Inhibitory sites in enzymes: zinc removal and reactivation by thionein.
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Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction. (+info)
Expression and regulation of 6-phosphofructo-2-kinase/fructose- 2,6-bisphosphatase isozymes in white adipose tissue.
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The aim of this work was to identify the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) isozyme(s) present in white adipose tissue. Ion-exchange chromatography of PFK-2 from rat epididymal fat pads yielded an elution pattern compatible with the presence of both the L (liver) and M (muscle) isozymes. This was consistent with a study of the phosphorylation of the purified adipose tissue enzyme by cAMP-dependent protein kinase, by specific labelling of the preparation with [2-32P]fructose 2,6-bisphosphate and by reaction with antibodies. Characterization of the PFK-2/FBPase-2 mRNAs showed that mature adipocytes express the mRNA that codes for the L isozyme and the two mRNAs that code for the M isozyme. Preadipocytes expressed mRNA that codes for the M isozyme. Incubation of rat epididymal fat pads with adrenaline stimulated glycolysis but decreased fructose 2,6-bisphosphate concentrations without significant inactivation of PFK-2. These results support previous findings showing that fructose 2,6-bisphosphate is not involved in the adrenaline-induced stimulation of glycolysis in white adipose tissue. (+info)
Cyclic AMP can decrease expression of genes subject to catabolite repression in Saccharomyces cerevisiae.
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External cyclic AMP (cAMP) hindered the derepression of gluconeogenic enzymes in a pde2 mutant of Saccharomyces cerevisiae, but it did not prevent invertase derepression. cAMP reduced nearly 20-fold the transcription driven by upstream activation sequence (UAS1FBP1) from FBP1, encoding fructose-1,6-bisphosphatase; it decreased 2-fold the activation of transcription by UAS2FBP1. Nuclear extracts from cells derepressed in the presence of cAMP were impaired in the formation of specific UASFBP1-protein complexes in band shift experiments. cAMP does not appear to act through the repressing protein Mig1. Control of FBP1 transcription through cAMP is redundant with other regulatory mechanisms. (+info)
Dehydroepiandrosterone suppresses the elevated hepatic glucose-6-phosphatase and fructose-1,6-bisphosphatase activities in C57BL/Ksj-db/db mice: comparison with troglitazone.
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The effect of dehydroepiandrosterone (DHEA) on the hepatic and muscle glucose metabolizing enzymes and on blood glucose were investigated in insulin-resistant diabetic C57BL/KsJ-db/db mice and their heterozygote littermates (db/+m). The results were compared with those after troglitazone administration under the same conditions. Despite hyperinsulinemia, hepatic glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphosphatase (FBPase) activities are higher in db/db than in db/+m mice. Dietary administration of DHEA and that of troglitazone for 15 days to respective groups of five mice each significantly decreased blood glucose in db/db mice and hepatic G6Pase and FBPase activities in both db/db and db/+m mice. Hepatic G6Pase and FBPase activities showed a linear relationship with blood glucose in all groups of mice, suggesting that the activities of G6Pase and FBPase are closely related to blood glucose levels. Because androstenedione, a DHEA metabolite, barely affected either of these enzyme activities or blood glucose in db/db mice, the actions of DHEA, which are similar to those of troglitazone, are presumed to be caused by DHEA itself. DHEA is considered to be a modulating agent for the activities of hepatic gluconeogenic enzymes in db/db mice. (+info)
Regulation of exopolysaccharide production by Lactococcus lactis subsp. cremoris By the sugar source.
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Lactococcus lactis produced more exopolysaccharide (EPS) on glucose than on fructose as the sugar substrate, although the transcription level of the eps gene cluster was independent of the sugar source. A major difference between cells grown on the two substrates was the capacity to produce sugar nucleotides, the EPS precursors. However, the activities of the enzymes required for the synthesis of nucleotide sugars were not changed upon growth on different sugars. The activity of fructosebisphosphatase (FBPase) was by far the lowest of the enzymes involved in precursor formation under all conditions. FBPase catalyzes the conversion of fructose-1, 6-diphosphate into fructose-6-phosphate, which is an essential step in the biosynthesis of sugar nucleotides from fructose but not from glucose. By overexpression of the fbp gene, which resulted in increased EPS synthesis on fructose, it was proven that the low activity of FBPase is indeed limiting not only for EPS production but also for growth on fructose as a sugar source. (+info)
Tissue-specific alternative splicing of rat brain fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase.
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We have reported the occurrence of eight splice variants of rat brain fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase (RB2K). In the present study, we quantified these splice variants in various tissues using a RNAse protection assay and found a tissue-specific pattern of alternative splicing of the RB2K gene. Splice variants containing exon F were specifically expressed in brain. Moreover, exons D and E were spliced in brain, skeletal muscle and heart. Consequently, eight, six, four and two splice variants were expressed in brain, skeletal muscle, heart and liver plus testis, respectively. These results suggest that distinct RB2K isoforms could be involved in regulation of glycolysis in a tissue-specific manner. (+info)
Redox signalling in the chloroplast: structure of oxidized pea fructose-1,6-bisphosphate phosphatase.
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Sunlight provides the energy source for the assimilation of carbon dioxide by photosynthesis, but it also provides regulatory signals that switch on specific sets of enzymes involved in the alternation of light and dark metabolisms in chloroplasts. Capture of photons by chlorophyll pigments triggers redox cascades that ultimately activate target enzymes via the reduction of regulatory disulfide bridges by thioredoxins. Here we report the structure of the oxidized, low-activity form of chloroplastic fructose-1, 6-bisphosphate phosphatase (FBPase), one of the four enzymes of the Calvin cycle whose activity is redox-regulated by light. The regulation is of allosteric nature, with a disulfide bridge promoting the disruption of the catalytic site across a distance of 20 A. Unexpectedly, regulation of plant FBPases by thiol-disulfide interchange differs in every respect from the regulation of mammalian gluconeogenic FBPases by AMP. We also report a second crystal form of oxidized FBPase whose tetrameric structure departs markedly from D(2) symmetry, a rare event in oligomeric structures, and the structure of a constitutively active mutant that is unable to form the regulatory disulfide bridge. Altogether, these structures provide a structural basis for redox regulation in the chloroplast. (+info)
Induction of a futile Embden-Meyerhof-Parnas pathway in Deinococcus radiodurans by Mn: possible role of the pentose phosphate pathway in cell survival.
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Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium Deinococcus radiodurans. Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation. Mn also facilitated glucose interactions with other nutrient components. These predictions were verified by in vivo and in vitro experiments. TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP). Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light. The presence of glucose also enhanced the radioresistance of the culture. Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in D. radiodurans. The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio. Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO(2). Mn-treated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light. Mn-treated cells were also more sensitive to UV light. Results suggested that metabolites of glucose generated from the PPP enhanced the survival of D. radiodurans. Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance. (+info)