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Papers by ana preller

Research paper thumbnail of The evolution of hexokinases

Archivos de biologia y medicina experimentales, 1987

Recent advances in the knowledge of the structural and functional aspects of the enzymes catalyzi... more Recent advances in the knowledge of the structural and functional aspects of the enzymes catalyzing sugar phosphorylation by ATP are reviewed. Hexokinases may exist, mainly in prokaryotes, as sugar-specific kinases (glucokinase, fructokinase, mannokinase) or as ubiquitous hexose-kinases which are relatively unspecific for the natural hexoses. Enzymes presenting intermediate specificity (e.g. mannofructokinases) have been also described. With a few exceptions, the molecular mass of a variety of hexokinases may be either 25 kDa, 50 kDa or 100 kDa. The smaller hexokinases have been found in some microorganisms whereas the 50 kDa enzymes are found (with only one exception) in most invertebrates and in a particular isozyme from vertebrates (hexokinase D). The 100 kDa enzymes are restricted to vertebrates (hexokinases A, B and C). These facts have led to the speculation that gene duplication events have played an important role in the evolutionary development of the hexokinases from prese...

Research paper thumbnail of In vivo ionic uptake through the skin of the South American toad Bufo arunco

Revue canadienne de biologie / éditée par l'Université de Montréal, 1971

Research paper thumbnail of Glucose utilization in vertebrates as a molecular probe for the study of evolution

Archivos de biología y medicina experimentales, 1979

Hexokinase isozymic profiles from the liver of 68 vertebrate species are presented. The compariso... more Hexokinase isozymic profiles from the liver of 68 vertebrate species are presented. The comparison of the diverse patterns observed, as well as the kinetic and physicochemical properties of the isozymes, reveals that the hexokinases from mammals are very similar to those from turtles and amphibians. The hexokinases from birds, lizards and snakes on the other hand are similar within themselves and different from the enzymes from mammals and amphibians. Liver pyruvate kinases show about the same behavior. The hexokinase system from vertebrate muscle however is very uniform in all the species studied consisting mainly of hexokinase B.

Research paper thumbnail of Cytoarchitecture of Caudiverbera caudiverbera stage VI oocytes: a light and electron microscope study

Anatomy and Embryology, 1999

The general characteristics and salient features of the full-grown stage VI Caudiverbera caudiver... more The general characteristics and salient features of the full-grown stage VI Caudiverbera caudiverbera oocyte at the light and electron microscopy level are described. The oocyte is a huge cell with radial symmetry and distinct polarity. A black animal hemisphere, rich in pigment granules and containing the nucleus, is clearly distinguished from the unpigmented white-yellowish vegetal hemisphere. The cell is surrounded by a highly invaginated plasma membrane, with numerous microvilli. The cortex underlying the plasma membrane contains cortical and pigment granules, mitochondria, rough endoplasmic reticulum and coated vesicles. Cytoskeletal components, such as actin filaments and microtubules, are also found in this region. The predominant structures, distributed throughout the cell, are the yolk platelets, which show a gradient in size with small platelets in the animal half and very large ones in the vegetal zone. Mitochondria are also very abundant in both hemispheres and clouds of these organelles are found in the perinuclear region, frequently associated with microtubules. Developed Golgi complexes are present in the cytoplasm and occasionally, annulate lamellae appear towards the inner zones. The nucleus is a large structure containing numerous nucleoli. The nuclear envelope is highly invaginated, especially at the side facing the vegetal pole. It is regularly perforated by large nuclear pores. Our results show that the structural organization of Caudiverbera oocytes, although similar to that of other amphibian oocytes, differs from them especially concerning the spatial distribution of several structural components.

Research paper thumbnail of Effect of salt depletion on sodium concentration in serum and urine of Bufo chilensis. Evidences for increased levels of neurohypophysial principles in their plasma

Comparative Biochemistry and Physiology Part A: Physiology, 1987

Salt-depleted toads Bufo chilensis were compared with animals maintained in NaCl solution and a c... more Salt-depleted toads Bufo chilensis were compared with animals maintained in NaCl solution and a control group with respect to Na+ content in serum and urine. 2. Plasma hydro-osmotic activity of the animals was measured by increased water transfer across the isolated urinary bladder of the frog (Cuudiuerbera caudiverbera). 3. Sodium in serum is not affected by pre-adaptation in distilled water. Urine Na+ is markedly reduced. 4. Plasma from depleted animals increases water transfer across the isolated urinary bladder. Immersion in NaCl solution did not have this effect. An increase in neurohypophysial hormones in the blood of the animals is postulated.

Research paper thumbnail of Glycogen synthesis in amphibian oocytes: evidence for an indirect pathway

Biochemical Journal, 1996

Glycogen is the main end product of glucose metabolism in amphibian oocytes. However, in the firs... more Glycogen is the main end product of glucose metabolism in amphibian oocytes. However, in the first few minutes after [U-14C]glucose microinjection most of the label is found in lactate. The burst of lactate production and the shape of the time curves for the labelling of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and glycogen suggest a precursor–product relationship of lactate with respect to glycogen and its intermediates. Expansion (by microinjection) of the pool of glycolytic intermediates, such as dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate or phosphoenolpyruvate, results in a marked decrease in [U-14C]glucose incorporation into glycogen. After co-injection of doubly labelled glucoses, extensive detritiation (93%) of the glucosyl units of glycogen was observed with [2-3H,U-14C]glucose and partial detritiation with [3-3H,U-14C]glucose (34%) or [5-3H,U-14C]glucose (46%). After injection of [6-3H,U-14C]glucose, a small but signific...

Research paper thumbnail of The separation and identification of picomole amounts of intermediates of glucose metabolism by high performance liquid chromatography on pellicular resins

Biological research, 1992

A column (CarboPac PA1, Dionex) containing an anion-exchange pellicular resin was used for the se... more A column (CarboPac PA1, Dionex) containing an anion-exchange pellicular resin was used for the separation of phosphoryl-hexoses derived from labeled glucose microinjected into individual frog oocytes or from cultures of Escherichia coli. Intermediates were identified by: a) comparison of retention times with those of authentic commercial compounds; b) the use of internal labeled standards; c) incubation of samples with specific enzymes and noting the disappearance of one radioactive peak and appearance of another at a new retention time.

Research paper thumbnail of Reflection confocal imaging of type I and type III isozymes of hexokinase in PC12 cells

Scanning, 2008

Reflection confocal microscopy was used to determine the intracellular distribution of Type I and... more Reflection confocal microscopy was used to determine the intracellular distribution of Type I and III isozymes of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) in PC12 cells; detection was by staining with diaminobenzidine as a substrate for horseradish peroxidase-conjugated antimouse immunoglobulins bound to isozyme-specific monoclonal antibodies. With both isozymes, detection of the staining pattern was significantly enhanced by reflection confocal imaging compared with viewing with transmitted brightfield optics. For Type I, prominent staining of cytoplasmic organelles having a distribution consistent with that of mitochondria was noted. For Type III, intense staining at the nuclear periphery was observed. A distinct punctate pattern along the nuclear surface implied a nonuniform distribution of the Type III hexokinase and may represent preferential association with nuclear pore structures. A study of technical factors involved in optimizing the reflection image was conducted. We demonstrate that both the choice of objective and the thickness of the mounting medium are critical to successful imaging, and we describe a simple test for assessing the suitability of objectives in any system.

Research paper thumbnail of Frog Oocytes: A Living Test Tube for Studies on Metabolic Regulation

IUBMB Life (International Union of Biochemistry and Molecular Biology: Life), 2001

This review is intended to illustrate how live frog oocytes may be advantageously used to address... more This review is intended to illustrate how live frog oocytes may be advantageously used to address the study of some problems of in vivo glucose metabolism. Glucose microinjected into the cells is preferentially committed to glycogen synthesis. We present evidence showing that both the direct and indirect pathways for polysaccharide deposition are operative in oocytes. A small amount of the injected glucose (<5%) is released as labeled CO 2 mainly through the pentose-P pathway. Coinjection of NADP C and glucose signi cantly stimulates 14 CO 2 production, half-maximal stimulation being obtained at 0.13 mM. Finally, we show the use of frog oocytes to measure in vivo the control coef cient of hexokinase on glycogen synthesis and the pentose-P pathway. A value of 0.7 was found for the control coef cient of hexokinase on glycogen synthesis, while the enzyme has no control at all over the pentose-P pathway. Therefore, the frog oocyte may be used as a living test tube for the study of almost any metabolic process of interest.

Research paper thumbnail of Hexokinase and not glycogen synthase controls the flux through the glycogen synthesis pathway in frog oocytes

FEBS Letters, 2013

Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycog... more Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycogen synthesis in vivo. We used metabolic control analysis (MCA) to determine the flux control coefficient for each of the enzymes involved in the pathway. Acute microinjection experiments in frog oocytes were specifically designed to change the endogenous activities of the enzymes, either by directly injecting increasing amounts of a given enzyme (HK, PGM and UGPase) or by microinjection of a positive allosteric effector (glc-6P for GS). Values of 0.61 ± 0.07, 0.19 ± 0.03, 0.13 ± 0.03, and À0.06 ± 0.08 were obtained for the flux control coefficients of hexokinase EC 2.7.1.1 (HK), phosphoglucomutase EC 5.4.2.1 (PGM), UDPglucose pyrophosphorylase EC 2.7.7.9 (UGPase) and glycogen synthase EC 2.4.1.11 (GS), respectively. These values satisfy the summation theorem since the sum of the control coefficients for all the enzymes of the pathway is 0.87. The results show that, in frog oocytes, glycogen synthesis through the direct pathway is under the control of hexokinase. Phosphoglucomutase and UDPG-pyrophosphorylase have a modest influence, while the control exerted by glycogen synthase is null.

Research paper thumbnail of Glycolysis is operative in amphibian oocytes

FEBS Letters, 1994

It is generally accepted that in frog full-grown oocytes glycolysis is absent and that carbon met... more It is generally accepted that in frog full-grown oocytes glycolysis is absent and that carbon metabolic flux is largely directed to glycogen synthesis. Use of an anion exchange pellicular resin for analytical resolution of intermediates in perchloric acid extracts of oocytes has allowed us to observe the formation of labelled lactate after microinjection of [u-i4C]glucose. Further, formation of ["P]ATP was observed after microinjection of 32P-labelled glucose-6-P, fructose-6-P or fructose-1,6-b&P, either in the presence or absence of 0.1 mM cyanide. The presence of phosphofructokinase activity, previously thought to be absent in oocytes, is also reported. These findings indicate that glycolysis to lactate is operative in frog oocytes.

Research paper thumbnail of Glycogen synthesis by the direct or indirect pathways depends on glucose availability: In vivo studies in frog oocytes

FEBS Letters, 2007

Besides the classic direct route, frog oocytes incorporate glucosyl units into glycogen by the so... more Besides the classic direct route, frog oocytes incorporate glucosyl units into glycogen by the so-called indirect pathway. The operation of both pathways depends on glucose availability. Below 0.5 mM glucose (calculated intracellular concentration), the indirect route accounts for 90% of polysaccharide formation, while the direct pathway supports 70% of total glucose incorporation when administered glucose is above 1.5 mM. A sigmoidal curve was obtained for the direct pathway with n H = 2.04, and half saturation was reached at 2.6 mM glucose. The curve for the indirect route presented an n H of 1.15 and an S 0.5 of 0.9 mM glucose.

Research paper thumbnail of Effects of long-term exposure to Cu2+ and Cd2+ on the pentose phosphate pathway dehydrogenase activities in the ovary of adult Bufo arenarum: possible role as biomarker for Cu2+ toxicity

Ecotoxicology and Environmental Safety, 2004

The effects of copper and cadmium on metabolism through the pentose phosphate pathway were evalua... more The effects of copper and cadmium on metabolism through the pentose phosphate pathway were evaluated in Bufo arenarum toad ovary. The effects of the two metals on dehydrogenases from this pathway were evaluated by three experiments: (1) in samples obtained from control females with addition of the metals to the reaction mixture (in vitro), (2) in samples obtained from control females and after long-term exposure of females to 4 and 100 mg/L of Cu or Cd in the incubation media (in vitro after exposure to the metals in vivo), and (3) 14 CO 2 production through the pentose phosphate pathway was evaluated after [U-14 C]glucose microinjection on ovulated oocytes (in vivo after microinjection of the metals). Results from (1) evidenced inhibition of both enzyme activities but only above 1.5 mM Cu and Cd added to the reaction mixture. In (2) both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities decreased in samples from the ovaries of females exposed in vivo to Cu, in a concentration-dependent manner (up to 90% in females exposed to 100 mg/L Cu: 2.1271.57 NADPH mmol/min mg protein  10 À5 vs 19.9778.54 in control females). Cd treatment of the toads only rendered an inhibitory effect on 6-phosphogluconate dehydrogenase activity after exposure to 4 mg/L of the bivalent cation. (3) In vivo 14 CO 2 evolution significantly decreased in oocytes coinjected with 6.3  10 À3 mM Cu (calculated intracellular final concentration of the metal injected) and radioactive glucose. Cu and Cd concentration in samples from exposed females were always under detection limit by particle-induced X-ray emission. The results presented here are in agreement with a role for both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities determination as biomarkers of effect and exposure for Cu but not for Cd toxicity.

Research paper thumbnail of Fructose-1,6-bisphosphatase in Stage VI Frog Oocytes: Evidence for an Active Enzyme in Vivo

Archives of Biochemistry and Biophysics, 1995

Research paper thumbnail of Regulatory Role of Fructose-2,6-bisP on Glucose Metabolism in Frog Oocytes:In VivoInhibition of Glycogen Synthesis

Archives of Biochemistry and Biophysics, 1997

Glycogen is the main product of glucose metabolism Glycogen synthesis following glucose microinje... more Glycogen is the main product of glucose metabolism Glycogen synthesis following glucose microinjection in fully grown amphibian oocytes (1, 2). Recently, we in frog oocytes proceeds preferentially by an indirect have reported that in frog oocytes, glycogen synthesis pathway involving gluconeogenesis from triose comafter labeled glucose microinjection occurs preferenpounds. Because of the known regulatory role of fructially by an ''indirect'' pathway (3) that involves glycolytose-2,6-bisP on glucose utilization in most vertebrate sis to three-carbon compounds which are then used by tissues we coinjected [U-14 C]glucose and fructose-2,6gluconeogenesis for the resynthesis of hexoses-P and bisP into oocytes and observed a marked inhibition of UDP-glucose, the proximate glycogen precursors. Such label incorporation into glycogen, with an I 50 value of an alternative pathway was reported a few years ago 2 mM, which is similar to the value measured for the in for mammalian liver (for reviews see 4-7), isolated hevitro inhibition of oocyte fructose-1,6-bisphosphatase. patocytes (8-10), and cultured astrocytes (11). The key Other hexoses-bisP were tested: 2,5-anhydromannitolgluconeogenic enzymatic activities, phosphoenolpyr-1,6-bisP was as effective as inhibitor as fructose-2,6uvate-carboxykinase and fructose-1,6-bisphosphatase bisP; glucose-1,6-bisP showed some effect although (FBPase), 3 are present in frog oocytes (3, 12). Oocyte 50% inhibition was obtained at a concentration 10 FBPase, as well as many other bisphosphatases, is times higher than with fructose-2,6-bisP; fructose-1,6strongly inhibited by fructose-2,6-bisP. Therefore, fruc-bisP had no effect at all. The inhibition pattern for tose-2,6-bisP could play an important role in the reguthe in vivo glycogen synthesis by these analogs closely lation of glucose utilization commited for glycogen synmatched the one obtained with partially purified oothesis through the indirect gluconeogenic pathway. cyte fructose-1,6-bisphosphatase. The intracellular In this paper we report the effect of microinjected concentration of fructose-2,6-bisP in unperturbed oofructose-2,6-bisP on glycogen synthesis in vivo and the cytes was found to be between 0.1 and 0.2 mM. Fruccellular concentration of the metabolite, together with tose-6-phosphate,2-kinase levels measured in oocyte fructose-6-phosphate,2-kinase activity. We have found homogenates were between 0.02 and 0.06 mU per gram of ovary. After 60 min incubation, fructose-2,6-bisP mi-that fructose-2,6-bisP does indeed control glycogen syncroinjected into the oocytes was almost completely de-thesis via the indirect pathway mainly through its acgraded, suggesting that fructose-2,6-bisphosphatase is tion upon oocyte FBPase. The physiological significance active in vivo. The results presented in this paper indiof these findings will be discussed. cate that fructose-2,6-bisP plays an important role in the in vivo regulation of glucose utilization in frog-MATERIALS AND METHODS grown oocytes.

Research paper thumbnail of A hexokinase-coupled radioassay for pyruvate kinase

Analytical Biochemistry, 1976

Research paper thumbnail of Frog oocyte glycogen synthase: enzyme regulation under in vitro and in vivo conditions

Archives of Biochemistry and Biophysics, 2003

Frog oocyte glycogen synthase properties differ significantly under in vitro or in vivo condition... more Frog oocyte glycogen synthase properties differ significantly under in vitro or in vivo conditions. The K m app for UDP-glucose in vivo was 1.4 mM (in the presence or absence of glucose-6-P). The in vitro value was 6 mM and was reduced by glucose-6-P to 0.8 mM. Under both conditions (in vitro and in vivo) V max was 0.2 mUnits per oocyte in the absence of glucose-6-P. V max in vivo was stimulated 2-fold by glucose-6-P, whereas, in vitro, a 10-fold increase was obtained. Glucose-6-P required for 50% activation in vivo was 15 lM and, depending on substrate concentrations, 50-100 lM in vitro. The prevailing enzyme obtained in vitro was the glucose-6-P-dependent form, which may be converted to the independent species by dephosphorylation. This transformation could not be observed in vivo. We suggest that enzyme activation by glucose-6-P in vivo is due to allosteric effects rather than to dephosphorylation of the enzyme. Regulatory mechanisms other than allosteric activation and covalent phosphorylation are discussed.

Research paper thumbnail of Localization of the type III isozyme of hexokinase at the nuclear periphery

Archives of Biochemistry and Biophysics, 1992

Research paper thumbnail of Measurement of glycogen synthase activity in crude extracts by CE

ELECTROPHORESIS, 2007

Glycogen synthase catalyzes the incorporation of UDP-glucose into glycogen. The activity of the e... more Glycogen synthase catalyzes the incorporation of UDP-glucose into glycogen. The activity of the enzyme is usually measured either by a spectrophotometric method or by a radioassay. The first one is not suitable because of the difficulties regarding the use of coupled enzymes in crude extracts, while the second is a time-consuming method involving glycogen isolation and manipulation of radioactivity. We have used a CZE technique as a novel approach to measure glycogen synthase activity. The separations were performed at 22 kV (36 mA) in uncoated capillaries (53 cm650 mm). Sample injection time was 30 s and nucleotides were monitored at 254 nm. Best resolution was achieved in 20 mM tetraborate buffer, pH 9.2. Curves of absorbance as a function of UDP and UDP-glucose concentration were linear. Enzyme activity in oocyte extracts was linear with respect to time (up to15 min) and enzyme concentration. The K m app. for UDP-glucose was 0.87 mM, a value identical to the one reported using the radioassay. CZE enables easy quantitation of compounds, high sensitivity, and automation of the process. Small sample sizes are required, interferences by auxiliary enzymes and manipulation of radioactivity are avoided, and analysis time is significantly diminished.

Research paper thumbnail of In vivo operation of the pentose phosphate pathway in frog oocytes is limited by NADP+ availability

FEBS Letters, 1999

Evolution of CO P from labelled glucose microinjected into frog oocytes in vivo may be ascribed t... more Evolution of CO P from labelled glucose microinjected into frog oocytes in vivo may be ascribed to the pentose-P pathway, as measured by radioactive CO P production from [1-IR C] and [6-IR C]glucose. Coinjection of NADP + and [ IR C]glucose significantly stimulated IR CO P production. The effect depends on the amount of NADP + injected, half maximal stimulation being obtained at 0.13 mM. The increase in CO P production was also observed with microinjected glucose-1-P, glucose-6-P or fructose-6-P used as substrates. Phenazine methosulfate, mimicked the effects of NADP +. A high NADPH/NADP + ratio of 4.3 was found in the cells, the intracellular concentration of NADP + being 19 W WM.

Research paper thumbnail of The evolution of hexokinases

Archivos de biologia y medicina experimentales, 1987

Recent advances in the knowledge of the structural and functional aspects of the enzymes catalyzi... more Recent advances in the knowledge of the structural and functional aspects of the enzymes catalyzing sugar phosphorylation by ATP are reviewed. Hexokinases may exist, mainly in prokaryotes, as sugar-specific kinases (glucokinase, fructokinase, mannokinase) or as ubiquitous hexose-kinases which are relatively unspecific for the natural hexoses. Enzymes presenting intermediate specificity (e.g. mannofructokinases) have been also described. With a few exceptions, the molecular mass of a variety of hexokinases may be either 25 kDa, 50 kDa or 100 kDa. The smaller hexokinases have been found in some microorganisms whereas the 50 kDa enzymes are found (with only one exception) in most invertebrates and in a particular isozyme from vertebrates (hexokinase D). The 100 kDa enzymes are restricted to vertebrates (hexokinases A, B and C). These facts have led to the speculation that gene duplication events have played an important role in the evolutionary development of the hexokinases from prese...

Research paper thumbnail of In vivo ionic uptake through the skin of the South American toad Bufo arunco

Revue canadienne de biologie / éditée par l'Université de Montréal, 1971

Research paper thumbnail of Glucose utilization in vertebrates as a molecular probe for the study of evolution

Archivos de biología y medicina experimentales, 1979

Hexokinase isozymic profiles from the liver of 68 vertebrate species are presented. The compariso... more Hexokinase isozymic profiles from the liver of 68 vertebrate species are presented. The comparison of the diverse patterns observed, as well as the kinetic and physicochemical properties of the isozymes, reveals that the hexokinases from mammals are very similar to those from turtles and amphibians. The hexokinases from birds, lizards and snakes on the other hand are similar within themselves and different from the enzymes from mammals and amphibians. Liver pyruvate kinases show about the same behavior. The hexokinase system from vertebrate muscle however is very uniform in all the species studied consisting mainly of hexokinase B.

Research paper thumbnail of Cytoarchitecture of Caudiverbera caudiverbera stage VI oocytes: a light and electron microscope study

Anatomy and Embryology, 1999

The general characteristics and salient features of the full-grown stage VI Caudiverbera caudiver... more The general characteristics and salient features of the full-grown stage VI Caudiverbera caudiverbera oocyte at the light and electron microscopy level are described. The oocyte is a huge cell with radial symmetry and distinct polarity. A black animal hemisphere, rich in pigment granules and containing the nucleus, is clearly distinguished from the unpigmented white-yellowish vegetal hemisphere. The cell is surrounded by a highly invaginated plasma membrane, with numerous microvilli. The cortex underlying the plasma membrane contains cortical and pigment granules, mitochondria, rough endoplasmic reticulum and coated vesicles. Cytoskeletal components, such as actin filaments and microtubules, are also found in this region. The predominant structures, distributed throughout the cell, are the yolk platelets, which show a gradient in size with small platelets in the animal half and very large ones in the vegetal zone. Mitochondria are also very abundant in both hemispheres and clouds of these organelles are found in the perinuclear region, frequently associated with microtubules. Developed Golgi complexes are present in the cytoplasm and occasionally, annulate lamellae appear towards the inner zones. The nucleus is a large structure containing numerous nucleoli. The nuclear envelope is highly invaginated, especially at the side facing the vegetal pole. It is regularly perforated by large nuclear pores. Our results show that the structural organization of Caudiverbera oocytes, although similar to that of other amphibian oocytes, differs from them especially concerning the spatial distribution of several structural components.

Research paper thumbnail of Effect of salt depletion on sodium concentration in serum and urine of Bufo chilensis. Evidences for increased levels of neurohypophysial principles in their plasma

Comparative Biochemistry and Physiology Part A: Physiology, 1987

Salt-depleted toads Bufo chilensis were compared with animals maintained in NaCl solution and a c... more Salt-depleted toads Bufo chilensis were compared with animals maintained in NaCl solution and a control group with respect to Na+ content in serum and urine. 2. Plasma hydro-osmotic activity of the animals was measured by increased water transfer across the isolated urinary bladder of the frog (Cuudiuerbera caudiverbera). 3. Sodium in serum is not affected by pre-adaptation in distilled water. Urine Na+ is markedly reduced. 4. Plasma from depleted animals increases water transfer across the isolated urinary bladder. Immersion in NaCl solution did not have this effect. An increase in neurohypophysial hormones in the blood of the animals is postulated.

Research paper thumbnail of Glycogen synthesis in amphibian oocytes: evidence for an indirect pathway

Biochemical Journal, 1996

Glycogen is the main end product of glucose metabolism in amphibian oocytes. However, in the firs... more Glycogen is the main end product of glucose metabolism in amphibian oocytes. However, in the first few minutes after [U-14C]glucose microinjection most of the label is found in lactate. The burst of lactate production and the shape of the time curves for the labelling of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and glycogen suggest a precursor–product relationship of lactate with respect to glycogen and its intermediates. Expansion (by microinjection) of the pool of glycolytic intermediates, such as dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate or phosphoenolpyruvate, results in a marked decrease in [U-14C]glucose incorporation into glycogen. After co-injection of doubly labelled glucoses, extensive detritiation (93%) of the glucosyl units of glycogen was observed with [2-3H,U-14C]glucose and partial detritiation with [3-3H,U-14C]glucose (34%) or [5-3H,U-14C]glucose (46%). After injection of [6-3H,U-14C]glucose, a small but signific...

Research paper thumbnail of The separation and identification of picomole amounts of intermediates of glucose metabolism by high performance liquid chromatography on pellicular resins

Biological research, 1992

A column (CarboPac PA1, Dionex) containing an anion-exchange pellicular resin was used for the se... more A column (CarboPac PA1, Dionex) containing an anion-exchange pellicular resin was used for the separation of phosphoryl-hexoses derived from labeled glucose microinjected into individual frog oocytes or from cultures of Escherichia coli. Intermediates were identified by: a) comparison of retention times with those of authentic commercial compounds; b) the use of internal labeled standards; c) incubation of samples with specific enzymes and noting the disappearance of one radioactive peak and appearance of another at a new retention time.

Research paper thumbnail of Reflection confocal imaging of type I and type III isozymes of hexokinase in PC12 cells

Scanning, 2008

Reflection confocal microscopy was used to determine the intracellular distribution of Type I and... more Reflection confocal microscopy was used to determine the intracellular distribution of Type I and III isozymes of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) in PC12 cells; detection was by staining with diaminobenzidine as a substrate for horseradish peroxidase-conjugated antimouse immunoglobulins bound to isozyme-specific monoclonal antibodies. With both isozymes, detection of the staining pattern was significantly enhanced by reflection confocal imaging compared with viewing with transmitted brightfield optics. For Type I, prominent staining of cytoplasmic organelles having a distribution consistent with that of mitochondria was noted. For Type III, intense staining at the nuclear periphery was observed. A distinct punctate pattern along the nuclear surface implied a nonuniform distribution of the Type III hexokinase and may represent preferential association with nuclear pore structures. A study of technical factors involved in optimizing the reflection image was conducted. We demonstrate that both the choice of objective and the thickness of the mounting medium are critical to successful imaging, and we describe a simple test for assessing the suitability of objectives in any system.

Research paper thumbnail of Frog Oocytes: A Living Test Tube for Studies on Metabolic Regulation

IUBMB Life (International Union of Biochemistry and Molecular Biology: Life), 2001

This review is intended to illustrate how live frog oocytes may be advantageously used to address... more This review is intended to illustrate how live frog oocytes may be advantageously used to address the study of some problems of in vivo glucose metabolism. Glucose microinjected into the cells is preferentially committed to glycogen synthesis. We present evidence showing that both the direct and indirect pathways for polysaccharide deposition are operative in oocytes. A small amount of the injected glucose (<5%) is released as labeled CO 2 mainly through the pentose-P pathway. Coinjection of NADP C and glucose signi cantly stimulates 14 CO 2 production, half-maximal stimulation being obtained at 0.13 mM. Finally, we show the use of frog oocytes to measure in vivo the control coef cient of hexokinase on glycogen synthesis and the pentose-P pathway. A value of 0.7 was found for the control coef cient of hexokinase on glycogen synthesis, while the enzyme has no control at all over the pentose-P pathway. Therefore, the frog oocyte may be used as a living test tube for the study of almost any metabolic process of interest.

Research paper thumbnail of Hexokinase and not glycogen synthase controls the flux through the glycogen synthesis pathway in frog oocytes

FEBS Letters, 2013

Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycog... more Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycogen synthesis in vivo. We used metabolic control analysis (MCA) to determine the flux control coefficient for each of the enzymes involved in the pathway. Acute microinjection experiments in frog oocytes were specifically designed to change the endogenous activities of the enzymes, either by directly injecting increasing amounts of a given enzyme (HK, PGM and UGPase) or by microinjection of a positive allosteric effector (glc-6P for GS). Values of 0.61 ± 0.07, 0.19 ± 0.03, 0.13 ± 0.03, and À0.06 ± 0.08 were obtained for the flux control coefficients of hexokinase EC 2.7.1.1 (HK), phosphoglucomutase EC 5.4.2.1 (PGM), UDPglucose pyrophosphorylase EC 2.7.7.9 (UGPase) and glycogen synthase EC 2.4.1.11 (GS), respectively. These values satisfy the summation theorem since the sum of the control coefficients for all the enzymes of the pathway is 0.87. The results show that, in frog oocytes, glycogen synthesis through the direct pathway is under the control of hexokinase. Phosphoglucomutase and UDPG-pyrophosphorylase have a modest influence, while the control exerted by glycogen synthase is null.

Research paper thumbnail of Glycolysis is operative in amphibian oocytes

FEBS Letters, 1994

It is generally accepted that in frog full-grown oocytes glycolysis is absent and that carbon met... more It is generally accepted that in frog full-grown oocytes glycolysis is absent and that carbon metabolic flux is largely directed to glycogen synthesis. Use of an anion exchange pellicular resin for analytical resolution of intermediates in perchloric acid extracts of oocytes has allowed us to observe the formation of labelled lactate after microinjection of [u-i4C]glucose. Further, formation of ["P]ATP was observed after microinjection of 32P-labelled glucose-6-P, fructose-6-P or fructose-1,6-b&P, either in the presence or absence of 0.1 mM cyanide. The presence of phosphofructokinase activity, previously thought to be absent in oocytes, is also reported. These findings indicate that glycolysis to lactate is operative in frog oocytes.

Research paper thumbnail of Glycogen synthesis by the direct or indirect pathways depends on glucose availability: In vivo studies in frog oocytes

FEBS Letters, 2007

Besides the classic direct route, frog oocytes incorporate glucosyl units into glycogen by the so... more Besides the classic direct route, frog oocytes incorporate glucosyl units into glycogen by the so-called indirect pathway. The operation of both pathways depends on glucose availability. Below 0.5 mM glucose (calculated intracellular concentration), the indirect route accounts for 90% of polysaccharide formation, while the direct pathway supports 70% of total glucose incorporation when administered glucose is above 1.5 mM. A sigmoidal curve was obtained for the direct pathway with n H = 2.04, and half saturation was reached at 2.6 mM glucose. The curve for the indirect route presented an n H of 1.15 and an S 0.5 of 0.9 mM glucose.

Research paper thumbnail of Effects of long-term exposure to Cu2+ and Cd2+ on the pentose phosphate pathway dehydrogenase activities in the ovary of adult Bufo arenarum: possible role as biomarker for Cu2+ toxicity

Ecotoxicology and Environmental Safety, 2004

The effects of copper and cadmium on metabolism through the pentose phosphate pathway were evalua... more The effects of copper and cadmium on metabolism through the pentose phosphate pathway were evaluated in Bufo arenarum toad ovary. The effects of the two metals on dehydrogenases from this pathway were evaluated by three experiments: (1) in samples obtained from control females with addition of the metals to the reaction mixture (in vitro), (2) in samples obtained from control females and after long-term exposure of females to 4 and 100 mg/L of Cu or Cd in the incubation media (in vitro after exposure to the metals in vivo), and (3) 14 CO 2 production through the pentose phosphate pathway was evaluated after [U-14 C]glucose microinjection on ovulated oocytes (in vivo after microinjection of the metals). Results from (1) evidenced inhibition of both enzyme activities but only above 1.5 mM Cu and Cd added to the reaction mixture. In (2) both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities decreased in samples from the ovaries of females exposed in vivo to Cu, in a concentration-dependent manner (up to 90% in females exposed to 100 mg/L Cu: 2.1271.57 NADPH mmol/min mg protein  10 À5 vs 19.9778.54 in control females). Cd treatment of the toads only rendered an inhibitory effect on 6-phosphogluconate dehydrogenase activity after exposure to 4 mg/L of the bivalent cation. (3) In vivo 14 CO 2 evolution significantly decreased in oocytes coinjected with 6.3  10 À3 mM Cu (calculated intracellular final concentration of the metal injected) and radioactive glucose. Cu and Cd concentration in samples from exposed females were always under detection limit by particle-induced X-ray emission. The results presented here are in agreement with a role for both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities determination as biomarkers of effect and exposure for Cu but not for Cd toxicity.

Research paper thumbnail of Fructose-1,6-bisphosphatase in Stage VI Frog Oocytes: Evidence for an Active Enzyme in Vivo

Archives of Biochemistry and Biophysics, 1995

Research paper thumbnail of Regulatory Role of Fructose-2,6-bisP on Glucose Metabolism in Frog Oocytes:In VivoInhibition of Glycogen Synthesis

Archives of Biochemistry and Biophysics, 1997

Glycogen is the main product of glucose metabolism Glycogen synthesis following glucose microinje... more Glycogen is the main product of glucose metabolism Glycogen synthesis following glucose microinjection in fully grown amphibian oocytes (1, 2). Recently, we in frog oocytes proceeds preferentially by an indirect have reported that in frog oocytes, glycogen synthesis pathway involving gluconeogenesis from triose comafter labeled glucose microinjection occurs preferenpounds. Because of the known regulatory role of fructially by an ''indirect'' pathway (3) that involves glycolytose-2,6-bisP on glucose utilization in most vertebrate sis to three-carbon compounds which are then used by tissues we coinjected [U-14 C]glucose and fructose-2,6gluconeogenesis for the resynthesis of hexoses-P and bisP into oocytes and observed a marked inhibition of UDP-glucose, the proximate glycogen precursors. Such label incorporation into glycogen, with an I 50 value of an alternative pathway was reported a few years ago 2 mM, which is similar to the value measured for the in for mammalian liver (for reviews see 4-7), isolated hevitro inhibition of oocyte fructose-1,6-bisphosphatase. patocytes (8-10), and cultured astrocytes (11). The key Other hexoses-bisP were tested: 2,5-anhydromannitolgluconeogenic enzymatic activities, phosphoenolpyr-1,6-bisP was as effective as inhibitor as fructose-2,6uvate-carboxykinase and fructose-1,6-bisphosphatase bisP; glucose-1,6-bisP showed some effect although (FBPase), 3 are present in frog oocytes (3, 12). Oocyte 50% inhibition was obtained at a concentration 10 FBPase, as well as many other bisphosphatases, is times higher than with fructose-2,6-bisP; fructose-1,6strongly inhibited by fructose-2,6-bisP. Therefore, fruc-bisP had no effect at all. The inhibition pattern for tose-2,6-bisP could play an important role in the reguthe in vivo glycogen synthesis by these analogs closely lation of glucose utilization commited for glycogen synmatched the one obtained with partially purified oothesis through the indirect gluconeogenic pathway. cyte fructose-1,6-bisphosphatase. The intracellular In this paper we report the effect of microinjected concentration of fructose-2,6-bisP in unperturbed oofructose-2,6-bisP on glycogen synthesis in vivo and the cytes was found to be between 0.1 and 0.2 mM. Fruccellular concentration of the metabolite, together with tose-6-phosphate,2-kinase levels measured in oocyte fructose-6-phosphate,2-kinase activity. We have found homogenates were between 0.02 and 0.06 mU per gram of ovary. After 60 min incubation, fructose-2,6-bisP mi-that fructose-2,6-bisP does indeed control glycogen syncroinjected into the oocytes was almost completely de-thesis via the indirect pathway mainly through its acgraded, suggesting that fructose-2,6-bisphosphatase is tion upon oocyte FBPase. The physiological significance active in vivo. The results presented in this paper indiof these findings will be discussed. cate that fructose-2,6-bisP plays an important role in the in vivo regulation of glucose utilization in frog-MATERIALS AND METHODS grown oocytes.

Research paper thumbnail of A hexokinase-coupled radioassay for pyruvate kinase

Analytical Biochemistry, 1976

Research paper thumbnail of Frog oocyte glycogen synthase: enzyme regulation under in vitro and in vivo conditions

Archives of Biochemistry and Biophysics, 2003

Frog oocyte glycogen synthase properties differ significantly under in vitro or in vivo condition... more Frog oocyte glycogen synthase properties differ significantly under in vitro or in vivo conditions. The K m app for UDP-glucose in vivo was 1.4 mM (in the presence or absence of glucose-6-P). The in vitro value was 6 mM and was reduced by glucose-6-P to 0.8 mM. Under both conditions (in vitro and in vivo) V max was 0.2 mUnits per oocyte in the absence of glucose-6-P. V max in vivo was stimulated 2-fold by glucose-6-P, whereas, in vitro, a 10-fold increase was obtained. Glucose-6-P required for 50% activation in vivo was 15 lM and, depending on substrate concentrations, 50-100 lM in vitro. The prevailing enzyme obtained in vitro was the glucose-6-P-dependent form, which may be converted to the independent species by dephosphorylation. This transformation could not be observed in vivo. We suggest that enzyme activation by glucose-6-P in vivo is due to allosteric effects rather than to dephosphorylation of the enzyme. Regulatory mechanisms other than allosteric activation and covalent phosphorylation are discussed.

Research paper thumbnail of Localization of the type III isozyme of hexokinase at the nuclear periphery

Archives of Biochemistry and Biophysics, 1992

Research paper thumbnail of Measurement of glycogen synthase activity in crude extracts by CE

ELECTROPHORESIS, 2007

Glycogen synthase catalyzes the incorporation of UDP-glucose into glycogen. The activity of the e... more Glycogen synthase catalyzes the incorporation of UDP-glucose into glycogen. The activity of the enzyme is usually measured either by a spectrophotometric method or by a radioassay. The first one is not suitable because of the difficulties regarding the use of coupled enzymes in crude extracts, while the second is a time-consuming method involving glycogen isolation and manipulation of radioactivity. We have used a CZE technique as a novel approach to measure glycogen synthase activity. The separations were performed at 22 kV (36 mA) in uncoated capillaries (53 cm650 mm). Sample injection time was 30 s and nucleotides were monitored at 254 nm. Best resolution was achieved in 20 mM tetraborate buffer, pH 9.2. Curves of absorbance as a function of UDP and UDP-glucose concentration were linear. Enzyme activity in oocyte extracts was linear with respect to time (up to15 min) and enzyme concentration. The K m app. for UDP-glucose was 0.87 mM, a value identical to the one reported using the radioassay. CZE enables easy quantitation of compounds, high sensitivity, and automation of the process. Small sample sizes are required, interferences by auxiliary enzymes and manipulation of radioactivity are avoided, and analysis time is significantly diminished.

Research paper thumbnail of In vivo operation of the pentose phosphate pathway in frog oocytes is limited by NADP+ availability

FEBS Letters, 1999

Evolution of CO P from labelled glucose microinjected into frog oocytes in vivo may be ascribed t... more Evolution of CO P from labelled glucose microinjected into frog oocytes in vivo may be ascribed to the pentose-P pathway, as measured by radioactive CO P production from [1-IR C] and [6-IR C]glucose. Coinjection of NADP + and [ IR C]glucose significantly stimulated IR CO P production. The effect depends on the amount of NADP + injected, half maximal stimulation being obtained at 0.13 mM. The increase in CO P production was also observed with microinjected glucose-1-P, glucose-6-P or fructose-6-P used as substrates. Phenazine methosulfate, mimicked the effects of NADP +. A high NADPH/NADP + ratio of 4.3 was found in the cells, the intracellular concentration of NADP + being 19 W WM.