Glucose and Fructose Metabolism in a Phosphoglucoisomeraseless Mutant of Saccharomyces cerevisiae

A mutant of Saccharomyces cerevisiae deficient in phosphoglucoisomerase (EC 5.3.1.9) is described. It does not grow on glucose or sucrose but does grow on galactose or maltose. Addition of glucose to cultures growing on fructose, mannose, or acetate arrests further growth without altering viability; removal of glucose permits resumption of growth. Glucose causes accumulation of nearly 30 mumoles of glucose-6-phosphate per g (wet weight) of cells and suppresses synthesis of ribonucleic acid. Inhibition of growth by glucose does not appear to be due to a loss of adenosine triphosphate or inorganic orthophosphate. The mutant, however, utilizes glucose-6-phosphate produced intracellularly. Release of carbon dioxide from specifically labeled glucose suggests a C-l preferential cleavage. The kinetics of glucose-6-phosphate accumulation during glucose utilization in the mutant is not consistent with the notion that the utilization of glucose is controlled by glucose-6-phosphate.     

Chemical Conversion of Ribonucleoside 5′-S-Methyl Phosphorothiolates into Ribonucleotide Anhydrides Including Adenosine 5′-Triphosphate and Uridine Diphosphate Glucose

Ribonucleotide anhydrides have been prepared from corresponding ribonucleoside 5′-S-methyl phosphorothiolates by demethylthiolation with iodine in dry pyridine at room temperature in the presence of appropriate phosphates such as inorganic orthophosphate, inorganic pyrophosphate or glucose 1-phosphate. Thus synthesis of ribonucleotide anhydrides have been achieved and three ribonucleoside 5′-triphosphates (ATP, CTP and UTP), three ribonucleoside 5′-diphosphates (ADP, CDP and UDP) and a pyrophosphate coenzyme (UDPG) have been synthesized and isolated as lithium salts by charcoal treatment followed by ion exchange chromatography.     

Control of pyruvate kinase activity during glycolysis and gluconeogenesis in Propionibacterium shermanii

The concentrations of glycolytic intermediates and ATP and the activities of certain glycolytic and gluconeogenic enzymes were determined in Propionibacterium shermanii cultures grown on a fully defined medium with glucose, glycerol or lactate as energy source. On all three energy sources, enzyme activities were similar and pyruvate kinase was considerably more active than the gluconeogenic enzyme pyruvate, orthophosphate dikinase, indicating the need for regulation of pyruvate kinase activity. The intracellular concentration of glucose 6-phosphate, a specific activator of pyruvate kinase in this organism, changed markedly according to both the nature and the concentration of the growth substrate: the concentration (7-10 mM) during growth with excess glucose or glycerol was higher than that (1-2 mM) during growth with lactate or at growth-limiting concentrations of glycerol or glucose. Other glycolytic intermediates, apart from pyruvate, were present at concentrations below 2 mM. Glucose 6-phosphate overcame inhibition of pyruvate kinase activity by ATP and inorganic phosphate. With 1 mM-ATP and more than 10 mM inorganic phosphate, a change in glucose 6-phosphate concentration from 1-2 mM was sufficient to switch pyruvate kinase from a strongly inhibited to a fully active state. The results provide a plausible mechanism for the regulation of glycolysis and gluconeogenesis in P. shermanii.     

Unidirectional transport of orthophosphate across the envelope of isolated cauliflower-bud amyloplasts

Isolated amyloplasts from cauliflower (Brassica oleracea L. var botrytis) buds are able to export orthophosphate unidirectionally into the incubation medium. This orthophosphate transport appears to be protein-mediated, as indicated by the following observations: (i) low temperature and the presence of inhibitors of protein-mediated transport reduced the rate of orthophosphate export, and (ii) the rate of orthophosphate export became saturated with rising internal substrate concentrations. Micromolar concentrations of 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid inhibited the rate of unidirectional orthophosphate export, thus indicating the involvement of the amyloplastic glucose-6-phosphate (Glc6P)translocator in the unidirectional export of orthophosphate. The effect of rising concentrations of orthophosphate upon the activity of ADP glucose pyrophosphorylase in desalted extracts was determined. Orthophosphate given in concentrations similar to those measured in the amyloplastic stroma under conditions of steady-state rates of Glc6P-dependent starch synthesis inhibited the activity of ADP-glucose pyrophosphorylase significantly. However, even under strong limiting substrate conditions the residual activity was sufficient to catalyze the flux of carbon into starch. The maximal rates of orthophosphate transport (in the counter-exchange mode) by isolated spinach (Spinacia oleracea L.) chloroplasts and by isolated cauliflower-bud amyloplasts were also determined. These rates were compared with the maximal rates of undirectional orthophosphate export by these plastids. From these measurements we can conclude that, compared with spinach chloroplasts, isolated amyloplasts of cauliflower exhibit a fivefold greater ratio of unidirectional orthophosphate transport to maximal rate of orthophosphate transport in the counter-exchange mode compared to spinach chloroplasts. The determined rate of maximal unidirectional orthophosphate export is sufficient to catalyze the release of additional inorganic phosphate liberated in the amyloplastic stroma during the process of Glc6P-dependent starch synthesis.     

Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts

Photoassimilation of ¹⁴CO₂ by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praealtum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction. Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. The amount of photoassimilated carbon partitioned into starch increased at both very low and high concentrations of orthophosphate. High concentrations of exogenous PGA also stimulated starch synthesis.

DHAP and PGA were the preferred forms of carbon exported to the medium, although indirect evidence suported hexose monophosphate export. The export of PGA and DHAP to the medium was stimulated by high exogenous orthophosphate, but depletion of chloroplastic reductive pentose phosphate intermediates did not occur. As a result only a relatively small inhibition in the rate of CO₂ assimilation occurred.

The rate of photoassimilation was stimulated by exogenous PGA, ribose 5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose 6-phosphate. Inhibition occurred with phosphoenolpyruvate and high concentrations of PGA and ribose 5-phosphate. PGA inhibition did not result from depletion of chloroplastic orthophosphate or from inhibition of ribulose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpyruvate were shown to interact with the orthophosphate translocator.

     

13C and 31P NMR studies on sugar metabolism in Bombyx mori and Philosamia cynthia ricini larvae

Studies were made on ¹³C and ³¹P NMR in larvae of two species of silkworm, Bombyx mori and Philosamia cynthia ricini, in vivo as well as in vitro to determine the pathways of glucose utilization, especially those to amino acids as components of silk fibroin. Results showed that the ¹³C of [1-¹³C]glucose administered orally into 5th instar larvae of both species was incorporated into glucose-1-phosphate, glucose-6-phosphate and trehalose. Serine, glutamate, glutamine, citrate, malate, trehalose and sorbitol-6-phosphate were detected in the hemolymphs of these larvae as metabolites of [1-¹³C]glucose. Two days after [1-¹³C]glucose administration, labeled alanine, glycine, serine, urea, glycogen, trehalose and glycerol were clearly detected in Bombyx larvae. Starvation caused rapid consumption of administered [1-¹³C]glucose with very little accumulation of ¹³C in glycogen or trehalose. In the in vivo³¹P NMR spectra of Bombyx larvae, ATP, arginine phosphate, sorbitol-6-phosphate, uridine diphosphoglucose, phosphoenolpyruvate and inorganic phosphate were detected with some sugar phosphates, such as glucose-1-phosphate and glucose-6-phosphate. During starvation, the intensity of the signal of inorganic phosphate increased and those of sugar phosphate other than sorbitol-6-phosphate decreased, but these changes were reversed by oral administration of glucose.     

Acetate kinase (pyrophosphate). A fourth pyrophosphate-dependent kinase from Entamoeba histolytica.

An enzyme from Entamoeba histolytica catalyzes the formation of acetyl phosphate and orthophosphate from acetate and inorganic pyrophosphate (PP_i), but it displays much greater activity in the direction of acetate formation. It has been purified 40-fold and separated from interfering enzyme activities by chromatography. Its reaction products have been quantitatively established. ATP cannot replace PP_i as phosphoryl donor in the direction of acetyl phosphate formation nor will any common nucleoside diphosphate replace orthophosphate as phosphoryl acceptor in the direction of acetate formation. The trivial name proposed for the new enzyme is acetate kinase (PP_i).     

A phosphohexomutase from the archaeon Sulfolobus solfataricus is covalently modified by phosphorylation on serine

A phosphoserine-containing peptide was identified from tryptic digests from Sulfolobus solfataricus P1 by liquid chromatography-tandem mass spectrometry. Its amino acid sequence closely matched that bracketing Ser-309 in the predicted protein product of open reading frame sso0207, a putative phosphohexomutase, in the genome of S. solfataricus P2. Open reading frame sso0207 was cloned, and its protein product expressed in Escherichia coli. The recombinant protein proved capable of interconverting mannose 1-phosphate and mannose 6-phosphate, as well as glucose 1-phosphate and glucose 6-phosphate, in vitro. It displayed no catalytic activity toward glucosamine 6-phosphate or N-acetylglucosamine 6-phosphate. Models constructed using the X-ray crystal structure of a homologous phosphohexomutase from Pseudomonas aeruginosa predicted that Ser-309 of the archaeal protein lies within the substrate binding site. The presence of a phosphoryl group at this location would be expected to electrostatically interfere with the binding of negatively charged phosphohexose substrates, thus attenuating the catalytic efficiency of the enzyme. Using site-directed mutagenesis, Ser-309 was substituted by aspartic acid to mimic the presence of a phosphoryl group. The V(max) of the mutationally altered protein was only 4% that of the unmodified form. Substitution of Ser-309 with larger, but uncharged, amino acids, including threonine, also decreased catalytic efficiency, but to a lesser extent--three- to fivefold. We therefore predict that phosphorylation of the enzyme in vivo serves to regulate its catalytic activity.     

Kinetic studies on microsomal glucose dehydrogenase in rat liver.

Glucose dehydrogenase from rat liver microsomes was found to react not only with glucose as a substrate but also with glucose 6-phosphate, 2-deoxyglucose 6-phosphate and galactose 6-phosphate. The relative maximum activity of this enzyme was 29% for glucose 6-phosphate, 99% for 2-deoxyglucose 6-phosphate, and 25% for galactose 6-phosphate, compared with 100% for glucose with NADP. The enzyme could utilize either NAD or NADP as a coenzyme. Using polyacrylamide gradient gel electrophoresis, we were able to detect several enzymatically active bands by incubation of the gels in a tetrazolium assay mixture. Each band had different Km values for the substrates (3.0 x 10(-5)M glucose 6-phosphate with NADP to 2.4M glucose with NAD) and for coenzymes (1.3 x 10(-6)M NAD with galactose 6-phosphate to 5.9 x 10(-5)M NAD with glucose). Though glucose 6-phosphate and galactose 6-phosphate reacted with glucose dehydrogenase, they inhibited the reaction of this enzyme only when either glucose or 2-deoxyglucose 6-phosphate was used as a substrate. The Ki values for glucose 6-phosphate with glucose as substrate were 4.0 x 10(-6)M with NAD, and 8.4 x 10(-6)M with NADP; for galactose 6-phosphate they were 6.7 x10(-6)M with NAD and 6.0 x 10(-6)M with NADP. The Ki values for glucose 6-phosphate with 2-deoxyglucose 6-phosphate as substrate were 6.3 x 10(-6)M with NAD and 8.9 x 10(-6)M with NADP; and for galactose 6-phosphate, 8.0 x 10(-6)M with NAD and 3.5 x 10(-6)M with NADP. Both NADH and NADPH inhibited glucose dehydrogenase when the corresponding oxidized coenzymes were used (Ki values: 8.0 x 10(-5)M by NADH and 9.1 x 10(-5)M by NADPH), while only NADPH inhibited cytoplasmic glucose 6-phosphate dehydrogenase (Ki: 2.4 x 10(-5)M). The results indicate that glucose dehydrogenase cannot directly oxidize glucose in vivo, but it might play a similar role to glucose 6-phosphate dehydrogenase. The differences in the kinetics of glucose dehydrogenase and glucose 6-phosphate dehydrogenase show that glucose 6-phosphate and galactose 6-phosphate could be metabolized in quite different ways in the microsomes and cytoplasm of rat liver.     

Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F₁ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - P_i inorganic orthophosphate Dedicated to Prof. Dr. Hans Grisebach on the occasion of his sixtieth birthday     

Purine nucleoside phosphorylase of rabbit liver. Mechanism of catalysis.

Initial velocity studies and product inhibition patterns for purine nucleoside phosphorylase from rabbit liver were examined in order to determine the predominant catalytic mechanism for the synthetic (forward) and phosphorolytic (reverse) reactions of the enzyme. Initial velocity studies in the absence of products gave intersecting or converging linear double reciprocal plots of the kinetic data for both the synthetic and phosphorolytic reactions of the enzyme. The observed kinetic pattern was consistent with a sequential mechanism, requiring that both substrates add to the enzyme before products may be released. The product inhibition patterns showed mutual competitive inhibition between guanine and guanosine as variable substrates and inhibitors. Ribose 1-phosphate and inorganic orthophosphate were also mutually competitive toward each other. Other combinations of substrates and products gave noncompetitive inhibition. Apparent inhibition constants calculated for guanine as competitive inhibitor and for ribose 1-phosphate as noncompetitive inhibitor of the enzyme, with guanosine as variable substrate, did not vary significantly with increasing concentrations of inorganic orthophosphate as fixed substrate. These results suggest that the mechanism was order and that substrates add to the enzyme in an obligatory order. Dead end inhibition studies carried out in the presence of the products guanine and ribose 1-phosphate, respectively, showed that the kinetically significant abortive ternary complexes of enzyme-guanine-inorganic orthophosphate (EQB) and enzyme-guanose-ribose 1-phosphate (EAP) are formed. The results of dead end inhibition studies are consistent with an obligatory order of substrate addition to the enzyme. The nucleoside or purine is probably the first substrate to form a binary complex with the enzyme, and with which inorganic orthophosphate or ribose 1-phosphate may interact as secondary substrates. The evidences presented in this investigation support an Ordered Theorell-Chance mechanism for the enzyme.     

Characterization of starch breakdown in the intact spinach chloroplast

Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia oleracea) chloroplast which had been preloaded with ¹⁴C-starch photosynthetically from ¹⁴CO₂. Starch breakdown was dependent upon inorganic phosphate and the ¹⁴C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of ¹⁴C from triose-phosphate to glycerate 3-phosphate. Iodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3-phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was concluded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic cleavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate.     

Membrane effects on hepatic microsomal glucose-6-phosphatase.

1) Rat liver microsomes exhibit only a weak hydrolyzing activity towards galactose 6-phosphate. Disruption of the microsomal vesicles does not change the apparent Michaelis constant for this substrate but enhances the apparent maximum velocity. 2) The inhibition of microsomal glucose-6-phosphatase (EC 3.1.3.9) by galactose 6-phosphate is of the competitive type in intact and disrupted microsomal vesicles, suggesting that both substrates are hydrolyzed by the same enzyme. 3) The high degree of latency found for the hydrolysis of galactose 6-phosphate compared to glucose 6-phosphate indicates the presence of a carrier for glucose 6-phosphate in the microsomal membrane. 4) Since glucose as a product is not trapped inside the microsomal vesicles, this sugar probably is able to penetrate the microsomal membrane.     

Multiple roles of glucose-6-phosphatases in pathophysiology: State of the art and future trends

Background

The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate to glucose and inorganic phosphate. The enzyme is a part of a multicomponent system that includes several integral membrane proteins; the catalytic subunit (G6PC) and transporters for glucose-6-phosphate, inorganic phosphate and glucose. The G6PC gene family presently includes three members, termed as G6PC, G6PC2, and G6PC3. Although the three isoforms show a moderate amino acid sequence homology, their membrane topology and catalytic site are very similar. The isoforms are expressed differently in various tissues. Mutations in all three genes have been reported to be associated with human diseases.

Scope of review

The present review outlines the biochemical features of the G6PC gene family products, the regulation of their expression, their role in the human pathology and the possibilities for pharmacological interventions.

Major conclusions

G6PCs emerge as integrators of extra- and intracellular glucose homeostasis. Beside the well known key role in blood glucose homeostasis, the members of the G6PC family seem to play a role as sensors of intracellular glucose and of intraluminal glucose/glucose-6-phosphate in the endoplasmic reticulum.

General significance

Since mutations in the three G6PC genes can be linked to human pathophysiological conditions, the better understanding of their functioning in connection with genetic alterations, altered expression and tissue distribution has an eminent importance.     

Glucose stimulation of heart phosphorylase phosphatase activity in vitro and in vivo

Summary To determine the mechanism of the glucose stimulation, glucose or glucose-6-phospate was added to dilute heart extracts in the presence or absence of AMP. The intracellular glucose, tissue glucose-6-phosphate, and tissue AMP concentrations were also determined in 24-h starved animals given glucose; 24-h starved animals given insulin as well as diabetic starved and diabetic starved insulin-treated animals were also studied.The A_0.5 for glucose stimulation of cardiac phosphorylase phosphatase activity was approximately 1 .2 mM. The A_0.5 for glucose-6-phosphate was approximately 0.02 mM. The glucose-6-phosphate concentration in all animals exceeded the Ao.5 by 10-fold. However, the intracellular glucose concentration in the glucose-treated, insulin-treated, diabetic, and diabetic insulin-treated rats was in the range of the A_0.5 for stimulation of phosphorylase phosphatase activity. AMP completely inhibited phosphorylase phosphatase activity at a concentration of 0.2 mM. Physiological concentrations of glucose and glucose-6-phosphate partially reversed this inhibition. Administration of glucose or insulin resulted in an increase in intracellular glucose concentration, an increase in tissue glucose-6-phosphate and a decrease in tissue AMP concentrations. These data suggest that glucose may be a physiological regulator of phosphorylase phosphatase in heart muscle as it is in liver.Recipient ofaMedical InvestigatorshipAward from theVeterans Administration.     

The effect of pH and temperature on the kinetics of native and altered glycogen phosphorylase.

Data on the effect of pH and temperature on the kinetics of rabbit muscle phosphorylases a and b and reduced phosphorylase b (α-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) with glycogen as the saturating and inorganic phosphate the variable substrate are presented. The kinetic profiles as a function of pH are similar for these enzyme species except that the positions of the pH-maximal velocity profiles for reduced phosphorylase b are relatively invariant in the 15 °–30 ° range, whereas the “native” phosphorylases exhibit a substantial shift of the lower pH limb of the profile toward the acid side when the temperature is lowered from 30 to 15 °C. It is proposed that a group with a pK near 6.0 at 30 °C determines the acid limb of maximal velocity profiles. The phosphoryl moiety of enzyme bound pyridoxal 5′-phosphate is suggested for this group. A conformational transition in the protein, which is somehow modified when the aldimine bond between protein and pyridoxal 5′-phosphate is reduced, is invoked to account for the large decrease of this acid side apparent pK for the ternary complex of native phosphorylases when the temperature is lowered. A group with a pK near 7.1 and a heat of ionization of about 8000 cal/mol determines the alkaline limb of maximal velocity profiles at 30 °C. An imidazoyl ring ionization of an enzyme histidyl group is proposed to account for this behavior. In the enzyme-glycogen binary complex, the apparent heat of ionization of this group has an anomalous value of about ?10,000 cal/ mol. It is suggested that a neighboring amino or arginyl guanidinium group is able to interact with the imidazoyl ring in the absence of bound inorganic phosphate to cause this anomalous behavior. The effect of pH on K_m for inorganic phosphate is simply explained by a group with a pK of 6.56 and low heat of ionization. The data are interpreted to indicate that the dianion of inorganic phosphate is the true substrate for all forms of phosphorylase. The kinetic results of this report are closely compared with other kinetic data in the literature on mammalian, plant, and bacterial α-glucan phosphorylases and general overall similarity is demonstrated. Various methods for analyzing pH-kinetic data for enzymes are briefly discussed, and the crucial difference in conclusions the choice of method can make is demonstrated with our data.     

Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Different gal Mutants

Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.     

On lysosomal fragility and induction of liver hexose-monophosphate dehydrogenases in the fasted-refed rat.

Young adult male rats were fasted for 3 days, then fed a glucose-rich diet, ad libitum. At the end of the fasting period, the specific activity of liver glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was decreased to 60% of control (nonfasted) levels. After 24 to 72 h of refeeding, the specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase increased seven- and twofold, respectively. During the fasting period, the liver lysosome fragility increased, as judged by increased release of bound acid phosphatase and β-N-acetylglucosammidase activity during standard homogenization. Three hours after feeding a carbohydrate-rich diet, a further increase in liver lysosomal fragility was observed that returned to control values prior to the induction of the dehydrogenases. Similarly, the susceptibility of liver lysosomes from fasted rats to increased fragility by the intraperitoneal injection of glucose or galactose was also observed. Prior starvation was not a requisite for labilization of lysosomal membranes by injected glucose, but induction of the pentose phosphate shunt dehydrogenase was not observed.In a group of 6-week old male rats fed a commercial pellet diet throughout, the injection of insulin caused no change in liver lysosomal fragility, though hypoglycemia resulted. Similar animals made diabetic by treatment with Streptozotocin and diabetic rats given insulin, showed no change in liver lysosmal fragility based on the percentage of free to total activities of β-N-acetylglucosaminidase, β-glucuronidase, β-galactosidase, and Cathespin D. However, when adult female rats were fasted for 24 h, then injected with sufficient insulin to produce hypoglycemia, liver lysosomal fragility, based on the release of β-N-acetylglucosaminidase during homogenization, increased nearly threefold. These studies demonstrate that stimulated lysosomal fragility can be initiated by refeeding fasted animals a carbohydrate-rich diet, by intraperitoneal injections of fasted rats with glucose or galactose, or by administering insulin alone to fasted rats. However, hyperglycemia induced by diabetogenic doses of Streptozotocin, or hypoglycemia induced in well-fed animals by insulin injection failed to elicit an enhanced liver lysosomal fragility. Whether induction of the enzymes of lipogenesis by rat liver is dependent upon a prior lysosomal membrane labilization remains to be determined.     

<Emphasis Type="Italic">PGM2</Emphasis> overexpression improves anaerobic galactose fermentation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium.