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P Labbe

Publications and source records attributed to P Labbe.

At least 37 records · Page 2Linked to original sources
PubMed1992

Iron Reduction and Trans Plasma Membrane Electron Transfer in the Yeast Saccharomyces cerevisiae.

The ferri-reductase activity of whole cells of Saccharomyces cerevisiae (washed free from the growth medium) was markedly increased 3 to 6 h after transferring the cells from a complete growth medium (preculture) to an iron-deficient growth medium (culture). This increase was prevented by the presence of iron, copper, excess oxygen, or other oxidative agents in the culture medium. The cells with increased ferri-reductase activity had a higher reduced glutathione content and a higher capacity to expose exofacial sulfhydryl groups. Plasma membranes purified from those cells exhibited a higher reduced nicotinamide adenine phosphate (NADPH)-dependent ferri-reductase specific activity. However, the intracellular levels of NADPH, NADH, and certain organic acids of the tricarboxylic acids cycle were unchanged, and the activity of NADPH-generating enzymes was not increased. Addition of Fe(III)-EDTA to iron-deprived and iron-rich cells in resting suspension resulted in a decrease in intracellular reduced glutathione in the case of iron-deprived cells and in an increase in organic acids and a sudden oxidation of NADH in both types of cells. The depolarizing effect of Fe(3+) was more pronounced in iron-rich cells. The metabolic pathways that may be involved in regulating the trans-plasma membrane electron transfer in yeast are discussed.

Journal Article↗
PubMed1991

The plasma membrane ferrireductase activity of Saccharomyces cerevisiae is partially controlled by cyclic AMP.

The plasma-membrane-bound ferrireductase activity of ras1 and ras2 mutants of Saccharomyces cerevisiae is not induced in response to iron limitation. This phenotype was suppressed by the bcy1 mutation in ras2 but not in ras1 mutants. The cellular haem content of ras-1-bearing strains decreased dramatically when cells were grown in semi-synthetic medium (low yeast extract content), which could account for their very low ferrireductase activity. The ferrireductase activity of cdc25 and cdc35 mutants dropped when the cells were shifted to a non-permissive temperature. This drop was prevented in the double mutant cdc35 sra5 by adding cyclic AMP to the growth medium. We propose that ferrireductase activity is under the control of a cyclic AMP-dependent protein phosphorylation.

Cell Membrane↗
PubMed1991

Kinetic studies on protoporphyrinogen oxidase inhibition by diphenyl ether herbicides.

Diphenyl ethers (DPEs) and related herbicides are powerful inhibitors of protoporphyrinogen oxidase, an enzyme involved in the biosynthesis of haems and chlorophylls. The inhibition kinetics of protoporphyrinogen oxidase of various origins by four DPEs, (methyl)-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid (acifluorfen and its methyl ester, acifluorfen-methyl), methyl-5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-chlorobenzoate (LS 820340) and methyl-5-[2-chloro-5-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid (RH 5348), were studied. The inhibitions of the enzymes from maize (Zea mays) mitochondrial and etiochloroplastic membranes and mouse liver mitochondrial membranes were competitive with respect to the substrate, protoporphyrinogen IX, for all four molecules. The relative efficiencies of the inhibitors were: acifluorfen-methyl greater than LS 820340 much greater than RH 5348 greater than or equal to acifluorfen. The four molecules showed mixed-competitive type inhibition of the enzyme from yeast mitochondria where acifluorfen, a carboxylic acid, had the same inhibitory activity as its methyl ester, acifluorfen-methyl, and both were much greater than that of LS 820340 and RH 5348.

Animals↗
PubMed1990

Iron-reductases in the yeast Saccharomyces cerevisiae.

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.

Cell Membrane↗
PubMed1989

Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides.

Diphenyl ether herbicides induce an accumulation of protoporphyrin IX in plant tissues. By analogy to human porphyria, the accumulation could be attributed to decreased (Mg or Fe)-chelatase or protoporphyrinogen oxidase activities. Possible effects of acifluorfen-methyl on these enzymes were investigated in isolated corn (maize, Zea mays) etioplasts, potato (Solanum tuberosum) and mouse mitochondria, and yeast mitochondrial membranes. Acifluorfen-methyl was strongly inhibitory to protoporphyrinogen oxidase activities whatever their origins [concn. causing 50% inhibition (IC50) = 4 nM for the corn etioplast enzyme]. By contrast, it was roughly 100,000 times less active on (Mg or Fe)-chelatase activities (IC50 = 80-100 microM). Our results lead us to propose protoporphyrinogen oxidase as a cellular target for diphenyl ether herbicides.

Chlorophyll↗
PubMed1989

Purification and properties of mouse liver coproporphyrinogen oxidase.

Coproporphyrinogen oxidase was purified to homogeneity from mouse liver. The specific activity of the pure enzyme was 3500 nmol.h-1.mg-1; its apparent molecular mass (35 kDa) was confirmed by immunological characterization of the enzyme in a trichloroacetic-acid-precipitated total-liver-protein extract. The native enzyme appeared to be a dimer of 70 kDa as determined by gel filtration under nondenaturating conditions. The Km value for coproporphyrinogen III was 0.3 microM. The purified enzyme was activated by neutral detergents and phospholipids (affecting both Vmax and Km) but inhibited by ionic detergents. Reactivity toward sulfhydryl agents suggested the possible involvement of (an) SH group(s) for the activity. When compared to the previously purified coproporphyrinogen oxidases (from bovine liver and yeast), the mouse liver coproporphyrinogen oxidase appears to share many common catalytic properties with both enzymes. However, its apparent molecular mass is very different from that of the bovine liver enzyme (71.6 kDa) but identical to that found for the yeast (Saccharomyces cerevisiae) enzyme.

Amino Acids↗
PubMed1989

Reductive and non-reductive mechanisms of iron assimilation by the yeast Saccharomyces cerevisiae.

Iron reduction and uptake was studied in wild-type and haem-deficient strains of Saccharomyces cerevisiae. Haem-deficient strains lacked inducible ferri-reductase activity and were unable to take up iron from different ferric chelates such as Fe(III)-citrate or rhodoturulic acid. In contrast, ferrioxamine B was taken up actively by the mutants as well as by the wild-type strains. At a low extracellular concentration, uptake was insensitive to ferrozine and competitively inhibited by Ga(III)-desferrioxamine B. Extracellular reductive dissociation of the siderophore occurred at higher extracellular concentrations. Two mechanisms appear to contribute to the uptake of ferrioxamine B by S. cerevisiae: one with high affinity, by which the siderophore is internalized as such and another with lower affinity by which iron is dissociated from the ligand prior to uptake.

Aminolevulinic Acid↗
PubMed1988

Purification and properties of ferrochelatase from the yeast Saccharomyces cerevisiae. Evidence for a precursor form of the protein.

Ferrochelatase was purified to homogeneity from yeast mitochondrial membranes and found to be a 40-kDa polypeptide with a pI at 6.3. Fatty acids were absolutely necessary to measure the activity in vitro. The Michaelis constants for protoporphyrin IX (9 x 10(-8) M), ferrous iron (1.6 x 10(-7) M), and zinc (9 x 10(-6) M) were determined on purified enzyme preparations in the presence of dithiothreitol. However, the Km for zinc was lower when measured in the absence of dithiothreitol (K-m(Zn2+) = 2.5 x 10(-7) M, Km(protoporphyrin) unchanged). The maximum velocities of the enzyme were 35,000 nmol of heme/h/mg of protein and 27,000 nmol of zinc-protoporphyrin/h/mg of protein. Antibodies against yeast ferrochelatase were raised in rabbits and used in studies on the biogenesis of the enzyme. Ferrochelatase is synthesized as a higher molecular weight precursor (Mr = 44,000) that is very rapidly matured in vivo to the Mr = 40,000 membrane-bound form. This precursor form of ferrochelatase was immunoprecipitated from in vitro translation (in a rabbit reticulocyte lysate system) of total yeast RNAs. The antibodies were used to characterize two yeast mutant strains deficient in ferrochelatase activity as being devoid of immunodetectable protein in vivo and ferrochelatase mRNA in vitro translation product. The N-terminal amino acid sequence of the purified protein has been established and was found to be frayed.

Amino Acid Sequence↗
PubMed1986

Purification and properties of coproporphyrinogen oxidase from the yeast Saccharomyces cerevisiae.

Coproporphyrinogen oxidase has been located in the cytosol of yeast cells. The enzyme was purified to homogeneity from a heme mutant strain exhibiting a high specific activity (15-20 enzyme units/mg soluble protein compared to 1-2 enzyme units/mg soluble protein of by the wild-type strain). The final preparation was homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Mr = 35,000) and isoelectrofocusing (pI = 6.2). Gel filtration on AcA 44 gave a relative molecular mass of 70,000. N-terminal amino-acid sequence analysis revealed a single polypeptide chain. Thus the enzyme appears to be a dimer with identical subunits. Two iron atoms/molecule of native protein were detected; they could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25. However the involvement of the iron atoms in the oxidative catalytic activity of the enzyme was not demonstrated. The Km value for coproporphyrinogen was 0.05 microM. The enzyme was active only when molecular oxygen was used as electron acceptor; no anaerobic activity could be detected. Thiol-directed reagents partially inhibited the enzyme, indicating that an SH group is required for activity. Yeast coproporphyrinogen oxidase was activated by phospholipids or neutral detergents as described for the bovine liver enzyme.

Amino Acid Sequence↗
PubMed1985

Fluorometric assays for coproporphyrinogen oxidase and protoporphyrinogen oxidase.

We describe fluorometric assays for two enzymes of the heme pathway, coproporphyrinogen oxidase and protoporphyrinogen oxidase. Both assays are based on measurement of protoporphyrin IX fluorescence generated from coproporphyrinogen III by the two consecutive reactions catalyzed by coproporphyrinogen oxidase and protoporphyrinogen oxidase. Both enzymatic activities are measured by recording protoporphyrin IX fluorescence increase in air-saturated buffer in the presence of EDTA (to inhibit ferrochelatase that can further metabolize protoporphyrin IX) and in the presence of dithiothreitol (that prevents nonenzymatic oxidation of porphyrinogens to porphyrins). Coproporphyrinogen oxidase (limiting) activity is measured in the presence of a large excess of protoporphyrinogen oxidase provided by yeast mitochondrial membranes isolated from commercial baker's yeast. These membranes are easy to prepare and are stable for at least 1 year when kept at -80 degrees C. Moreover they ensure maximum fluorescence of the generated protoporphyrin (solubilization effect), avoiding use of a detergent in the incubation medium. The fluorometric protoporphyrinogen oxidase two-step assay is closely related to that already described (J.-M. Camadro, D. Urban-Grimal, and P. Labbe, 1982, Biochem. Biophys. Res. Commun. 106, 724-730). Protoporphyrinogen is enzymatically generated from coproporphyrinogen by partially purified yeast coproporphyrinogen oxidase. The protoporphyrinogen oxidase reaction is then initiated by addition of the membrane fraction to be tested. However, when very low amounts of membrane are used, low amounts of Tween 80 (less than 1 mg/ml) have to be added to the incubation mixture to solubilize protoporphyrin IX in order to ensure optimal fluorescence intensity. This detergent has no effect on the rate of the enzymatic reaction when used at concentrations less than 2 mg/ml. Activities ranging from 0.1 to 4-5 nmol protoporphyrin formed per hour per assay are easily and reproducibly measured in less than 30 min.

Animals↗
PubMed1982

Kinetic studies of ferrochelatase in yeast. Zinc or iron as competing substrates.

Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) has been studied in yeast mitochondrial membranes with special reference to zinc-chelatase and iron-chelatase activities. Using physiological substrates (protoporphyrin IX, Fe(II) and Zn(II), anaerobic conditions of incubation and direct spectrophotometric assay, apparent Km values smaller than those previously described were found for the membrane-bound enzyme. Fe(II) but not Fe(III) was a strong competitive inhibitor of zinc-chelatase activity, while Zn(II) was a slight competitive inhibitor of iron-chelatase activity. These results could point to modes of control of ferrochelatase activity in yeast. We suggest that reduced supply of Fe(II) may explain the in vivo accumulation of zinc-protoporphyrin in yeast cells incubated under 'resting' conditions.

Anaerobiosis↗