Ana Preller

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

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 microg/L of Cu or Cd in the incubation media (in vitro after exposure to the metals in vivo), and (3) 14CO2 production through the pentose phosphate pathway was evaluated after [U-14C]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 microg/L Cu: 2.12 +/- 1.57 NADPH micromol/min microg protein x 10(-5) vs 19.97 +/- 8.54 in control females). Cd treatment of the toads only rendered an inhibitory effect on 6-phosphogluconate dehydrogenase activity after exposure to 4 microg/L of the bivalent cation. (3) In vivo 14CO2 evolution significantly decreased in oocytes coinjected with 6.3 x 10(-3) 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.

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

Evolution of CO2 from labelled glucose microinjected into frog oocytes in vivo may be ascribed to the pentose‐P pathway, as measured by radioactive CO2 production from [1‐14C] and [6‐14C]glucose. Coinjection of NADP+ and [14C]glucose significantly stimulated 14CO2 production. The effect depends on the amount of NADP+ injected, half maximal stimulation being obtained at 0.13 mM. The increase in CO2 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 μM.

Frog Oocytes: A Living Test Tube for Studies on Metabolic Regulation

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 CO2 mainly through the pentose‐P pathway. Coinjection of NADP+ and glucose significantly stimulates 14CO2 production, half‐maximal stimulation being obtained at 0.13 mM. Finally, we show the use of frog oocytes to measure in vivo the control coefficient of hexokinase on glycogen synthesis and the pentose‐P pathway. A value of 0.7 was found for the control coefficient 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.

Frog oocyte glycogen synthase: enzyme regulation under in vitro and in vivo conditions ☆

Frog oocyte glycogen synthase properties differ significantly under in vitro or in vivo conditions. The K(mapp) for UDP-glucose in vivo was 1.4mM (in the presence or absence of glucose-6-P). The in vitro value was 6mM and was reduced by glucose-6-P to 0.8mM. Under both conditions (in vitro and in vivo) V(max) was 0.2 m Units 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 microM and, depending on substrate concentrations, 50-100 microM 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.

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

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.

Glycolysis is operative in amphibian oocytes.

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‐14C]glucose. Further, formation of [32P]ATP was observed after microinjection of 32P‐labelled glucose‐6‐P, fructose‐6‐P or fructose‐1,6‐bis‐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.

A hexokinase-coupled radioassay for pyruvate kinase.

Abstract A procedure for the assay of low activities of pyruvate kinase (0.01 to 4 mIU) is described. The method consists of coupling the formation of ATP by the pyruvate kinase reaction to hexokinase in the presence of uniformly labeled [14C]glucose. The labeled glucose 6-phosphate thus formed is easily separated from the unreacted glucose using small columns of Dowex 1-X8 formate and detected by liquid scintillation spectrometry. Chromatographic patterns of pyruvate kinases from 25 mg of rat liver, 3.5 mg of frog oocyte, and 0.5 mg of the whole body of the fruit fly Drosophila melanogaster are presented as illustrations of the sensitivity of the radioassay.

Hexokinase and not glycogen synthase controls the flux through the glycogen synthesis pathway in frog oocytes.

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 −0.06 ± 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.