Purification and properties of heme oxygenase from rat liver microsomes.

Heme oxygenase was purified to apparent homogeneity from liver microsomes of rats which had been treated with either cobaltous chloride or hemin to induce heme oxygenase in the liver and the purified preparations from either rats showed an apparent molecular weight of about 200,000 when estimated by gel filtration on a column of Sephadex G-200, and gave a minimum molecular weight of about 32,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hepatic heme oxygenase could bind heme to form a heme . heme oxygenase complex showing an absorption peak at 405 nm, and the extinction coefficient at 405 nm of the heme . heme oxygenase complex was 140 mM-1 cm-1. The heme bound to the hepatic heme oxygenase protein was easily converted to biliverdin when the complex was incubated with the NADPH-cytochrome c reductase system in air. The hepatic heme oxygenase appears to have characteristics essentially similar to those of the splenic heme oxygenase (Yoshida, T., and Kikuchi, G. (1978) J. Biol. Chem. 253, 4224 and 4230). The heme oxygenase preparation which was purified from the cobalt-treated rats contained a small amount of cobaltic protoporphyrin, indicating that cobalt protoporphyrin was synthesized in these rats.     

Structural studies on bovine spleen heme oxygenase. Immunological and structural diversity among mammalian heme oxygenase enzymes.

Heme oxygenase is an Mr 32,000 microsomal enzyme which catalyzes the rate-limiting step in the oxidative catabolism of heme to yield equimolar quantities of biliverdin IX alpha, carbon monoxide, and iron. In the present investigation, evidence is presented suggesting that immunochemical and structural differences exist between bovine spleen heme oxygenase and heme oxygenase enzymes from other mammalian species. Using an antibody directed against bovine spleen heme oxygenase, enzyme-linked immunosorbent assays, Western blotting experiments, and cell-free translation immunoprecipitation studies showed that bovine spleen heme oxygenase is only weakly immunochemically related to heme oxygenase from rat spleen. This observation was supported by the fact that a rat spleen heme oxygenase cDNA probe did not hybridize significantly to bovine spleen heme oxygenase mRNA in Northern analyses nor to restriction fragments containing the bovine heme oxygenase gene in Southern analyses. Tryptic peptides were prepared from bovine spleen heme oxygenase and the amino acid sequences of nine peptides comprising 94 amino acid residues were determined, providing the first information on the primary structure of bovine spleen heme oxygenase. Comparison of the sequences of these tryptic peptides with regions of the deduced amino acid sequences of rat spleen and human macrophage heme oxygenase revealed sequence similarities ranging from 55 to 100%. Several peptides displaying the highest degree of sequence similarity were found to occur in regions of the heme oxygenase molecule postulated to contain the heme binding site, indicating that despite the immunochemical and apparent structural differences between bovine spleen heme oxygenase and the rat and human enzymes, functionally important amino acid residues have been conserved in the evolution of mammalian heme oxygenase genes.     

Solubilization and partial purification of heme oxygenase from rat liver.

Hepatic microsomal heme oxygenase was solubilized, partially purified, and characterized from Co2+-treated rats. The enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibited a minimum molecular weight of greater than or equal to 68,000. The solubilized enzyme was totally devoid of contamination with cytochrome P-450 or b5. The requirement for reduced pyridine nucleotides was absolute, and ascorbate could not support heme oxidative activity. However, both TPNH and DPNH could serve as electron donors, with TPNH being more effective. The presence of an appropriate flavoprotein reductase was essential for heme oxidation. The enzyme had an apparent Km of 40 micrometer, a pH optimum of 7.5, and lost substantial activity upon freezing and thawing. Methemoglobin was 30% as effective a substrate for the enzyme as was heme. Free porphyrins could not serve as substrates for the enzyme. The activity of the enzyme was inhibited by HgCl2, p-chloromercuribenzoate, iodoacetamide, mercaptoethanol, and dithiothrietol indicating that free -SH group(s) is necessary for enzyme activity.     

Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform

Recently we reported on the presence of two isoforms of heme oxygenase in rat liver microsomes, referred to as HO-1 and HO-2, and that only HO-1 is inducible (Maines, M. D., Trakshel, G. M., and Kutty, R. K. (1986) J. Biol. Chem. 261, 411-419). Presently we report on the detection of two isoforms of the enzyme in rat testis and purification to near homogeneity of the noninducible isoform, HO-2. A comparative characterization of the liver HO-1 and the testicular HO-2 is also provided. The relative abundance of the isoforms in the two organs was dissimilar. In the testis, the predominant form was HO-2, and only minute amounts of HO-1 were detected. In the liver, however, a 1:2 ratio of HO-1 to HO-2 was noted. The activity of HO-2 in both organs was refractory to cadmium, an inducer of the hepatic HO-1. Under nondenaturing electrophoresis conditions, HO-2 showed a higher mobility than HO-1; on a sodium dodecyl sulfate-polyacrylamide gel, HO-2 displayed a higher monomeric Mr. The apparent Mr values for HO-2 and HO-1 were 36,000 and 30,000, respectively. The isoforms differed in immunochemical properties. Antiserum to the liver HO-1 did not recognize the testicular HO-2 when examined by double immunodiffusion or by Western immunoblotting. HO-2 was more sensitive to heat inactivation than HO-1. When exposed at 65 degrees C (10 min), 70% of HO-1 activity was retained; however, nearly 80% of HO-2 activity was lost. The apparent Km values for heme for HO-1 and HO-2 were 0.24 and 0.40 microM, respectively. HO-1 and HO-2 had similar requirements for cofactor and flavoprotein reductase and were inhibited by heme-ligands (CO, KCN, NaN3). HO-2 utilized as substrate, Fe-protoporphyrin, Fe-hematoporphyrin, and Fe-hematoporphyrin acetate; it did not degrade intact purified rat liver cytochromes b5 and P-450 LM2, catalase, cytochrome c, hemoglobin, or myoglobin.     

Purification and characterization of an alpha-ketoisocaproate oxygenase of rat liver

Rat liver contains a cytosolic alpha-ketoisocaproate oxygenase which oxidatively decarboxylates and hydroxylates alpha-ketoisocaproate to form beta-hydroxyisovalerate. This oxygenase was purified to near homogeneity. The oxygenase is unstable during purification, unless 5% monothioglycerol is added. The purified enzyme is stable in the presence of 5% monothioglycerol for 3 weeks at 4 degrees C and at least 10 weeks at -80 degrees C. The molecular weight of the alpha-ketoisocaproate oxygenase as determined to be 46,000 and 51,000 using denaturing and nondenaturing conditions, respectively, indicating a monomer. The alpha-ketoisocaproate oxygenase requires Fe2+; other metal ions did not replace Fe2+. Ascorbate activates the enzyme at subsaturating levels of Fe2+, by regenerating Fe2+. The activity is markedly affected by the type of buffer used. For example, the oxygenase activity increased 2- to 3-fold when 0.1 M maleate was used. Iron chelators, such as ADP and EDTA, are inhibitory. The ratio of decarboxylation of 1 mM alpha-[1-14C] ketoisocaproate (as measured by 14CO2 release) to decarboxylation of 1 mM alpha-[1-14C]keto-gamma-methiolbutyrate is 1.0 for all purification fractions, indicating that a single enzyme catalyzes the decarboxylation of both substrates. The apparent Km and Vmax values of the alpha-ketoisocaproate oxygenase using optimized assay conditions are 0.32 mM and 130 nmol/min/mg of protein for alpha-ketoisocaproate and 1.9 mM and 247 nmol/min/mg of protein for alpha-keto-gamma-methiolbutyrate. The principal product of the purified alpha-ketoisocaproate oxygenase, using alpha-ketoisocaproate as a substrate, is beta-hydroxyisovalerate, although small amounts of a compound, which has the chromatographic properties of isovalerate, are also produced.     

Comparison of liver mixed-function oxygenase and antioxidant enzymes in vertebrates

We performed a comparative analysis of cytochrome P450, cytochrome b5, MFO associated enzymes and cytosolic antioxidant enzymes in hepatic microsomes and cytosolic fractions prepared from five animal species representing three vertebrate classes living in tropical conditions (Brazil). The data obtained show that rats have higher hepato-somatic index, specific cytochrome b5 concentration, and NADPH-dependent cytochrome c (P450) activity compared to ectothermic species, SOD activity similar to those in amphibians, and specific concentration of cytochrome P450 and catalase activity lower than in a toad, but higher than in fishes and a frog. Our data indicate that tropical fishes may have reduced xenobiotic-metabolizing ability compared to the rat and amphibians. In contrast to fish and rat, amphibians have a low ratio (< 0.5) of cytochrome b5 concentration to that of P450. Most species showed cytochrome b5 sensitivity to oxygen. Thus, the use of sodium dithionate as a reducer, rather than NADPH, may be preferential in b5 determinations.     

Purification and characterization of the enzymes of bile acid conjugation from fish liver

1. The two steps in bile acid conjugation have been studied in subcellular fractions of liver from three species of fish; vermillion rockfish, canary rockfish and ling codfish. 2. The bile acid: coenzyme A (CoA) ligase activity in homogenates and isolated microsomes is undetectable due to indeterminate factors. 3. A purification scheme is presented which eliminates the interfering factors. The purified ligase was found to have a lower affinity for bile acids as compared to the mammalian form and to be present in much lower titer. 4. Since it appears to be the rate controlling enzyme in all species, it is expected that the rate of bile acid conjugation is much slower in non-mammalian liver as compared to mammalian liver. 5. The bile acid-CoA:taurine N-acyltransferase was found to exist as a dimer of molecular weight 100,000, in contrast to the monomeric mammalian forms. 6. The only major kinetic difference is that the fish liver forms have rates of glycine conjugation which are only 1-2% of the rate with taurine, in part due to a very high Km for glycine.     

Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia

An enzymatic system has been isolated that catalyzes dihydroxylation of phthalate to form 1,2-dihydroxy-4,5-dicarboxy-3,5-cyclohexadiene with consumption of NADH and O2. This system is comprised of two proteins: a flavo-iron-sulfur protein with NADH-dependent oxidoreductase activity and a nonheme iron protein with oxygenase activity. Phthalate oxygenase is a large (approximately 217 kDa) protein composed of apparently identical 48-kDa monomers. The active enzyme has one Rieske-type [2Fe-2S] center and one mononuclear iron/monomer. Removal of the mononuclear iron by incubation with EDTA or with o-phenanthroline inhibits oxygenation; ferrous ion completely restores activity. No other metals are effective. Phthalate oxygenase is specific for phthalate or other closely related compounds. However, only phthalate is tightly coupled to NADH oxidation and O2 consumption with a stoichiometry of 1:1:1. Phthalate oxygenase is chemically competent to oxygenate phthalate when artificially supplied with reducing equivalents and O2. Phthalate oxygenase reductase is required, however, for efficient catalytic activity. The reductase is a monomeric 34-kDa flavo-iron-sulfur protein containing FMN and a plant-ferredoxin-type [2Fe-2S] center in a 1:1 ratio. Phthalate oxygenase reductase is specific for NADH but can pass electrons to a variety of acceptors, including: phthalate oxygenase, cytochrome c, ferricyanide, and dichlorophenolindophenol. This system is similar to other bacterial oxygenase systems involved in aromatic degradation including: benzoate dioxygenase, toluene dioxygenase, benzene dioxygenase, and 4-methoxybenzoate demethoxylase. However, phthalate oxygenase can be isolated in large quantities and is more stable than most other such systems.     

Crystal structure of heme oxygenase from the gram-negative pathogen Neisseria meningitidis and a comparison with mammalian heme oxygenase-1

We report the crystal structure of heme oxygenase from the pathogenic bacterium Neisseria meningitidis at 1.5 A and compare and contrast it with known structures of heme oxygenase-1 from mammalian sources. Both the bacterial and mammalian enzymes share the same overall fold, with a histidine contributing a ligand to the proximal side of the heme iron and a kinked alpha-helix defining the distal pocket. The distal helix differs noticeably in both sequence and conformation, and the distal pocket of the Neisseria enzyme is substantially smaller than in the mammalian enzyme. Key glycine residues provide the flexibility for the helical kink, allow close contact of the helix backbone with the heme, and may interact directly with heme ligands.     

Purification and characterization of avian liver mevalonate-5-pyrophosphate decarboxylase

Mevalonate-5-pyrophosphate decarboxylase [ATP:5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33] has been purified 5800 times from chicken liver and obtained in a stable and highly purified form. The protein is a dimer of molecular weight 85400 +/- 1941, and its subunits were not resolved by gel electrophoresis in denaturing conditions. The purified enzyme does not require the presence of SH-containing reagents for either activity or stability. The enzyme shows a high specificity for adenosine 5'-triphosphate (ATP) and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 4.0 to 6.5. Inhibitory effects for the enzyme activity were detected by citrate, phthalate, and phosphate. The isoelectric point, as determined by column chromatofocusing, is 4.8. The kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.0141 mM and 0.504 mM have been obtained for mevalonate-5-pyrophosphate and ATP, respectively.     

Purification and characterization of diacetyl-reducing enzymes from Staphylococcus aureus.

A method was developed to purify diacetyl-reducing enzymes from Staphylococcus aureus. Two enzymes capable of catalysing diacetyl reduction were isolated, neither of which turned out to be a specific diacetyl reductase. One of them is a lactate dehydrogenase similar to the one from Staphylococcus epidermidis, which accepts diacetyl, although poorly. The other one uses as coenzyme beta-NAD and reduces uncharged alpha-dicarbonyls with more than three carbon atoms (especially the alpha-diketones diacetyl and pentane-2,3-dione), producing the L(+) form of the corresponding alpha-hydroxycarbonyls. This enzyme has an Mr of 68,000 and is, most probably, a monomer. Its optimum pH is 6.0. Its shows a high affinity for NADH and a rather low one for diacetyl, which, at least in vitro, does not seem to be as good a substrate as pentane-2,3-dione. We propose for it the systematic name L-alpha-hydroxyketone:NAD+ oxidoreductase and the recommended name of alpha-diketone reductase (NAD). We also suggest that the diacetyl reductase entry in the I.U.B. classification be suppressed.     

Degradation of heme by a soluble peptide of heme oxygenase obtained from rat liver microsomes by mild trypsinization.

A tryptic peptide of heme oxygenase obtained after solubilization of rat liver microsomes by mild trypsin treatment was purified. The purified peptide gave only a single protein band with a molecular mass of 28 kDa on SDS/PAGE. 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-O2 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. An NADPH-cytochrome-P-450 reductase system composed of NADPH and detergent-solubilized NADPH-cytochrome-P-450 reductase readily reduced the ferric heme bound to the tryptic peptide, but failed to transfer the second electron required for rapid heme degradation, suggesting that the hydrophobic domain of heme oxygenase is important for receiving the second electron from the reductase.     

Expression of heme oxygenase and its RNA in mouse liver after injection of heme and splenectomy.

Heme is known to activate the HO (heme oxygenase) gene in cultured cells, but little is known about the effect of heme on the HO gene in intact organisms. The expressions of HO and its RNA in mouse liver were measured using mouse HO cDNA and HO antibody after injection of heme or splenectomy. The antibody was prepared against a beta-galactosidase-HO hybrid protein made in Escherichia coli. The HO mRNA level increased to a maximum 15 h after heme injection. In contrast, expression of HO was maximal about 45 h after heme injection. Essentially the same results were obtained in mice after splenectomy. These results suggest that the HO gene in mouse liver was activated by the injection of heme and splenectomy.     

Purification and characterization of ubiquitin from mammalian testis

Ubiquitin was extracted from testis of 4 mammals and purified to homogeneity by gel filtration chromatography. Amino acid compositions and NH2-terminal sequences were found to be identical in the 4 species and with calf thymus ubiquitin. Ubiquitin conformation was shown to be very sensitive to oxidation. Improved methods for radioimmunoassay of ubiquitin in tissue extracts are also discussed.     

Crystal structure of the dioxygen-bound heme oxygenase from Corynebacterium diphtheriae: implications for heme oxygenase function

HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen association and dissociation rate constants are 5 microm(-1) s(-1) and 0.22 s(-1), respectively, yielding an O(2) affinity of 21 microm(-1), which is approximately 20 times greater than that of mammalian myoglobins. However, the affinity of HmuO for CO is only 3-4-fold greater than that for mammalian myoglobins, implying the presence of strong hydrogen bonding interactions in the distal pocket of HmuO that preferentially favor O(2) binding. Resonance Raman spectra show that the Fe-O(2) vibrations are tightly coupled to porphyrin vibrations, indicating the highly bent Fe-O-O geometry that is characteristic of the oxy forms of heme oxygenases. In the crystal structure of the oxy form the Fe-O-O angle is 110 degrees, the O-O bond is pointed toward the heme alpha-meso-carbon by direct steric interactions with Gly-135 and Gly-139, and hydrogen bonds occur between the bound O(2) and the amide nitrogen of Gly-139 and a distal pocket water molecule, which is a part of an extended hydrogen bonding network that provides the solvent protons required for oxygen activation. In addition, the O-O bond is orthogonal to the plane of the proximal imidazole side chain, which facilitates hydroxylation of the porphyrin alpha-meso-carbon by preventing premature O-O bond cleavage.     

Purification and characterization of N-acetylglucosaminyl sulfotransferase from chick corneas

Sulfation is an important conjugation pathway in deactivating thyroid hormones, keeping the proper hormonal balance, and increasing the rate of thyroid hormone metabolism. We have identified, cloned, and characterized a sulfotransferase (SULT) that is capable of thyroid hormone conjugation in the dog. This enzyme, designated cSULT1B1, displays a strong identity (>84%) to the human ST1B2 enzyme. However, cSULT1B1 displays less identity, about 73%, to mouse and rat orthologs. In addition, the canine enzyme is three amino acids shorter than the rodent ones but has the same length as the human ortholog, 296 amino acids. The bacterial expressed and partial purified cSULT1B1 enzyme sulfates p-nitrophenol and 1-naphtol, but not dopamine. The thyroid hormones 3,3'-diiodothyronine and 3,5,3'-triiodothyronine are efficiently sulfated. 3,3',5'-Triiodothyronine is sulfated to lesser degree while sulfation of 3,5'-diiodothyronine and 3,3',5,5'-tetraiodothyronine cannot be detected. The cSULT1B1 is found in the colon (highest level), kidney and small intestine in dogs, but surprisingly not in the male dog liver although low levels of immunoreactivity were detected in the female dog liver. The male dog expresses more of SULT1B1 enzyme in the lower part of the small intestine while the female dog displays an opposite pattern of expression. These results describe the cloning and characterization of a canine thyroid hormone sulfating enzyme that is more closely related to the human ortholog than to the rodent thyroid sulfating enzymes.