Presence of nonoxidative enzymes of the pentose phosphate shunt in Tetrahymena.

Tetrahymena pyriformis, strain HSM, do not have glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, but contain transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, and ribokinase. The nonoxidative enzymes of the pentose phosphate shunt function in metabolism as indicated by the incorporation of label from [1-14C]ribose into CO2 and glycogen and by the increase in total glycogen content of cultures supplemented with ribose.     

Metabolism of glucose by unicellular blue-green algae

Summary A facultative photo- and chemoheterotroph, the unicellular bluegreen alga Aphanocapsa 6714, dissimilates glucose with formation of CO₂ as the only major product. A substantial fraction of the glucose consumed is assimilated and stored as polyglucose (probably glycogen). The oxidation of glucose proceeds through the pentose phosphate pathway. The first enzyme of this pathway, glucose-6-phosphate dehydrogenase, is partly inducible. In addition, the rate of glucose oxidation is controlled, at the level of glucose-6-phosphate dehydrogenase function, by the intracellular level of an intermediate of the Calvin cycle, ribulose-1,5-diphosphate, which is a specific allosteric inhibitor of this enzyme. As a consequence, the rate of glucose oxidation is greatly reduced by illumination, an effect reversed by the presence of DCMU, an inhibitor of photosystem II.Two obligate photoautotrophs, Synechococcus 6301 and Aphanocapsa 6308, produce CO₂ from glucose at extremely low rates, although their levels of pentose pathway enzymes and of hexokinase are similar to those in Aphanocapsa 6714. Failure to grow with glucose appears to reflect the absence of an effective glucose permease. A general hypothesis concerning the primary pathways of carbon metabolism in blue-green algae is presented.Abbreviations A (U)DPG ADP-glucose or UDP-glucose - G-1-P glucose-1-phosphate - G-6-P glucose-6-phosphate - G_(int.) intracellular glucose - F-6-P fructose-6-phosphate - 6-PG 6-phosphogluconate - Ru-5-P ribulose-5-phosphate - RUDP ribulose-1,5-diphosphate - PGA 3-phosphoglycerate - GAP glyceraldehyde-3-phosphate     

Significance of the non-oxidative route of the pentose phosphate pathway for supplying carbon to the purine-nucleotide pathway in <Emphasis Type="Italic">Corynebacterium ammoniagenes</Emphasis>

To evaluate the strategy of supplying ribose 5-phosphate to the purine-nucleotide pathway exclusively via the nonoxidative route, the glucose 6-phosphate dehydrogenase gene zwf was disrupted in inosine- and 5′-xanthylic acid-producers of Corynebacterium ammoniagenes. In both producers, interruption of the oxidative route caused a decrease in production yields of about 50%. Attempts to increase the capacity of the nonoxidative route through overexpression of the transketolase or transaldolase gene in the zwf mutants led to no discernable effects on production, indicating that, in C. ammoniagenes, the nonoxidative route alone cannot provide sufficient ribose 5-phosphate for high-level production, although nonoxidative synthesis of the precursor is possible. Electronic Publication     

Unusual binding sites for horseradish peroxidase on the surface of cultured and isolated mammalian cells

Summary Binding sites for horseradish peroxidase (HRP), with unusual properties, were detected on the surface of cultured and isolated cells after the cells (on cover slips) had been quickly dried, fixed in cold methanol, and postfixed in a paraformaldehyde solution. The reaction for surface-bound HRP was suppressed by micromolar concentrations of glycoproteins such as invertase, equine luteinizing hormone (eLH) or human chorionic gonadotropin (hCG). The reaction was also suppressed by 20 mM CDP, UDP, GTP, NAD, and ribose 5-phosphate. Two to six times higher concentrations of GMP, fructose 1-phosphate, galactose 6 phosphate, mannose 6-phosphate, fructose 6-phosphate, and glucose 6-phosphate were required to suppress the binding eaction. AMP, ATP, heparin, mannan, and eight non-phosphorylated sugars showed relatively low competing potencies but fucoidin and -lactalbumin were strong inhibitors. No addition of Ca²⁺ was required for the binding of HRP to the cell surface. However, calcium-depleted, inactive HRP did not compete with the binding of native (calcium-containing) HRP whereas H₂O₂-inactivated HRP suppressed the binding. GTP, NAD, ribose 5-phosphate, and EGTA accelerated the release of previously-bound HRP from the cell surface whereas glycoproteins (invertase, cLH, and hCG) did not do se. Addition of Ca²⁺ to GTP, NAD, ribose 5-phosphate or to EGTA prevented the accelerated release of HRP from the cell surface. It is suggested that calciam, present either in the surface membrane or in HRP itself, is involved in the binding of HRP to the cell surface and in the inhibition of binding by GTP, NAD, and ribose 5-phosphate. It is also suggested that -lactalbumin, GTP, UDP, and CDP compete with the binding of HRP to a glycosyltransferase on the cell surface.     

Characterization and Regulatory Properties of Glucose-6-Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The K_m value for glucose-6-phosphate is 1.6 × 10^?4 and 6.3 × 10^?4M at low (1.0–6.0 × 10^?4M) and high (6.0–30.0 × 10^?4M) concentrations of the substrate, respectively. The K_m value for NADP⁺ is 1.4 × 10^?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg²⁺, and Mn²⁺. In the presence of NADPH, the plot of activity vs. NADP⁺ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg²⁺ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.     

Subcellular distribution of enzymes of the oxidative pentose phosphate pathway in root and leaf tissues

The subcellular distribution of enzymes of the oxidative pentose phosphate pathway was studied in plants. Root and leaf tissues from several species were separated by differential centrifugation into plastidic and cytosolic fractions. In all tissues studied, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in both plastidic and cytosolic compartments. In maize and pea root, and spinach and pea leaf, the non-oxidative enzymes of the pentose phosphate pathway (transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase) appear to be restricted to the plastid. In tobacco leaf and root, however, the non-oxidative enzymes were found in the cytosolic as well as the plastidic compartments. In the absence of ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epimerase in the cytosol, the product of the oxidative limb of the pathway (ribulose 5-phosphate) must be transported into a compartment capable of utilizing it. Ribulose 5-phosphate was supplied to isolated intact pea root plastids and was shown to be capable of supporting nitrite reduction. The kinetics of ribulose 5-phosphate-driven nitrite reduction in isolated pea root plastids suggested that the metabolite was translocated across the plastid envelope in a carrier-mediated transport process, indicating the presence of a translocator capable of transporting pentose phosphates.Keywords: Pentose phosphate, subcellular, plastid, ribulose 5-phosphate, compartmentation      

Modelling C3 photosynthesis: A sensitivity analysis of the photosynthetic carbon-reduction cycle

A model of the C ₃ photosynthetic system is developed which describes the sensitivity of the steadystate rate of carbon dioxide assimilation to changes in the activity of several enzymes of the system. The model requires measurements of the steady-state rate of carbon dioxide assimilation, the concentrations of several intermediates in the photosynthetic system, and the concentration of the active site of ribulose 1,5-bisphosphate carboxyalse/oxygenase (Rubisco). It is shown that in sunflowers (Helianthus annuus L.) at photon flux densities that are largely saturating for the rate of photosynthesis, the steady-stete rate of carbon dioxide assimilation is most sensitive to Rubisco activity and, to a lesser degree, to the activities of the stromal fructose, 6-bisphosphatase and the enzymes catalysing sucrose synthesis. The activities of sedoheptulose 1,7-bisphosphatase, ribulose 5-phosphate kinase, ATP synthase and the ADP-glucose pyrophosphorylase are calculated to have a negligible effect on the flux under the high-light conditions. The utility of this analysis in developing simpler models of photosynthesis is also discussed.Abbreviations c _i intercellular CO₂ concentration - C _infP ^supJ control coefficient for enzyme P with respect to flux J - DHAP dihydroxyacetonephosphate - E4P erythrose 4-phosphate - F6P fructose 6-phosphate - FBP fructose 1,6-bisphosphate - FBPase fructose 1,6-bisphosphatase - G3P glyceraldehyde 3-phosphate - G1P glucose 1-phosphate - G6P glucose 6-phosphate - Pi inorganic phosphate - PCR photosynthetic carbon reduction - PGA 3-phosphoglyceric acid - PPFD photosynthetically active photon flux density - R _n ^J response coefficient for effector n with respect to flux J - R5P ribose 5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - Ru5P ribulose 5-phosphate - RuBP ribulose 1,5-bisphosphate - S7P sedoheptulose 7-phosphate - SBP sedoheptulose 1,7-bisphosphate - SBPase sedoheptulose 1,7-bisphosphatase - SPS sucrose-phosphate synthase - Xu5P xylulose 5-phosphate - _n ^P elasticity coefficient for effector n with respect to the catalytic velocity of enzyme P This research was funded by an Australian Research Council grant to I.E.W. and was undertaken during a visity by K.A.M. to the James Cook University of North Queensland. The expert help of Glenys Hanley and Mick Kelly is greatly appreciated.     

Oxygen-18 studies of the mechanism of pyrophosphoryl group transfer catalyzed by phosphoribosylpyrophosphate synthetase.

The formation of phosphoribosylpyrophosphate (PRPP) and adenosine 5′-monophosphate (AMP) from ribose 5-phosphate and adenosine 5′-triphosphate, catalyzed by purified PRPP synthetase from Salmonella typhimurium, was conducted in ¹⁸O-enriched water. The products were isolated, and inorganic phosphate was isolated from AMP and the pyrophosphoryl moiety of PRPP. Oxygen-18 was incorporated into PRPP but not into AMP. These results indicate that PRPP synthesis proceeds with scission of a βPO bond of adenosine 5′-triphosphate. Oxygen-18 enters PRPP by prior exchange of H₂¹⁸O into ribose 5-phosphate; the rate of this exchange was measured by combined gas chromatography-mass spectrometry of the trimethylsilyl derivative of ribose 5-phosphate.     

Purification and Properties of Glucose-6-Phosphate Dehydrogenase from Bacillus subtilis Spores

Glucose-6-phosphate dehydrogenase [d-glucose-6-phosphate: NADP oxidoreductase, EC. 1. 1. 1. 49] obtained from spores of Bacillus subtilis PCI 219 strain was partially purified by filtration on Sephadex G-200, ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-25 (about 54-fold). The optimum pH for stability of this enzyme was about 6.3 and the optimum pH for the reaction about 8.3. The apparent K_m values of the enzyme were 5.7 × 10^–4 M for glucose-6-phosphate and 2.4 × 10^–4 M for nicotinamide adenine dinucleotide phosphate (NADP). The isoelectric point was about pH 3.9. The enzyme activity was unaffected by the addition of Mg⁺⁺ or Ca⁺⁺. The inactive glucoses-6-phosphate dehydrogenase obtained from the spores heated at 85 C for 30 min was not reactivated by the addition of ethylenediaminetetraacetic acid, dipicolinic acid or some salts unlike inactive glucose dehydrogenase.     

Enzymes of glucose metabolism in normal mouse pancreatic islets

1. Glucose-phosphorylating and glucose 6-phosphatase activities, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP⁺-linked isocitrate dehydrogenase, `malic' enzyme and pyruvate carboxylase were assayed in homogenates of normal mouse islets. 2. Two glucose-phosphorylating activities were detected; the major activity had K<sub>m</sub> 0.075mm for glucose and was inhibited by glucose 6-phosphate (non-competitive with glucose) and mannoheptulose (competitive with glucose). The other (minor) activity had a high K<sub>m</sub> for glucose (mean value 16mm) and was apparently not inhibited by glucose 6-phosphate. 3. Glucose 6-phosphatase activity was present in amounts comparable with the total glucose-phosphorylating activity, with K<sub>m</sub> 1mm for glucose 6-phosphate. Glucose was an inhibitor and the inhibition showed mixed kinetics. No inhibition of glucose 6-phosphate hydrolysis was observed with mannose, citrate or tolbutamide. The inhibition by glucose was not reversed by mannoheptulose. 4. 6-Phosphogluconate dehydrogenase had K<sub>m</sub> values of 2.5 and 21μm for NADP<sup>+</sup> and 6-phosphogluconate respectively. 5. Glucose 6-phosphate dehydrogenase had K<sub>m</sub> values of 4 and 22μm for NADP<sup>+</sup> and glucose 6-phosphate. The K<sub>m</sub> for glucose 6-phosphate was considerably below the intra-islet concentration of glucose 6-phosphate at physiological extracellular glucose concentrations. The enzyme had no apparent requirement for cations. Of a number of possible modifiers of glucose 6-phosphate dehydrogenase, only NADPH was inhibitory. The inhibition by NADPH was competitive with NADP<sup>+</sup> and apparently mixed with respect to glucose 6-phosphate. 6. NADP<sup>+</sup>–isocitrate dehydrogenase was present but the islet homogenate contained little, if any, `malic' enzyme. The presence of pyruvate carboxylase was also demonstrated. 7. The results obtained are discussed with reference to glucose phosphorylation and glucose 6-phosphate oxidation in the intact mouse islet, and the possible nature of the β-cell glucoreceptor mechanism.     

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an <Emphasis Type="Italic">E. coli</Emphasis> strain disabled in the oxidative pentose phosphate pathway

Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (¹³C, ¹⁵N, and/or ²H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-¹³C-glycerol and ¹⁵N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, ¹³C-sodium formate combined with ¹³C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to ¹J_C5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1′/C3′/C5′ positions within the ribose rings have reduced labeling. Addition of ¹³C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1′/C3′/C5′ positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and ¹⁵N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs.     

Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly,Eurosta solidaginis

The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate glyceraldehyde glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate glycerol-3-phosphate glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the -glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.Abbreviations 6PGDH 6-Phosphogluconate dehydrogenase - DHAP dihydroxy acetone phosphate - F6P fructose-6-phosphate - F6Pase fructose-6-phospha-tase - FBPase fructose-bisphosphatase - G3P glycerol-3-phosphate - G3Pase glycerol-3-phosphate phophatase - G3PDH glycerol-3-phosphate dehydrogenase - G6P glucose-6-phosphate - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - GAK glyceraldehyde kinase - GAP glyceraldehyde-3-phosphate - GAPase glyceraldehyde-3-phosphatase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glycerol dehydrogenase - GPase glycogen phosphorylase - HMS hexose monophosphate shunt - LDH lactate dehydrogenase - NADP-IDH NADP⁺-dependent isocitrate dehydrogenase - PDHald polyol dehydrogenase, glyceraldehyde activity - PDHgluc polyol dehydrogenase, glucose activity - PFK phosphofructokinase - PGI phosphoglucoisomerase - PGK phosphoglycerate kinase - PGM phosphoglucomutase - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride - SoDH sorbitol dehydrogenase - V _max maximal enzyme activity - ww wet weight     

Oxidation of C1-compounds in Pseudomonas C

Extracts of Pseudomonas C grown on methanol as sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD⁺) and formate dehydrogenase (NAD⁺) were also detected in the extracts.The addition of d-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD⁺ or NADP⁺. In addition, the oxidation of [¹⁴C]formaldehyde to CO₂, by extracts of Pseudomonas C, increased when d-ribulose 5-phosphate was present in the assay mixtures.The amount of radioactivity found in CO₂, was 6.8-times higher when extracts of methanol-grown Pseudomona C were incubated for a short period of time with [1-¹⁴C]glucose 6-phosphate than with [U-¹⁴C]glucose 6-phosphate.These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO₂ both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.     

Recycling of carbon in the oxidative pentose phosphate pathway in non-photosynthetic plastids

Recycling of carbon in the oxidative pentose phosphate pathway (OPPP) of intact pea root plastids has been studied. The synthesis of dihydroxyacetone phosphate (DHAP) and evolution of CO₂ was followed in relation to nitrite reduction. A close coupling was observed between all three measured fluxes which were linear for up to 60 min and dependent upon the integrity of the plastids. However, the quantitative relationship between 1-¹⁴CO₂ evolution from glucose 6-phosphate and nitrite reduction varied with available hexose phosphate concentration. When 10 mM glucose 6-phosphate was supplied to intact plastids a stoichiometry of 1.35 was observed between ¹⁴CO₂ evolution and nitrite reduction. As exogenous glucose 6-phosphate was decreased this value fell, becoming 0.47 in the presence of 0.2 mM glucose 6-phosphate, indicative of considerable recycling of carbon. This conclusion was reinforced when using [2-¹⁴C]glucose-6-phosphate. The measured release of 2-¹⁴CO₂ was consistent with the data for 1-¹⁴CO₂, suggesting complete recycling of carbon in the OPPP. Ribose 5-phosphate was also able to support nitrite reduction and DHAP production. A stoichiometry of 2 NO ₂ ^? reduced: 1 DHAP synthesised was observed at concentrations of 1 mM ribose 5-phosphate or less. At concentrations of ribose 5-phosphate greater than 1 mM this stoichiometry was lost as a result of enhanced DHAP synthesis without further increase in nitrite reduction. It is suggested that this decoupling from nitrite reduction is a function of excess substrate entering directly into the non-oxidative reactions of the OPPP, and may be useful when the demand for OPPP products is not linked to the demand for reductant. The significance of recycling in the OPPP is discussed in relation to the coordination of nitrate assimilation with carbohydrate oxidation in roots and with the utilisation of carbohydrate by other pathways within plastids.     

d-Mannitol dehydrogenase and d-mannitol-1-phosphate dehydrogenase in Platymonas subcordiformis: some characteristics and their role in osmotic adaptation

d-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and d-mannitol dehydrogenase (EC 1.1.1.67) were estimated in a cell-free extract of the unicellular alga Platymonas subcordiformis Hazen (Prasinophyceae), d-Mannitol dehydrogenase had two activity maxima at pH 7.0 and 9.5, and a substrate specifity for d-fructose and NADH or for d-mannitol and NAD⁺. The K _m values were 43 mM for d-fructose and 10 mM for d-mannitol. d-Mannitol-1-phosphate dehydrogenase had a maximum activity at pH 7.5 and was specific for d-fructose 6-phosphate and NADH. The K _m value for d-fructose 6-phosphate was 5.5 mM. The reverse reaction with d-mannitol 1-phosphate as substrate could not be detected in the extract. After the addition of NaCl (up to 800 mM) to the enzyme assay, the activity of d-mannitol dehydrogenase was strongly inhibited while the activity of d-mannitol-1-phosphate dehydrogenase was enhanced. Under salt stress the K _m values of the d-mannitol dehydrogenase were shifted to higher values. The K _m value for d-fructose 6-phosphate as substrate for d-mannitol-1-phosphate dehydrogenase remained constant. Hence, it is concluded that in Platymonas the d-mannitol pool is derectly regulated via alternative pathways with different activities dependent on the osmotic pressure.Abbreviations Fru6P d-fructose 6-phosphate - Mes 2-(N-morpholino)ethanesulfonic acid - MT-DH d-mannitol-dehydrogenase - MT1P-DH d-mannitol-1-phosphate dehydrogenase - Pipes 1,4-piperazinediethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Ribose Utilization by Veillonella alcalescens

The utilization of ribose by Veillonella alcalescens has been further investigated. Nonfermentation of ribose is not a result of a phosphorylation lesion since ribose-phosphorylating activity was measured in cell extracts. Resting cells accumulated ribose-5-phosphate and nucleotides when ¹⁴C-ribose was provided; no other sugar phosphates were detectable. Resting cells that were shifted to growth conditions polymerized rather than degraded the accumulated ribose compounds. Cell extracts contained a fructose diphosphate phosphatase. Ribose-5-phosphate, glucose-6-phosphate, and fructose-6-phosphate were not hydrolyzed. It is postulated that the nonfermentation of ribose is not due to any metabolic lesions, but is a consequence of metabolic control at the fructose diphosphate level of glycolysis.     

The pentose phosphate pathway of glucose metabolism. Measurement of the non-oxidative reactions of the cycle

Methods for the quantitative determination of ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase, transketolase and transaldolase in tissue extracts are described. The determinations depend on the measurement of glyceraldehyde 3-phosphate by using the coupled system triose phosphate isomerase, α-glycero-phosphate dehydrogenase and NADH. By using additional purified enzymes transketolase, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase conditions could be arranged so that each enzyme in turn was made rate-limiting in the overall system. Transaldolase was measured with fructose 6-phosphate and erythrose 4-phosphate as substrates, and again glyceraldehyde 3-phosphate was measured by using the same coupled system. Measurements of the activities of the non-oxidative reactions of the pentose phosphate pathway were made in a variety of tissues and the values compared with those of the two oxidative steps catalysed by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.     

Characterization of mannitol metabolism in the mangrove red algaCaloglossa leprieurii (Montagne) J.Agardh

A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheK _m values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD⁺. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aK _m value of 1.2 mM and a high requirement of Mg²⁺ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedK _m values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD⁺. For the non-specific HK, only theK _m values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.Abbreviations F6P fructose-6-phosphate - HK hexokinase - Mt1P mannitol-1-phosphate - Mt1PDH mannitol-1-phosphate dehydrogenase - Mt1Pase mannitol-1-phosphatase - MtDH mannitol dehydrogenase     

Anoxia and freezing exposures stimulate covalent modification of enzymes of carbohydrate metabolism in Littorina littorea

The effects of anoxia (N₂ atmosphere at 5 °C) or freezing (at-8 °C) exposure in vivo on the activities of five enzymes of carbohydrate metabolism were assessed in foot muscle and hepatopancreases of the marine periwinkle Littorina littorea. Changes in glycogen phosphorylase, glycogen synthetase, pyruvate kinase and pyruvate dehydrogenase under either stress were generally consistent with covalent modification of the enzymes to decrease enzyme activity and/or convert the enzyme to a less active form. However, no evidence for a similar covalent modification of phosphofructokinase was found. The metabolic effects of freezing and anoxia were generally similar, suggesting that a primary contributor to freezing survival is the implementation of anaerobic metabolism and metabolic arrest mechanisms that also promote anoxia survival in marine molluses. However, in hepatopancreas phosphorylase was activated and pyruvate kinase remained in two enzyme forms in freezing-exposed snails, contrary to the results for anoxic animals. Ion exchange chromatography on DE-52 Sephadex revealed the presence of two forms of pyruvate kinase in both tissues of control L. littorea, eluting at 30–50 mmol·1^-1 KCl (peak I) or 90–110 mmol·1^-1 KCl (peak II). Anoxia exposure converted pyruvate kinase in both tissues to the peak I form, as did freezing for foot muscle pyruvate kinase. Kinetic analysis showed that peak I pyruvate kinase had lower affinities for substrates, phosphoenolpyruvate and ADP, and was very strongly inhibited by l-alanine compared with the peak II enzyme. Peak I pyruvate kinase had an I ₅₀ value for l-alanine of 0.38 mmol·1^-1, whereas peak II pyruvate kinase was unaffected by l-alanine evenat 40 mmol·1^-1. In vitro incubation of extracts from control foot muscle under conditions promoting phosphorylation or dephosphorylation identified the peak I and II forms as the low and high phosphate forms, respectively. This result for L. littorea pyruvate kinase was highly unusual and contrary to the typical effect of anoxia on pyruvate kinase in marine molluscs which is to stimulate the phosphorylation of pyruvate kinase and, thereby, convert the enzyme to a less active form.Abbreviations AABS p-(p-aminophenylazo)benzene sulphonic acid - F2, 6P fructose-2,6-bisphosphate - F6P fructose-6-phosphate - G6P glucose-6-phosphate - GP glycogen phosphorylase - GS glycogen synthase - I ₅₀ inhibitor concentration reducing enzyme velocity by 50% - MR metabolic rate - PDH pyruvate dehydrogenase - PEP phosphoenopyruvate - PFK phosphofructokinase - PK pyruvate kinase - SW sea water - F _a air temperature - TCA trichloroacetic acid - UDPG uridine-diphosphate glucose - WW wet weight