RESUMO
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.
Assuntos
Frutosedifosfatos/farmacologia , Glucose/metabolismo , Glicogênio/biossíntese , Oócitos/metabolismo , Animais , Anuros , Radioisótopos de Carbono , Feminino , Cinética , Oócitos/efeitos dos fármacos , Fosfofrutoquinase-2 , Monoéster Fosfórico Hidrolases/metabolismo , Técnica de Diluição de Radioisótopos , Fosfatos Açúcares/farmacologiaRESUMO
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.
Assuntos
Biologia , Ética Médica , Sociedades Médicas/normas , Chile , Comissão de ÉticaRESUMO
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.
Assuntos
Anuros/metabolismo , Glicogênio/biossíntese , Oócitos/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Gluconeogênese , Glucose/administração & dosagem , Glucose/metabolismo , Glicólise , Técnicas In Vitro , Cinética , Lactatos/metabolismo , Ácido Láctico , Microinjeções , Oócitos/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (GTP)/antagonistas & inibidores , Ácidos Picolínicos/farmacologiaRESUMO
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.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glucose/metabolismo , Animais , Anuros , Escherichia coli/metabolismo , Feminino , Hexoses/isolamento & purificação , Microinjeções , Oócitos/metabolismo , Resinas Vegetais , Fatores de TempoRESUMO
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.
Assuntos
Evolução Molecular , Hexoquinase/genética , Regulação Alostérica , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucosefosfato Desidrogenase/biossíntese , Glucosefosfato Desidrogenase/farmacologia , Hexoquinase/metabolismo , Invertebrados/genética , Invertebrados/metabolismo , Cinética , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Vertebrados/genética , Vertebrados/metabolismoRESUMO
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.
Assuntos
Compartimento Celular , Glucose/metabolismo , Complexos Multienzimáticos/metabolismo , Oócitos/metabolismo , Animais , Anuros , Dióxido de Carbono/metabolismo , Glicogênio/biossíntese , Hexoquinase/metabolismo , Via de Pentose FosfatoRESUMO
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.
Assuntos
Metabolismo dos Carboidratos , Compartimento Celular , Glicólise , Fosfotransferases/metabolismo , Animais , Citosol/enzimologia , Eritrócitos/enzimologia , Euglena gracilis , Humanos , Isoenzimas/metabolismo , Fígado/enzimologia , Proteínas Musculares/metabolismo , Músculos/enzimologia , Fosfofrutoquinase-1/metabolismo , Coelhos , RatosRESUMO
Glucose phosphorylating activities were measured in liver extracts from chicks at several developmental stages. Enzyme activity levels in supernates were low (about 0.16 units/g liver) from day 10th of egg incubation until the 17th day, at which time a transient increase to 0.5 units/g was observed. At hatching, the levels were again low (0.15 units/g) compared to adult levels (0.9 units/g). Particulate hexokinase activity was rather constant from day 10th to adulthood (about 0.3 units/g). Chromatography of liver supernates in DEAE-cellulose columns revealed the presence of four hexokinases in embryos up to day 15th of incubation. From that day onwards, the least retained from (hexokinase 4) was no longer found. The most retained form (hexokinase 1) disappeared at hatching, at which time a pattern consisting of hexokinases 2 and 3 was found to be very similar to the adult profile. The four isozymes were characterized as low Km glucose hexokinase of broad sugar specificities and molecular weights of about 100,000. Particulate hexokinase activity of embryonic chick liver was found to be composed of the same isozymes observed in cytosolic extracts. Incubation of particles with glucose 6-P or ATP failed to release hexokinase activity.
Assuntos
Embrião de Galinha/enzimologia , Hexoquinase/metabolismo , Isoenzimas/metabolismo , Fígado/enzimologia , Animais , Fracionamento Celular , Cromatografia DEAE-Celulose , Glucose/metabolismo , Crescimento , Cinética , Glicogênio Hepático/análise , Radioimunoensaio , Ratos , Fatores de TempoAssuntos
Glucose/metabolismo , Hexoquinase/genética , Fígado/enzimologia , Músculos/enzimologia , Anfíbios , Animais , Aves , Bovinos , Embrião de Galinha , Cromatografia DEAE-Celulose , Cricetinae , Peixes , Hexoquinase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Mamíferos , Fosfotransferases/classificação , Filogenia , Coelhos , Ratos , Répteis , Especificidade da EspécieRESUMO
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.