Mannose inhibits Arabidopsis germination via a hexokinase-mediated step.
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Low concentrations of the glucose (Glc) analog mannose (Man) inhibit germination of Arabidopsis seeds. Man is phosphorylated by hexokinase (HXK), but the absence of germination was not due to ATP or phosphate depletion. The addition of metabolizable sugars reversed the Man-mediated inhibition of germination. Carbohydrate-mediated regulation of gene expression involving a HXK-mediated pathway is known to be activated by Glc, Man, and other monosaccharides. Therefore, we investigated whether Man blocks germination through this system. By testing other Glc analogs, we found that 2-deoxyglucose, which, like Man, is phosphorylated by HXK, also blocked germination; no inhibition was observed with 6-deoxyglucose or 3-O-methylglucose, which are not substrates for HXK. Since these latter two sugars are taken up at a rate similar to that of Man, uptake is unlikely to be involved in the inhibition of germination. Furthermore, we show that mannoheptulose, a specific HXK inhibitor, restores germination of seeds grown in the presence of Man. We conclude that HXK is involved in the Man-mediated repression of germination of Arabidopsis seeds, possibly via energy depletion. (+info)
Role of glutaraldehyde in calcification of porcine aortic valve fibroblasts.
(18/8540)
Glutaraldehyde-treated porcine aortic valve xenografts frequently fail due to calcification. Calcification in the prostheses begins intracellularly. In a previous study, various types of cell injury to canine valvular fibroblasts, including glutaraldehyde treatment, led to calcification. An influx of extracellular Ca2+ into the phosphate-rich cytosol was theorized to be the mechanism of calcification. To test the Ca2+ influx theory, cytosolic Ca2+ and Pi concentrations were assessed in glutaraldehyde-treated porcine aortic valve fibroblasts, and their relationship to a subsequent calcification was studied. Glutaraldehyde caused an immediate and sustained massive cytosolic Ca2+ increase that was dose dependent and a several-fold increase in Pi. Calcification of cells followed within a week. The earliest calcification was observed in blebs formed on glutaraldehyde-treated cells. Live control cells or cells fixed with glutaraldehyde in Ca2+-free solution did not calcify under the same conditions. Concomitant increases in Ca2+ and Pi in glutaraldehyde-treated cells appear to underlie the mechanism of calcification, and the presence of extracellular Ca2+ during glutaraldehyde fixation promotes calcification. (+info)
Enzyme-mononucleotide interactions: three different folds share common structural elements for ATP recognition.
(19/8540)
Three ATP-dependent enzymes with different folds, cAMP-dependent protein kinase, D-Ala:D-Ala ligase and the alpha-subunit of the alpha2beta2 ribonucleotide reductase, have a similar organization of their ATP-binding sites. The most meaningful similarity was found over 23 structurally equivalent residues in each protein and includes three strands each from their beta-sheets, in addition to a connecting loop. The equivalent secondary structure elements in each of these enzymes donate four amino acids forming key hydrogen bonds responsible for the common orientation of the "AMP" moieties of their ATP-ligands. One lysine residue conserved throughout the three families binds the alpha-phosphate in each protein. The common fragments of structure also position some, but not all, of the equivalent residues involved in hydrophobic contacts with the adenine ring. These examples of convergent evolution reinforce the view that different proteins can fold in different ways to produce similar structures locally, and nature can take advantage of these features when structure and function demand it, as shown here for the common mode of ATP-binding by three unrelated proteins. (+info)
An electrophoretic study of the reversible binding of phosphate to ovalbumin.
(20/8540)
An electrophoretic method is described for the measurement of relatively weak interaction of ions with proteins, and illustrated with the ovalbumin-phosphate system in 0.1I buffers, pH6.1. Results indicate that under these conditions the interaction of a single dibasic phosphate ion with ovalbumin is described by an association constant of approx. 250M-1. (+info)
Growth and production kinetics of a teicoplanin producing strain of Actinoplanes teichomyceticus.
(21/8540)
The growth and production kinetics of a teicoplanin producing strain of Actinoplanes teichomyceticus (ATCC 31121) was investigated during batch cultivations on defined media. The growth was characterised by two exponential growth phases (EGPs), with a higher specific growth rate in the first than in the second phase. Also the specific rate of formation of teicoplanin was significantly lower in the second phase than in the first phase. This two-phased growth pattern was suggested to be caused by inhibition of growth by teicoplanin accumulated. Furthermore high concentrations of ammonia or phosphate reduced both the specific growth rate in the first EGP and the total production of teicoplanin. (+info)
ATP and phosphate reciprocally affect subunit association of human recombinant High Km 5'-nucleotidase. Role for the C-terminal polyglutamic acid tract in subunit association and catalytic activity.
(22/8540)
IMP-specific, High Km 5'-nucleotidase (EC 3.1.3.5) is an ubiquitous enzyme, the activity of which is highly regulated by substrate, ATP, and inorganic phosphate. The cDNA encoding this enzyme has recently been cloned and found to contain a unique stretch of nine glutamic and four aspartic acid residues at the C-terminus. To study the effects of this acidic tail, and of ATP and inorganic phosphate on enzyme function, we generated several structural modifications of the 5'-nucleotidase cDNA, expressed the corresponding proteins in Escherichia coli and compared their molecular and kinetic properties. As with the enzyme purified from human placenta, all recombinant proteins were activated by ATP and inhibited by inorganic phosphate. Although the S0.5-values were higher, the specific activities of the purified protein variants (except that truncated at the C-terminus) were similar. The molecular mass of the full-length enzyme subunit has been estimated at 57.3 kDa and the molecular mass of the native protein, as determined by gel-filtration chromatography, was estimated to be 195 kDa. Increasing the concentration of NaCl to 0.3 M promoted oligomerization of the protein and the formation of aggregates of 332 kDa. ATP induced further oligomerization to 715 kDa, while inorganic phosphate reduced the estimated molecular mass to 226 kDa. In contrast to the truncation of 30 amino acids at the N-terminus, which did not alter enzyme properties, the removal of the polyglutamic/aspartic acid tail of 13 residues at the C-terminus caused profound kinetic and structural changes, including a 29-fold decrease in specific activity and a significant increase in the sensitivity to inhibition by inorganic phosphate in the presence of AMP. Structurally, there was a dramatic loss of the ability to form oligomers at physiological salt concentration which was only partially restored by the addition of NaCl or ATP. These data suggest an important function of the polyglutamic acid tract in the process of association and dissociation of 5'-nucleotidase subunits. (+info)
ATP-synthase of Rhodobacter capsulatus: coupling of proton flow through F0 to reactions in F1 under the ATP synthesis and slip conditions.
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A stepwise increasing membrane potential was generated in chromatophores of the phototrophic bacterium Rhodobacter capsulatus by illumination with short flashes of light. Proton transfer through ATP-synthase (measured by electrochromic carotenoid bandshift and by pH-indicators) and ATP release (measured by luminescence of luciferin-luciferase) were monitored. The ratio between the amount of protons translocated by F0F1 and the ATP yield decreased with the flash number from an apparent value of 13 after the first flash to about 5 when averaged over three flashes. In the absence of ADP, protons slipped through F0F1. The proton transfer through F0F1 after the first flash contained two kinetic components, of about 6 ms and 20 ms both under the ATP synthesis conditions and under slip. The slower component of proton transfer was substantially suppressed in the absence of ADP. We attribute our observations to the mechanism of energy storage in the ATP-synthase needed to couple the transfer of four protons with the synthesis of one molecule of ATP. Most probably, the transfer of initial protons of each tetrad creates a strain in the enzyme that slows the translocation of the following protons. (+info)
Genes coding for phosphotransacetylase and acetate kinase in Sinorhizobium meliloti are in an operon that is inducible by phosphate stress and controlled by phoB.
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Recent work in this laboratory has shown that the gene coding for acetate kinase (ackA) in Sinorhizobium meliloti is up-regulated in response to phosphate limitation. Characterization of the region surrounding ackA revealed that it is adjacent to pta, which codes for phosphotransacetylase, and that these two genes are part of an operon composed of at least two additional genes in the following order: an open reading frame (orfA), pta, ackA, and the partial sequence of a gene with an inferred peptide that has a high degree of homology to enoyl-ACP reductase (fabI). Experiments combining enzyme assays, a chromosomal lacZ::ackA transcriptional fusion, complementation analysis with cosmid subclones, and the creation of mutations in pta and ackA all indicated that the orfA-pta-ackA-fabI genes are cotranscribed in response to phosphate starvation. Primer extension was used to map the position of the phosphate starvation-inducible transcriptional start sites upstream of orfA. The start sites were found to be preceded by a sequence having similarity to PHO boxes from other phosphate-regulated genes in S. meliloti and to the consensus PHO box in Escherichia coli. Introduction of a phoB mutation in the wild-type strain eliminated elevated levels of acetate kinase and phosphotransacetylase activities in response to phosphate limitation and also eliminated the phosphate stress-induced up-regulation of the ackA::lacZ fusion. Mutations in either ackA alone or both pta and ackA did not affect the nodulation or nitrogen fixation phenotype of S. meliloti. (+info)