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).

Measurement of glycogen synthase activity in crude extracts by CE.

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.

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.

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).

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.

In vivo measurements of control coefficients for hexokinase and glucose-6-phosphate dehydrogenase in Xenopus laevis oocytes.

Hexokinase and glucose-6-phosphate dehydrogenase activities were increased in Xenopus laevis oocytes by microinjection of commercial pure enzymes. The effect of increased fractional activities on glycogen synthesis or on the production of 14CO(2) (the oxidative portion of the pentose phosphate pathway) was investigated by microinjection of [1-(14)C]glucose and measurements of the radioactivity in glycogen and CO(2). Control coefficients calculated from the data show that hexokinase plays an important role in the control of glycogen synthesis (control coefficient=0.

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-(14)C] and [6-(14)C]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.

Regulatory role of fructose-2,6-bisP on glucose metabolism in frog oocytes: in vivo inhibition of glycogen synthesis.

Glycogen synthesis following glucose microinjection in frog oocytes proceeds preferentially by an indirect pathway involving gluconeogenesis from triose compounds. Because of the known regulatory role of fructose-2,6-bisP on glucose utilization in most vertebrate tissues we coinjected [U-14C]glucose and fructose-2,6-bisP into oocytes and observed a marked inhibition of label incorporation into glycogen, with an I50 value of 2 microM, which is similar to the value measured for the in vitro inhibition of oocyte fructose-1,6-bisphosphatase. Other hexoses-bisP were tested: 2,5-anhydromannitol-1,6-bisP was as effective as inhibitor as fructose-2,6-bisP; glucose-1,6-bisP showed some effect although 50% inhibition was obtained at a concentration 10 times higher than with fructose-2,6-bisP; fructose-1,6-bisP had no effect at all.

[Ethics code of the Chilean Biological Society].

The Chilean Biological Society has approved an ethics code for researchers, elaborated by its Ethic Committee. The text, with 16 articles, undertakes the main ethical problems that researchers must solve, such as institutional, professional or societal ethics, scientific fraud, breaches in collaborative work, relationships between researchers, participation in juries and committees, ethical breaches in scientific publications, scientific responsibility and punishments. This code declares its respect and valorization of all life forms and adheres to international biomedical ethical codes.

Glycogen synthesis in amphibian oocytes: evidence for an indirect pathway.

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.

Fructose-1,6-bisphosphatase in stage VI frog oocytes: evidence for an active enzyme in vivo.

The characterization of fructose-1,6-bisphosphatase in stage VI oocytes from the frog Caudiverbera caudiverbera, as well as the in vivo activity, is reported. The enzyme has a subunit molecular weight of approximately 43,500, has an apparent Km value of 17 microM for fructose-1,6-bisP, and is inhibited by substrate concentrations beyond 100 microM. AMP and fructose-2,6-bisP are strong inhibitors of oocyte fructose-1,6-bisphosphatase activity with Ki values of 9 and 2 microM respectively.

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.

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

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.

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits.

Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C.

The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.

The evolution of hexokinases.

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.

Amino acid sequence homology between yeast hexokinases and rat hexokinase C.

Automated Edman degradation of seven purified tryptic peptides from Novikoff hepatoma hexokinase C revealed amino acid sequences that could be easily aligned within the primary structure of yeast hexokinases. This high degree of structural homology suggests a common evolutionary origin for mammalian and yeast hexokinases. Some of the sequenced peptides overlapped with each other, as well as with regions of the sequence of yeast hexokinases, suggesting that during evolution the 100,000 molecular weight subunit mammalian hexokinases may have resulted from gene duplication followed by gene fusion from a pre-vertebrate 50,000 molecular weight hexokinase ancestor.

Hexokinase A from mammalian brain: comparative peptide mapping and immunological studies with monoclonal antibodies.

Immunological reactivity of partially purified hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.

Search for compartments of glucose metabolism in the microinjected frog oocyte.

Microinjection of frog oocytes allows the modification of intracellular levels of substrates, intermediates, cofactors and enzymes. Use of labeled glucose at specific positions has led us to conclude that oocytes utilize glucose mainly for glycogen synthesis and to a lesser extent for the pentose-P pathway. Glycolysis, glycogenolysis and gluconeogenesis are not operative in these cells.

Allosteric inhibition of brain hexokinase by glucose 6-phosphate in the reverse reaction.

A study of the reverse reaction of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.

[The organization of metabolism : subcellular localization of glycolytic enzymes].

The subject of cellular metabolic organization (with focus on carbohydrate metabolism) is reviewed. The existence of a "soluble" phase in the cell is considered unlikely. A note of caution regarding metabolic compartmentation as shown by isotope studies is presented.

Comparative studies on glucose phosphorylating isoenzymes from vertebrates--VIII. Immunochemical studies on mammalian hexokinases A.

An immune serum elicited in a rabbit by injection of homogeneous brain hexokinase A was shown to be specific for the antigen. Other rat hexokinase isoenzymes (hexokinases B, C or D) did not present cross-reaction when tested by immunoinhibition of enzyme activity, double immunodiffusion and immunoadsorbent columns. The enzyme activity of hexokinase A from several mammals (rodents, lagomorphs, artiodactyls) was partially inhibited by the immune serum.

A modified form of mitochondrial hexokinase produced by ATP-induced solubilization.

Mitochondrial hexokinases from several rat tissues were analyzed by DEAE-cellulose chromatography. Solubilization by glucose-6-P or Triton X-100 released hexokinases A and B. Solubilization by ATP resulted in a decrease of hexokinase A and the concomitant appearance of a new fraction of lower net charge (hexokinase Am) which readily reverts to hexokinase A by dialysis or dilution.