A plastid terminal oxidase associated with carotenoid desaturation during chromoplast differentiation.

The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and zeta-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development.

RGD-dependent growth of maize calluses and immunodetection of an integrin-like protein.

When maize calluses are grown in the presence of the RGD peptide, important morphological changes are observed indicating the presence of a likely RGD-binding receptor. Polyclonal antibodies generated against the human beta1 integrin subunit, the platelet integrin alphaIIbeta3 (P23) and antibodies specific for either the beta3 platelet chain or the alphaIIb polypeptide cross-react with glycoproteins in Western blot analyses. Immunoprecipitation assays indicate that this maize integrin-like protein shares structural similarities with the animal alphaIIbeta3 complex. We also show that AcAt2, a polyclonal antibody raised against Arabidopsis proteins purified on an RGD column, interacts with a maize protein.

A plant surface protein sharing structural properties with animal integrins.

Using a polyclonal antibody (P23) generated against the human platelet integrin aIIb beta3 and a FITC-conjugate secondary antibody, fluorescence is observed at the surface of protoplasts isolated from Arabidopsis thaliana and Rubus fruticosus. Arabidopsis thaliana cells grown in suspension culture containing P23 and glycylarginylglycylaspartylserine (GRGDS), a synthetic peptide containing the RGD sequence found in many extracellular matrix adhesive proteins demonstrated aberrant cell wall/plasma membrane interactions and organization. When glycoproteins from these plants, purified on a concanavalin A Sepharose 4B, were subjected to SDS/PAGE and Western blotting, under reduced and non-reduced conditions, immunoblots probed with P23 revealed bands in both species. A shift in electrophoretic mobility is observed to different apparent molecular mass when no reducing agent is present. When purified by immunoaffinity chromatography on anti-aIIb beta3 Sepharose or Sepharose linked to the synthetic peptide D-Arg-Gly-Asp-Trp, the major antigenic components detected migrate at 30 kDa and 60 kDa in the first experiment and 60 kDa in the second one. Only the 60-kDa component is immunodetected with antibodies specific for either the beta3 platelet chain or the aIIb polypeptide, suggesting the presence of two polypeptides co-migrating. To address more precisely the structure of this complex in plants, competition assays were performed. A significant inhibition is observed with CS3 a monoclonal antibody that interacts with the complexed form aIIb beta3 but not the dissociated subunits. Further structural similarities with the animal aIIb beta3 complex is demonstrated with Western blotting detection after plant glycoproteins immunoprecipitation with CS3 in absence or presence of 5 mM EDTA to dissociate the complex. We also present data on the characterization of a polyclonal antibody, named AcAt2, raised against Arabidopsis glycocoproteins purified by affinity chromatography on a D-RGDW column and eluted with the same peptide, that specifically interacts with the animal aIIb beta3 receptor.

Purification and characterization of a phytase (myo-inositol-hexakisphosphate phosphohydrolase) accumulated in maize (Zea mays) seedlings during germination.

Phytase (myo-inositol-hexakisphosphate phosphohydrolase, EC 3.1.3.8) has been purified from 5-7-day-old maize (Zea mays) seedlings, using a four-step purification procedure. The native protein has a molecular mass of about 76 kDa and is built up from two 38 kDa subunits. The pH and temperature optima of the purified enzyme were respectively 4.8 and 55 degrees C. The apparent Km for phytate was estimated to be 117 microM. Like other acidic phytases, the maize seedling enzyme exhibited a broad affinity for various phosphorylated substrates and especially for penta- and tri-phosphate esters of myo-inositol. The amino acid composition of the h.p.l.c.-purified protein indicated a high hydrophobicity (44% non-polar amino acids). Rabbit antibodies were produced in response to maize seedling phytase. Western-blot analyses clearly demonstrate that the increase of phytase activity observed during the first 7 days of germination corresponded to an accumulation of the protein in maize seedlings. Phytase accumulated essentially in the shoots (mesocotyl plus coleoptiles.

Uptake of iron from ferric-citrate in the cyanobacteria Synechocystis PCC6803.

Iron is an essential element for bacterial growth. Its intracellular concentration is mainly controlled at the uptake level, which is mediated by iron-dicitrate transport in various prokaryotes. We report here that the cyanobacterium Synechocystis PCC6803 takes up iron from ferric citrate. This process is dependent of the iron:citrate ratio, with a better efficiency for the highest concentration of citrate. The iron-citrate complex is the substrate of this active uptake, inhibited in part by the uncoupler FCCP. Iron starvation activates this uptake. These data suggest the existence of a specific ferric citrate receptor on the cyanobacterial membrane.

Purification, characterization and function of bacterioferritin from the cyanobacterium Synechocystis P.C.C. 6803.

Storage and buffering of iron is achieved by a class of proteins, the ferritins, widely distributed throughout the living kingdoms. All ferritins have in common their three-dimensional structure and their ability to store large amounts of iron in their central cavity. However, eukaryotic ferritins from plants and animals and bacterioferritins have no sequence similarity, and besides non-haem iron bacterioferritins contain haem residues whereas eukaryotic ferritins do not. In this paper we report the first purification and characterization of a bacterioferritin from a cyanobacterium. It has a molecular mass of 400 kDa and is built up from 19 kDa subunits. Its N-terminal sequence shows 73% identity with that of the Escherichia coli bacterioferritin subunit. It contains 2300 atoms of iron and 1500 molecules of phosphate per ferritin molecule and 0.25 haem residue per subunit; the alpha-peak of the cytochrome has its maximum at 559 nm. In contrast with what is known for eukaryotic ferritins, we found that bacterioferritin from Synechocystis is not inducible by iron under the conditions that we have tested and that it has a constant concentration whatever the iron status of the cells, even at very low iron concentration. Bacterioferritin from Synechocystis P.C.C. 6803 is fully assembled in vivo and it is shown by labelling with 59Fe that it is able to load iron in vitro as well as in vivo. Bacterioferritin from Synechocystis is shown to have an iron-buffering function while the bulk of cellular iron is found associated with a pool of low-molecular-mass electronegative molecules. The role of Synechocystis bacterioferritin in iron metabolism is discussed.

Photoreduction and incorporation of iron into ferritins.

Pea seed ferritin is able to incorporate ferrous iron into the mineral core. Fe2+ may be formed by reduction of exogenous Fe3+ with ascorbate or by photoreduction by ferritin and by ferric citrate. In our experimental conditions the bulk of the photoreduction is carried out by ferritin, which is able to photoreduce its endogenous iron. Citrate does not enhance the photoreduction capacity of ferritin, and exogenous ferric citrate improves the yield of the reaction by about 30%. The mineral core of the ferritin is shown to photoreduce actively, and the protein shell does not participate directly in the photoreduction. Low light intensities and low concentration of reducing agents do not allow a release of iron from ferritins, but induce a 'redox mill' of photoreduction and simultaneous ferroxidase-mediated incorporation. High ascorbate concentrations induce the release of ferritin iron. These reactions are accompanied by the correlated occurrence of damage caused by radicals arising from Fenton reactions, leading to specific cleavages in the 28 kDa phytoferritin subunit. This damage caused by radicals occurs during the oxidative incorporation into the mineral core and is prevented by o-phenanthroline or by keeping the samples in the dark.

Mechanism of the transition from plant ferritin to phytosiderin.

Soluble and insoluble forms of ferritins have been purified from dry pea seeds by gel filtration. The insoluble form is called phytosiderin by analogy with animal hemosiderin. Native gel electrophoresis of these two forms have shown that the soluble one (ferritin) is homogenous in size and more compact than the insoluble one (phytosiderin) which is heterogenous in size. However, when iron is removed from these two classes of molecules (apoferritin), they have the same mobility in isopycnic centrifugations. Polyacrylamide-sodium dodecyl sulfate gel electrophoresis revealed a difference in their subunit composition: ferritin molecules are built up from a 28-kDa subunit and phytosiderin from a 26.5-kDa subunit. Partial proteolysis using a Staphylococcus aureus protease indicates a strong relationship between these two polypeptides. Intermediates between these two forms have also been characterized and are composed of both subunits in various amounts. Ferritin and phytosiderin are both able to incorporate iron in vitro into their mineral core. It is also shown that in vitro iron exchange induces ferritin degradation. This degradation is prevented by inhibitors of the Fenton cycle (iron chelates like o-phenanthroline and desferrioxamine B) and reduced by Tris, a radical scavenger. Under in vitro conditions of controlled radical damage the 28-kDa subunit is converted into the 26.5-kDa subunit. Purification of the 28-kDa subunit has allowed us to determine the NH2-terminal sequence. The NH2 extremity of the 26.5-kDa subunit is heterogenous, but the sequence of its main component is identical to the sequence of the 28-kDa subunit downstream residue Leu-21. These data indicate that the 26.5-kDa subunit is produced by radical mediated damage leading to a series of cleavages in the NH2 terminal part of the 28-kDa subunit.

Evidence for a translation-mediated attenuation of a spinach chloroplast rDNA operon.

The presence of potential hairpin structures H1, H2, H3 in the leader region of a spinach rDNA operon led us to postulate that this operon is regulated by premature termination. The mechanism would be controlled by the presence or absence of ribosomes translating a leader peptide. In vitro synchronized transcription by E. coli RNA polymerase shows that pauses do occur in the leader region. By their sizes, the transient transcripts could correspond to pauses on H1 and H2 as predicted by the model in the absence of ribosomes. The complete leader sequence (pKOPH) and the leader sequence with the hairpin structures deleted (pKOP) have been used to the GalK gene in the pK01 plasmid. The resulting plasmids have been used to transform a GalK- E. coli strain. Measurements of GalK expression show that the promoter region of spinach chloroplast rDNA is neither subjected to the growth rate nor to the stringent control. However, under growth conditions leading to an excess of free ribosomes, the expression of GalK gene appears systematically to be reduced in pKOPH when compared with that of pKOP. These results are consistent with a role of the leader region in a translation-mediated attenuation of the chloroplast rDNA expression.

[Regulation of catalase synthesis in Proteus mirabilis]. / Régulation de la synthèse de la catalase chez Proteus mirabilis.

During the log-phase growth of Proteus mirabilis the specific activity of catalase decreases, while at the beginning of or during the stationary phase an increase takes place which is abolished by inhibitors of nucleic acid or protein synthesis. Glucose in the culture medium has no appreciable effect on the level of enzyme synthesis nor does the passage of bacteria to anaerobiosis bring any noticeable change. Successive additions of hydrogen peroxide up to weak final concentrations (0.2--0.5 mM) stimulate catalase synthesis. Determination of the enzyme in vivo reveals but a weak proportion of the total catalase which can only be titrated after the breakdown of cells. The titrable enzyme in vivo represents, as an order of magnitude, the activity found associated with the cell wall, in an easily released form after the mechanical separation of the inner and outer membranes. Thus, bacteria can act upon exogenous peroxide only through a peripheral catalase while they possess in a masked form an important reserve of cytoplasmic enzyme.

Regulation of phenylalanine oxidase synthesis in Proteus mirabilis.

Cells of Proteus mirabilis could oxidize L-phenylalanine to phenylpyruvate only when grown in the presence of a number of amino acids, particularly, L-alanine, L-asparagine, L-glutamate, and L-glutamine. Production of phenylalanine oxidase was slowly lost upon growth in a minimal medium containing ammonium ions as a nitrogen source but was reversed by the addition of casein hydrolysate. Oxidase activity as well as a phenylalanine-dichlorophenolindophenol (DCIP) reductase activity increased in P. mirabilis only during cell multiplication. Both rifampin and nalidixic acid caused inhibition of oxidase synthesis. A phenylalanine-active transport was found to be operative when bacteria were grown in the absence of added amino acids. After anaerobic growth, cells of P. mirabilis had lost their ability to carry the phenylalanine oxidase reaction when assayed in the presence of air, and nitrate could not be used as an electron acceptor for the oxidation of phenylalanine. However, some phenylalanine-dichlorophenolindophenol reductase activity was still present in anaerobic bacteria at the early stage of cell multiplication.