RESUMEN
Neutrophils rely predominantly on glycolytic metabolism for their biological functions, including reactive oxygen species (ROS) production. Although pyruvate kinase M2 (PKM2) is a glycolytic enzyme known to be involved in metabolic reprogramming and gene transcription in many immune cell types, its role in neutrophils remains poorly understood. Here, we report that PKM2 regulates ROS production and microbial killing by neutrophils. Zymosan-activated neutrophils showed increased cytoplasmic expression of PKM2. Pharmacological inhibition or genetic deficiency of PKM2 in neutrophils reduced ROS production and Staphylococcus aureus killing in vitro. In addition, this also resulted in phosphoenolpyruvate (PEP) accumulation and decreased dihydroxyacetone phosphate (DHAP) production, which is required for de novo synthesis of diacylglycerol (DAG) from glycolysis. In vivo, PKM2 deficiency in myeloid cells impaired the control of infection with Staphylococcus aureus. Our results fill the gap in the current knowledge of the importance of lower glycolysis for ROS production in neutrophils, highlighting the role of PKM2 in regulating the DHAP and DAG synthesis to promote ROS production in neutrophils.
Asunto(s)
Neutrófilos , Piruvato Quinasa , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neutrófilos/metabolismo , Fosforilación , GlucólisisRESUMEN
The shrimp Litopenaeus vannamei is the main farmed crustacean worldwide. This shrimp suffers environmental changes in oxygen availability that affect its energy metabolism. Pyruvate kinase (PK) catalyzes the last reaction of glycolysis and is key for the regulation of glycolysis and gluconeogenesis. There is ample knowledge about mammalian PK, but in crustaceans, the information is very scarce. In this study, we analyzed in silico the structures of the PK gene and protein. Also, the effects of hypoxia on gene expression, enzymatic activity, glucose, and lactate in hepatopancreas and muscle were analyzed. The PK gene is 15,103 bp and contains 11 exons and 10 introns, producing four mRNA variants by alternative splicing and named PK1, PK2, PK3 and PK4, that results in two proteins with longer C-terminus and two with a 12 bp insertion. The promoter contains putative binding sites for transcription factors (TF) that are typically involved in stress responses. The deduced amino acid sequences contain the classic domains, binding sites for allosteric effectors and potential reversible phosphorylation residues. Protein modeling indicates a homotetramer with highly conserved structure. The effect of hypoxia for 6 and 12 h showed tissue-specific patterns, with higher expression, enzyme activity and lactate in muscle, but higher glucose in hepatopancreas. Changes in response to hypoxia were detected at 12 h in expression with induction in muscle and reduction in hepatopancreas, while enzyme activity was maintained, and glucose and lactate decreased. These results show rapid changes in expression and metabolites, while enzyme activity was maintained to cope with short-term hypoxia.
Asunto(s)
Penaeidae , Piruvato Quinasa , Animales , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Hipoxia/genética , Hipoxia/metabolismo , Oxígeno/metabolismo , Glucosa/metabolismo , Lactatos , Penaeidae/metabolismo , Mamíferos/metabolismoRESUMEN
Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are K+-dependent, whereas those with K117 are K+-independent. In a phylogenetic tree, 80% of the sequences with E117 are occupied by T113/K114/T120 and 77% of those with K117 possess L113/Q114/(L,I,V)120. This work aims to understand these residues' contribution to the K+-independent pyruvate kinases using the K+-dependent rabbit muscle enzyme. Residues 117 and 120 are crucial in the differences between the K+-dependent and -independent mutants. K+-independent activity increased with L113 and Q114 to K117, but L120 induced structural differences that inactivated the enzyme. T120 appears to be key in folding the protein and closure of the lid of the active site to acquire its active conformation in the K+-dependent enzymes. E117K mutant was K+-independent and the enzyme acquired the active conformation by a different mechanism. In the K+-independent apoenzyme of Mycobacterium tuberculosis, K72 (K117) flips out of the active site; in the holoenzyme, K72 faces toward the active site bridging the substrates through water molecules. The results provide evidence that two different mechanisms have evolved for the catalysis of this reaction.
Asunto(s)
Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Piruvato Quinasa/ultraestructura , Secuencia de Aminoácidos/genética , Animales , Apoenzimas/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico , Ácido Glutámico/metabolismo , Lisina/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Filogenia , Potasio/metabolismo , Conformación Proteica , ConejosRESUMEN
Pyruvate kinase (PK), encoded by the PKLR gene, is a key player in glycolysis controlling the integrity of erythrocytes. Due to Plasmodium selection, mutations for PK deficiency, which leads to hemolytic anemia, are associated with resistance to malaria in sub-Saharan Africa and with susceptibility to intracellular pathogens in experimental models. In this case-control study, we enrolled 4,555 individuals and investigated whether PKLR single nucleotide polymorphisms (SNPs) putatively selected for malaria resistance are associated with susceptibility to leprosy across Brazil (Manaus-North; Salvador-Northeast; Rondonópolis-Midwest and Rio de Janeiro-Southeast) and with tuberculosis in Mozambique. Haplotype T/G/G (rs1052176/rs4971072/rs11264359) was associated with leprosy susceptibility in Rio de Janeiro (OR = 2.46, p = 0.00001) and Salvador (OR = 1.57, p = 0.04), and with tuberculosis in Mozambique (OR = 1.52, p = 0.07). This haplotype downregulates PKLR expression in nerve and skin, accordingly to GTEx, and might subtly modulate ferritin and haptoglobin levels in serum. Furthermore, we observed genetic signatures of positive selection in the HCN3 gene (xpEHH>2 -recent selection) in Europe but not in Africa, involving 6 SNPs which are PKLR/HCN3 eQTLs. However, this evidence was not corroborated by the other tests (FST, Tajima's D and iHS). Altogether, we provide evidence that a common PKLR locus in Africans contribute to mycobacterial susceptibility in African descent populations and also highlight, for first, PKLR as a susceptibility gene for leprosy and TB.
Asunto(s)
Malaria/genética , Polimorfismo de Nucleótido Simple , Piruvato Quinasa/genética , Adulto , Brasil , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Desequilibrio de Ligamiento , Modelos Logísticos , Masculino , Persona de Mediana Edad , Mozambique , Piruvato Quinasa/deficiencia , Adulto JovenRESUMEN
Investigation of human genes under pathogen-driven selection as Plasmodium sp. has pinpointed genetic variants that participate in the adaptation to the environment and/or are related to severities of human diseases. The current study examined an example of an evolutionary trade-off in which genetic variants in the PKLR gene putatively selected for malaria resistance influence the susceptibility to mycobacterial diseases (leprosy and tuberculosis) in Brazilian population and Mozambique. A complete characterization of the biological effect of those risk variants may clarify the role of the PKLR gene in leprosy and tuberculosis. Deciphering the genetic basis of mycobacterial diseases has implications for the identification of true high-risk individuals in order to optimize screening strategies. Furthermore, the trade-off mechanism discussed in this work might occur in other central genes of immune response and biochemical pathways, controlling the susceptibility to other infectious diseases.
Asunto(s)
Humanos , Masculino , Femenino , Adolescente , Adulto , Persona de Mediana Edad , Piruvato Quinasa/genética , Polimorfismo de Nucleótido Simple , Malaria/genética , Mozambique , Haplotipos , Brasil , Predisposición Genética a la Enfermedad , Frecuencia de los GenesRESUMEN
BACKGROUND: Pyruvate kinase deficiency (PKD) is a rare recessive congenital hemolytic anemia caused by mutations in the PKLR gene. The disease shows a marked variability in clinical expression. We studied the molecular features of nine unrelated Argentinian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase deficiency. DESIGN AND METHODS: Routine hematologic investigations were performed to rule out other causes of chronic hemolytic anemia. Sanger sequencing and in-sílico analysis were carried out to identify and characterize the genetics variants. RESULTS: Six different novel missense variants were detected among the 18 studied alleles: c.661 G > C (Asp221His), c.956 G > T (Gly319Val), c.1595 G > C (Arg532Pro), c.347 G > A (Arg116Gln), c.1232 G > T (Gly411Val), c.1021G > A (Gly341Ser). Structural implications of amino-acid substitutions were correlated with the clinical phenotypes seen in the probands. CONCLUSIONS: This is the first comprehensive report on molecular characterization of pyruvate kinase deficiency in Argentina and the second from South America that would contribute to our knowledge on the distribution and frequency of PKLR variants in our population but also offer new insights into the interpretation of the effect of PKLR variants and phenotype.
Asunto(s)
Alelos , Anemia Hemolítica Congénita no Esferocítica/genética , Mutación Missense , Piruvato Quinasa/deficiencia , Errores Innatos del Metabolismo del Piruvato/genética , Adolescente , Adulto , Sustitución de Aminoácidos , Argentina , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Piruvato Quinasa/genéticaAsunto(s)
Anemia/genética , Eliptocitosis Hereditaria/genética , Glucosafosfato Deshidrogenasa/genética , Mutación , Piruvato Quinasa/genética , Anemia/diagnóstico , República Dominicana , Salud de la Familia , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Recién Nacido , Patrón de Herencia/genética , Masculino , Enfermedades Raras/diagnóstico , Enfermedades Raras/genética , Gemelos Monocigóticos/genéticaRESUMEN
The mosquito Aedes aegypti is vector of several viruses including yellow fever virus, dengue virus chikungunya virus and Zika virus. One of the major problems involving these diseases transmission is that A. aegypti embryos are resistant to desiccation at the end of embryogenesis, surviving and remaining viable for several months inside the egg. Therefore, a fine metabolism control is essential to support these organisms throughout this period of resistance. The carbohydrate metabolism has been shown to be of great importance during arthropod embryogenesis, changing dramatically in order to promote growth and differentiation and in periods of resistance. This study investigated fundamental aspects of glucose metabolism in three stages of A. aegypti egg development: pre-desiccated, desiccated, and rehydrated. The activities of regulatory enzymes in carbohydrate metabolism such as pyruvate kinase, hexokinase and glucose 6-phosphate dehydrogenase were evaluated. We show that these activities were reduced in A. aegypti desiccated eggs, suggesting a decreased activity of glycolytic and pentose phosphate pathway. In contrast, gluconeogenesis increased in desiccated eggs, which uses protein as substrate to synthesize glucose. Accordingly, protein amount decreased during this stage, while glucose levels increased. Glycogen content, a major carbohydrate reserve in mosquitoes, was evaluated and shown to be lower in desiccated and rehydrated eggs, indicating it was used to supply energy metabolism. We observed a reactivation of carbohydrate catabolism and an increased gluconeogenesis after rehydration, suggesting that controlling glucose metabolism was essential not only to survive the period of desiccation, but also for subsequent larvae hatch. Taken together, these results contribute to a better understanding of metabolism regulation in A. aegypti eggs during desiccation periods. Such regulatory mechanisms enable higher survival rate and consequently promote virus transmission by these important disease vectors, making them interesting subjects in the search for novel control methods.
Asunto(s)
Aedes/crecimiento & desarrollo , Aedes/fisiología , Embrión no Mamífero/fisiología , Metabolismo Energético , Gluconeogénesis , Glucólisis , Aedes/embriología , Aedes/enzimología , Animales , Desecación , Embrión no Mamífero/enzimología , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/metabolismo , Hexoquinasa/genética , Hexoquinasa/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Larva/enzimología , Larva/crecimiento & desarrollo , Larva/fisiología , Estado de Hidratación del Organismo , Vía de Pentosa Fosfato , Filogenia , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Estrés Fisiológico , Análisis de SupervivenciaRESUMEN
Several evidences have suggested the involvement of enzymes belonging to the phosphotransfer network, formed by creatine kinase (CK), pyruvate kinase (PK) and adenylate kinase (AK), as well the oxidative stress on the pathogenesis of infectious diseases associated with the central nervous system (CNS). Thus, the aim of this study was to evaluate whether listeriosis alters the brain energy metabolism and/or causes oxidative stress in different brain structures of cattle experimentally infected by Listeria monocytogenes. The cytosolic CK activity was inhibited in the cerebral cortex, cerebellum, brainstem and hippocampus of infected animals compared to uninfected animals, while the mitochondrial CK activity was increased. The PK activity was inhibited in all brain structures of infected animals, while the AK activity was unchanged. Na+, K+-ATPase activity decreased in the cerebral cortex, cerebellum and hippocampus of animals infected by L. monocytogenes. Regarding the oxidative strees variables, the cerebellum and brainstem of infected animals showed increased thiobarbituric acid reactive substances, while the catalase activity was inhibited. Glutathione S-transferarase was inhibited in the cerebral cortex and brainstem of infected animals, and it was increased in the cerebellum. L. monocytogenes was quantified in the liver (nâ¯=â¯5/5) and cerebral cortex (nâ¯=â¯4/5) of the infected cattle. Based on these evidences, the nucleocytoplasmic communication between CK isoenzymes was insufficient to avoid an impairment of cerebral bioenergetics. Moreover, the inhibition on brain PK activity caused an impairment in the communication between sites of ATP generation and ATP utilization. The lipid peroxidation and alteration on antioxidant status observed in some brain structures were also involved during the disease. In summary, these alterations contribute to disease pathogenesis linked to CNS during cattle listeriosis.
Asunto(s)
Adenilato Quinasa/metabolismo , Encéfalo/enzimología , Enfermedades de los Bovinos/enzimología , Creatina Quinasa/metabolismo , Listeria monocytogenes/fisiología , Listeriosis/veterinaria , Piruvato Quinasa/metabolismo , Adenilato Quinasa/genética , Animales , Antioxidantes/metabolismo , Encéfalo/metabolismo , Encéfalo/microbiología , Bovinos , Enfermedades de los Bovinos/metabolismo , Enfermedades de los Bovinos/microbiología , Creatina Quinasa/genética , Forma Mitocondrial de la Creatina-Quinasa/genética , Forma Mitocondrial de la Creatina-Quinasa/metabolismo , Metabolismo Energético , Listeriosis/enzimología , Listeriosis/metabolismo , Listeriosis/microbiología , Oxidantes/metabolismo , Estrés Oxidativo , Fosforilación , Piruvato Quinasa/genéticaRESUMEN
Several studies have been demonstrated that phosphotransfer network, through the adenylate kinase (AK) and pyruvate kinase (PK) activities, allows for new perspectives leading to understanding of disease conditions associated with disturbances in energy metabolism, metabolic monitoring and signalling. In this sense, the aim of this study was to evaluate whether experimental infection by Aeromonas caviae alters hepatic AK and PK activities of silver catfish Rhamdia quelen. Hepatic AK and PK activities decreased in infected animals compared to uninfected animals, as well as the hepatic adenosine triphosphate (ATP) levels. Also, a severe hepatic damage was observed in the infected animals due to the presence of dilation and congestion of vessels, degeneration of hepatocytes and loss of liver parenchyma architecture and sinusoidal structure. Therefore, we have demonstrated, for the first time, that experimental infection by A. caviae inhibits key enzymes linked to the communication between sites of ATP generation and ATP utilization. Moreover, the absence of a reciprocal compensatory mechanism between these enzymes contributes directly to hepatic damage and for a severe energetic imbalance, which may contribute to disease pathophysiology.
Asunto(s)
Aeromonas caviae/fisiología , Bagres , Enfermedades de los Peces/enzimología , Proteínas de Peces/genética , Infecciones por Bacterias Gramnegativas/veterinaria , Hígado/enzimología , Adenilato Quinasa/genética , Adenilato Quinasa/metabolismo , Animales , Metabolismo Energético , Enfermedades de los Peces/virología , Proteínas de Peces/metabolismo , Infecciones por Bacterias Gramnegativas/enzimología , Infecciones por Bacterias Gramnegativas/virología , Hígado/virología , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismoRESUMEN
BACKGROUND: During the last two decades many efforts have been directed towards obtaining efficient microbial processes for the production of shikimic acid (SA); however, feeding high amounts of substrate to increase the titer of this compound has invariably rendered low conversion yields, leaving room for improvement of the producing strains. In this work we report an alternative platform to overproduce SA in a laboratory-evolved Escherichia coli strain, based on plasmid-driven constitutive expression of six genes selected from the pentose phosphate and aromatic amino acid pathways, artificially arranged as an operon. Production strains also carried inactivated genes coding for phosphotransferase system components (ptsHIcrr), shikimate kinases I and II (aroK and aroL), pyruvate kinase I (pykF) and the lactose operon repressor (lacI). RESULTS: The strong and constitutive expression of the constructed operon permitted SA production from the beginning of the cultures, as evidenced in 1 L batch-mode fermentors starting with high concentrations of glucose and yeast extract. Inactivation of the pykF gene improved SA production under the evaluated conditions by increasing the titer, yield and productivity of this metabolite compared to the isogenic pykF+ strain. The best producing strain accumulated up to 43 g/L of SA in 30 h and relatively low concentrations of acetate and aromatic byproducts were detected, with SA accounting for 80% of the produced aromatic compounds. These results were consistent with high expression levels of the glycolytic pathway and synthetic operon genes from the beginning of fermentations, as revealed by transcriptomic analysis. Despite the consumption of 100 g/L of glucose, the yields on glucose of SA and of total aromatic compounds were about 50% and 60% of the theoretical maximum, respectively. The obtained yields and specific production and consumption rates proved to be constant with three different substrate concentrations. CONCLUSIONS: The developed production system allowed continuous SA accumulation until glucose exhaustion and eliminated the requirement for culture inducers. The obtained SA titers and yields represent the highest reported values for a high-substrate batch process, postulating the strategy described in this report as an interesting alternative to the traditionally employed fed-batch processes for SA production.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Glucosa/metabolismo , Vía de Pentosa Fosfato/genética , Fosfotransferasas/metabolismo , Piruvato Quinasa/metabolismo , Ácido Shikímico/metabolismo , Reactores Biológicos , Fermentación , Fosfotransferasas/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Piruvato Quinasa/genéticaRESUMEN
We identified the pyruvate kinase liver/red cell enzyme gene mutation of 8 children previously diagnosed with pyruvate kinase deficiency who were living in a remote town in the western United States. Six were found to be homozygous for the mutation 1529G-A (510 Arg-Gln). Two previously thought to have pyruvate kinase deficiency did not, because they were heterozygous.
Asunto(s)
Mutación , Piruvato Quinasa/deficiencia , Piruvato Quinasa/genética , Anemia Hemolítica Congénita/etiología , Colelitiasis/etiología , Tamización de Portadores Genéticos , Pérdida Auditiva/etiología , Homocigoto , Humanos , Hiperbilirrubinemia/etiología , Ictericia/etiología , Población Rural , Estados UnidosRESUMEN
We report X-ray structures of pyruvate kinase from Leishmania mexicana (LmPYK) that are trapped in different conformations. These, together with the previously reported structure of LmPYK in its inactive (T-state) conformation, allow comparisons of three different conformers of the same species of pyruvate kinase (PYK). Four new site point mutants showing the effects of side-chain alteration at subunit interfaces are also enzymatically characterised. The LmPYK tetramer crystals grown with ammonium sulphate as precipitant adopt an active-like conformation, with sulphate ions at the active and effector sites. The sulphates occupy positions similar to those of the phosphates of ligands bound to active (R-state) and constitutively active (nonallosteric) PYKs from several species, and provide insight into the structural roles of the phosphates of the substrates and effectors. Crystal soaking in sulphate-free buffers was found to induce major conformational changes in the tetramer. In particular, the unwinding of the Aalpha6' helix and the inward hinge movement of the B domain are coupled with a significant widening (4 A) of the tetramer caused by lateral movement of the C domains. The two new LmPYK structures and the activity studies of site point mutations described in this article are consistent with a developing picture of allosteric activity in which localised changes in protein flexibility govern the distribution of conformer families adopted by the tetramer in its active and inactive states.
Asunto(s)
Leishmania mexicana/enzimología , Estructura Cuaternaria de Proteína , Piruvato Quinasa/química , Sulfatos/química , Adenosina Trifosfato/química , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismoRESUMEN
Here we present a biochemical and molecular biology study of the enzyme pyruvate kinase (PYK) from the parasitic protozoa Leishmania donovani. The PYK gene was cloned, mutagenised and over expressed and its kinetic parameters determined. Like in other kinetoplastids, L. donovani PYK is allosterically stimulated by the effector fructose 2,6 biphosphate and not by fructose 1,6 biphosphate. When the putative effector binding site of L. donovani PYK was mutagenised, we obtained two mutants with extreme kinetic behavior: Lys453Leu, which retained a sigmoidal kinetics and was little affected by the effector; and His480Gln, which deployed a hyperbolic kinetics that was not changed by the addition of the effector. Molecular Dynamics (MD) studies revealed that the mutations not only altered the effector binding site of L. donovani PYK but also changed the folding of its domain C.
Asunto(s)
Leishmania donovani/enzimología , Leishmania donovani/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , ADN Protozoario/genética , ADN Protozoario/aislamiento & purificación , Genoma , Glucólisis , Hexoquinasa/genética , Hexoquinasa/metabolismo , Humanos , Cinética , Leishmaniasis Visceral , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
Pyruvate kinase (PK) deficiency is a rare red cell glycolytic enzymopathy. The purpose of the present investigation was to offer prenatal diagnosis for PK deficiency to a couple who had a previous child with severe enzyme deficiency and congenital non-spherocytic hemolytic anemia. PK deficiency was identified in the family by assaying the enzyme activity in red cells. Chorionic villus sampling was performed in an 11-week gestation and the mutation was located in exon 10 of the PKLR gene characterized by polymerase chain reaction and using restriction endonuclease digestion with the MspI enzyme, which was confirmed by DNA sequencing on the ABI 310 DNA sequencer. Both the parents were heterozygous for the 1436G-->A [479 Arg-->His] mutation in exon 10 and the proband was homozygous for this mutation. The fetus was also heterozygous for this mutation and the pregnancy was continued. Prenatal diagnosis allowed the parents with a severely affected child with PK deficiency to have the reproductive choice of having the fetus tested in a subsequent pregnancy.
Asunto(s)
Anemia Hemolítica Congénita no Esferocítica/diagnóstico , Mutación , Diagnóstico Prenatal/métodos , Piruvato Quinasa/deficiencia , Piruvato Quinasa/genética , Anemia Hemolítica/genética , Anemia Hemolítica Congénita no Esferocítica/genética , Análisis Mutacional de ADN , Enzimas de Restricción del ADN/metabolismo , Exones , Femenino , Homocigoto , Humanos , India , Masculino , Embarazo , Primer Trimestre del EmbarazoRESUMEN
A substrate cycle composed of phosphofructo 1-kinase I (PFK) and fructose 1,6 bisphosphatase I (FBPase) has been proposed in rat spermatids. This substrate cycle can explain the ability of glucose to induce a decrease in intracellular ATP, a phenomenon that was related to regulation of [Ca(2+)]i in these cells. In spite of the importance of this metabolic cycle, the expression and activities of the enzymes that compose such cycle have not been systematically studied in spermatogenic cells. Here, we show that PFK and FBPase activities were present in pachytene spermatocytes and round spermatids extracts. Expression of PFK at the mRNA and protein levels showed a relatively similar expression in spermatogenic cells, but a stronger expression in Sertoli cells. Instead, expression of FBPase at the mRNA and protein levels was stronger in round and elongating spermatids as compared to other spermatogenic cells. A similar pattern was observed when evidencing FBPase activity by a NADPH-nitroblue tetrazolium-linked cytochemical assay in isolated pachytene spermatocytes and round spermatids. Rat spermatids also showed the ability to convert lactate to fructose- and glucose-6-P, indicating that both glycolytic and gluconeogenic fluxes are present in these cells. Our results indicate that a coordinated expression of key substrate cycle enzymes, at the level of PFK/FBPase, appear in the last stages of spermatogenic cell differentiation, suggesting that the co-regulation of these enzymes are required for the ability of these cells to respond to glucose and induce metabolic and Ca(2+) signals that can be important for sperm development and function.
Asunto(s)
Fructosa-Bifosfatasa/metabolismo , Regulación Enzimológica de la Expresión Génica , Gluconeogénesis , Glucólisis , Fosfofructoquinasa-1 Tipo Muscular/metabolismo , Espermatogénesis/fisiología , Espermatozoides/enzimología , Testículo/enzimología , Animales , Fructosa-Bifosfatasa/genética , Fructosafosfatos/metabolismo , Glucosa/metabolismo , Glucosa-6-Fosfato/metabolismo , Técnicas In Vitro , Ácido Láctico/metabolismo , Masculino , Fosfofructoquinasa-1 Tipo Muscular/genética , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Células de Sertoli/enzimología , Ciclo del Sustrato , Testículo/citologíaRESUMEN
Pyruvate kinase (PK) deficiency is a rare red cell glycolytic enzymopathy. The purpose of the present investigation was to offer prenatal diagnosis for PK deficiency to a couple who had a previous child with severe enzyme deficiency and congenital non-spherocytic hemolytic anemia. PK deficiency was identified in the family by assaying the enzyme activity in red cells. Chorionic villus sampling was performed in an 11-week gestation and the mutation was located in exon 10 of the PKLR gene characterized by polymerase chain reaction and using restriction endonuclease digestion with the MspI enzyme, which was confirmed by DNA sequencing on the ABI 310 DNA sequencer. Both the parents were heterozygous for the 1436G-->A [479 Arg-->His] mutation in exon 10 and the proband was homozygous for this mutation. The fetus was also heterozygous for this mutation and the pregnancy was continued. Prenatal diagnosis allowed the parents with a severely affected child with PK deficiency to have the reproductive choice of having the fetus tested in a subsequent pregnancy.
Asunto(s)
Humanos , Masculino , Femenino , Embarazo , Anemia Hemolítica Congénita no Esferocítica/diagnóstico , Diagnóstico Prenatal/métodos , Mutación , Piruvato Quinasa/deficiencia , Piruvato Quinasa/genética , Anemia Hemolítica Congénita no Esferocítica/genética , Anemia Hemolítica/genética , Análisis Mutacional de ADN , Enzimas de Restricción del ADN/metabolismo , Homocigoto , Primer Trimestre del Embarazo , Exones , IndiaRESUMEN
The activity of the enzymes of the central metabolic pathways has been the subject of intensive analysis; however, the Entner-Doudoroff (ED) pathway has only recently begun to attract attention. The metabolic response to edd gene knockout in Escherichia coli JM101 and PTS- Glc+ was investigated in gluconate and glucose batch cultures and compared with other pyruvate kinase and PTS mutants previously constructed. Even though the specific growth rates between the strain carrying the edd gene knockout and its parent JM101 and PTS- Glc+ edd and its parent PTS- Glc+ were very similar, reproducible changes in the specific consumption rates and biomass yields were obtained when grown on glucose. These results support the participation of the ED pathway not only on gluconate metabolism but on other metabolic and biochemical processes in E. coli. Despite that gluconate is a non-PTS carbohydrate, the PTS- Glc+ and derived strains showed important reductions in the specific growth and gluconate consumption rates. Moreover, the overall activity of the ED pathway on gluconate resulted in important increments in PTS- Glc+ and PTS- Glc+ pykF mutants. Additional results obtained with the pykA pykF mutant indicate the important contribution of the pyruvate kinase enzymes to pyruvate synthesis and energy production in both carbon sources.
Asunto(s)
Escherichia coli/metabolismo , Gluconatos/metabolismo , Glucosa/metabolismo , Aldehído-Liasas/análisis , Biomasa , Escherichia coli/crecimiento & desarrollo , Eliminación de Gen , Hidroliasas/análisis , Hidroliasas/genética , Fosfotransferasas/genética , Piruvato Quinasa/genéticaRESUMEN
The expression of aldolase A and B isoenzyme transcripts was confirmed by RT-PCR in rat kidney and their cell distribution was compared with characteristic enzymes of the gluconeogenic and glycolytic metabolic pathway: fructose-1,6-bisphosphatase (FBPase), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate kinase (PK). We detected aldolase A isoenzyme in the thin limb and collecting ducts of the medulla and in the distal tubules and glomerula of the cortex. The same pattern of distribution was found for PK, but not for aldolase B, PEPCK, and FBPase. In addition, co-localization studies confirmed that aldolase B, FBPase, and PEPCK are expressed in the same proximal cells. This segregated cell distribution of aldolase A and B with key glycolytic and gluconeogenic enzymes, respectively, suggests that these aldolase isoenzymes participate in different metabolic pathways. In order to test if FBPase interacts with aldolase B, FBPase was immobilized on agarose and subjected to binding experiments. The results show that only aldolase B is specifically bound to FBPase and that this interaction was specifically disrupted by 60 microM Fru-1,6-P2. These data indicate the presence of a modulated enzyme-enzyme interaction between FBPase and isoenzyme B. They affirm that in kidney, aldolase B specifically participates, along the gluconeogenic pathway and aldolase A in glycolysis.
Asunto(s)
Fructosa-Bifosfatasa/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Glucosa/metabolismo , Isoenzimas/metabolismo , Riñón/enzimología , Animales , Cromatografía de Afinidad , Detergentes/metabolismo , Fructosa-Bifosfato Aldolasa/genética , Gluconeogénesis , Glucólisis , Isoenzimas/genética , Riñón/citología , Complejos Multienzimáticos , Octoxinol/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Ratas , Ratas Wistar , PorcinosRESUMEN
We report the kinetic characterization of a previously unidentified pyruvate kinase (PK) activity in extracts from Entamoeba histolytica trophozoites. This activity was about 74% of the activity of pyruvate phosphate dikinase. EhPK differed from most PKs in that its pH optimum was 5.5-6.5 and was inhibited by high PEP concentrations (1-5mM); these are concentrations at which PK is usually assayed. The optimal temperature was above 40 degrees C with negligible activity below 20 degrees C. EhPK exhibited hyperbolic kinetics with respect to both PEP (K(m) = 0.018 mM) and ADP (K(m) = 1.05 mM). However, it exhibited a sigmoidal behavior with respect to PEP at sub-saturating ADP concentrations. EhPK did not require monovalent cations for activity. Fructose-1,6 bisphosphate was a potent non-essential activator; it increased the affinity for ADP without modification of the V(max) or the affinity for PEP. Phosphate, citrate, malate, and alpha-ketoglutarate significantly inhibited EhPK activity. A putative EhPK gene fragment found in EhDNA was analyzed. The data indicate that E. histolytica trophozoites contain an active PK, which might contribute to the generation of glycolytic ATP for parasite survival.