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Biomedical subjects

I Chérel

Publications and source records attributed to I Chérel.

7 recordsLinked to original sources
PubMed2000

Biochemical characterization of the Arabidopsis K+ channels KAT1 and AKT1 expressed or co-expressed in insect cells.

KAT1 and AKT1 belong to the multigenic family of the inwardly rectifying Shaker-like plant K+ channels. They were biochemically characterized after expression in insect cells using recombinant baculoviruses. The channels were solubilized from microsomal fractions prepared from infected cells (among eight different detergents only one, L-alpha-lysophosphatidylcholine, was efficient for solubilization), and purified to homogeneity using immunoaffinity (KAT1) or ion-exchange and size exclusion (AKT1) techniques. The following results were obtained with the purified polypeptides: (i) neither KAT1 nor AKT1 was found to be glycosylated; (ii) both polypeptides were mainly present as homotetrameric structures, supporting the hypothesis of a tetrameric structure for the functional channels; (iii) no heteromeric KAT1/AKT1 assembly was detected when the two polypeptides were co-expressed in insect cells. The use of the two-hybrid system in yeast also failed to detect any interaction between KAT1 and AKT1 polypeptides. Because of these negative results, the hypothesis that plant K+-channel subunits are able to co-assemble without any discrimination, previously put forward based on co-expression in Xenopus oocytes of various K+-channel subunits (including KAT1 and AKT1), has still to be supported by independent approaches. Co-localization of channel subunits within the same plant tissue/cell does not allow us to conclude that the subunits form heteromultimeric channels.

Animals↗
PubMed1997

Tetramerization of the AKT1 plant potassium channel involves its C-terminal cytoplasmic domain.

All plant channels identified so far show high conservation throughout the polypeptide sequence except in the ankyrin domain which is present only in those closely related to AKT1. In this study, the architecture of the AKT1 protein has been investigated. AKT1 polypeptides expressed in the baculovirus/Sf9 cells system were found to assemble into tetramers as observed with animal Shaker-like potassium channel subunits. The AKT1 C-terminal intracytoplasmic region (downstream from the transmembrane domain) alone formed tetrameric structures when expressed in Sf9 cells, revealing a tetramerization process different from that of Shaker channels. Tests of subfragments from this sequence in the two-hybrid system detected two kinds of interaction. The first, involving two identical segments (amino acids 371-516), would form a contact between subunits, probably via their putative cyclic nucleotide-binding domains. The second interaction was found between the last 81 amino acids of the protein and a region lying between the channel hydrophobic core and the putative cyclic nucleotide-binding domain. As the interacting regions are highly conserved in all known plant potassium channels, the structural organization of AKT1 is likely to extend to these channels. The significance of this model with respect to animal cyclic nucleotide-gated channels is also discussed.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of nitrate reductase-deficient mutants in tomato (Lycopersicon esculentum Mill.).

Five nitrate reductase-deficient mutants of tomato were isolated from an M2 population after ethylmethanesulphonate (EMS) seed treatment by means of selection for chlorate resistance. All mutations were monogenic and recessive and complementation analysis revealed that they were non-allelic. Biochemical and molecular characterization of these mutants showed that four of them are cofactor mutants while one is an apoenzyme mutant.

Autoradiography↗
PubMed1990

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia.

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.

Journal Article↗
PubMed1987

Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants.

Messenger RNAs encoding the nitrate reductase apoenzyme from tobacco can be translated in a cell-free system. Poly(A)+ mRNA fractions from the 23-32 S area of a sucrose gradient were used to build a cDNA library in the expression vector gt11 with an efficiency of cloning of approximately 10(4) recombinants/ng mRNA. Recombinant clones were screened with a rabbit polyclonal antibody directed against the corn nitrate reductase, which cross reacts specifically with the nitrate reductases from dicotyledons. Among 240000 recombinant plaques, eight clones were isolated containing inserts of sizes ranging from 1.6 kb to 2.1 kb and sharing sequence homologies. Seven of these clones contained a common internal 1.6 kb EcoRI fragment. The identity of these clones was confirmed as follows. A fusion protein of 170 kDa inducible by IPTG and recognized by the rabbit nitrate reductase antibody was expressed by a lysogen derived from one of the recombinants. The antibodies binding the fused protein were eluted and shown to be inhibitory to the catalytic activity of tobacco nitrate reductase. Two monoclonal antibodies directed against nitrate reductase were also able to bind the hybrid protein. The 1.6 kb EcoRI fragment was sequenced by the method of Sanger. The open reading frame corresponding to a translational fusion with the -galactosidase coding sequence of the vector shared strong homology at the amino acid level with the heme-binding domain of proteins of the cytochrome b5 superfamily and with human erythrocyte cytochrome b5 reductase. When the 1.6 kb EcoRI fragment was used as a probe for Northern blot experiments a signal corresponding to a 3.5 kb RNA was detected in tobacco and in Nicotiana plumbaginifolia mRNA preparations but no cross-hybridization with corn mRNAs was detected. The probe hybridized with low copy number sequences in genomic blots of tobacco DNA.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of Nicotiana plumbaginifolia nitrate reductase-deficient mutants: genetic and biochemical analysis of the NIA complementation group.

Two hundred and eleven nitrate reductase-deficient mutants (NR-) were isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures by chlorate selection and regenerated into plant. More than 40% of these clones were classified as cnx and presumed to be affected in the biosynthesis of the molybdenum cofactor, the remaining clones being classified as nia mutants. A genetic analysis of the regenerated plants confirmed this proportion of nia and cnx clones. All mutants regenerated were found to carry monogenic recessive mutations that impaired growth on nitrate as sole nitrogen source. Mutants propagated by grafting on N. tabacum systematically displayed a chlorotic leaf phenotype. This chlorosis was therefore related to the NR deficiency. The observation of leaves with NR- chlorotic sectors surrounded by NR+ wild-type tissues suggests that an NR deficiency is not corrected by diffusible factors. Periclinal chimeras between wild-type tobacco and the NR- graft were also observed. In this type of chimeric tissue chlorosis was no longer detectable when NR+ cells were in the secondmost (L2) layer, but was still detectable when NR- cells were in the secondmost layer. The genetic analysis of nia mutants revealed that they belong to a single complementation group. However three nia mutants were found to complement some of the other nia mutants. The apoenzyme of nitrate reductase was immunologically detected in several nia mutants but not in other members of this complementation group. Some of the nia mutants, although they were NR-, still displayed methylviologen-nitrate reductase activity at a high level. These data show that the nia complementation group corresponds to the structural gene of nitrate reductase. Some of the mutations affecting this structural gene result in the overproduction of an inactive nitrate reductase, suggesting a feedback regulation of the level of the apoenzyme in the wild type.

Chimera↗