Activities of glucose metabolic enzymes in human preantral follicles: in vitro modulation by follicle-stimulating hormone, luteinizing hormone, epidermal growth factor, insulin-like growth factor I, and transforming growth factor beta1.
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Modulation of glucose metabolic capacity of human preantral follicles in vitro by gonadotropins and intraovarian growth factors was evaluated by monitoring the activities of phosphofructokinase (PFK) and pyruvate kinase (PK), two regulatory enzymes of the glycolytic pathway, and malate dehydrogenase (MDH), a key mitochondrial enzyme of the Krebs cycle. Preantral follicles in classes 1 and 2 from premenopausal women were cultured separately in vitro in the absence or presence of FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF-I), or transforming growth factor beta1 (TGFbeta1) for 24 h. Mitochondrial fraction was separated from the cytosolic fraction, and both fractions were used for enzyme assays. FSH and LH significantly stimulated PFK and PK activities in class 1 and 2 follicles; however, a 170-fold increase in MDH activity was noted for class 2 follicles that were exposed to FSH. Although both EGF and TGFbeta1 stimulated glycolytic and Krebs cycle enzymes for class 1 preantral follicles, TGFbeta1 consistently stimulated the activities of both glycolytic enzymes more than that of EGF. IGF-I induced PK and MDH activities in class 1 follicles but negatively influenced PFK activity for class 1 follicles. In general, only gonadotropins consistently stimulated both glycolytic and Krebs cycle enzyme activities several-fold in class 2 follicles. These results suggest that gonadotropins and ovarian growth factors differentially influence follicular energy-producing capacity from glucose. Moreover, gonadotropins may either directly influence glucose metabolism in class 2 preantral follicles or do so indirectly through factors other than the well-known intraovarian growth factors. Because growth factors modulate granulosa cell mitosis and functionality, their role on energy production may be related to specific cellular activities. (+info)
Genetic and biochemical characterization of phosphofructokinase from the opportunistic pathogenic yeast Candida albicans.
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We have used the two PFK genes of Saccharomyces cerevisiae encoding the alpha and beta-subunit of the enzyme phosphofructokinase (Pfk) as heterologous probes to isolate fragments of the respective genes from the dimorphic pathogenic fungus Candida albicans. The complete coding sequences were obtained by combining sequences of chromosomal fragments and fragments obtained by inverse polymerase chain reaction (PCR). The CaPFK1 and CaPFK2 comprise open reading frames of 2961 bp and 2838 bp, respectively, encoding Pfk subunits with deduced molecular masses of 109 kDa and 104 kDa. The genes presumably evolved by a duplication event from a prokaryotic type ancestor, followed by another duplication. Heterologous expression in S. cerevisiae revealed that each gene alone was able to complement the glucose-negative phenotype of a pfk1 pfk2 double mutant. In vitro Pfk activity in S. cerevisiae was not only obtained after coexpression of both genes, but also in conjunction with the respective complementary subunits from S. cerevisiae. This indicates the formation of functional hetero-oligomers consisting of C. albicans and S. cerevisiae Pfk subunits. In C. albicans, specific Pfk activity was shown to decrease twofold upon induction of hyphal growth. CaPfk cross-reacts with a polyclonal antiserum raised against ScPfk and displays similar allosteric properties, i.e. inhibition by ATP and activation by AMP and fructose 2,6-bisphosphate. (+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)
Interaction of neuronal nitric-oxide synthase and phosphofructokinase-M.
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Neurons that express neuronal nitric-oxide synthase (nNOS) are resistant to NO-induced neurotoxicity; however, the mechanism by which these neurons are protected is not clear. To identify proteins possibly involved in this process, we performed affinity chromatography with the nNOS PDZ domain, a N-terminal motif that mediates protein interactions. Using this method to fractionate soluble tissue extracts, we identified the muscle isoform of phosphofructokinase (PFK-M) as a protein that binds to nNOS both in brain and skeletal muscle. PFK-M interacts with the PDZ domain of nNOS, and nNOS-PFK-M binding can be competed by peptides that bind to the PDZ domain of nNOS. We found that nNOS is significantly associated with PFK-M in skeletal muscle because nNOS can be immunodepleted from cytosolic skeletal muscle extracts using an antibody directed against PFK-M. In brain, nNOS and PFK-M are both enriched in synaptosomes, and specifically, in the synaptic vesicle fraction, where they can interact. At the cellular level, PFK-M is enriched in neurons that express nNOS protein. As fructose-1, 6-bisphosphate, the product of PFK activity, is neuroprotective, the interaction of nNOS and PFK may contribute to neuroprotection of nNOS positive cells. (+info)
Differences in phosphofructokinase regulation in normal and tumor rat thyroid cells.
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The kinetic and molecular properties of a phosphofructokinase derived from a transplantable rat thyroid tumor lacking regulatory control on the glycolytic pathway were studied. The properties of the near-purified enzyme (specific activity 140 units/mg) were compared with those of phosphofructokinase from normal rat thyroid (specific activity 134 units/mg). The electrophoretic mobilities and gel elution behavior of these two enzymes were almost similar. The thyroid tumor phosphofructokinase showed, however, a greater degree of size and/or shape heterogeneity in the presence of ATP than the normal thyroid enzyme, as determined by gel filtration and sucrose density gradient centrifugation. Kinetic studies below pH 7.4 showed a sigmoid response curve for both enzymes when the velocity was determined at 1 mM ATP with varying levels of fructose-6-P. The interaction coefficient, however, was 4.2 and 2.6 for normal and tumor thyroid phosphofructokinase, respectively. Ammonium sulfate decreased the cooperative interactions with the substrate fructose-6-P in both enzymes. The thyroid tumor enzyme, however, was less sensitive to the inhibition by ATP and by citrate. The reversal of citrate inhibition by cyclic 3':5'-adenosine monophosphate was also less effective with the thyroid tumor phosphofructokinase, while the protective effect of fructose-6-P was stronger. The difference in citrate inhibition between tumor and normal thyroid enzyme was not strongly affected by varying the MgCl2 concentration up to 10 mM. It is concluded that the complex allosteric regulation typical of the normal thyroid phosphofructokinase is still present in the enzyme isolated from the thyroid tumor tissue. The latter, however, is more loosely controlled by its physiological effectors, such as ATP, citrate, and cyclic AMP. (+info)
Role of pfkA and general carbohydrate catabolism in seed colonization by Enterobacter cloacae.
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Enterobacter cloacae A-11 is a transposon mutant of strain 501R3 that was deficient in cucumber spermosphere colonization and in the utilization of certain carbohydrates (D. P. Roberts, C. J. Sheets, and J. S. Hartung, Can. J. Microbiol. 38:1128-1134, 1992). In vitro growth of strain A-11 was reduced or deficient on most carbohydrates that supported growth of strain 501R3 but was unaffected on fructose, glycerol, and all amino acids and organic acids tested. Colonization by strain A-11 was significantly reduced (P +info)
A single point mutation leads to an instability of the hetero-octameric structure of yeast phosphofructokinase.
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Yeast phosphofructokinase is an oligomeric enzyme whose detectable activity in vitro depends on its hetero-octameric structure. Here we provide data demonstrating that an alanine residue at positions 874 (for the PFK1-encoded alpha-subunit) or 868 (for the PFK2-encoded beta-subunit) is crucial to achieve this structure. Thus subunits carrying substitutions by either aspartate or lysine of this residue cause a lack of phosphofructokinase activity in vitro and signals of the subunits are poorly detectable in Western blots. Size-exclusion HPLC in conjunction with ELISA detection of the enzyme protein confirmed that no functional octamer is produced in such mutants. Our data suggest that the mutant subunits, not being assembled, tend to aggregate and subsequently become degraded. Substitution of the alanine by valine in either subunit leads to a reduction in specific activities, as expected from a conservative exchange. The kinetic data of the latter mutant revealed a higher affinity to the substrate fructose 6-phosphate, a lower extent of ATP inhibition and a lower degree of activation by fructose 2,6-bisphosphate. In addition, the affinity of mutants carrying a valine instead of an alanine in either the alpha- or the beta-subunit to fructose 2, 6-bisphosphate was increased. As no X-ray data on eukaryotic phosphofructokinases are available yet, our data provide the first evidence that a non-charge amino acid at position 874 or 868 is essential for the formation of the functional oligomer. This conclusion is substantiated by comparison with the structure of the well-known prokaryotic enzyme. (+info)
Generalization of the theory of transition times in metabolic pathways: a geometrical approach.
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Cell metabolism is able to respond to changes in both internal parameters and boundary constraints. The time any system variable takes to make this response has relevant implications for understanding the evolutionary optimization of metabolism as well as for biotechnological applications. This work is focused on estimating the magnitude of the average time taken by any observable of the system to reach a new state when either a perturbation or a persistent variation occurs. With this aim, a new variable, called characteristic time, based on geometric considerations, is introduced. It is stressed that this new definition is completely general, being useful for evaluating the response time, even in complex transitions involving periodic behavior. It is shown that, in some particular situations, this magnitude coincides with previously defined transition times but differs drastically in others. Finally, to illustrate the applicability of this approach, a model of a reaction mediated by an allosteric enzyme is analyzed. (+info)