (GT)n repeats in the promoter region. These repeats modulate the transcription of the heme oxygenase-1 gene, and the longer repeat sequences are known to reduce the transcription. We detected a significant difference in the allele frequencies of each number of (GT)n repeats between Japanese and German populations. However, we could not find a relation between those polymorphisms and neonatal hyperbilirubinemia. We next analyzed the state of Hb switching of the γ- to β-globin chain and the phenotype of γ-globin chain isoforms in cord blood. We found no relation between fetal Hb composition and neonatal hyperbilirubinemia. Further studies are required to elucidate genetic or environmental factors in neonatal hyperbilirubinemia in Japanese infants.

Importance of histidine residue 25 of rat heme oxygenase for its catalytic activity

A truncated, soluble, and enzymatically active rat heme oxygenase lacking its membrane-associative, 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.

Imaging of oxygen gradients in monolayer cultured cells using green fluorescent protein

Gradients of Po(2) between capillary blood and mitochondria are the driving force for diffusional O(2) delivery in tissues. Hypoxic microenvironments in tissues that result from diffusional O(2) gradients are especially relevant in solid tumors because they have been related to a poor prognosis. To address the impact of tissue O(2) gradients, we developed a novel technique that permits imaging of intracellular O(2) levels in cultured cells at a subcellular spatial resolution. This was done, with the sensitivity to O(2) ≤3%, by the O(2)-dependent red shift of green fluorescent protein (AcGFP1) fluorescence. Measurements were carried out in a confluent monolayer of Hep3B cells expressing AcGFP1 in the cytoplasm. To establish a two-dimensional O(2) diffusion model, a thin quartz glass slip was placed onto the monolayer cells to prevent O(2) diffusion from the top surface of the cell layer. The magnitude of the red shift progressively increased as the distance from the gas coverslip interface increased. It reached an anoxic level in cells located at ∼220 μm and ∼690 μm from the gas coverslip boundary at 1% and 3% gas phase O(2), respectively. Thus the average O(2) gradient was 0.03 mmHg/μm in the present tissue model. Abolition of mitochondrial respiration significantly dampened the gradients. Furthermore, intracellular gradients of the red shift in mitochondria-targeted AcGFP1 in single Hep3B cells suggest that the origin of tissue O(2) gradients is intracellular. Findings in the present two-dimensional O(2) diffusion model support the crucial role of tissue O(2) diffusion in defining the O(2) microenvironment in individual cells.

Protein expressed by the ho2 gene of the cyanobacterium Synechocystis sp. PCC 6803 is a true heme oxygenase. Properties of the heme and enzyme complex.

Two isoforms of a heme oxygenase gene, ho1 and ho2, with 51% identity in amino acid sequence have been identified in the cyanobacterium Synechocystis sp. PCC 6803. Isoform‐1, Syn HO‐1, has been characterized, while isoform‐2, Syn HO‐2, has not. In this study, a full‐length ho2 gene was cloned using synthetic DNA and Syn HO‐2 was demonstrated to be highly expressed in Escherichia coli as a soluble, catalytically active protein. Like Syn HO‐1, the purified Syn HO‐2 bound hemin stoichiometrically to form a heme–enzyme complex and degraded heme to biliverdin IXα, CO and iron in the presence of reducing systems such as NADPH/ferredoxin reductase/ferredoxin and sodium ascorbate. The activity of Syn HO‐2 was found to be comparable to that of Syn HO‐1 by measuring the amount of bilirubin formed. In the reaction with hydrogen peroxide, Syn HO‐2 converted heme to verdoheme. This shows that during the conversion of hemin to α‐meso‐hydroxyhemin, hydroperoxo species is the activated oxygen species as in other heme oxygenase reactions. The absorption spectrum of the hemin–Syn HO‐2 complex at neutral pH showed a Soret band at 412 nm and two peaks at 540 nm and 575 nm, features observed in the hemin‐Syn HO‐1 complex at alkaline pH, suggesting that the major species of iron(III) heme iron at neutral pH is a hexa‐coordinate low spin species. Electron paramagnetic resonance (EPR) revealed that the iron(III) complex was in dynamic equilibrium between low spin and high spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components. These observations suggest that the structure of the heme pocket of the Syn HO‐2 is different from that of Syn HO‐1.

Histidine 20, the Crucial Proximal Axial Heme Ligand of Bacterial Heme Oxygenase Hmu O from Corynebacterium diphtheriae

The hemin complex of Hmu O, a 24-kDa soluble heme degradation enzyme in Corynebacterium diphtheriae, is coordinated axially to a neutral imidazole of a proximal histidine residue in Hmu O. To identify which of the eight histidines in Hmu O is the proximal heme ligand, we have constructed and expressed the plasmids for eight His → Ala Hmu O mutants. Reconstituted with hemin, the active site structures and enzymatic activity of these mutants have been examined by EPR, resonance Raman, and optical absorption spectroscopy. EPR of the NO-bound ferrous heme-Hmu O mutant complexes reveals His20 as the proximal heme ligand in Hmu O, and this is confirmed by resonance Raman results from the ligand-free ferrous heme-H20A. All eight His → Ala mutants bind hemin stoichiometrically, proving that none of the histidines is essential for hemin-Hmu O formation. However, His20 is crucial to Hmu O catalysis. Its absence by point mutation has inhibited the conversion of hemin to biliverdin. The ferric heme-H20A complex is pentacoordinate. Resonance Raman of the CO-bound ferrous heme-H20A corroborates this and reveals an Fe-C-O bending mode, δ(Fe-C-O), the first reported for a pentacoordinate CO-bound hemeprotein. The appearance of δ(Fe-C-O) in C. diphtheriae Hmu O H20A but not mammalian HO-1 mutant H25A indicates that the heme environment between the two heme oxygenases is different.

Primary structure of rat brain protein carboxyl methyltransferase deduced from cDNA sequence.

Two cDNA clones for protein carboxyl methyltransferase were isolated from a rat brain cDNA library in lambda gt 11 with synthetic oligonucleotides as probes. The two clones differ in size, but the nucleotide sequence including the whole coding region of the shorter cDNA is completely identical with the corresponding sequence of the longer cDNA. The open reading frame encodes a polypeptide of 227 amino acid residues, with a molecular weight of 24,626. This molecular weight is comparable to those reported for other protein carboxyl methyltransferases from several animals, which were determined by gel filtration chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Occurrence of differentiated keratin peptide(K1) in cultured human squamous cell carcinomas

To date, the largest keratin peptide(K1, 68 KD) has been absent in cultured human squamous cell carcinomas. Using a low salt aqueous solution, not containing high salt and Triton X-100, as a washing buffer for keratin extraction, followed by two dimensional polyacrylamide gel electrophoresis, immunological techniques and Northern blot analysis, we demonstrated K1 peptide in two kinds of cultured human squamous cell carcinomas. Until now keratin extraction has been done using high salt/Triton X-100 solution during which K1 peptide may be removed together developed an affinity with the buffer. Many investigators may have therefore overlooked K1.