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     

Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules

Subcellular organelle fractionation of nitrogen-fixing nodules of soybean (Glycine max (L.) Merr.) indicates that a number of enzymes involved in the assimilation of ammonia into amino acids and purines are located in the proplastids. These include asparagine synthetase (EC 6.3.1.1), phosphoribosyl amidotransferase (EC 2.4.2.14), phosphoglycerate dehydrogenase (EC 1.1.1.95), serine hydroxymethylase (EC 2.1.2.1), and methylene-tetrahydrofolate dehydrogenase (EC 1.5.1.5). Of the two isoenzymes of asparate aminotransferase (EC 2.6.1.1) in the nodule, only one was located in the proplastid fraction. Both glutamate synthase (EC 1.4.1.14) and triosephosphate isomerase (EC 5.3.1.1) were associated at least in part with the proplastids. Glutamine synthetase (EC 6.3.1.2) and xanthine dehydrogenase (EC 1.2.1.37) were found in significant quantities only in the soluble fraction. Phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) was found mostly in the soluble fraction, although small amounts of it were detected in other organelle fractions. These results together with recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of ammonium assimilation, amide synthesis and uredie biogenesis in the nodule.Abbreviations FH₄ tetrahydrofolic acid - PRPP 5-phospho--D-ribose 1-pyrophosphate - PRPP synthetase ribosephosphate pyrophosphokinase (phosphoribosylpyrophosphate synthetase)     

Regulation of flavin biosynthesis in the methylotrophic yeast Hansenula polymorpha

Under various conditions of growth of the methylotrophic yeast Hansenula polymorpha, a tight correlation was observed between the levels of flavin adenine dinucleotide (FAD)-containing alcohol oxidase, and the levels of intracellularly bound FAD and flavin biosynthetic enzymes. Adaptation of the organism to changes in the physiological requirement for FAD was by adjustment of the levels of the enzymes catalyzing the last three steps in flavin biosynthesis, riboflavin synthetase, riboflavin kinase and flavin mononucleotide adenylyltransferase. The regulation of the synthesis of the latter enzymes in relation to that of alcohol oxidase synthesis was studied in experiments involving addition of glucose to cells of H. polymorpha growing on methanol in batch cultures or in carbon-limited continuous cultures. This resulted not only in selective inactivation of alcohol oxidase and release of FAD, as previously reported, but invariably also in repression/inactivation of the flavin biosynthetic enzymes. In further experiments involving addition of FAD to the same type of cultures it became clear that inactivation of the latter enzymes was not caused directly by glucose, but rather by free FAD that accumulated intracellularly. In these experiments no repression or inactivation of alcohol oxidase occurred and it is therefore concluded that the synthesis of this enzyme and the flavin biosynthetic enzymes is under separate control, the former by glucose (and possibly methanol) and the latter by intracellular levels of free FAD.Abbreviations FAD Flavin adenine dinucleotide - FMN riboflavin-5-phosphate; flavin mononucleotide - Rf riboflavin     

Experimental protein malnutrition in primates: Cytochemical studies on the cerebellum of the squirrel monkey,Saimiri Sciureus

Synopsis The cerebellum of healthy and malnourished squirrel monkeys was studied histopathologically and cytochemically for a number of important enzymes such as phosphatases (acid and alkaline phosphatase, ATPase, thiamine pyrophosphatase), esterases (simple esterase and acetylcholinesterase), dehydrogenases (succinate, malate and isocitrate dehydrogenase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase), monoamine oxidase and phosphorylase. The Purkinje cells, stellate and basket cells were found to be more sensitive to protein malnutrition compared to the other types of cells in the cerebellum. An increase in the number of dark cells with large amounts of ribonucleoprotein complex in the Purkinje cell layer of the extremely malnourished animals sacrificed after 15 weeks on a low protein diet may be significant and may reflect either an abnormal metabolic process or an interruption in the axonal transport of RNA complex. This may also be directly related to a significant reduction in the level of oxidative enzymes, especially those of the tricarboxylic acid cycle, these being the main source of energy stored in ATP. At the same time the level of lysosomal enzymes, which are responsible for the catalysis of the different degradation reactions, is greatly increased and indicates cellular catabolism. The present investigations point to the probability that the neurons adapt to the changed environment by beginning to utilize structural proteins for their basic metabolism.     

The effect of gyrase inhibitors and cyclic AMP on induction and glucose repression of the 6-hydroxy-nicotine oxidases in Arthrobacter oxidans

The induction by d,l-nicotine of the enantiozymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase in Archrobacter oxidans was differently affected by the inhibitors of Escherichia coli gyrase, novobiocin and nalidixic acid. These compounds inhibited 6-hydroxy-L-nicotine oxidase induction slightly, but led to an increase in the level of 6-hydroxy-D-nicotine oxidase activity. Furthermore, the specific repression by glucose of 6-hydroxy-D-nicotine oxidase synthesis was not abolished by the addition of cAMP but by that of novobiocin.Abbreviations 6-HDNO 6-hydroxy-D-nicotine oxidase - 6-HLNO 6-hydroxy-L-nicotine oxidase - cAMP cyclic 3,5-adenosine monophosphate - Enzymes Adenylate cyclase - ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1) - cAMP-phosphodiesterase 3:5-cyclic-nucleotide 5-nucleotido-hydrolase (EC 3.1.4.17) - DNA gyrase DNA topoisomerase II (EC 5.99) - DNA polymerase deoxynucleosidetriphosphate: DNA desoxynucleotidyl-transferase (EC 2.7.7.7) - 6-hydroxy-L-nicotine oxidase 6-hydroxy-L-nicotine: oxygen oxidoreductase (EC 1.5.3.5) - 6-hydroxy-D-nicotine oxidase 6-hydroxy-D-nicotine: oxygen oxidoreductase (EC 1.5.3.6) - -lactamase penicillin amido--lactamhydrolase (EC 3.5.2.6) - nicotine dehydrogenase nicotine: (acceptor)6-oxidoreductase (hydroxylating) (EC 1.5.99.4)     

Regulation of the glucolytic enzymes inPseudomonas putida

Summary The synthesis of glucose catabolizing enzymes is under inductive control inPseudomonas putida. Glucose, gluconate and 2-ketogluconate are the best nutritional inducers of these enzymes. Mutants unable to catabolize gluconate or 2-ketogluconate synthesized relatively high levels of glucose dehydrogenase and gluconate-6P dehydrase activities when grown in the presence of these substrates. This identifies both compounds as true inducers of these enzymes. KDGP aldolase is induced by its substrate, as evidenced by the inability of mutant cells unable to form KDGP to produce this enzyme at levels above the basal one. A 3-carbon compound appears to be the inducer of glyceraldehyde-3P dehydrogenase. This pattern of regulation suggests that there is a low degree of coordinate control in the synthesis of the glucolytic enzymes byP. putida. This is also supported by the lack of proportionality found in the levels of two enzymes governed by the same inducers, glucose dehydrogenase and gluconate-6P dehydrase, in cells grown on different conditions.Abbrevitions P phosphate - KDGP 2-Keto-3-deoxygluconate-6-phosphate - GDH glucose dehydrogenase - GNDH gluconate dehydrogenase - GK glucokinase - GNK gluconokinase - KGK ketogluconokinase - KGR 2-Ketogluconate-6-phosphate reductase - GPDH glucose-6-phosphate dehydrogenase - GNPD gluconate-6-phosphate dehydrase - KDGPA 2-Keto-3-deoxygluconate-6-phosphate aldolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase     

Modifications by chronic intermittent hypoxia and drug treatment on skeletal muscle metabolism

The energy metabolism was evaluated in gastrocnemius muscle from 3-month-old rats subjected to either mild or severe 4-week intermittent normobaric hypoxia. Furthermore, 4-week treatment with CNS-acting drugs, namely, -adrenergic (-yohimbine), vasodilator (papaverine, pinacidil), or oxygen-increasing (almitrine) agents was performed. The muscular concentration of the following metabolites was evaluated: glycogen, glucose, glucose 6-phosphate, pyruvate, lactate, lactateto-pyruvate ratio; citrate, -ketoglutarate, succinate, malate; aspartate, glutamate, alanine; ammonia; ATP, ADP, AMP, creatine phosphate. Furthermore the Vmax of the following muscular enzymes was evaluated: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase; citrate synthase, malate dehydrogenase; total NADH cytochrome c reductase; cytochrome oxidase. The adaptation to chronic intermittent normobaric mild or severe hypoxia induced alterations of the components in the anaerobic glycolytic pathway [as supported by the increased activity of lactate dehydrogenase and/or hexokinase, resulting in the decreased glycolytic substrate concentration consistent with the increased lactate production and lactate-to-pyruvate ratio] and in the mitochondrial mechanism [as supported by the decreased activity of malate dehydrogenase and/or citrate synthase resulting in the decreased concentration of some key components in the tricarboxylic acid cycle]. The effect of the concomitant pharmacological treatment suggests that the action of CNS-acting drugs could be also related to their direct influence on the muscular biochemical mechanisms linked to energy transduction.     

Symplasmic and apoplasmic radial ion transport in plant roots

Enzymes of starch synthesis and degradation were identified in crude extracts of the unicellular green alga Dunaliella marina (Volvocales). By polyacrylamide gel electrophoresis and specific staining for enzyme activities, 4 multiple forms of starch synthase, 2 amylases, and at least 2 forms of -glucan phosphorylase were visible. Using specific -glucans incorporated into the gel before electrophoresis we have tentatively correlated -amylase and -amylase with both hydrolytic activities. The activities of -glucan phosphorylase and amylase(s) were measured quantitatively in crude extracts, and the concomitant action of -glucan phosphorylase and amylase(s) was found to account for the fastest rate of starch mobilization observed in vivo. Isolated chloroplasts retained both typical plastid marker enzymes and ADPglucose pyrophosphorylase, starch synthase, amylase(s), and -glucan phosphorylase to a similar percentage. Gel electrophoretic analysis followed by staining for enzyme activity of a stromal fraction resulted in a pattern of multiple forms of starch-metabolizing enzymes analogous to that found in a crude extract. We interpret the combined data as indicating the exclusive location in vivo of starch-metabolizing enzymes in chloroplasts of D. marina.Abbreviations Chl chlorophyll - DEAE-dextran diethylaminoethyl-dextran - DDT dithiothreitol - EDTA ethylenediamine tetraacetic acid - FBPase fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11 - G1P glucose 1-phosphate - G6P-DH glucose 6-phosphate dehydrogenase, EC 1.1.1.49 - HEPES N-2-hydroxyethylpiperazine-N-ethanesulphonic acid - MES 2-(N-morpholino)ethanesulphonic acid - P_i inorganic orthophosphate - RuBP carboxylase ribulose-1,5-bisphosphate carboxylase, EC 4.1.1.39     

Seasonal variation of enzyme activities in Laminaria hyperborea

The patterns of seasonal variation of enzyme levels in the brown alga Laminaria hyperborea (Gunn.) Fosl. have been investigated for the following enzymes: Ribulosebisphosphate-carboxylase (EC 4.1.1.39), phosphoenolpyruvate-carboxykinase (EC 4.1.1.32), glyceraldehyde-3-phosphate-dehydrogenase (NADP dep., EC 1.2.1.12), malate-dehydrogenase (NAD dep., EC 1.1.1.37), L-aspartate-2-oxoglutarate aminotransferase (EC 2.6.1.1), and mannitol-l-phosphate-dehydrogenase (EC 1.1.1.17). The first four enzymes exhibit a circannual periodicity, characterized by a pronounced spring-maximum of enzyme activity in April and May. As a consequence, the phylloid can maintain high metabolic rates from early spring on, although water temperature has then only slightly risen above the annual minimum. This findings is discussed in relationship to the growth- and developmental cycle of L. hyperborea and to the seasonal variation of photosynthesis and light-independent CO₂-fixation. The seasonal pattern, outlined above, correlates well with the circannual fluctuations of the nitrogen content of the sea and with the variation of the internal nitrogen- and nitrate-content of the alga. This coincidence may indicate that nitrogen levels play an important role in the regulation of enzyme activities and, hence, the metabolic capacities of L. hyperborea.Abbreviations PEPCK phosphoenolpyruvate carboxykinase (EC 4.1.1.32) - RUBPC ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - GAPDH (NADP dep.) glyceraldehyde-3-phosphate dehydrogenase (NADP dependent) (EC 1.2.1.12) - MDH (NAD dep.) malate dehydrogenase (NAD dependent) (EC 1.1.1.37) - AAT L-aspartate-2-oxoglutarate aminotransferase (EC 2.6.1.1) - Mannitol-1-P DH mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) - LIF lightindependent CO₂-fixation - DHAP dihydroacetone phosphate - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - OAA oxaloacetate     

Glycerol and dihydroxyacetone dissimilation in Desulfovibrio strains

During growth on glycerol two marine Desulfovibrio strains that can grow on an unusually broad range of substrates contained high activities of glycerol kinase, NAD(P)-independent glycerol 3-phosphate dehydrogenase and the other enzymes necessary for the conversion of dihydroxyacetone phosphate to pyruvate. Glycerol dehydrogenase and a specific dihydroxyacetone kinase were absent. During growth on dihydroxyacetone, glycerol kinase is involved in the initial conversion of this compound to dihydroxyacetone phosphate which is then further metabolized. Some kinetic properties of the partially purified glycerol kinase were determined. The role of NAD as electron carrier in the energy metabolism during growth of these strains on glycerol and dihydroxyacetone is discussed.Glycerol also supported growth of three out of four classical Desulfovibrio strains tested. D. vulgaris strain Hildenborough grew slowly on glycerol and contained glycerol kinase, glycerol 3-phosphate dehydrogenase and enzymes for the dissimilation of dihydroxyacetone phosphate. In D. gigas which did not grow on glycerol the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase were absent in lactate-grown cells.Abbreviations DHA dihydroxyacetone - DHAP dihydroxyacetone phosphate - G3P glycerol 3-phosphate - GAP glyceraldehyde 3-phosphate - 3-PGA 3-phosphoglycerate - 2-PGA 2-phosphoglycerate - 2,3-DPGA 2,3-diphosphoglycerate - PEP phosphoenolpyruvate - DH dehydrogenase - GK glycerol kinase - DHAK dihydroxyacetone kinase - TIM triosephosphate isomerase - PGK 3-phosphoglycerate kinase - PK pyruvate kinase - LDH lactate dehydrogenase - DTT dithiotreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid - PIPES piperazine-1,1-bis(2-ethane sulfonic acid) - BV²⁺/BV⁺ oxidized/reduced benzylviologen - PMS phenazine methosulfate - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide     

Activities of Enzymes of Carbohydrate and Energy Metabolism of the Spores of the Microsporidian, Nosema grylli

ABSTRACT. The presence of 14 enzymes was investigated using purified spores of the microsporidian Nosema grylli from fat body of the crickets Gryllus bimaculatus . Glucose 6-phosphate dehydrogenase (EC 1.1.1.49), phosphoglucomutase (EC 5.4.2.2), phosphoglucose isomerase (EC 5.3.1.9), fructose 6-phosphate kinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), 3-phosophoglycerate kinase (EC 2.7.2.3), pyruvate kinase (EC 2.7.1.40) and glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) were detected with activities of 15 ± 1, 7 ± 1, 1,549 ± 255, 10 ± 1, 5 ± 1, 16 ± 4, 6 ± 1 and 16 ± 2 nmol/min. mg protein, respectively. Hexokinase (EC 2.7.1.1), NAD-dependent malate dehydrogenase (EC 1.1.1.37), malic enzyme (EC 1.1.1.40), lactate dehydrogenase (EC 1.1.1.27), alcohol dehydrogenase (EC 1.1.1.1) and succinate dehydrogenase (EC 1.3.99.1) were not detectable. These results suggest the catabolism of carbohydrates in microsporidia occurs via the Embden-Meyerhof pathway. Glycerol 3-phosphate dehydrogenase may reoxidize NADH which is produced by glyceraldehyde 3-phosphate dehydrogenase in glycolysis.     

Regulation of methylamine and formaldehyde metabolism in Arthrobacter P1

The regulation of methylamine and formaldehyde metabolism in Arthrobacter P1 was investigated in carbonlimited continuous cultures. To avoid toxic effects of higher formaldehyde concentrations, formaldehyde-limited cultures were established in smooth substrate transitions from choline-limitation. Evidence was obtained that the synthesis of enzymes involved in the conversion of methylamine into formaldehyde and in formaldehyde fixation is induced sequentially in this organism. Compared to growth with methylamine the molar growth yield on formaldehyde was approximately 30% higher. This difference is mainly due to the expenditure of energy for the uptake of methylamine from the medium.The addition of a pulse of a heterotrophic substrate, glucose or acetate, to C₁ substrate-limited continuous cultures resulted in relief of carbon limitation and transient synthesis of increasing amounts of cell material. Concomitantly, a significant decrease in the specific activities of hexulose phosphate synthase was observed. However, the total activity of hexulose phosphate synthase in these cultures remained clearly in excess of that required to fix the formaldehyde that became available in time. The observed strong decrease in the specific activities of this RuMP cycle enzyme strongly suggests that its synthesis is controlled via catabolite repression exerted by the metabolism of heterotrophic substrates.Abbreviations HPS 3-Hexulose-6-phosphate synthase - HPI 3-hexulose-6-phosphate isomerase - RuMP ribulose monophosphate     

Further enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the male rat

Summary The segmentation of the proximal tubules of the male rat kidney was studied by means of enzyme histochemical reactions. Soluble oxidoreductases (glucose 6-phosphate dehydrogenase, -glycerophosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, NAD- and NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase) were demonstrated using methods which reduce enzyme diffusion (incubating in presence of polyvinyl alcohol) and eliminate interference from tissue tetrazolium reductases. Less soluble or insoluble enzymes (glucose 6-phosphatase, -hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases) were demonstrated by incubation in conventional watery media.Segmental differences were observed in respect to all enzymes studied, and most reactions clearly visualized the three segments known to exist from ultrastructural as well as previous histochemical studies: The pars convoluta includes the first (P₁) and most of the second (P₂) segment. The transition to the third segment (P₃) is in the beginning of the pars recta. Also these reactions revealed a difference between the first part of the P₃, which runs through the cortex in the medullary rays, and the terminal part transversing the outer stripe of the medulla. In most instances intensity of reaction decreased in the last portion of the P₃.A number of the enzymes studied were mainly or solely localized to the P₃ (glucose 6-phosphate dehydrogenase, -glycerophosphate dehydrogenases, -hydroxybutyrate dehydrogenase, 3-hydroxysteroid dehydrogenase, decarboxylating malate dehydrogenase and uridine diphosphate glucose dehydrogenase). Some possible functional implications of the findings are discussed.Supported by grants from Fonden til Lægevidenskabens Fremme and the Danish Medical Research Council. — Mr. Kaj L. Pedersen is thanked for valuable photographic assistance.     

P-31 NMR saturation transfer experiments in Chlamydomonas reinhardtii : evidence for the NMR visibility of chloroplastidic Pi

ATP synthesis and consumption in respiring cells of the green alga Chlamydomonas reinhardtii were measured with ³¹P in vivo NMR saturation transfer experiments to determine the intracellular compartmentation of inorganic phosphate. Most of the observed flux towards ATP synthesis was catalyzed by the coupled enzymes glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK). The attribution of the measured flux to these enzymes is supported by the observation, that (i) the magnetization transfer was strongly reduced by iodoacetate, an irreversible inhibitor of GAPDH and that (ii) the unidirectional flux was much greater than the net flux through the mitochondrial F₀F₁-ATPase as determined by oxygen consumption measurements. In Chlamydomonas, glycolysis is divided into a chloroplastidic and a cytosolic part with the enzymes GAPDH/PGK being located in the chloroplast stroma (Klein 1986). The ³¹P-NMR signal of inorganic phosphate must, therefore, originate from the chloroplast. The life time of the magnetic label transferred to P_i by these enzymes is too short for it to be transported to the cytosol via the phosphate translocator of the chloroplast envelope. When the intracellular compartmentation of P_i was taken into consideration the calculated unidirectional ATP synthesis rate was equal to the consumption rate, indicating operation of GAPDH/PGK near equilibrium. The assignment of most of the intracellular P_i to the chloroplast is in contradiction to earlier reports, which attributed the Pi signal to the cytosol. This is of special interest for the use of the chemical shift of the P_i signal as an intracellular pH-marker in plant cells.Abbreviations 3-PGA 3-phosphoglycerate - CW continuous wave - dG6P 2-deoxyglucose-6-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - MO equilibrium z-magnetization - M₀ instantaneous z-magnetization after selective saturation for time t - MDP methylene-diphosphonic acid - PDE phosphodiester - PGK phosphoglycerate kinase - P_i inorganic orthophosphate - polyP polyphosphate - T₁ longitudinal relaxation time - ₁ longitudinal relaxation time with chemical exchange - TCA cycle tricarboxylic acid cycle Correspondence to: A. Mayer     

Sequestration of arginine by polyphosphate in vacuoles of yeast (Saccharomyces cerevisiae)

The conditions for synthesis, purification, and properties of tryptophanase by a marine organism (Vibrio K-7) were studied. Tryptophanase was induced by tryptophan and its analogs, and partially repressed by 0.5% glucose or glycerol. NaCl (0.4M) was required for optimal growth and tryptophanase activity in whole cells. The enzyme was purified to 92% homogeneity by heat treatment, hydroxyapatite chromatography and fractionation with ammonium sulfate. This tryptophanase has been found to have kinetic properties similar to the tryptophanase from other microorganisms. It carries out both , -elimination reactions (using tryptophan, serine, cysteine and S-methyl-cysteine as substrates) and -replacement reactions (forming tryptophan from indole and serine, cysteine or S-methyl-cysteine). The enzyme has a sedimentation coefficient of 9.2S and requires pyridoxal 5-phosphate as a cofactor. The optimal pH for the tryptophanase reaction is pH 8.0.Nonstandard Abbreviations PLP pyridoxal 5-phosphate - TPase tryptophanase - TSase tryptophan synthase - DHase dehydratase - TCA tricarboxylic acid - BSA bovine serum albumin Preliminary reports of this work have been presented (M. J. Klug and R. D. DeMoss, Bacteriol. Proc. 1971, p. 132; D. D. Whitt and R. D. DeMoss, Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, p. 148)     

Heterotrophic capacities of Plectonema boryanum

Acquisition of the dark heterotrophic growth capacity on glucose in Plectonema boryanum involves both adaptation and enrichment of a fast-growing genotype. The adaptation includes induction of functions involved in glucose incorporation and increase in glucose-6-phosphate dehydrogenase activity. Photosynthetic products are implicated in the control of both systems. Efficient energy conversion in the dark, as measured by cyanophage multiplication, correlates in time with the increase in potential for glucose incorporation while heterotrophic growth capacity correlates with the increase in glucose-6-phosphate dehydrogenase activity. The lower efficiency of heterotrophic growth compared to photoautotrophic growth is discussed in light of the conservation of the photosynthetic potency in the heterotrophic cells.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - G6P glucose-6-phosphate - NADP nicotinamide adenine dinucleotide phosphate - NTG N-methyl-N-nitro-N-nitrosoguanidine - RUDP ribulose-1,5-diphosphate - TCA trichloroacetic acid Dedicated to Prof. R. Y. Stanier on the occasion of his 60th birthday     

Physiology and metabolism of pathogenic neisseria: tricarboxylic acid cycle activity in Neisseria gonorrhoeae.

Tricarboyxlic acid cycle activity was examined in Neisseria gonorrhoeae CS-7. The catabolism of glucose in N. gonorrheae by a combination of the Entner-Doudoroff and pentose phosphate pathways resulted in the accumulation of acetate, which was not further catabolized until the glucose was depleted or growth became limiting. Radiorespirometric studies revealed that the label in the 1 position of acetate was converted to CO2 at twice the rate of the label in the 2 position, indicating the presence of a tricarboxylic acid cycle. Growth on glucose markedly reduced the levels of all tricarboxylic acid cycle enzymes except citrate synthase (EC 4.1.3.7). Extracts of glucose-grown cells contained detectable levels of all tricarboxylic acid cycle enzymes except aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), and a pyridine nucleotide-dependent malate dehydrogenase (EC 1.1.1.37). Extracts of cells capable of oxidizing acetate lacked only the pyridine nucleotide-dependent malate dehydrogenase. In lieu of this enzyem, a particulate pyridine nucleotide-independent malate oxidase (EC 1.1.3.3) was present. This enzyme required flavin adenine dinucleotide for activity and appeared to be associated with the electron transport chain. Radiorespirometric studies utilizing labeled glutamate demonstrated that a portion of the tricarboxylic acid cycle functioned during glucose catabolism. In spite of the presence of all tricarboxylic acid cycle enzymes, N. gonorrhoeae CS-7 was unable to grow in medium supplemented with cycle intermediates.     

Gluconeogenesis from pyruvate in the hyperthermophilic archaeon Pyrococcus furiosus: involvement of reactions of the Embden-Meyerhof pathway

The hyperthermophilic archaeon Pyrococcus furiosus was grown on pyruvate as carbon and energy source. The enzymes involved in gluconeogenesis were investigated. The following findings indicate that glucose-6-phosphate formation from pyruvate involves phosphoenolpyruvate synthetase, enzymes of the Embden-Meyerhof pathway and fructose-1,6-bisphosphate phosphatase.Cell extracts of pyruvate-grown P.furiosus contained the following enzyme activities: phosphoenolpyruvate synthetase (0.025 U/mg, 50 °C), enolase (0.9 U/mg, 80 °C), phosphoglycerate mutase (0.13 U/mg, 55 °C), phosphoglycerate kinase (0.01 U/mg, 50 °C), glyceraldehyde-3-phosphate dehydrogenase reducing either NADP⁺ or NAD⁺ (NADP⁺: 0.019 U/mg, NAD⁺: 0.009 U/mg; 50 °C), triosephosphate isomerase (1.4 U/mg, 50 °C), fructose-1,6-bisphosphate aldolase (0.0045 U/mg, 55 °C), fructose-1,6-bisphosphate phosphatase (0.026 U/mg, 75 °C), and glucose-6-phosphate isomerase (0.22 U/mg, 50 °C). Kinetic properties (V _max values and apparent K _m values) of the enzymes indicate that they operate in the direction of sugar synthesis. The specific enzyme activities of phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (NADP⁺-reducing) and fructose-1,6-bisphosphate phosphatase in pyruvate-grown P. furiosus were by a factor of 3, 10 and 4, respectively, higher as compared to maltose-grown cells suggesting that these enzymes are induced under conditions of gluconeogenesis. Furthermore, cell extracts contained ferredoxin: NADP⁺ oxidoreductase (0.023 U/mg, 60 °C); phosphoenolpyruvate carboxylase (0.018 U/mg, 50 °C) acts as an anaplerotic enzyme.Thus, in P. furiosus sugar formation from pyruvate involves reactions of the Embden-Meyerhof pathway, whereas sugar degradation to pyruvate proceeds via a modified non-phosphorylated Entner-Doudoroff pathway.     

Oxidative enzymes in the pancreatic islets of normal and obese-hyperglycemic mice

Summary Histochemical data are presented concerning distributions of succinic dehydrogenase (SD), lactic dehydrogenase (LD), diphosphopyridine nucleotide diaphorase (DPND), triphosphopyridine nucleotide diaphorase (TPND) and glucose-6-phosphate dehydrogenase (G-6-PD) in the pancreas from the American variety of obese-hyperglycemic mice (AO-mice) and their lean litter mates (AN-mice).A high LD activity was found in the exocrine parenchyma, while the reaction in the islet tissue and the duct epithelium was only weak. A considerable reaction for DPND was noted throughout the pancreas. SD activity was slightly more pronounced in the acinar tissue and duct epithelium as compared to the islet tissue, where only a moderate activity appeared. Strong reactions for TPND and G-6-PD were found in the islet cells and duct epithelium, while the activity in the exocrine parenchyma was less pronounced. The hyperactive islet B cells in the AO-mice showed no obvious differences in enzyme activity and distribution compared to that of the AN-mice. The enzyme pattern of the A cells could not be clearly distinguished from that in the B cells.The results suggest the existence at least in the B cells of the mice islet tissue of an active hexosemonophosphate shunt. The probable significance of the hexosemonophosphate shunt for insulin synthesis is briefly discussed.The following abbreviations are used DPN Diphosphopyridine nucleotide - DPND Diphosphopyridine nucleotide diaphorase - DPNH Diphosphopyridine nucleotide, reduced form - EM Embden-Meyerhof - G-6-PD Glucose-6-phosphate dehydrogenase - HMP Hexose monophosphate - LD Lactic dehydrogenase - MTT 3,5-diphenyl-2-(4,5-dimethyl-thiazol-2-yl) tetrazolium bromide - Nitro-BT 2,2-di-p-nitrophenyl-5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-biphenylene)-ditetrazolium chloride - PVP Polyvinyl pyrrolidone (M. W. 11 000) - SD Succinic dehydrogenase - TPN Triphosphopyridine nucleotide - TPND Triphosphopyridine nucleotide diaphorase - TPNH Triphosphopyridine nucleotide, reduced form     

Non-PTS uptake and subsequent metabolism of glucose in Pediococcus halophilus as demonstrated with a double mutant defective in phosphoenolpyruvate:mannose phosphotransferase system and in phosphofructokinase

Pediococcus halophilus possesses phosphoenolpyruvate:mannose phosphotransferase system (man:PTS) as a main glucose transporter. A man:PTS defective (man:PTS^d) strain X-160 could, however, utilize glucose. A possible glucose-transport mechanism other than PTS was studied with the strain X-160 and its derivative, man:PTS^d phosphofructokinase defective (PFK^–) strain M-13. Glucose uptake by X-160 at pH 5.5 was inhibited by any of carbonylcyanide m-chlorophenylhydrazone, nigericin, N,N-dicyclohexylcarbodiimide, or iodoacetic acid. The double mutant M-13 could still transport glucose and accumulated intracellularly a large amount of hexose-phosphates (ca. 8 mM glucose 6-phosphate and ca. 2 mM fructose 6-phosphate). Protonophores also inhibited the glucose transport at pH 5.5, as determined by the amounts of accumulated hexose-phosphates (< 4 mM). These showed involvement of proton motive force (P) in the non-PTS glucose transport. It was concluded that the non-PTS glucose transporter operated in concert with hexokinase or glucokinase for the metabolism of glucose in the man:PTS^d strain.Abbreviations BM basal medium - BM-G basal medium containing glucose - CM complex medium - man:PTS phosphoenolpyruvate:mannose phosphotransferase system - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - P proton motive force - pH transmembrane pH gradient - transmembrane electrical potential difference - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PIPES piperazine-N,N-bis(-ethanesulfonic acid) - MES 4-morpholineethanesulfonic acid - G-6-P glucose 6-phosphate - F-6-P fructose 6-phosphate - FDP fructose 1,6-bisphosphate - EMP Embden-Meyerhof-Parnas pathway - PFK phosphofructokinase - GK glucokinase - HK hexokinase - IAA iodoacetic acid - II^man enzyme II component of man:PTS