Biomedical subjects

J Eichel

Publications and source records attributed to J Eichel.

9 recordsLinked to original sources

PubMed2001

Protein fingerprinting as a complementary tool for the classification of Pseudomonas bacteria.

A collection of total 42 bacterial strains belonging to the genus Pseudomonas were characterised based on protein fingerprinting using sodium dodecyl sulphate polyacrylamide gel electrophoregrams of cell-free extracts. Densitometrical analysis revealed unique and distinct profiles characteristic of the studied species. This comparison differentiated the isolates into four main clusters and twelve subclusters. The obtained protein patterns have proved to be an effective and reliable method both for the classification of bacteria and for showing similarities and variability among them.

Bacterial Proteins↗

PubMed2000

Metabolic instability of Escherichia coli cyclopropane fatty acid synthase is due to RpoH-dependent proteolysis.

Cyclopropane fatty acids (CFAs) are generally synthesized as bacterial cultures enter stationary phase. In Escherichia coli, the onset of CFA synthesis results from increased transcription of cfa, the gene encoding CFA synthase. However, the increased level of CFA synthase activity is transient; the activity quickly declines to the basal level. We report that the loss of CFA activity is due to proteolytic degradation dependent on expression of the heat shock regulon. CFA synthase degradation is unaffected by mutations in the lon, clpP, and groEL genes or by depletion of the intracellular ATP pools. It seems likely that CFA synthase is the target of an unidentified energy-independent heat shock regulon protease. This seems to be the first example of heat shock-dependent degradation of a normal biosynthetic enzyme.

Adenosine Triphosphatases↗

PubMed2000

Plant methionine synthase: new insights into properties and expression.

We investigated the enzyme methionine synthase (MSY) in Catharanthus roseus. The properties were characterized with purified protein isolated either from plant cell cultures or after heterologous expression in Escherichia coli. The protein was a monomer and accepted both the triglutamate (CH3-H4PteGlu3, apparent Km = 80 microM) and the monoglutamate (CH3-H4PteGlu1, apparent Km = 350 microM) of methyl-5,6,7,8-tetrahydropteroate as methyl donor, with a ratio of approximately 90:1 in favor of the triglutamate. Both activities required inorganic phosphate, but with different kinetics, and both were dependent on reducing agents. The activity required zinc, as shown by depletion and reconstitution experiments. Mg2+ had no effect on the activity. Two MSY isoforms purified from parsley cell cultures revealed the same properties as the C. roseus enzyme, however, the parsley proteins had no detectable activity with the monoglutamate substrate. The second part of the work compared the expression of the three enzymes of the methyl cycle (MSY, S-adenosyl-L-methionine synthetase, S-adenosyl-L-homocysteine hydrolase). In cell cultures, all three enzymes were present under all conditions investigated, with small changes at the protein level and more pronounced changes at the RNA level. Studies with seedlings revealed a low expression of all three enzymes in cotyledons, when compared to hypocotyls and radiculas. Immunohistochemical experiments indicated that MSY expression in cotyledons is cell-type specific, with the strongest signals detected in the upper epidermis.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1999

Effect of ppGpp on Escherichia coli cyclopropane fatty acid synthesis is mediated through the RpoS sigma factor (sigmaS).

Strains of Escherichia coli carrying mutations at the relA locus are deficient in cyclopropane fatty acid (CFA) synthesis, a phospholipid modification that occurs as cultures enter stationary phase. RelA protein catalyzes the synthesis of guanosine-3',5'-bisdiphosphate (ppGpp); therefore, ppGpp was a putative direct regulator of CFA synthesis. The nucleotide could act by increasing either the activity or the amount of CFA synthase, the enzyme catalyzing the lipid modification. We report that the effect of RelA on CFA synthesis is indirect. In vitro and in vivo experiments show no direct interaction between ppGpp and CFA synthase activity. The relA effect is due to ppGpp-engendered stimulation of the synthesis of the alternative sigma factor, RpoS, which is required for function of one of the two promoters responsible for expression of CFA synthase.

Bacterial Proteins↗

PubMed1997

Three differentially expressed S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization.

We describe the molecular and functional characterization of three closely related S-adenosyl-L-methionine synthetase (SAMS) isoenzymes from Catharanthus roseus (Madagascar periwinkle). The genes are differentially expressed in cell cultures during growth of the culture and after application of various stresses (elicitor, nutritional down-shift, increased NaCl). Seedlings revealed organ-specific expression and differential gene regulation after salt stress. A relationship analysis indicated that plant SAMS group in two main clusters distinguished by characteristic amino acid exchanges at specific positions, and this suggested differences in the enzyme properties or the regulation. SAMS1 and SAMS2 are of type I and SAMS3 is of type II. The properties of the isoenzymes were compared after heterologous expression of the individual enzymes, but no significant differences were detected in a) optima for temperature (37 to 45 degrees C) or pH (7 to 8.3); b) dependence on cations (divalent: Mg2+, Mn2+, Co2+; monovalent: K+, NH4+, Na+); c) K(m)s for ATP and L-methionine; d) inhibition by reaction products (S-adenosyl-L-methionine, PPi, Pi), by the reaction intermediate tripolyphosphate, and by the substrate analogues ethionine and cycloleucine; e) response to metabolites from the methyl cycle (L-homocysteine) or from related pathways (L-ornithine, putrescine, spermidine, spermine); f) native protein size (gel permeation chromatography). The results represent the first characterization of plant SAMS isoenzyme properties with individually expressed proteins. The possibility is discussed that the isoenzyme differences reflect specificities in the association with enzymes that use S-adenosyl-L-methionine.

Cloning, Molecular↗

PubMed1995

Vitamin-B12-independent methionine synthase from a higher plant (Catharanthus roseus). Molecular characterization, regulation, heterologous expression, and enzyme properties.

Methionine synthases catalyze the formation of methionine by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine. This reaction is the last step in L-methionine biosynthesis, and it also serves to regenerate the methyl group of S-adenosylmethionine, a cofactor required for biological methylation reactions. We describe the cloning, expression and characterization of a methionine synthase from the higher plant Catharanthus roseus. cDNAs were identified that encoded a protein of 85 kDa sharing 50% identify with the cobalamin-independent methionine synthase from Escherichia coli (MetE) and 41% identity with a partial sequence of a yeast homolog of MetE. The C. roseus protein was expressed at high levels in E. coli. The enzyme accepts the triglutamate form of methyltetrahydrofolate as a methyl donor but not the monoglutamate form, and it does not require S-adenosylmethionine or cobalamin for activity. The properties indicate that the enzyme is a cobalamin-independent methionine synthase (EC 2.1.1.14). In contrast to the E. coli MetE, the plant protein does not require phosphate or magnesium ions for activity. Immunoblots of plants extracts showed that the protein was localized in the cytosol, and was present in a variety of plant species. A nutritional downshift of the C. roseus cell culture revealed a strong, transient transcriptional activation, but no significant increment in the total level of the protein. The availability of the protein and the cDNA now provide tools to investigate the complexities of methionine biosynthesis in plants.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1993

HSP90 homologue from Madagascar periwinkle (Catharanthus roseus): cDNA sequence, regulation of protein expression and location in the endoplasmic reticulum.

We describe cDNAs for a HSP90 homologue from Catharanthus roseus and studies on the regulation of expression. The largest cDNA (2670 bp) coded for a protein of 817 amino acids with a calculated size of 93,491 Da and a pI of 4.61. It contained a eucaryotic secretory signal, the endoplasmic reticulum (ER) targeting and retention signal (Lys-Asp-Glu-Leu), and the HSP90 protein family signature with one conservative exchange (Asn-Lys-Asp-Ile-Phe-Leu instead of Asn-Lys-Glu-Ile-Phe-Leu). RNA blots revealed a transcript of 2.8-2.9 kb, and genomic DNA blots suggested a single gene. The expression was analysed with antiserum against a fusion protein expressed in Escherichia coli. Immunoblots revealed a protein of 93 +/- 1.5 kDa (often a doublet) only in the membrane fraction, and sucrose density gradients suggested association with the ER. The protein was constitutively expressed in C. roseus cell cultures grown at 25 degrees C, and expression was apparently unaffected by various stress conditions, such as heat, high sucrose, elicitor from Phytophthora megasperma or yeast extract. It was not detectable in young C. roseus plants at room temperature, and heat shock for several hours at 37 degrees C was necessary to obtain detectable expression. In maize (Zea mays), a cross-reacting protein was detectable in cell cultures, but not in young plants. The results suggested that the cloned protein is not a major component in the heat shock response. We propose a chaperone role in the assembly and processing of cell wall components and other secreted proteins, i.e. functions that are very active in cells with a high rate of growth and division.

Amino Acid Sequence↗

PubMed1989

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Allied Health Personnel↗

PubMedDate not supplied

Variability of the fatty acid composition during development of the green microalga Apatococcus constipatus.

The green microalga Apatococcus constipatus was investigated for its fatty acid composition using GC and MS techniques. Considerable variations were found in individual fatty acid contents according to the stage of culture development. A set of saturated fatty acid homologues was distinguished as the main component regardless of the culture age. The occurrence of some uncommon fatty acids in minor amounts, such as hydroxylated ones, was found to be characteristic of the studied species. Depending on the development stage, those compounds were detected either only during initial growth phases or throughout the cultivation time.

Chlorophyta↗