Juan Slebe T. - Academia.edu (original) (raw)

Papers by Juan Slebe T.

Research paper thumbnail of Introduction of a tryptophan reporter group into different regions of the frutose-l,6-bisphosphatase for the study of ligand binding and conformational dynamics

Biochemical Society Transactions, Oct 1, 2000

Research paper thumbnail of Over-expression of muscle glycogen synthase in human diabetic nephropathy

Histochemistry and Cell Biology, 2014

glycogen amount and cell death was observed. Based on a previous transcriptome study on human dia... more glycogen amount and cell death was observed. Based on a previous transcriptome study on human diabetic kidney disease, significant differences in the expression of genes involved in glycogen metabolism were analyzed. We propose that glucose, but not insulin, is the main modulator of MGS activity in HK2 cells, suggesting that blood glucose control is the best approach to modulate renal glycogeninduced damage during long-term diabetes.

Research paper thumbnail of Relacion Del Sindrome Diabetico Con La Expresion y Localizacion Celular De Glicogeno Sintasa, Fosfoenolpiruvato Carboxiquinasa y FRUCTOSA-1, 6-BISFOSTASA, Enzimas Claves en La Homeostasis De La Glucosa

Research paper thumbnail of Carbamylation of aspartate transaminase and the pK value of the active site lysyl residue

Journal of Biological Chemistry, 1976

Abnormal lysyl residues can be detected in aspartate transaminase by following the rate of reacti... more Abnormal lysyl residues can be detected in aspartate transaminase by following the rate of reaction of amino groups with KN'%O and the rate of enzymatic inactivation. Peptide isolation subsequent to carbamylation of the apoenzyme produces a peptide which is absent in the carbamylated holoenzyme. The composition of the carbamylated peptide matches that of a tryptic peptide containing the active site Lys-258. The holoenzyme retains full catalytic activity after carbamylation of its NHz-terminal alanine and lysyl residues other than Lys-258, which is protected by aldimine formation with pyridoxal phosphate. Apoenzyme prepared from KNCO-treated holoenzyme (apoenzyme') is susceptible to further carbamylation at Lys-258 with irreversible loss of catalytic activity. Carbamylation of the active site lysyl residue is 25 to 50 times more rapid than that of the other 18 lysyl residues of aspartate transaminase. The kinetics of inactivation by KNCO at different pH values served to determine the pH-independent second order rate constant (k) and the pK of the amino group of Lys-258. These values are pK = 7.98 & 0.08 and k = 146 * 5 M-k', which are similar to the values determined for carbamylation of the NH,terminal groups of human hemoglobin (

Research paper thumbnail of Selective thiol group modification renders fructose-1,6-bisphosphatase insensitive to fructose 2,6-bisphosphate inhibition

Journal of Biological Chemistry, Jun 1, 1987

Limited treatment of native pig kidney fructose-1,6-bisphosphatase (50 microM enzyme subunit) wit... more Limited treatment of native pig kidney fructose-1,6-bisphosphatase (50 microM enzyme subunit) with [14C]N-ethylmaleimide (100 microM) at 30 degrees C, pH 7.5, in the presence of AMP (200 microM) results in the modification of 1 reactive cysteine residue/enzyme subunit. The N-ethylmaleimide-modified fructose-1,6-bisphosphatase has a functional catalytic site but is no longer inhibited by fructose 2,6-bisphosphate. The enzyme derivative also exhibits decreased affinity toward Mg2+. The presence of fructose 2,6-bisphosphate during the modification protects the enzyme against the loss of fructose 2,6-bisphosphate inhibition. Moreover, the modified enzyme is inhibited by monovalent cations, as previously reported (Reyes, A., Hubert, E., and Slebe, J.C. (1985) Biochem. Biophys. Res. Commun. 127, 373-379), and does not show inhibition by high substrate concentrations. A comparison of the kinetic properties of native and N-ethylmaleimide-modified fructose-1,6-bisphosphatase reveals differences in some properties but none is so striking as the complete loss of fructose 2,6-bisphosphate sensitivity. The results demonstrate that fructose 2,6-bisphosphate interacts with a specific allosteric site on fructose-1,6-bisphosphatase, and they also indicate that high levels of fructose 1,6-bisphosphate inhibit the enzyme by binding to this fructose 2,6-bisphosphate allosteric site.

Research paper thumbnail of Affinity labelling of fructose-1, 6-bisphosphatase with adenosine 5'-monophosphate 2' 3'-dialdehyde

Research paper thumbnail of Selective chemical modification and 19F NMR in the assignment of a pK value to the active site lysyl residue in aspartate transaminase

Journal of Biological Chemistry, 1978

Research paper thumbnail of Estructura y Funcion De Aspartato Aminotransferasa Mitocondrial: Efecto De Mutagenesis Sitio Dirigida Sobre Su Estabilidad, Plegamiento e Importacion a La Matriz Mitocondrial

Research paper thumbnail of Glutathione depletion in spermatogonia-type germ cells: Autophagy and Ago2 function

de reunión: P09-005-SP Fecha de publicación: JUL 2015 Ver información de revista Información del ... more de reunión: P09-005-SP Fecha de publicación: JUL 2015 Ver información de revista Información del autor Direcciones: [ 1 ] Univ Austral Chile, Inst Bioquim & Microbiol, Valdivia, Chile [ 2 ] Univ Regensburg, Lab RNA Biol, Biochem Ctr Regensburg BZR, D-93053 Regensburg, Germany [ 3 ] Univ Chile, Fac Ciencias Quim & Farmaceut, Adv Ctr Chron Dis ACCDiS, Santiago, Chile [ 4 ] Univ Chile, Fac Med, Santiago, Chile Editorial WILEY-BLACKWELL, 111 RIVER ST, HOBOKEN 07030-5774, NJ USA Categorías / Clasificación Áreas de investigación: Biochemistry & Molecular Biology Categorías de Web of Science: Biochemistry & Molecular Biology Información del documento Tipo de documento: Meeting Abstract Idioma: English Número de acceso: WOS:000362570602010 ISSN: 1742-464X eISSN: 1742-4658 Información de la revista • Impact Factor: Journal Citation Reports®

Research paper thumbnail of Stereochemistry of holoaspartate transaminase after modification of the active site Lys-258

Journal of Biological Chemistry, 1979

Proton incorporation at position C4 of the substrate-coenzyme Schiff base of aspartate transamina... more Proton incorporation at position C4 of the substrate-coenzyme Schiff base of aspartate transaminase is a stereospecific process. After carbamylation of the active site Lys-258, the stereospecificity of the reaction in 2H2O is retained. By a correlation method, it is shown that addition occurs from the si side of the complex and the pyridoxamine phosphate produced is deuterated at position pro-S of the pyridoxamine methylene group. These results constitute a demonstration for the stereochemstry of a half-transamination process of the phosphorylated coenzyme under single turnover conditions. They also illustrate that free Lys-258 is not required to maintain stereospecificity and cast doubts on the implication of this residue as a participant in C4 proton addition during catalysis by the native form of this mammalian enzyme.

Research paper thumbnail of Fluorine-19 as a covalent active site-directed magnetic resonance probe in aspartate transaminase

Journal of Biological Chemistry, 1976

Phosphopyridoxyl trifluoroethylamine has been synthesized as an active site-directed lsF NMR prob... more Phosphopyridoxyl trifluoroethylamine has been synthesized as an active site-directed lsF NMR probe for aspartate transaminase. This coenzyme derivative adds stoichiometrically to the apotransaminase as observed by both fluorescence and circular dichroism measurements. The fluorinated phosphopyridoxamine derivative, when bound to the apotransaminase, will not dissociate upon extensive dialysis or passage through Sephadex G-25. The compound behaves as a pyridoxamine phosphate derivative and not as a coenzyme .substrate complex, since both competing anions and dicarboxylic acid inhibitors still bind to the phosphopyridoxyl trifluoroethylamine enzyme. The 19F NMR spectra of the enzyme-bound phosphopyridoxyl trifluoroethylamine were measured as a function of pH, ionic strength, and temperature. The 19F NMR of the enzyme-bound coenzyme derivative revealed no predetermined asymmetry in the subunits of aspartate transaminase in solution in terms of differences in chemical shift or resonance line shape between the two environments. A pH-dependent chemical shift change of the single 19F resonance was observed, which is consistent with the influence of a single ionization with an apparent pK, of 8.4 in 0.10 M KC1 at 30". Increasing the ionic strength resulted in increasing values for the observed pK,, the highest recorded value was 9.1 in 3.0 M KCl. The temperature dependence of the pH titration of the chemical shift gives AH' of ionization of 10.5 kcal/mol. The evidence suggests a possible c-amino group, electrostatically affected by positive charges, being responsible for the titration effect of the active site-bound fluorine derivative of pyridoxamine phosphate.

Research paper thumbnail of Gluconeogenesis-Linked Glycogen Metabolism Is Important in the Achievement of In Vitro Capacitation of Dog Spermatozoa in a Medium Without Glucose1

Biology of Reproduction, 2004

In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metab... more In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [ 14 C]glycogen after the addition to l-CCM with [ 14 C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.

Research paper thumbnail of Stereochemistry of holoaspartate transaminase after modification of the active site Lys-258

The Journal of biological chemistry, Jan 10, 1979

Proton incorporation at position C4 of the substrate-coenzyme Schiff base of aspartate transamina... more Proton incorporation at position C4 of the substrate-coenzyme Schiff base of aspartate transaminase is a stereospecific process. After carbamylation of the active site Lys-258, the stereospecificity of the reaction in 2H2O is retained. By a correlation method, it is shown that addition occurs from the si side of the complex and the pyridoxamine phosphate produced is deuterated at position pro-S of the pyridoxamine methylene group. These results constitute a demonstration for the stereochemstry of a half-transamination process of the phosphorylated coenzyme under single turnover conditions. They also illustrate that free Lys-258 is not required to maintain stereospecificity and cast doubts on the implication of this residue as a participant in C4 proton addition during catalysis by the native form of this mammalian enzyme.

Research paper thumbnail of Estudio De Nuevos Mecanismos De Regulacion De La Fructosa 1,6-BISFOSFATASA: Formacion De Complejos Metabolicos, Compartimentacion Celular y Union Asimetrica Del Sustrato y Del Efector FRUCTOSA-2,6-BISFOSFATO a Los Sitios Activos De

Research paper thumbnail of The Many Faces of Carbohydrate Metabolism in Male Germ Cells: From Single Molecules to Active Polymers

International Journal of Medical and Surgical Sciences, 2018

Spermatogenesis is a complex physiological process that involves cell proliferation, meiotic divi... more Spermatogenesis is a complex physiological process that involves cell proliferation, meiotic division and a final cell differentiation of post-meiotic cells into spermatozoa. During this process male germ cells also undergo a metabolic differentiation process, in which post-meiotic spermatogenic cells (spermatids) but not meiotic spermatogenic cells (spermatocytes) respond differentially to D-glucose metabolism, glucose transporters (GLUTs) distribution and utilization of non-hexose substrates, such as lactate/pyruvate or dihydroxyacetone. These differences might be explained by the requirement for a specific metabolic process to support cell differentiation or in some cases, cell viability. In addition, though glycogen is considered to be the main glucose store, in male germ cells this polymer may play a novel role in cell proliferation, acting as a new marker for apoptotic events in testicular tissue via a yet unknown mechanism. In this article, we summarize the main metabolic cha...

Research paper thumbnail of Fluorinated amino acids and phosphopyridoxyl fluoroamino acids as reversible active site directed inhibitors of aspartate transaminase

Biochemical and Biophysical Research Communications, 1975

Research paper thumbnail of Novel expression of liver FBPase in Langerhans islets of human and rat pancreas

Journal of Cellular Physiology, 2005

Several reports have indicated the absence of gluconeogenic enzymes in pancreatic islet cells. In... more Several reports have indicated the absence of gluconeogenic enzymes in pancreatic islet cells. In contrast, here we demonstrate that liver fructose-1,6-bisphosphatase (FBPase) is highly expressed both in human and rat pancreas. Interestingly, pancreatic FBPase is active and functional, and is inhibited by AMP and fructose-2,6-bisphosphate (Fru-2,6-P2). These results suggest that FBPase may participate as a component of a metabolic sensing mechanism present in the pancreas. Immunolocalization analysis showed that FBPase is expressed both in human and rat Langerhans islets, specifically in beta cells. In humans, FBPase was also located in the canaliculus and acinar cells. These results indicate that FBPase coupled with phosphofructokinase (PFK) plays a crucial role in the metabolism of pancreatic islet cells. The demonstration of gluconeogenic recycling of trioses as a new metabolic signaling pathway may contribute to our understanding of the differences between the insulin secretagogues trioses, fructose, and glucose in pancreas.

Research paper thumbnail of Effect of Tacrolimus on Activity and Expression of P-Glycoprotein and ATP-Binding Cassette Transporter A5 (ABCA5) Proteins in Hematoencephalic Barrier Cells

Biological & Pharmaceutical Bulletin, 2008

Research paper thumbnail of Potassium activation and its relationship to a highly reactive cysteine residue in fructose 1,6-bisphosphatase

Archives of Biochemistry and Biophysics, 1986

Research paper thumbnail of Temperature, but not excess of glycogen, regulate post-mortem AMPK activity in muscle of steer carcasses

Post-mortem muscle temperature affects the rate of decline in pH in a linear manner from 37.5 °C ... more Post-mortem muscle temperature affects the rate of decline in pH in a linear manner from 37.5 °C down near 0 °C, and this pH decline is correlated with the enzymatic degradation of glycogen to lactate. This transformation occurs in an anaerobic context that includes the metabolic splice between glycogenolysis and glycolysis; and both processes are strongly upregulated by AMPK enzyme. In this study we reported changes (0.5 h and 24 h post-mortem) in muscle glycogen concentration, lactate and AMPK activity from 12 samples of Longissimus dorsi from 38 steers that produced high pH (>5.9) and normal pH (<5.8) carcasses at 24 h post-mortem. Moreover, we evaluated changes in AMPK activity in samples from both categories incubated at 37, 25, 17 and 5 °C and supplemented with exogenous glycogen. Finally, we analysed if there were structural differences between polymers from both categories. Our analyses show that enzymatic AMPK activity was significantly higher at 17 °C than at 37 °C o...

Research paper thumbnail of Introduction of a tryptophan reporter group into different regions of the frutose-l,6-bisphosphatase for the study of ligand binding and conformational dynamics

Biochemical Society Transactions, Oct 1, 2000

Research paper thumbnail of Over-expression of muscle glycogen synthase in human diabetic nephropathy

Histochemistry and Cell Biology, 2014

glycogen amount and cell death was observed. Based on a previous transcriptome study on human dia... more glycogen amount and cell death was observed. Based on a previous transcriptome study on human diabetic kidney disease, significant differences in the expression of genes involved in glycogen metabolism were analyzed. We propose that glucose, but not insulin, is the main modulator of MGS activity in HK2 cells, suggesting that blood glucose control is the best approach to modulate renal glycogeninduced damage during long-term diabetes.

Research paper thumbnail of Relacion Del Sindrome Diabetico Con La Expresion y Localizacion Celular De Glicogeno Sintasa, Fosfoenolpiruvato Carboxiquinasa y FRUCTOSA-1, 6-BISFOSTASA, Enzimas Claves en La Homeostasis De La Glucosa

Research paper thumbnail of Carbamylation of aspartate transaminase and the pK value of the active site lysyl residue

Journal of Biological Chemistry, 1976

Abnormal lysyl residues can be detected in aspartate transaminase by following the rate of reacti... more Abnormal lysyl residues can be detected in aspartate transaminase by following the rate of reaction of amino groups with KN'%O and the rate of enzymatic inactivation. Peptide isolation subsequent to carbamylation of the apoenzyme produces a peptide which is absent in the carbamylated holoenzyme. The composition of the carbamylated peptide matches that of a tryptic peptide containing the active site Lys-258. The holoenzyme retains full catalytic activity after carbamylation of its NHz-terminal alanine and lysyl residues other than Lys-258, which is protected by aldimine formation with pyridoxal phosphate. Apoenzyme prepared from KNCO-treated holoenzyme (apoenzyme') is susceptible to further carbamylation at Lys-258 with irreversible loss of catalytic activity. Carbamylation of the active site lysyl residue is 25 to 50 times more rapid than that of the other 18 lysyl residues of aspartate transaminase. The kinetics of inactivation by KNCO at different pH values served to determine the pH-independent second order rate constant (k) and the pK of the amino group of Lys-258. These values are pK = 7.98 & 0.08 and k = 146 * 5 M-k', which are similar to the values determined for carbamylation of the NH,terminal groups of human hemoglobin (

Research paper thumbnail of Selective thiol group modification renders fructose-1,6-bisphosphatase insensitive to fructose 2,6-bisphosphate inhibition

Journal of Biological Chemistry, Jun 1, 1987

Limited treatment of native pig kidney fructose-1,6-bisphosphatase (50 microM enzyme subunit) wit... more Limited treatment of native pig kidney fructose-1,6-bisphosphatase (50 microM enzyme subunit) with [14C]N-ethylmaleimide (100 microM) at 30 degrees C, pH 7.5, in the presence of AMP (200 microM) results in the modification of 1 reactive cysteine residue/enzyme subunit. The N-ethylmaleimide-modified fructose-1,6-bisphosphatase has a functional catalytic site but is no longer inhibited by fructose 2,6-bisphosphate. The enzyme derivative also exhibits decreased affinity toward Mg2+. The presence of fructose 2,6-bisphosphate during the modification protects the enzyme against the loss of fructose 2,6-bisphosphate inhibition. Moreover, the modified enzyme is inhibited by monovalent cations, as previously reported (Reyes, A., Hubert, E., and Slebe, J.C. (1985) Biochem. Biophys. Res. Commun. 127, 373-379), and does not show inhibition by high substrate concentrations. A comparison of the kinetic properties of native and N-ethylmaleimide-modified fructose-1,6-bisphosphatase reveals differences in some properties but none is so striking as the complete loss of fructose 2,6-bisphosphate sensitivity. The results demonstrate that fructose 2,6-bisphosphate interacts with a specific allosteric site on fructose-1,6-bisphosphatase, and they also indicate that high levels of fructose 1,6-bisphosphate inhibit the enzyme by binding to this fructose 2,6-bisphosphate allosteric site.

Research paper thumbnail of Affinity labelling of fructose-1, 6-bisphosphatase with adenosine 5'-monophosphate 2' 3'-dialdehyde

Research paper thumbnail of Selective chemical modification and 19F NMR in the assignment of a pK value to the active site lysyl residue in aspartate transaminase

Journal of Biological Chemistry, 1978

Research paper thumbnail of Estructura y Funcion De Aspartato Aminotransferasa Mitocondrial: Efecto De Mutagenesis Sitio Dirigida Sobre Su Estabilidad, Plegamiento e Importacion a La Matriz Mitocondrial

Research paper thumbnail of Glutathione depletion in spermatogonia-type germ cells: Autophagy and Ago2 function

de reunión: P09-005-SP Fecha de publicación: JUL 2015 Ver información de revista Información del ... more de reunión: P09-005-SP Fecha de publicación: JUL 2015 Ver información de revista Información del autor Direcciones: [ 1 ] Univ Austral Chile, Inst Bioquim & Microbiol, Valdivia, Chile [ 2 ] Univ Regensburg, Lab RNA Biol, Biochem Ctr Regensburg BZR, D-93053 Regensburg, Germany [ 3 ] Univ Chile, Fac Ciencias Quim & Farmaceut, Adv Ctr Chron Dis ACCDiS, Santiago, Chile [ 4 ] Univ Chile, Fac Med, Santiago, Chile Editorial WILEY-BLACKWELL, 111 RIVER ST, HOBOKEN 07030-5774, NJ USA Categorías / Clasificación Áreas de investigación: Biochemistry & Molecular Biology Categorías de Web of Science: Biochemistry & Molecular Biology Información del documento Tipo de documento: Meeting Abstract Idioma: English Número de acceso: WOS:000362570602010 ISSN: 1742-464X eISSN: 1742-4658 Información de la revista • Impact Factor: Journal Citation Reports®

Research paper thumbnail of Stereochemistry of holoaspartate transaminase after modification of the active site Lys-258

Journal of Biological Chemistry, 1979

Proton incorporation at position C4 of the substrate-coenzyme Schiff base of aspartate transamina... more Proton incorporation at position C4 of the substrate-coenzyme Schiff base of aspartate transaminase is a stereospecific process. After carbamylation of the active site Lys-258, the stereospecificity of the reaction in 2H2O is retained. By a correlation method, it is shown that addition occurs from the si side of the complex and the pyridoxamine phosphate produced is deuterated at position pro-S of the pyridoxamine methylene group. These results constitute a demonstration for the stereochemstry of a half-transamination process of the phosphorylated coenzyme under single turnover conditions. They also illustrate that free Lys-258 is not required to maintain stereospecificity and cast doubts on the implication of this residue as a participant in C4 proton addition during catalysis by the native form of this mammalian enzyme.

Research paper thumbnail of Fluorine-19 as a covalent active site-directed magnetic resonance probe in aspartate transaminase

Journal of Biological Chemistry, 1976

Phosphopyridoxyl trifluoroethylamine has been synthesized as an active site-directed lsF NMR prob... more Phosphopyridoxyl trifluoroethylamine has been synthesized as an active site-directed lsF NMR probe for aspartate transaminase. This coenzyme derivative adds stoichiometrically to the apotransaminase as observed by both fluorescence and circular dichroism measurements. The fluorinated phosphopyridoxamine derivative, when bound to the apotransaminase, will not dissociate upon extensive dialysis or passage through Sephadex G-25. The compound behaves as a pyridoxamine phosphate derivative and not as a coenzyme .substrate complex, since both competing anions and dicarboxylic acid inhibitors still bind to the phosphopyridoxyl trifluoroethylamine enzyme. The 19F NMR spectra of the enzyme-bound phosphopyridoxyl trifluoroethylamine were measured as a function of pH, ionic strength, and temperature. The 19F NMR of the enzyme-bound coenzyme derivative revealed no predetermined asymmetry in the subunits of aspartate transaminase in solution in terms of differences in chemical shift or resonance line shape between the two environments. A pH-dependent chemical shift change of the single 19F resonance was observed, which is consistent with the influence of a single ionization with an apparent pK, of 8.4 in 0.10 M KC1 at 30". Increasing the ionic strength resulted in increasing values for the observed pK,, the highest recorded value was 9.1 in 3.0 M KCl. The temperature dependence of the pH titration of the chemical shift gives AH' of ionization of 10.5 kcal/mol. The evidence suggests a possible c-amino group, electrostatically affected by positive charges, being responsible for the titration effect of the active site-bound fluorine derivative of pyridoxamine phosphate.

Research paper thumbnail of Gluconeogenesis-Linked Glycogen Metabolism Is Important in the Achievement of In Vitro Capacitation of Dog Spermatozoa in a Medium Without Glucose1

Biology of Reproduction, 2004

In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metab... more In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [ 14 C]glycogen after the addition to l-CCM with [ 14 C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.

Research paper thumbnail of Stereochemistry of holoaspartate transaminase after modification of the active site Lys-258

The Journal of biological chemistry, Jan 10, 1979

Proton incorporation at position C4 of the substrate-coenzyme Schiff base of aspartate transamina... more Proton incorporation at position C4 of the substrate-coenzyme Schiff base of aspartate transaminase is a stereospecific process. After carbamylation of the active site Lys-258, the stereospecificity of the reaction in 2H2O is retained. By a correlation method, it is shown that addition occurs from the si side of the complex and the pyridoxamine phosphate produced is deuterated at position pro-S of the pyridoxamine methylene group. These results constitute a demonstration for the stereochemstry of a half-transamination process of the phosphorylated coenzyme under single turnover conditions. They also illustrate that free Lys-258 is not required to maintain stereospecificity and cast doubts on the implication of this residue as a participant in C4 proton addition during catalysis by the native form of this mammalian enzyme.

Research paper thumbnail of Estudio De Nuevos Mecanismos De Regulacion De La Fructosa 1,6-BISFOSFATASA: Formacion De Complejos Metabolicos, Compartimentacion Celular y Union Asimetrica Del Sustrato y Del Efector FRUCTOSA-2,6-BISFOSFATO a Los Sitios Activos De

Research paper thumbnail of The Many Faces of Carbohydrate Metabolism in Male Germ Cells: From Single Molecules to Active Polymers

International Journal of Medical and Surgical Sciences, 2018

Spermatogenesis is a complex physiological process that involves cell proliferation, meiotic divi... more Spermatogenesis is a complex physiological process that involves cell proliferation, meiotic division and a final cell differentiation of post-meiotic cells into spermatozoa. During this process male germ cells also undergo a metabolic differentiation process, in which post-meiotic spermatogenic cells (spermatids) but not meiotic spermatogenic cells (spermatocytes) respond differentially to D-glucose metabolism, glucose transporters (GLUTs) distribution and utilization of non-hexose substrates, such as lactate/pyruvate or dihydroxyacetone. These differences might be explained by the requirement for a specific metabolic process to support cell differentiation or in some cases, cell viability. In addition, though glycogen is considered to be the main glucose store, in male germ cells this polymer may play a novel role in cell proliferation, acting as a new marker for apoptotic events in testicular tissue via a yet unknown mechanism. In this article, we summarize the main metabolic cha...

Research paper thumbnail of Fluorinated amino acids and phosphopyridoxyl fluoroamino acids as reversible active site directed inhibitors of aspartate transaminase

Biochemical and Biophysical Research Communications, 1975

Research paper thumbnail of Novel expression of liver FBPase in Langerhans islets of human and rat pancreas

Journal of Cellular Physiology, 2005

Several reports have indicated the absence of gluconeogenic enzymes in pancreatic islet cells. In... more Several reports have indicated the absence of gluconeogenic enzymes in pancreatic islet cells. In contrast, here we demonstrate that liver fructose-1,6-bisphosphatase (FBPase) is highly expressed both in human and rat pancreas. Interestingly, pancreatic FBPase is active and functional, and is inhibited by AMP and fructose-2,6-bisphosphate (Fru-2,6-P2). These results suggest that FBPase may participate as a component of a metabolic sensing mechanism present in the pancreas. Immunolocalization analysis showed that FBPase is expressed both in human and rat Langerhans islets, specifically in beta cells. In humans, FBPase was also located in the canaliculus and acinar cells. These results indicate that FBPase coupled with phosphofructokinase (PFK) plays a crucial role in the metabolism of pancreatic islet cells. The demonstration of gluconeogenic recycling of trioses as a new metabolic signaling pathway may contribute to our understanding of the differences between the insulin secretagogues trioses, fructose, and glucose in pancreas.

Research paper thumbnail of Effect of Tacrolimus on Activity and Expression of P-Glycoprotein and ATP-Binding Cassette Transporter A5 (ABCA5) Proteins in Hematoencephalic Barrier Cells

Biological & Pharmaceutical Bulletin, 2008

Research paper thumbnail of Potassium activation and its relationship to a highly reactive cysteine residue in fructose 1,6-bisphosphatase

Archives of Biochemistry and Biophysics, 1986

Research paper thumbnail of Temperature, but not excess of glycogen, regulate post-mortem AMPK activity in muscle of steer carcasses

Post-mortem muscle temperature affects the rate of decline in pH in a linear manner from 37.5 °C ... more Post-mortem muscle temperature affects the rate of decline in pH in a linear manner from 37.5 °C down near 0 °C, and this pH decline is correlated with the enzymatic degradation of glycogen to lactate. This transformation occurs in an anaerobic context that includes the metabolic splice between glycogenolysis and glycolysis; and both processes are strongly upregulated by AMPK enzyme. In this study we reported changes (0.5 h and 24 h post-mortem) in muscle glycogen concentration, lactate and AMPK activity from 12 samples of Longissimus dorsi from 38 steers that produced high pH (>5.9) and normal pH (<5.8) carcasses at 24 h post-mortem. Moreover, we evaluated changes in AMPK activity in samples from both categories incubated at 37, 25, 17 and 5 °C and supplemented with exogenous glycogen. Finally, we analysed if there were structural differences between polymers from both categories. Our analyses show that enzymatic AMPK activity was significantly higher at 17 °C than at 37 °C o...