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Some of the unusual molecular and catalytic properties of a high molecular weight dihydro-orotate dehydrogenase (DHOD) from Neurospora crassa have been determined. Comparison of the properties of this enzyme with the properties of the soluble biosynthetic enzyme of prokaryotes has revealed several important differences. The fungal enzyme is located in a mitochondrial membrane in a position consistent with linkage with the respiratory chain through ubiquinone (Miller, R. W.: Arch. Biochem, Biophys. 146, 256-270 (1971)). Release of the enzyme from the membrane results in a solubilized protein complex containing bound lipids and inactive hydrophobic proteins. Non-specific protein aggregation is minimized during purification by Triton-X-100 and phospholipase treatments. The catalytically active enzyme has an apparent molecular weight of 210 000. In contrast to soluble DHOD preparations the high molecular weight enzyme has no endogenous dihydro-orotate oxidase (EC 1.3.3.1) activity and is relatively insensitive to inactivation by sulfhydryl-reactive reagents in the presence of dihydro-orotate (DHO). The enzyme activity is highly sensitive to conditions causing oxidation of flavin mononucleotide (FMN). The activity cannot be restored by cysteine or other means. FMN is present in all purified preparations in a bound, non-fluorescent (reduced) form until dihydro-orotic acid is removed or oxidized. Catalytic efficiency of the purified enzyme was 12 000 mol DHO oxidized per minute per mole FMN. This high turnover rate is due in part to the small flavin content of the purified enzyme, equivalent to 1 mol FMN per 120 000 g of catalytically active protein. Iron was detected in the purified enzyme by atomic absorption spectroscopy but labile sulfide was absent. Thenoyltrifluoroacetone, an iron chelator, only partially inhibited DHO oxidation regardless of electron acceptor. Fatty acids interact with a hydrophobic site of the enzyme in non-competitive fashion but under certain conditions appear to significantly alter the Km for ubiquinone. Orotate, by comparison, is a purely competitive inhibitor. Both types of inhibitor may function to regulate the biosynthesis of orotate in vivo. Superoxide anion is not produced in significant quantities by the DHO-reduced enzyme unless both ubiquinone and a suitable single electron carrier such as phenazine methosulfate are present. DHOD has been proposed as a source of superoxide anion in mammalian mitochondria (Forman, H. J. & Kennedy, J. A.: J. Biol. Chem. 250, 4322-4326 (1975)).
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PMID:A high molecular weight dihydro-orotate dehydrogenase of Neurospora crassa. Purification and properties of the enzyme. 13 Jan 99

F1-ATPase isolated from rat liver mitochondria has been found to contain approximately 1 mole of FAD and 6 g atoms of nonheme iron per mole of enzyme.
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PMID:Rat liver mitochondrial F1-ATPase, an FAD containing ferroprotein. 15 59

Soluble extracts of Plectonema boryanum have been shown to contain a single, electrophoretically distinct, superoxide dismutase. The enzyme has been isolated and has been found to be an iron-containing enzyme similar to that described from the periplasm of Escherichia coli. It contains 1 Fe3+/mole of enzyme. The molecular weight was approximately 36 500, and the enzyme appeared to be composed of two subunits of equal size joined by non-covalent interactions. ESR data are presented, as are the results of amino acid analysis.
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PMID:The purification and properties of superoxide dismutase from a blue-green alga. 16 32

Two proteins (P1 and P2, with weights of 57,500 and 27,500 respectively) were isolated from Euglena gracilis. Both proteins show cyanide-insensitive superoxide dismutase activity in the "classical" superoxide dismutase assay, using xanthine-xanthine oxidase as O2.- generator. If O2.- is generated chemically (autoxidation of reduced anthraquinone), photochemically (illuminated riboflavine) or pulse radiolytically, only protein P1 but not P2 shows SOD activity. Protein P1 contains 1 g atom (determined: 0.82) iron (no Mn or Cu) per mole protein and may thus be defined as iron-superoxide dismutase. Protein P2, showing the spectral properties of a flavoprotein, exhibits the activities of ferredoxin-NADP-oxidoreductase and "diaphorase". The cyanide-insensitive SOD-activity of this Diaphorase" in the xanthine oxidase-assay for superoxide dismutase makes this classical and commonly used test unreliable for assay cyanide insensitive SOD activities. The existence of the "prokaryote-type" of superoxide dismutase (Fe-SOD) in Euglena gracilis is exceptional for an eukaryotic, autotrophically grown organisms.
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PMID:Cyanide insensitive iron superoxide dismutase in Euglena gracilis. Comparison of the reliabilities of different test systems for superoxide dismutases. 22 43

Two techniques have been applied to the determination of the number and type (2-Fe, 4-Fe) of iron-sulfur centers in the iron-sulfur flavoprotein succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1]. One procedure uses p-CF3C6H4SH as an extrusion reagent and Fourier transform 19F nuclear magentic resonance as the method of detection and quantitation of extruded cores of these centers in the form of [Fe2S2(SRF)4]2- and [Fe4S4(SRF)4]2- (RF = p-C6H4CF3). The second procedure, interprotein core transfer, involves thiol displacement of iron-sulfur cores followed by specific core transfer to the apoproteins of Bacillus polymyxa ferredoxin and adrenodoxin. Detection and quantitation are accomplished by electron paramagnetic resonance of reduced proteins at low temperatures. Both procedures clearly show that succinate dehydrogenase contains two dimeric (Fe2S2) and one tetrameric (Fe4S4) centers per mole of histidyl flavin, accounting for all eight nonheme iron and eight labile sulfur atoms found by chemical analysis. These results remove uncertainties created by the less than stoichiometric amounts of binuclear centers detected by electron paramagnetic resonance after dithionite reduction and provide secure characterization of the iron-sulfur centers in this enzyme.
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PMID:Characterization of the iron-sulfur centers in succinate dehydrogenase. 22 82

The Co- and Ru-substituted derivatives of adrenal iron-sulfur protein (adrenodoxin) were prepared from its apoprotein in the presence of urea, dithiothreitol, Na2S, and metal ions. Both metal-substituted proteins had 2 g-atoms each of metal and labile sulfur per mole of protein. The Co derivative had optical absorption maxima at 257, 264, 470, and 1430 nm with shoulders at 275, 280, 300, and 380 nm. The molar extinction coefficient per Co atom was 2.200 M-1 cm-1 at 470 nm. The Ru derivative had a broad maximum at 500 nm with a molar extinction coefficient of approximately 100 M-1 cm-1 per Ru atom. The visible chromophore of the Co- and Ru-substituted proteins with mercurials revealed that the saturation levels are 8.6 and 8.4 mol of mercurial/mol of protein. The values agree with that of the native protein within experimental errors. The tyrosyl residue at position 82 displayed a broad anomalous emission at 335 and 331 nm for the Co- and Ru-substituted proteins, respectively, as well as in the case of the native protein. There was no electron paramagnetic resonance signal of the Co derivative in a wide magnetic field at 77 degrees K. Additionally, the Co and Ru derivatives had no enzymatic activity toward NADPH-cytochrome c reduction in the presence of adrenal diaphorase (adrenodoxin reductase). There was no indication that Mn, Ni, Cu, and Os are incorporated into the apoprotein in the presence of urea. Incorporation of Fe into the protein was examined in the presence of Co or Ru. In a system containing both Fe and Ru, Fe was exclusively incorporated into the protein. In contrast to this, the reaction products from a system containing both Fe and Co were found to consist of both Fe and Co derivatives at approximately equimolar quantity.
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PMID:Cobalt and ruthenium replacement for iron in adrenal iron-sulfur protein (adrenodoxin). Preparation and some properties. 23 19

Yeast aconitase [citrate (isocitrate) hydro-lyase, ED 4.2.1.3], inductively formed by Candida iipolytica in the presence of fluoroacetate, was purified approximately 100-fold by Sephadex G-100 gel filtration and DEAE-Sephadex column chromatography, yielding dark-brown needle crystals. The crystalline aconitase was homogenious as judged by polyacrylamide gel electrophoresis and sedimentation by ultracentrifugation. The enzyme showed maximal activity at pH 8.0 and at 55 degrees. It has an S20, W of 5.03 S, a molecular weight of 68,500 and an isolectric point of pH 4.2. The presence of 2.10 moles of iron per mole of the enzyme was demonstrated by atomic absorption spectroscopy.
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PMID:The aconitase of yeast. II. Crystallization and general properties of yeast aconitase. 23 89

Using the technique of affinity chromatography on a myo-inositol-substituted Sepharose, the myo-inositol oxygenase from rat kidneys was purified to homogeneity. The active enzyme contains iron, most probably in its divalent form. Electrophoresis on polyacrylamide gel containing sodium dodecylsulphate causes the cleavage of the enzyme protein into apparently identical subunits with a molecular weight of approximately 17,000. The smallest active unit consists of 4 subunits, and is in a pH-dependent equilibium with species consisting of 8, 12, and 16 subunits, respectively, which all show the same specific enzyme activity. In the presence of oxygen the enzyme is highly unstable; at the early stages of inactivation it can be reactivated by reducing agents like NaBH4. Under anaerobic conditions or under the influence of Fe2-chelating agents, the enzyme is also inactivated; this inactivation is caused by the loss of iron and concomitant cleavage into the subunits. It can be reversed by incubation with FeSO4 in the presence of air. If myo-inositol and FeSO4 are present, the reactivation involves an oligomerization to the species with 16 subunits with the uptake of 8 gram-atoms of iron per mole of this species. The enzyme reaction follows Michaelis-Menten kinetics; the Michaelis constants are 4.5 x 10(-2)M for myo-inositol and 9.5 x 10(-6)M for oxygen.
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PMID:myo-Inositol oxygenase from rat kidneys. I: Purification by affinity chromatography; physical and catalytic properties. 43

The molar absorbance of cyanmethemoglobin at 540 nm was measured in 35 species of mammals, birds, and reptiles. Values obtained were compared with those measured on human cyanmethemoglobin by the same procedure. There was no significant difference (P greater than or equal to 0.1) between the human and animal blood values observed in 29 samples. In six cases (4 mammals, 1 reptile, and 1 bird), the significant difference (P greater than or equal to 0.05) did not exceed 5% of the human value. The practice of applying the accepted absorbance of 1.10 x 10(4) M-1 cm-1 per mole of iron in human cyanmethemeglobin for the analysis of animal blood will not cause more than 5% uncertainty. Such results are satisfactory for use in clinical veterinary medicine except for those research projects concerned with animal hemoglobin function and structure.
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PMID:Molar absorbance of cyanmethemoglobin from blood of different animals. 47 42

Myoglobin (Mb) was isolated from canine skeletal muscle by a novel heat denaturation-gel filtration-ion exchange chromatography procedure. The purified major Mb was homogeneous by gel electrophoretic and ultracentrifugal analysis, and the sedimentation coefficient at infinite dilution (S degrees 20, w) was 1.9 S. The molecular weight by sedimentation equilibrium was 1.72 X 10(4) and was essentially identical with the values by the iron analysis (1.80 X 10(4) and the amino acid composition (1.78 X 10(4). The spectroscopic properties of deoxy-, oxy-, carbonmonoxy- and met-derivatives of the Mb were determined in ultraviolet, Soret and visible regions. The pK' of acid-alkaline transition of the met-Mb was estimated as 8.80+/-0.04 (25 degrees) from the pH-dependent spectral change. The oxygen equilibrium studies revealed complete absence of such allosteric properties as heme-heme interaction, anion effect and the Bohr effect which were always present in normal mammalian hemoglobins. Oxygen tension for the half-oxygenation was 0.48 mmHg (20 degrees) and its temperature-dependent change gave the delta H degrees of -15.7 Kcal/mole.
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PMID:[A study on the physiological basis of training effect--with special reference to myoglobin. I. Isolation and properties of myoglobin from dog skeletal muscle (author's transl)]. 54 71


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