Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrificans grown on glucose/nitrate

Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ l-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD⁺-and NADP⁺-linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg²⁺ requirement. Kinetics for both NAD(P)⁺ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD⁺-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP⁺-linked activity. Neither of the two activities are inhibited by 100 M NADH but both are inhibited by NADPH. The NAD⁺-linked activity is far more sensitive to inhibition by NADPH than the NADP⁺-linked activity.     

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     

<Emphasis Type="Italic">Thermotoga maritima</Emphasis> TM0298 is a highly thermostable mannitol dehydrogenase

Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases. To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol dehydrogenase. T. maritima mannitol dehydrogenase (TmMtDH) is optimally active between 90 and 100 °C and retains 63% of its activity at 120 °C but shows no detectable activity at room temperature. Its kinetic inactivation follows a first-order mechanism, with half-lives of 57 min at 80 °C and 6 min at 95 °C. Although TmMtDH has a higher V _max with NADPH than with NADH, its catalytic efficiency is 2.2 times higher with NADH than with NADPH and 33 times higher with NAD⁺ than with NADP⁺. This cofactor specificity can be explained by the high density of negatively charged residues (Glu193, Asp195, and Glu196) downstream of the NAD(P) interaction site, the glycine motif. We demonstrate that TmMtDH contains a single catalytic zinc per subunit. Finally, we provide the first proof of concept that mannitol can be produced directly from glucose in a two-step enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60 °C.     

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.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Histochemical observations on the exoerythrocytic malaria parasite Plasmodium berghei in rat liver

Summary Enzyme histochemical methods were performed on sporozoite infected liver tissue of rats in order to gain insight into the nutrition and metabolism of exoerythrocytic forms of Plasmodium berghei. The following enzymes were demonstrated in the hepatocytic stages of the parasites, obtained 41 and 48 h after inoculation of sporozoites: acid phosphatase, cytochrome oxidase, NADH-tetrazolium reductase, succinate dehydrogenase, NAD⁺ and NADP⁺ dependent isocitrate dehydrogenase, NADP⁺-dependent malate dehydrogenase, lactate dehydrogenases, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenases and -glycerol-phosphate dehydrogenase. The results suggest that a conventional Embden-Meyerhoff pathway, pentose phosphate pathway and Krebs' citric acid cycle may in part be present in these exoerythrocytic parasites. Alkaline phosphatase, nucleoside polyphosphatase, 5nucleotidase. glucose-6-phosphatase, -glucan phosphorylase, NAD⁺ dependent malate dehydrogenase, amino-peptidase M and non-specific esterases were not detected by our techniques in the parasite. The enzyme distribution of this intrahepatocytic malaria parasite revealed by histochemistry is compared with the enzyme distribution in the other phases of the parasite's life cycle.This study was made possible by grants from the Jan Dekker Foundation for Biomedical Research and the Niels Stensen Foundation, The Netherlands, to the first author     

The influence of freezing and freeze-drying of tissue specimens on enzyme activity

Summary In the presented study the influence of freezing and freeze-drying on enzyme activity is described. Attention is paid to 16 enzymes which can be used for quantitative enzyme histochemical techniques.With the exception of succinate dehydrogenase only, no significant inactivation during freezing and freeze-drying procedures could be demonstrated with lactate dehydrogenase, malate dehydrogenase (NAD⁺), malate dehydrogenase (decarboxylating) (NADP⁺), isocitrate dehydrogenase (NADP⁺), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADH-oxydoreductase, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome c oxidase, phosphoglucomutase, glucosephosphate isomerase, glucose-6-phosphatase, acid phosphatase, -glucuronidase and non specific aryl esterase. Therefore the results supply a sound foundation for those quantitative enzyme histochemical techniques in which tissue specimens are frozen or frozen-dried before enzyme estimations are performed.     

Photokinetic microanalysis of NADP+, using bacterial luciferase

Summary Bioluminescence photokinetic assay of NADP⁺ is described, using the glucose-6-phosphate dehydrogenase reaction for conversion to its reduced form and subsequent measurement of this with luciferase extracts of Vibria fisherii. The analyses were applied to the determination of the activity of minute amounts of glutathione reductase using NADP⁺ as measurable product and for nucleotide assay in cell samples of 0.5–10 g dry weight. The sensitivity was sufficient for determining 0.5 picomoles NADP⁺.Previously, FMN, NADH, NAD⁺ and NADH have been analysed with the bacterial luciferase system. Its applicability has now been extended by the assay of NADP⁺.     

Interactions between magnesium ions,pH, glucose-6-phosphate,and NADPH/NADP+ ratios in the modulation of chloroplast glucose-6-phosphate dehydrogenase in vitro

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg²⁺, proton, and substrate concentrations. Mg²⁺ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg²⁺-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP⁺ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg²⁺. Increasing the NADPH/NADP⁺ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg²⁺ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP⁺ ratio, enzyme activity is not affected by the Mg²⁺ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - G-6-PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - PPC pentose phosphate cycle     

Activity of NAD-dependent glucose-6-phosphate dehydrogenase in yeast-like organisms

Summary From tested yeast-like organisms, onlyGeotrichum candidum showed the same activity of glucose-6-phosphate dehydrogenase with both NAD⁺ and NADP⁺. i. e. 0.017–0.019 mol NADH/min. mg dry weight of cell free extracts. Omission of Mg⁺⁺ in the reaction mixture did not influence the activity of the enzyme in the presence of NAD⁺. Cell free extracts ofEndomyces magnusii showed only low activity of this enzyme and the ratio of its activity in the presence of NAD⁺ and NADP⁺, respectively, varied in individual cultures.Rhodotorula glutinis showed only an NADP⁺-dependent activity.     

Production of NADPH in the mannitol cycle and its relation to polyketide formation in Alternaria alternata.

The enzymes mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, mannitol dehydrogenase and hexokinase participate in an enzymatic cycle in the fungus Alternaria alternata. One turn of the cycle gives the net result: NADH + NADP+ + ATP leads to NAD+ + NADPH + ADP + Pi. The cycle alone can meet the total need of NADPH formation for fat synthesis in the organism. A polyketide producing strain of A. alternata shows a lower mannitol oxidation as well as a lower fat synthesis than a nonproducing mutant, supporting the hypothesis that polyketide formation is favoured at limiting NADPH production. It is further suggested that the mannitol cycle is regulating the glycolytic flux by substrate withdrawal from phosphofructokinase.     

Polyol Dehydrogenases in the Soluble Fraction of Acetobacter melanogenum

Polyol dehydrogenases of Acetobacter melanogenum were investigated. Three polyol dehydrogenases, i. e. NAD⁺-linked d-mannitol dehydrogenase, NAD⁺-linked sorbitol dehydrogenase and NADP⁺-linked d-mannitol dehydrogenase, in the soluble fraction of the organism were purified 12-fold, 8-fold and 88-fold, respectively, by fractionation with ammonium sulfate and DEAE-cellulose column chromatography. NAD⁺-linked sorbitol dehydrogenase reduced 5-keto-d-fructose (5KF) to l-sorbose in the presence of NADH, whereas NADP⁺-linked d-mannitol dehydrogenase reduced the same substrate to d-fructose in the presence of NADPH. It was also shown that NAD⁺-linked d-mannitol dehydrogenase was specific for the interconversion between d-mannitol and d-fructose and that this enzyme was very unstable in alkaline conditions.     

Diurnal changes in adenylates and nicotinamide nucleotides in sugar beet leaves

Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers at 25°C, with a photon flux density of 500 mol m^-2s^-1. Measurements were made of net CO₂ exchange, leaf adenylates (ATP, ADP and AMP), and leaf nicotinamide nucleotides (NAD⁺, NADP⁺, NADH, NADPH), over the diurnal period (16h light/8 h dark) and during photosynthetic induction. All the measurements were carried out on recently expanded leaves from 5-week-old plants. When the lights were switched on in the growth chamber, the rate of photosynthetic CO₂ uptake, and the levels of leaf ATP and NADPH increased to a maximum in 30 min and remained there throughout the light period. The increase in ATP over the first few minutes of illumination was associated with the phosphorylation of ADP to ATP and the increase in NADPH with the reduction of NADP⁺; subsequently, the increase in ATP was associated with an increase in total adenylates while the increase in NADPH was associated with an accumulation of NADP⁺ and NADPH due to the light-driven phosphorylation of NAD⁺ to NADP⁺. On return to darkness, ATP and NADPH values decreased much more slowly, requiring 2 to 4 hours to reach minimum values. From these results we suggest that (i) the total adenylate and NADPH and NADP⁺ (but not NAD⁺ and NADH) pools increase following exposure to light; (ii) the increase in pool size is not accompanied by any large change in the energy or redox states of the system; and (iii) the measured ratios of ATP/ADP and NADPH/NADP⁺ for intact leaves are low and constant during steady-state illumination.Abbreviations AEC adenylate energy charge - DHAP dihydroxyacetone phosphate - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide - PES phenazine ethosulfate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - PFD photon flux density - Ru5P ribulose-5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly regulated by the ratio of NADPH/NADP⁺, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP⁺ ratio. With a ratio of NADPH/NADP⁺ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited.This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP⁺ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions.Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP⁺ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP⁺.It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP⁺ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolite unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP⁺ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

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.     

The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria

1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase and NADH–cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP⁺-linked) and NADPH–cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, α-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.     

Effect of phosphon-D on photosynthetic light reactions and on reactions of the oxidative and reductive pentose phosphate cycle in a reconstituted spinach (Spinacia oleracea L.) chloroplast system

Phosphon-D (tributyl-2, 4-dichlorobenzylphosphonium chloride), known as an inhibitor of gibberellin biosynthesis, enhances photosynthetic electron transport by up to 200%, with Fe(CN) ₆ ^3- and NADP⁺ being the electron acceptors. Maximum stimulation is reached at phosphon-D concentrations around 2–5 M. At the same time photosynthetic ATP formation is gradually inhibited. Phosphon-D concentrations over 0.1 mM inhibit electron transport. The uncoupling activity of phosphon-D is manifested by inhibition of noncyclic ATP synthesis and by stimulation of light-induced electron flow. The inhibition of ATP synthesis drastically decreases photosynthetic carbon assimilation in a reconstituted spinach chloroplast system. The two ATP-dependent kinase reactions of the reductive pentose phosphate cycle become the rate-limiting steps. On the other hand a stimulated photoelectron transport increases the NADPH/NADP⁺ ratio, resulting in a drastic inhibition of chloroplast glucose-6-phosphate dehydrogenase (EC 1.1.1.49), the key enzyme of the oxidative pentose phosphate cycle. When light-induced electron flow is inhibited by high phosphon-D concentrations and the NADPH/NADP⁺ ratio is low, the light-dependent inhibition of glucose-6-phosphate dehydrogenase is gradually abolished.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride - B-Nine N-dimethylaminosuccinamic acid - CCC (2-chloroethyl)-trimethylammonium chloride - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DCPIP dichlorophenolindophenol - G-6-PDH glucose-6-phosphate dehydrogenase - FBP fructose bisphosphate - F-6-P fructose-6-phosphate - 3-PGA 3-phosphoglyceric acid - Posphon-D tributyl-2,4-dichlorobenzylphosphonium chloride - PMP pentose monophosphates - PPC pentose phosphate cycle - RuBP ribulose bisphosphate - Ru-5-P ribulose-5-phosphate Dedicated to Prof. Dr. Drs.h.c. Adolf Butenandt on the occasion of his 75. birthday     

Mannitol Metabolism in Lentinus edodes, the Shiitake Mushroom

Mannitol metabolism was evaluated in fruiting bodies of Lentinus edodes. Cell extracts were prepared from fruiting bodies, and key enzymes involved in mannitol metabolism were assayed, including hexokinase, mannitol dehydrogenase, mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, and fructose-6-phosphatase. Mannitol dehydrogenase, fructose-6-phosphatase, mannitol-1-phosphatase, and hexokinase activities were found in extracts of fruiting bodies. However, mannitol-1-phosphate dehydrogenase activity was not detected. Mycelial cultures were grown in an enriched liquid medium, and enzymes of the mannitol cycle were assayed in cell extracts of rapidly growing cells. Mannitol-1-phosphate dehydrogenase activity was also not found in mycelial extracts. Hence, evidence for a complete mannitol cycle both in vegetative mycelia and during mushroom development was lacking. The pathway of mannitol synthesis in L. edodes appears to utilize fructose as an intermediate.     

Selective Inhibition by Actinomycin D of the Synthesis in Photosynthetic and Non-photosynthetic Enzymes During the Greening of Etiolated Bean Leaves

The effect of actinomycin D on the synthesis of the photosynthetic apparatus during illumination of etiolated leaves of Phaseolus vulgaris was studied. The increase of chlorophyll content and of the activities of some photosynthetic enzymes (NADPH diaphorase, ferredoxin, NADP⁺ glyceraldehyde-3-phosphate dehydrogenase) was compared with simultaneous measurements of the level of other enzymes not considered associated with photosynthesis (ornithine transcarbamylase, glucose-6-phosphate dehydrogenase, NAD⁺ glyceraldehyde-3-phosphate dehydrogenase).