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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.

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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 microA) in uncoated capillaries (53 cmx50 microm). 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.

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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.

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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.

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En drosophila melanogaster, los genes estructurales que codifican para hexoquinasas A y B, se ubican estrechamente ligados en el cromosoma I (x). Estos loci corresponden a la región citológica 8D4.EI en el mapa citogenético de dicho cromosoma. Con el objetivo de determinar la ubicación exacta de los loci para hexoquinasas A y B se realizó un análisis molecular, mediante el uso de cromosomas artificiales de levadura, que contienen DNA de la sección 8 del cromosoma x. Para tal efecto, se aisló DNA cromosómico intacto de siete clones YAC de s. cerevisiae que cubren dicha región. El DNA fue resuelto mediante electroforesis de campo pulsado y analizado por hibridación con DNA del plásmido pB322 y con un fragmento de DNA de 0, 67 Kb de d. melanogaster, amplificado por PCR con partidores heterólogos específicos para hexoquinasas. Los resultados de estos experimentos permitieron concluir que, la sonda homóloga es capaz de hibridar con el YAC II, lo cual coincide con la cobertura de este clon en la región citológica esperada

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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

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