236 resultados para PHOSPHOENOLPYRUVATE CARBOXYLASE


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Imaging of photochemical yield of photosystem II (PSII) computed from leaf chlorophyll fluorescence images and gas-exchange measurements were performed on Rosa rubiginosa leaflets during abscisic acid (ABA) addition. In air ABA induced a decrease of both the net CO2 assimilation (An) and the stomatal water vapor conductance (gs). After ABA treatment, imaging in transient nonphotorespiratory conditions (0.1% O2) revealed a heterogeneous decrease of PSII photochemical yield. This decline was fully reversed by a transient high CO2 concentration (7400 μmol mol−1) in the leaf atmosphere. It was concluded that ABA primarily affected An by decreasing the CO2 supply at ribulose-1,5-bisphosphate carboxylase/oxygenase. Therefore, the An versus intercellular mole fraction (Ci) relationship was assumed not to be affected by ABA, and images of Ci and gs were constructed from images of PSII photochemical yield under nonphotorespiratory conditions. The distribution of gs remained unimodal following ABA treatment. A comparison of calculations of Ci from images and gas exchange in ABA-treated leaves showed that the overestimation of Ci estimated from gas exchange was only partly due to heterogeneity. This overestimation was also attributed to the cuticular transpiration, which largely affects the calculation of the leaf conductance to CO2, when leaf conductance to water is low.

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The aim of this study was to investigate the interactions between cytokinin, sugar repression, and light in the senescence-related decline in photosynthetic enzymes of leaves. In transgenic tobacco (Nicotiana tabacum) plants that induce the production of cytokinin in senescing tissue, the age-dependent decline in NADH-dependent hydroxypyruvate reductase (HPR), ribulose-1,5-bisphosphate carboxylase/oxygenase, and other enzymes involved in photosynthetic metabolism was delayed but not prevented. Glucose (Glc) and fructose contents increased with leaf age in wild-type tobacco and, to a greater extent, in transgenic tobacco. To study whether sugar accumulation in senescing leaves can counteract the effect of cytokinin on senescence, discs of wild-type leaves were incubated with Glc and cytokinin solutions. The photorespiratory enzyme HPR declined rapidly in the presence of 20 mm Glc, especially at very low photon flux density. Although HPR protein was increased in the presence of cytokinin, cytokinin did not prevent the Glc-dependent decline. Illumination at moderate photon flux density resulted in the rapid synthesis of HPR and partially prevented the negative effect of Glc. Similar results were obtained for the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. It is concluded that sugars, cytokinin, and light interact during senescence by influencing the decline in proteins involved in photosynthetic metabolism.

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We develop a heuristic model for chaperonin-facilitated protein folding, the iterative annealing mechanism, based on theoretical descriptions of "rugged" conformational free energy landscapes for protein folding, and on experimental evidence that (i) folding proceeds by a nucleation mechanism whereby correct and incorrect nucleation lead to fast and slow folding kinetics, respectively, and (ii) chaperonins optimize the rate and yield of protein folding by an active ATP-dependent process. The chaperonins GroEL and GroES catalyze the folding of ribulose bisphosphate carboxylase at a rate proportional to the GroEL concentration. Kinetically trapped folding-incompetent conformers of ribulose bisphosphate carboxylase are converted to the native state in a reaction involving multiple rounds of quantized ATP hydrolysis by GroEL. We propose that chaperonins optimize protein folding by an iterative annealing mechanism; they repeatedly bind kinetically trapped conformers, randomly disrupt their structure, and release them in less folded states, allowing substrate proteins multiple opportunities to find pathways leading to the most thermodynamically stable state. By this mechanism, chaperonins greatly expand the range of environmental conditions in which folding to the native state is possible. We suggest that the development of this device for optimizing protein folding was an early and significant evolutionary event.

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Photosynthetic carbon metabolism is initiated by ribulose-bisphosphate carboxylase/oxygenase (Rubisco), which uses both CO2 and O2 as substrates. One 2-phosphoglycolate (P-glycolate) molecule is produced for each O2 molecule fixed. P-glycolate has been considered to be metabolized exclusively via the oxidative photosynthetic carbon cycle. This paper reports an additional pathway for P-glycolate and glycolate metabolism in the chloroplasts. Light-dependent glycolate or P-glycolate oxidation by osmotically shocked chloroplasts from the algae Dunaliella or spinach leaves was measured by three electron acceptors, methyl viologen (MV), potassium ferricyanide, or dichloroindophenol. Glycolate oxidation was assayed with 3-(3,4)-dichlorophenyl)-1,1-dimethylurea (DCMU) as oxygen uptake in the presence of MV at a rate of 9 mol per mg of chlorophyll per h. Washed thylakoids from spinach leaves oxidized glycolate at a rate of 22 mol per mg of chlorophyll per h. This light-dependent oxidation was inhibited completely by SHAM, an inhibitor of quinone oxidoreductase, and 75% by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which inhibits electron transfer from plastoquinone to the cytochrome b6f complex. SHAM stimulated severalfold glycolate excretion by algal cells, Dunaliella or Chlamydomonas, and by isolated Dunaliella chloroplasts. Glycolate and P-glycolate were oxidized about equally well to glyoxylate and phosphate. On the basis of results of inhibitor action, the possible site which accepts electrons from glycolate or P-glycolate is a quinone after the DCMU site but before the DBMIB site. This glycolate oxidation is a light-dependent, SHAM-sensitive, glycolate-quinone oxidoreductase system that is associated with photosynthetic electron transport in the chloroplasts.

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The crystal structure of pyruvate phosphate dikinase, a histidyl multiphosphotransfer enzyme that synthesizes adenosine triphosphate, reveals a three-domain molecule in which the phosphohistidine domain is flanked by the nucleotide and the phosphoenolpyruvate/pyruvate domains, with the two substrate binding sites approximately 45 angstroms apart. The modes of substrate binding have been deduced by analogy to D-Ala-D-Ala ligase and to pyruvate kinase. Coupling between the two remote active sites is facilitated by two conformational states of the phosphohistidine domain. While the crystal structure represents the state of interaction with the nucleotide, the second state is achieved by swiveling around two flexible peptide linkers. This dramatic conformational transition brings the phosphocarrier residue in close proximity to phosphoenolpyruvate/pyruvate. The swiveling-domain paradigm provides an effective mechanism for communication in complex multidomain/multiactive site proteins.

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Leaves of the C4 plant maize have two major types of photosynthetic cells: a ring of five large bundle sheath cells (BSC) surrounds each vascular bundle and smaller mesophyll cells (MC) lie between the cylinders of bundle sheath cells. The enzyme ribulose bisphosphate carboxylase/oxygenase is encoded by nuclear rbcS and chloroplast rbcL genes. It is not present in MC but is abundant in adjacent BSC of green leaves. As reported previously, the separate regions of rbcS-m3, which are required for stimulating transcription of the gene in BSC and for suppressing expression of reporter genes in MC, were identified by an in situ expression assay; expression was not suppressed in MC until after leaves of dark-grown seedlings had been illuminated for 24 h. Now we have found that transient expression of rbcS-m3 reporter genes is stimulated in BSC via a red/far-red reversible phytochrome photoperception and signal transduction system but that blue light is required for suppressing rbcS-m3 reporter gene expression in MC. Blue light is also required for the suppression system to develop in MC. Thus, the maize gene rbcS-m3 contains certain sequences that are responsive to a phytochrome photoperception and signal transduction system and other regions that respond to a UVA/blue light photoperception and signal transduction system. Various models of "coaction" of plant photoreceptors have been advanced; these observations show the basis for one type of coaction.

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Chemotactic responses in Escherichia coli are typically mediated by transmembrane receptors that monitor chemoeffector levels with periplasmic binding domains and communicate with the flagellar motors through two cytoplasmic proteins, CheA and CheY. CheA autophosphorylates and then donates its phosphate to CheY, which in turn controls flagellar rotation. E. coli also exhibits chemotactic responses to substrates that are transported by the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS). Unlike conventional chemoreception, PTS substrates are sensed during their uptake and concomitant phosphorylation by the cell. The phosphoryl groups are transferred from PEP to the carbohydrates through two common intermediates, enzyme I (EI) and phosphohistidine carrier protein (HPr), and then to sugar-specific enzymes II. We found that in mutant strains HPr-like proteins could substitute for HPr in transport but did not mediate chemotactic signaling. In in vitro assays, these proteins exhibited reduced phosphotransfer rates from EI, indicating that the phosphorylation state of EI might link the PTS phospho-relay to the flagellar signaling pathway. Tests with purified proteins revealed that unphosphorylated EI inhibited CheA autophosphorylation, whereas phosphorylated EI did not. These findings suggest the following model for signal transduction in PTS-dependent chemotaxis. During uptake of a PTS carbohydrate, EI is dephosphorylated more rapidly by HPr than it is phosphorylated at the expense of PEP. Consequently, unphosphorylated EI builds up and inhibits CheA autophosphorylation. This slows the flow of phosphates to CheY, eliciting an up-gradient swimming response by the cell.

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Vitamin E in the reduced, alpha-tocopherol form shows very modest anticlotting activity. By contrast, vitamin E quinone is a potent anticoagulant. This observation may have significance for field trials in which vitamin E is observed to exhibit beneficial effects on ischemic heart disease and stroke. Vitamin E quinone is a potent inhibitor of the vitamin K-dependent carboxylase that controls blood clotting. A newly discovered mechanism for the inhibition requires attachment of the active site thiol groups of the carboxylase to one or more methyl groups on vitamin E quinone. The results from a series of model reactions support this interpretation of the anticlotting activity associated with vitamin E.

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Nuclear-encoded proteins targeted to the chloroplast are typically synthesized with N-terminal transit peptides which are proteolytically removed upon import. Structurally related proteins of 145 and 143 kDa copurify with a soluble chloroplast processing enzyme (CPE) that cleaves the precursor for the major light-harvesting chlorophyll a/b binding protein and have been implicated in the maturation of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and acyl carrier protein. The 145- and 143-kDa proteins have not been found as a heterodimer and thus may represent functionally independent isoforms encoded by separate genes. Here we describe the primary structure of a 140-kDa polypeptide encoded by cDNAs isolated by using antibodies raised against the 145/143-kDa doublet. The 140-kDa polypeptide contains a transit peptide, and strikingly, a His-Xaa-Xaa-Glu-His zinc-binding motif that is conserved in a recently recognized family of metalloendopeptidases, which includes Escherichia coli protease III, insulin-degrading enzyme, and subunit beta of the mitochondrial processing peptidase. Identity of 25-30%, concentrated near the N terminus of the 140-kDa polypeptide, is found with these proteases. Expression of CPE in leaves is not light dependent. Indeed, transcripts are present in dark-grown plants, and the 145/143-kDa doublet and proteolytic activity are both found in etioplasts, as well as in root plastids. Thus, CPE appears to be a necessary component of the import machinery in photosynthetic and nonphotosynthetic tissues, and it may function as a general stromal processing peptidase in plastids.

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Holocarboxylase synthetase (HCS) catalyzes the biotinylation of the four biotin-dependent carboxylases in human cells. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS is targeted to the mitochondria and cytoplasm. We isolated 21 human HCS cDNA clones, in four size classes of 2.0-4.0 kb, by complementation of an Escherichia coli birA mutant defective in biotin ligase. Expression of the cDNA clones promoted biotinylation of the bacterial biotinyl carboxyl carrier protein as well as a carboxyl-terminal fragment of the alpha subunit of human propionyl-CoA carboxylase expressed from a plasmid. The open reading frame encodes a predicted protein of 726 aa and M(r) 80,759. Northern blot analysis revealed the presence of a 5.8-kb major species and 4.0-, 4.5-, and 8.5-kb minor species of poly(A)+ RNA in human tissues. Human HCS shows specific regions of homology with the BirA protein of E. coli and the presumptive biotin ligase of Paracoccus denitrificans. Several forms of HCS mRNA are generated by alternative splicing, and as a result, two mRNA molecules bear different putative translation initiation sites. A sequence upstream of the first translation initiation site encodes a peptide structurally similar to mitochondrial presequences, but it lacks an in-frame ATG codon to direct its translation. We anticipate that alternative splicing most likely mediates the mitochondrial versus cytoplasmic expression, although the elements required for directing the enzyme to the mitochondria remain to be confirmed.

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Senescence-associated coordination in amounts of enzymes localized in different cellular compartments were determined in attached leaves of young wheat (Triticum aestivum L. cv. Arina) plants. Senescence was initiated at the time of full leaf elongation based on declines in total RNA and soluble protein. Removal of N from the growth medium just at the time of full leaf elongation enhanced the rate of senescence. Sustained declines in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), and a marked decrease in the rbcS transcripts, just after full leaf elongation indicated that Rubisco synthesis/degradation was very sensitive to the onset of senescence. Rubisco activase amount also declined during senescence but the proportion of rca transcript relative to the total poly A RNA pool increased 3-fold during senescence. Thus, continued synthesis of activase may be required to maintain functional Rubisco throughout senescence. N stress led to declines in the amount of proteins located in the chloroplast, the peroxisome and the cytosol. Transcripts of the Clp protease subunits also declined in response to N stress, indicating that Clp is not a senescence-specific protease. In contrast to the other proteins, mitochondrial NADH-glutamate dehydrogenase (EC 1.4.1.2) was relatively stable during senescence and was not affected by N stress. During natural senescence with adequate plant nitrate supply the amount of nitrite reductase (EC 1.7.7.1) increased, and those of glutamine synthetase (EC 1.4.7.1) and glutamate synthase (EC 6.3.1.2) were stable. These results indicated that N assimilatory capacity can continue or even increase during senescence if the substrate supply is maintained. Differential stabilities of proteins, even within the same cellular compartment, indicate that proteolytic activity during senescence must be highly regulated.

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Our objective was to determine the coordination of transcript and/or protein abundances of stromal enzymes during leaf senescence. First trifolioliate leaves of Phaseolus vulgaris L. plants were sampled beginning at the time of full leaf expansion; at this same time, half of the plants were switched to a nutrient solution lacking N. Total RNA and soluble protein abundances decreased after full leaf expansion whereas chlorophyll abundance remained constant; N stress enhanced the decline in these traits. Abundances of ribulose-1,5-bisposphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39), Rubisco activase and phosphoribulokinase (Ru5P kinase; EC 2.7.1.19) decreased after full leaf expansion in a coordinated manner for both treatments. In contrast, adenosine diphosphate glucose (ADPGlc) pyrophosphorylase (EC 2.7.7.27) abundance was relatively constant during natural senescence but did decline similar to the other enzymes under N stress. Northern analyses indicated that transcript abundances for all enzymes declined markedly on a fresh-weight basis just after full leaf expansion. This rapid decline was particularly strong for the Rubisco small subunit (rbcS) transcript. The decline was enhanced by N stress for rbcS and Rubisco activase (rca), but not for Ru5P kinase (prk) and ADPGlc pyrophosphorylase (agp). Transcripts of the Clp protease subunits clpC and clpP declined in abundance just after full leaf expansion, similar to the other mRNA species. When Northern blots were analyzed using equal RNA loads, rbcS transcripts still declined markedly just after full leaf expansion whereas rca and clpC transcripts increased over time. The results indicated that senescence was initiated near the time of full leaf expansion, was accelerated by N stress, and was characterized by large decline in transcripts of stromal enzymes. The decreased mRNA abundances were in general associated with steadily declining stromal protein abundances, with ADPGlc pyrophosphorylase being the notable exception. Transcript analyses for the Clp subunits supported a recent report (Shanklin et al., 1995, Plant Cell 7: 1713--1722) indicating that the Clp protease subunits were constitutive throughout development and suggested that ClpC and ClpP do not function as a senescence-specific proteolytic system in Phaseolus.

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Background/Aims: There is increasing interest in the influence of excess body weight and associated metabolic factors on the liver. In patients with non-alcoholic steatohepatitis, lower levels of adiponectin were associated with higher grades of hepatic steatosis and necroinflammatory activity, suggesting a pathophysiological role for this adipokine in liver disease. Methods: We studied 194 consecutive patients with untreated chronic HCV, to assess the relationship between adiponectin and its receptors and hepatic steatosis, fibrosis and inflammation. Results: Significant negative correlations between serum adiponectin and male gender, body mass index and serum insulin were observed. However, there was no association between serum adiponectin and stage of fibrosis and lower levels of serum adiponectin were associated with the presence of steatosis in males only. In contrast, there was a significant increase in serum adiponectin and hepatic adiponectin immunoreactivity with increasing inflammation. The hepatic mRNA expression of the adiponectin receptors, AdipoR1 and AdipoR2, displayed significant but opposite associations with phosphoenolpyruvate carboxykinase (PEPCK) gene expression, a substitute marker of hepatic insulin sensitivity. Conclusions: In patients with chronic HCV, adiponectin was associated with steatosis only in males and was paradoxically increased with inflammation. Our results suggest that the role of adiponectin in chronic liver diseases may be linked to gender and etiology. (c) 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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The chicken ovalbumin upstream promoter-transcription factors ( COUP-TFs) are orphan members of the nuclear hormone receptor ( NR) superfamily. COUP-TFs are involved in organogenesis and neurogenesis. However, their role in skeletal muscle ( and other major mass tissues) and metabolism remains obscure. Skeletal muscle accounts for similar to 40% of total body mass and energy expenditure. Moreover, this peripheral tissue is a primary site of glucose and fatty acid utilization. We utilize small interfering RNA ( siRNA)-mediated attenuation of Coup-TfI and II ( mRNA and protein) in a skeletal muscle cell culture model to understand the regulatory role of Coup-Tfs in this energy demanding tissue. This targeted NR repression resulted in the significant attenuation of genes that regulate lipid mobilization and utilization ( including Ppar alpha, Fabp3, and Cpt-1). This was coupled to reduced fatty acid beta-oxidation. Additionally we observed significant attenuation of Ucp1, a gene involved in energy expenditure. Concordantly, we observed a 5-fold increase in ATP levels in cells with siRNA-mediated repression of Coup-TfI and II. Furthermore, the expression of classical liver X receptor ( LXR) target genes involved in reverse cholesterol transport ( Abca1 and Abcg1) were both significantly repressed. Moreover, we observed that repression of the Coup-Tfs ablated the activation of Abca1, and Abcg1 mRNA expression by the selective LXR agonist, T0901317. In concordance, Coup-Tf-siRNA-transfected cells were refractory to Lxr-mediated reduction of total intracellular cholesterol levels in contrast to the negative control cells. In agreement Lxr-mediated activation of the Abca1 promoter in Coup-Tf-siRNA cells was attenuated. Collectively, these data suggest a pivotal role for Coup-Tfs in the regulation of lipid utilization/cholesterol homeostasis in skeletal muscle cells and the modulation of Lxr-dependent gene regulation.

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Background: Protein phosphorylation is an extremely important mechanism of cellular regulation. A large-scale study of phosphoproteins in a whole-cell lysate of Saccharomyces cerevisiae has previously identified 383 phosphorylation sites in 216 peptide sequences. However, the protein kinases responsible for the phosphorylation of the identified proteins have not previously been assigned. Results: We used Predikin in combination with other bioinformatic tools, to predict which of 116 unique protein kinases in yeast phosphorylates each experimentally determined site in the phosphoproteome. The prediction was based on the match between the phosphorylated 7-residue sequence and the predicted substrate specificity of each kinase, with the highest weight applied to the residues or positions that contribute most to the substrate specificity. We estimated the reliability of the predictions by performing a parallel prediction on phosphopeptides for which the kinase has been experimentally determined. Conclusion: The results reveal that the functions of the protein kinases and their predicted phosphoprotein substrates are often correlated, for example in endocytosis, cytokinesis, transcription, replication, carbohydrate metabolism and stress response. The predictions link phosphoproteins of unknown function with protein kinases with known functions and vice versa, suggesting functions for the uncharacterized proteins. The study indicates that the phosphoproteins and the associated protein kinases represented in our dataset have housekeeping cellular roles; certain kinases are not represented because they may only be activated during specific cellular responses. Our results demonstrate the utility of our previously reported protein kinase substrate prediction approach (Predikin) as a tool for establishing links between kinases and phosphoproteins that can subsequently be tested experimentally.