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.

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=0.7) but its influence on the control of the pentose phosphate pathway is almost nil (control coefficient=-0.01). Glucose-6-phosphate dehydrogenase injections did not affect the production of 14CO(2) by the pentose phosphate pathway, indicating that other factors control the operation of this pathway. In addition, an almost null control of this enzyme on glycogen synthesis flux was observed.

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 0.13 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 microM.

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. The inhibition pattern for the in vivo glycogen synthesis by these analogs closely matched the one obtained with partially purified oocyte fructose-1,6-bisphosphatase. The intracellular concentration of fructose-2,6-bisP in unperturbed oocytes was found to be between 0.1 and 0.2 microM. Fructose-6-phosphate,2-kinase levels measured in oocyte homogenates were between 0.02 and 0.06 mU per gram of ovary. After 60 min incubation, fructose-2,6-bisP microinjected into the oocytes was almost completely degraded, suggesting that fructose-2,6-bisphosphatase is active in vivo. The results presented in this paper indicate that fructose-2,6-bisP plays an important role in the in vivo regulation of glucose utilization in frog-grown oocytes.

[Ethics code of the Chilean Biological Society]. / Código de ética de la Sociedad de Biología de Chile.

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. It declares that all knowledge, created or obtained by researchers is mankind's heritage.

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. 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 significant and reproducible detritiation (13%) in glycogen was observed. Co-injection of [U-14C]glucose and 3-mercaptopicolinate resulted in marked inhibition of glycogen labelling. Half-maximal inhibition was observed at 0.58 mM 3-mercaptopicolinate, which agrees with the IC50 value (0.47 mM) for the inhibition in vitro of phosphoenolpyruvate carboxykinase activity. We concluded that in frog oocytes most of the glucosyl units are incorporated into glycogen by an indirect pathway involving breakdown of glucose to lactate, which is then converted into glycogen via gluconeogenesis. Both processes, glycolytic degradation of glucose to lactate and subsequent reconversion of the latter into hexose phosphates and glycogen, occur in the same cell.

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. Inhibition by AMP is cooperative with a nH value of 2.2. In vivo fructose-1,6-bisphosphatase activity was demonstrated by microinjection of [U-14C]- or [6-32P]fructose-1,6-bisP and subsequent chromatographic separation and identification of labeled products. The relevance of these findings for the metabolism of glucose in frog oocytes is discussed.

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.

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. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.

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 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.

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.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 present day organisms. The fact that the 100 kDa hexokinases are allosterically inhibited by the product, glucose 6-P, may indicate that a duplicated active site has evolved to a regulatory binding site. Comparisons of the amino acid sequence of a few peptides from hexokinase C are presented to support the gene duplication hypothesis. Also, partial sequence comparisons of vertebrate hexokinases with the sequences of two hexokinase isozymes from yeast show strong similarities suggesting a rather slow amino acid substitution rate of homologous genes.

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 2.7.1.1) from brain of several vertebrate species has been compared by using enzyme-linked immunosorbent assay and seven monoclonal antibodies raised against the rat brain enzyme. The epitopes recognized by three of these antibodies have been rather widely conserved among the species examined (rat, mouse, guinea pig, rabbit, cat, dog, sheep, cow, pig, chicken), while this was not the case for the epitopes recognized by the other antibodies, which differed markedly in their distribution among these species. The domain structure of these enzymes has been examined by peptide mapping (after limited tryptic digestion) in conjunction with immunoblotting techniques employing monoclonal antibodies. The results indicate that the overall domain structure of these enzymes is similar to that previously described for rat brain hexokinase A, but that there are significant differences in the size of these domains in enzymes from different species.

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. The subject of compartmentation of glucose utilization has been addressed in this paper. First, we show that microinjection of glucose results in a 30-fold increase of carbon incorporation into glycogen when compared to oocytes incubated at saturating glucose concentrations. On the other hand, carbon incorporation into CO2, remains at about the same levels in both conditions Second, microinjection of NADP+ increases CO2 release and inhibits glycogen synthesis from glucose. Third, co-injection of unlabeled intermediates affects differentially glycogen synthesis and CO2 production from labeled glucose. Finally, microinjection of pure yeast hexokinase stimulates markedly 14CO2 release and inhibits glycogen synthesis. We conclude that two separate pools of glucose-6-P exists in oocytes: one pool is committed to the pathway of glycogen synthesis while a second pool serves as substrate for the operation of the pentose-P pathway.

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 2.7.1.1) has been performed using a photometric method based on a mutarotase-glucose oxidase-peroxidase-chromogen system to trap and visualize glucose, plus a glycerol kinase-glycerol system to trap ATP. Glucose 6-phosphate or 2-deoxyglucose 6-phosphate were used as phosphoryl donors at different concentrations of ADP. Variation of glucose 6-phosphate concentrations resulted in a biphasic curve from which apparent Km and Ki values of ca. 0.2 mM were calculated. In contrast, variation of 2-deoxyglucose 6-phosphate concentrations resulted in Michaelian kinetics with an apparent Km of 2 mM. The Km value for MgADP was 16 mM irrespective of the nature and concentration of the hexose 6-phosphate substrate. These results are fully consistent with an allosteric site for glucose 6-phosphate as an explanation for the inhibition of animal hexokinases by glucose 6-P and further indicate that the maximal rate is the parameter affected. From these observations and previous knowledge, the possible occurrence in animal hexokinases of a regulatory site for ATP to account for the competition between glucose 6-phosphate and ATP in the forward reaction is postulated.

[The organization of metabolism : subcellular localization of glycolytic enzymes]. / Organización del metabolismo: localización subcelular de enzimas glicolíticas.

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. Emphasis is given to the description of experiments purporting to show the influence of protein crowding, interactions between glycolytic enzymes, and reversible association to structural proteins or to organelles. The transient nature of those associations is considered to be particularly relevant to the organization and regulation of glycolysis. The proposed role of isozymes as structural determinants of metabolic compartmentation is stressed. A few studies based on immunocytochemical observations of glycolytic enzymes are briefly described. A list of questions whose answers are badly needed 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. In the case of mouse enzyme, the amount of serum required to inhibit 50% of the activity was five-fold higher than in the case of the rat enzyme. Enzymes from cow or sheep brain were only marginally affected. Hexokinases A isolated from various mammals, tested against the rat enzyme, showed faint lines of precipitation and marked spurs in double immunodiffusion plates even when enzymes from closely related rodents were analyzed. Immunoadsorbent columns, on the other hand, were able to retain most of the activity of hexokinases A from the mammals studied. Micro-complement fixation tests showed that hexokinases A from mammals outside the Order Rodentia were only partially recognized by the anti-hexokinase Arat serum. The results suggest that amino acid substitutions on the hexokinase A molecule have occurred at a rather fast rate.