Partial purification and characterization of a uracil DNA N-glycosidase from Bacillus subtilis.

A uracil specific DNA N-glycosidase activity has been partially purified from crude extracts of Bacillus subtilis. The enzyme has a molecular weight of approximately 24 000 with no subunit structure. It has no requirement for any known cofactors but is inhibited in the presence of Co2+, Fe2+, or Zn2+. The enzyme is specific for uracil in single- and double-stranded deoxyribonucleopolymers and does not release free uracil from RNA or from poly(rU):poly(dA). In addition, neither Udr, dUMP, nor dUTP is recognized as substrate. The enzyme will attack small poly(dU) oligomers but the minimum size recognized as substrate is (pU)4. This enzyme may have a role in the repair (by base excision) or uracil in DNA arising either by incorporation during DNA synthesis or by deamination of cytosine in DNA.     

Oxygen-based free radical generation by ferrous ions and deferoxamine

Deferoxamine accelerates the autooxidation of iron as measured by the rapid disappearance of Fe2+, the associated appearance of Fe3+, and the uptake of oxygen. Protons are released in the reaction. The formation of H2O2 was detected by the horseradish peroxidase-catalyzed oxidation of scopoletin, and the formation of hydroxyl radicals (OH.) was suggested by the formation of the OH. spin trap adduct (DMPO/OH). with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the generation of the methyl radical adduct on the further addition of dimethyl sulfoxide. (DMPO/OH). adduct formation was inhibited by catalase but not by superoxide dismutase. The oxidant formed converted iodide to a trichloroacetic acid-precipitable form (iodination) and was bactericidal to logarithmic phase Escherichia coli. Both iodination and bactericidal activity was inhibited by catalase and by OH. scavengers, but not by superoxide dismutase. Iodination was optimal in 5 x 10(-4) M acetate buffer, pH 5.0, and when the Fe2+ and deferoxamine concentrations were equimolar at 10(-4) M. Fe2+ could not be replaced by Fe3+, Co2+, Zn2+, Ca2+, Mg2+, or Mn2+, or deferoxamine by EDTA, diethylenetriaminepentaacetic acid, or bathophenanthroline. These findings indicate that Fe2+ and deferoxamine can act as an oxygen radical generating system, which may contribute to its biological effects in vitro and in vivo.     

Effects of cations on guanylate cyclase of sea urchin sperm.

Mn2+ and to some degree Fe2+, but not Mg+, Ca2+, ba2+, Sr2+, Co2+, Ni2+, La3+, or Fe3+ were able to serve as effective metal cofactors for sea urchin sperm guanylate cyclase. The apparent Michaelis constant for Mn2+ in the presence of 0.25 mM MnGTP was 0.23 mM. In the presence of a fixed free mn2+ concentration, variation in mngTP resulted in sigmoid velocity-substrate plots and in reciprocal plots that were concave upward. These positive cooperative patterns were observed at both pH 7.0 and 7.8 and in the presence or absence of Triton X-100. When Mn2+ and GTP were equimolar, Ca2+, Ba2+, Sr2+, and Mg2+ increased apparent guanylate cyclase activity. This increase in enzyme activity at least could be accounted for partially by an increase in free Mn2+ concentration caused by the complex formation of GTP with the added metals. However, even at relatively low GTP concentrations and with Mn2+ concentrations in excess of GTP, Ca2+, Sr2+, and Ba2+ significantly increased guanosine 3':5'-monophosphate production. As the total GTP concentration was increased, the degree of stimulation in the presence of Ca2+ decreased, despite maintenance of a fixed total concentration of Ca2+ and a fixed free concentration of Mn2+, suggesting that the concentration of CaGTP and MnGTP were determining factors in the observed response. The concave upward reciprocal plots of velocity against MnGTP concentration were changed to linear plots in the presence of CaGTP or SrGTP. These results suggest that sea urchin sperm guanylate cyclase contains multiple nucleotide binding sites and that stimulation of guanosine 3':5'-monophosphate synthesis by Ca2+, Sr2+, and perhaps other metals may reflect interaction of a metal-GTP complex with enzyme as either an effector or a substrate.     

Metal ion requirements for sequence-specific endoribonuclease activity of the Tetrahymena ribozyme

A shortened form of the self-splicing intervening sequence RNA of Tetrahymena thermophila acts as an enzyme, catalyzing sequence-specific cleavage of RNA substrates. We have now examined the metal ion requirements of this reaction. Mg2+ and Mn2+ are the only metal ions that by themselves give RNA enzyme activity. Atomic absorption spectroscopy indicates that Zn, Cu, Co, and Fe are not present in amounts equimolar to the RNA enzyme and when added to reaction mixtures do not facilitate cleavage. Thus, these ions can be eliminated as cofactors for the reaction. While Ca2+ has no activity by itself, it alleviates a portion of the Mg2+ requirement; 1 mM Ca2+ reduces the Mg2+ optimum from 2 to 1 mM. These results, combined with studies of the reactivity of mixtures of metal ions, lead us to postulate that two classes of metal ion binding sites are required for catalysis. Class 1 sites have more activity with Mn2+ than with Mg2+, with the other divalent ions and Na+ and K+ having no activity. It is not known if ions located at class 1 sites have specific structural roles or are directly involved in active-site chemistry. Class 2 sites, which are presumably structural, have an order of preference Mg2+ greater than or equal to Ca2+ greater than Mn2+ and Ca2+ greater than Sr2+ greater than Ba2+, with Zn2+, Cu2+, Co2+, Na+, and K+ giving no detectable activity over the concentration range tested.     

The nicotinamide adenine dinucleotides as allosteric effectors of human hemoglobin

The oxygen binding properties of human hemoglobin are appreciably altered by the nicotinamide dinucleotides NADH, NADP+, and NADPH. These cofactors are important in the control of many metabolic pathways and in providing reductive potential for a number of enzymatic reactions, including in vivo reduction of methemoglobin. Specific binding of these cofactors to hemoglobin and their potential for acting as allosteric modifiers of hemoglobin function have not been previously recognized. Detailed oxygen binding studies utilizing a thin-layer method suggest that the nicotinamide dinucleotides bind with high affinity to the deoxyhemoglobin tetramer at the beta chain anion-binding site and stabilize the low affinity "T-state" conformation. Stripped Hb A in 0.05 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 6.5, at 20 degrees C is half-saturated at a pO2 of 1.6 mm Hg. In the presence of 0.5 mM NADH, NADP+, or NADPH, the P50 is raised to 3.8, 7.1, and 12.5 mm Hg, respectively. The Bohr factor for stripped Hb A in 0.05 M HEPES buffer is sensitive to these effectors and is raised from 0.25 to about 0.65 by the addition of NADPH. The data suggest that routine use of these effectors in studies of human hemoglobin variants or the allosteric mechanism of Hb A be considered carefully. The relatively low intraerythrocytic levels of the nicotinamide dinucleotides in relation to hemoglobin dictate that these cofactors cannot significantly affect in vivo oxygen delivery. However, the converse is theoretically possible. The binding of the cofactors to hemoglobin and the preferential binding of their reduced forms may affect cofactor-dependent metabolic processes in red blood cells.     

Cys-His proteases are among the wired proteins of the cell

Integrated cell protein degradation can be paced by the transfer of reductive energy, as revealed by experimental agents of informative actions. The peptidolytic pair of Cys-His proteases can undergo oxidative reactions to inactive derivatives and inhibitory metal binding. Proton-dependent ionizations can modify ongoing activity. If the reaction rate of a Cys-His protease were found responsive to the ranges of metal/redox/proton factors regulated within the cell, then these factors might serve to link the peptidolytic reaction rate to cell controls. Here, cathepsin B (cat B) was found to be inhibited by Zn2+, Fe3+, and Cu2+ (1-50 microM) under excess GSH or DTT protease activators (6 mM). Under DTT or GSH (6 mM) the initial inhibitory action of Zn2+ is stable indefinitely; however, the inhibitory actions of Fe3+ and Cu2+ are reversed over approximately 1h. The 12-14 min half time of reversal of initial protease inhibition is correlated with the measured reduction of Fe3+ to Fe2+ by DTT or GSH (pH 5.5 or 6.5). Endogenous Fe2+ concentrations (100 microM) inhibit cat B only marginally. However, the inhibitory threshold of several microM Fe3+ is only a few percent oxidation of the endogenous pool. Without metals cat B reaction is reportedly proportional to GSH concentration, and is inhibited by increasing GSSG/GSH redox ratio. Following activation with GSH, cat B can be influenced by Fe3+/Fe2+, Cu2+/Cu+, and GSSG/GSH ratios and concentrations. Results are interpreted in relation to properties of the thiolate-imidazolium pair as illustrated by Dock modeling of their shared Fe3+ binding. It is proposed that the interaction of Cys-His with 1 electron transition between Fe2+ and Fe3+ serves as a sensor, signal integrator and switch wiring cat B reaction rate to the transfer of reductive energy in the presence of excess GSH. Speciated metals might also serve among electron acceptors transferring from reduced protease to oxygen. Results provide a model for pharmacologic redox switching of protease functions with metal-interactive drugs, and other nano-technology engineering.     

Alpha-oxidation of 3-methyl-substituted fatty acids in rat liver.

3-Methyl-substituted fatty acids are first oxidatively decarboxylated (alpha-oxidation) before they are degraded further via beta-oxidation. We synthesized [1-14C]phytanic and 3-[1-14C]methylmargaric acids in order to study their alpha-oxidation in isolated rat hepatocytes, rat liver homogenates and subcellular fractions. alpha-Oxidation was measured as the production of radioactive CO2. In isolated hepatocytes, maximal rates of alpha-oxidation amounted to 7 and 10 nmol/min x 10(8) cells with phytanic acid and 3-methylmargaric acid, respectively. At equimolar substrate concentrations, alpha-oxidation of branched fatty acids was approximately 10- to 15-fold slower than the beta-oxidation of the straight chain palmitate. In whole liver homogenates, rates of alpha-oxidation that equaled 60 to 70% of those observed in the hepatocytes were obtained. Optimum rates required O2, NADPH, Fe3+, and ATP. Fe3+ could be replaced by Fe2+ and ATP could be replaced by a number of other phosphorylated nucleosides and even inorganic phosphate without loss of activity. NADH could substitute for NADPH but not always with full restoration of activity. A variety of other cofactors and metal ions was either inhibitory or without effect. Scavengers of reactive oxygen species, known to be formed during the NADPH-dependent microsomal reduction of ferric-phosphate complexes, were without effect on alpha-oxidation. No evidence was found for the accumulation of NADPH-dependent or Fe(3+)-dependent reaction intermediates. Subcellular fractionation of liver homogenates demonstrated that alpha-oxidation was located predominantly, if not exclusively, in the endoplasmic reticulum. alpha-Oxidation, measured in microsomal fractions, was not inhibited by CO, cytochrome c, or ferricyanide, indicating that NADPH cytochrome P450 reductase and cytochrome P450 are not involved in alpha-oxidation. Our results indicate that, contrary to current belief, alpha-oxidation is catalyzed by the endoplasmic reticulum. The cofactor requirements suggest that alpha-oxidation involves the reduction of Fe3+ by electrons from NADPH and that it is stimulated by phosphate ions and nucleotides.     

Isolation and properties of a particulate fraction for the desaturation of palmitic acid from Alcaligenes faecalis.

A particulate fraction obtained from Alcaligenes faecalis could desaturate palmitic acid to palmitoleic acid. NADPH, ATP, CoA, Fe2+ and Mg2+ were essential cofactors for the reaction. The desaturation showed an absolute requirement for O2. Metal ions like Mn2+, Mo6+ and Cu2+ did not affect the desaturation, while Zn2+ was inhibitory. Sulfhydryl agents such as cysteine, glutathione and beta-mercaptoethanol had no effect, but SH-blocking agents like HgCl2 and p-hydroxymercuribenzoate inhibited the reaction. Azide and cyanide strongly inhibited the reaction while CO had no effect. The presence of a b-type cytochrome in the enzyme preparation was confirmed by the spectral studies on the reaction of enzyme with NADPH. Involvement of b-type cytochrome in the desaturation reaction was demonstrated by the reoxidation of b-type cytochrome initially reduced with NADPH, by the addition of palmitic acid and other cofactors. The pH optimum for the enzyme activity was 7.4. The optimum temperature for enzyme activity was 25 degrees C and maximum activity was obtained at the end of 45 min.     

Occurrence of Lipid Peroxidation in Brain Microsomes in the Presence of NADH and Vanadate

Oxidation of NADH by rat brain microsomes was stimulated severalfold on addition of vanadate. During the reaction, vanadate was reduced, oxygen was consumed, and H2O2 was generated with a stoichiometry of 1:1 for NADH/O2, as in the case of other membranes. Extra oxygen was found to be consumed over that needed for H2O2 generation specifically when brain microsomes were used. This appears to be due to the peroxidation of lipids known to be accompanied by a large consumption of oxygen. Occurrence of lipid peroxidation in brain microsomes in the presence of NADH and vanadate has been demonstrated. This activity was obtained specifically with the polymeric form of vanadate and with NADH, and was inhibited by the divalent cations Cu2+, Mn2+, and Ca2+, by dihydroxyphenolic compounds, and by hemin in a concentration-dependent fashion. In the presence of a small concentration of vanadate, addition of an increasing concentration of Fe2+ gave increasing lipid peroxidation. After undergoing lipid peroxidation in the presence of NADH and vanadate, the binding of quinuclidinyl benzylate, a muscarinic antagonist, to brain membranes was decreased.