Control of diauxic growth of Azotobacter vinelandii on acetate and glucose.

Batch cultures of Azotobacter vinelandii were inoculated with cells pregrown on either acetate or glucose. When they were subsequently grown on a mixture of acetate and glucose, typical diauxic growth was observed, with preferential uptake of acetate in the first and glucose in the second phase of growth. Extracts from acetate-pregrown cells exhibited high acetate kinase activity in the first phase of growth. This activity decreased and activities of the two glucose enzymes glucose 6-phosphate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase increased in the second phase. Extracts from glucose-pregrown cells exhibited high initial activities of the two glucose enzymes, which decreased while acetate kinase activity increased in the first phase of growth. Again, in the second phase, activities of the two glucose enzymes increased and acetate kinase activity decreased. In any case, isocitrate dehydrogenase activity varied only slightly and unspecifically. The differences in enzyme activity and the constancy of isocitrate dehydrogenase were confirmed by experiments with either acetate- or glucose-limited chemostats. In chemostats in which both of the substrates were limiting, all of the enzymes displayed significant activities. Glucose 6-phosphate dehydrogenase activity was inhibited by acetyl coenzyme A and acetyl phosphate but not by acetate. It is proposed that diauxic growth is based on the control of enzymes involved in acetate or glucose dissimilation by which acetate or its metabolites control the expression and activity of glucose enzymes.     

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu2+

Citr⁺Lactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu²⁺. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu²⁺, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu²⁺. On the other hand, the Y_Glu value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu²⁺ enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu²⁺ stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PP_i. In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.     

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.     

Effect of dichloroacetate and glucagon on the incorporation of labeled substrates into glucose and on pyruvate dehydrogenase in hepatocytes from fed and starved rats

Dichloroacetate (2 mm) stimulated the conversion of [1-¹⁴C]lactate to glucose in hepatocytes from fed rats. In hepatocytes from rats starved for 24 h, where the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio is elevated, dichloroacetate inhibited the conversion of [1-¹⁴C]lactate to glucose. Dichloroacetate stimulated ¹⁴CO₂ production from [1-¹⁴C]lactate in both cases. It also completely activated pyruvate dehydrogenase and increased flux through the enzyme. The addition of β-hydroxybutyrate, which elevates the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio, changed the metabolism of [1-¹⁴C]lactate in hepatocytes from fed rats to a pattern similar to that seen in hepatocytes from starved rats. Thus, the effect of dichloroacetate on labeled glucose synthesis from lactate appears to depend on the mitochondrial oxidation-reduction state of the hepatocytes. Glucagon (10 nm) stimulated labeled glucose synthesis from lactate or alanine in hepatocytes from both fed and starved rats and in the absence or presence of dichloroacetate. The hormone had no effect on pyruvate dehydrogenase activity whether or not the enzyme had been activated by dichloroacetate. Thus, it appears that pyruvate dehydrogenase is not involved in the hormonal regulation of gluconeogenesis. Glucagon inhibited the incorporation of 10 mm [1-¹⁴C]pyruvate into glucose in hepatocytes from starved rats. This inhibition has been attributed to an inhibition of pyruvate dehydrogenase by the hormone (Zahlten et al., 1973, Proc. Nat. Acad. Sci. USA70, 3213–3218). However, dichloroacetate did not prevent the inhibition of glucose synthesis. Nor did glucagon alter the activity of pyruvate dehydrogenase in homogenates of cells that had been incubated with 10 mm pyruvate in the absence or presence of dichloroacetate. Thus, the inhibition by glucagon of pyruvate gluconeogenesis does not appear to be due to an inhibition of pyruvate dehydrogenase.     

Inhibition of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in sea urchin eggs by palmitoyl-coenzyme a and reversal by polyamines

Palmitoyl-CoA inhibited crude glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the eggs of the sea urchin, Hemicentrotus pulcherrimus. Fifty percent inhibition of the glucose 6-phosphate dehydrogenase in the supernatant of unfertilized eggs was obtained with 0.43 ± 0.05 μm palmitoyl-CoA, and of 6-phosphogluconate dehydrogenase with 4.41 ± 0.20 μm palmitoyl-CoA. Also, these enzymes in fertilized eggs 30 min after fertilization were inhibited by palmitoyl-CoA almost as much as in unfertilized eggs. Na-Palmitate, coenzyme A, acetyl-CoA, palmitoylcarnitine, and carnitine failed to exert any inhibitory effect on the activities of these dehydrogenases. The intracellular concentration of long-chain fatty acyl-CoA in unfertilized eggs (3.08 ± 0.33 nmol/10⁶ eggs) was high enough for the inhibition of these enzymes, and decreased following fertilization to a low level (1.49 ± 0.08 nmol/10⁶ eggs 30 min after fertilization). Spermine and spermidine canceled the inhibition of these enzymes by palmitoyl-CoA. In view of the inhibition of glucose 6-phosphate dehydrogenase and of 6-phosphogluconate dehydrogenase by palmitoyl-CoA, these dehydrogenases in the pentose monophosphate cycle are probably inhibited in unfertilized eggs by long-chain fatty acyl-CoA and released from the inhibited state by both the decrease in the level of long-chain acyl-CoA and the increase in the level of polyamines following fertilization.     

The effect of 4-hydroxy-2, 3-trans-pent-en-1-al and maleylaldehyde on some mitochondrial functions

Low concentrations of HPE and MLA inhibited state 3 respiration of rat liver mitochondria in the presence of different NAD⁺-dependent substrates. MLA appeared to be more active than HPE. High aldehyde concentrations inhibited the state 3 respiration with succinate. The restraint of succinate oxidation by HPE and MLA and of glutamate plus malate oxidation by MLA correlated with the inhibition of succinate and glutamate dehydrogenase activites, respectively. HPE inhibited glutamate dehydrogenase at concentrations higher than those affecting glutamate oxidation. Malate dehydrogenase activity was slightly sensitive to HPE and MLA. Both aldehydes inhibited NADH oxidation by freeze-thawed mitochondria. These results suggest the existence of a site particularly sensitive to aldehydes in the electron transport chain between the specific NAD⁺-linked dehydrogenases and ubiquinone.     

Induction of cyclosporine-sensitive mitochondrial permeability transition pore by substrates forming acetyl-CoA under normal conditions and in type 2 diabetes

Oxidation of pyruvate and palmitoylcarnitine in mitochondria is accompanied by the formation of acetyl-CoA, with its possible participation in the acetylation of various proteins and enzymes that may lead to the inhibition of their functions. This paper studies the effect of the excess of these substrates on respiration and induction of mitochondrial permeability transition pore (MPTP) in mitochondria and liver homogenates of healthy, obese, and type 2 diabetic (T2D) rats and mice. Both substrates produced a reversible inhibition of respiration and induced the opening of MPTP sensitive to cyclosporin A. Induction of MPTP in mitochondria was further activated by calcium ions and inhibited by the NO donor SNAP and NAD–a coenzyme and activator of deacetylation reactions. In obese and T2D animals, the opening of MPTP was stimulated by lower concentrations of L-palmitoylcarnitine than in healthy animals. In these pathologies, an activation effect on the MPTP induction was produced by ammonium ions, in the presence of which the concentration of L-palmitoylcarnitine required for the pore opening was reduced more than twofold. In liver homogenates, an added arginine reduced the probability of the MPTP formation. Analysis of mathematical models has shown that, due to the inhibition of pyruvate dehydrogenase kinase (PDK) by pyruvate, phosphorylation of pyruvate dehydrogenase (PDH) is strongly reduced, and this makes it possible to produce acetyl CoA in a wide range of pyruvate concentrations. The data obtained show that excess substrates that produce acetyl-CoA increase the probability of the MPTP opening, especially in pathologies associated with obesity and T2D. The ability of NO and NAD to inhibit MPTP indicates the participation of phosphorylation and acetylation/deacetylation reactions in this process.     

Purification of porcine liver pyruvate dehydrogenase complex and characterization of its catalytic and regulatory properties.

Procedures are described for isolating highly purified porcine liver pyruvate and α-ketoglutarate dehydrogenase complexes. Rabbit serum stabilized these enzyme complexes in mitochondrial extracts, apparently by inhibiting lysosomal proteases. The complexes were purified by a three-step procedure involving fractionation with polyethylene glycol, pelleting through 12.5% sucrose, and a second fractionation under altered conditions with polyethylene glycol. Sedimentation equilibrium studies gave a molecular weight of 7.2 × 10⁶ for the liver pyruvate dehydrogenase complex. Kinetic parameters are presented for the reaction catalyzed by the pyruvate dehydrogenase complex and for the regulatory reactions catalyzed by the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. For the overall catalytic reaction, the competitive K_i to K_m ratio for NADH versus NAD⁺ and acetyl CoA versus CoA were 4.7 and 5.2, respectively. Near maximal stimulations of pyruvate dehydrogenase kinase by NADH and acetyl CoA were observed at NADH:NAD⁺ and acetyl CoA:CoA ratios of 0.15 and 0.5, respectively. The much lower ratios required for enhanced inactivation of the complex by pyruvate dehydrogenase kinase than for product inhibition indicate that the level of activity of the regulatory enzyme is not directly determined by the relative affinity of substrates and products of catalytic sites in the pyruvate dehydrogenase complex. In the pyruvate dehydrogenase kinase reaction, K⁺ and NH⁺₄ decreased the K_m for ATP and the competitive inhibition constants for ADP and (β,γ-methylene)adenosine triphosphate. Thiamine pyrophosphate strongly inhibited kinase activity. A high concentration of ADP did not alter the degree of inhibition by thiamine pyrophosphate nor did it increase the concentration of thiamine pyrophosphate required for half-maximal inhibition.     

Analysis of calorimetric data for isodesmic self-associating systems.

ATP and respiration (NADH)-driven NAD(P)⁺ transhydrogenase (EC 1.6.1.1) activities are low in membranes from Escherichia coli cultured on yeast extract medium (17 and 21 nmol/min × mg) but high on glucose (82 and 142 nmol/min × mg). The ATPase and respiratory activities in both cases appeared comparable. Growth of the bacteria in yeast extract medium followed by washing and replacement into a glucose medium showed that after 3 h the energy-linked and energy-independent NAD(P)⁺ transhydrogenase (reduction of acetylpyridine NAD⁺ by NADPH) activities had appeared simultaneously. Incorporation of chloramphenicol or omission of glucose in the induction medium resulted in no increase in these activities indicating that de novo protein synthesis is required for the induction of energy-linked and -independent NAD(P)⁺ transhydrogenase. It was found that the K_m values for acetylpyridine NAD⁺ and NADPH for the energy-independent reaction in membranes from glucose grown cells (143 and 62 μm) were similar to those in membranes from cells grown on glucose-yeast extract (135 and 45 μm), respectively, but the maximum velocity at infinite acetyl pyridine NAD⁺ and NADPH increased from 353 to 2175 nmol/min × mg. Furthermore, the membrane-bound NAD(P)⁺ transhydrogenase in glucose-yeast extract grown cells showed substrate inhibition at high NADPH and low acetyl pyridine NAD⁺ levels. Further kinetic data demonstrate that the mechanism of the energy-independent NAD(P)⁺ transhydrogenase in E. coli is similar to that of the mitochondrial enzyme and exhibits similar responses to competitive inhibitors at the NAD⁺ and NADPH sites.     

The inhibition of acetate oxidation by ethanol in Acetobacter aceti

Ethanol grown Acetobacter aceti differed from acetate grown. In ethanol grown cells, acetate uptake, caused by the oxidation of acetate, was completely inhibited by ethanol, in acetate grown cells only to 20%. This was correlated with a 65-fold higher specific activity of the membrane bound NAD(P)-independent alcohol dehydrogenase in ethanol grown than in acetate grown cells. In comparison with ethanol grown cells, acetate grown cells showed a 3-fold higher acetate respiration rate and 3-fold higher specific activities of some tricarboxylic acid cycle enzymes tested. Both adaptations were due to induction by the homologous and not to repression by the heterologous growth substrate. A. aceti showed a membrane bound NAD(P)-independent malate dehydrogenase and no activity of a soluble NAD(P)-dependent one, as was known before from A. xylinum. A hypothesis was proposed explaining the observed inhibition of malate dehydrogenase and of functioning of the tricarboxylic acid cycle in the presence of ethanol or butanol or glucose by a competition of two electron currents for a common link in the convergent electron transport chains. The electrons coming from the quinoproteins, alcohol dehydrogenase and glucose dehydrogenase on the one side and those coming from the flavoproteins, malate dehydrogenase and succinate dehydrogenase via ubiquinonecytochrome c reductase on the other side are meeting at cytochrome c. Here the quinoproteins may be favoured by higher affinity and so inhibit the flavoproteins. Inhibition could be alleviated in the cell free system by increasing the oxygen supply.Dedicated to Professor Carl Martius on the occasion of his 80th birthday, March 1st 1986     

NADPH/NADP+ ratio: regulatory implications in yeast glyoxylic acid cycle

Summary The utilization by yeast of two carbon sources is carried out through the operation of the glyoxylic acid cycle. Kinetic data from the isocitrate transforming enzymes suggest that the flow of isocitrate through the glyoxylic acid cycle depends upon the inhibition of the isocitrate decarboxylating enzymes. Both isocitrate dehydrogenases are inhibited by a mixture of glyoxylate + oxaloacetate, but for the reasons described in the text we consider that this inhibition is of no physiological significance. On the other hand, we have found that NADPH is a competitive inhibitor of NADP-isocitrate dehydrogenase with respect to NADP⁺, with a K_I similar to its K_M. It also produces an additive effect on the NADH-produced inhibition of NAD-isocitrate dehydrogenase. We propose NADPH as the compound that channels the utilization of isocitrate into the glyoxylic acid cycle. This is supported by the finding of an increased NADPH/NADP⁺ ratio in acetate grown yeast with respect to glucose grown cells.     

NADP+-specific 2-oxoglutarate dehydrogenase in denitrifying Pseudomonas species

Several denitrifying Pseudomonas strains contained an NADP⁺-specific 2-oxoglutarate dehydrogenase, in contrast to an NAD⁺-specific pyruvate dehydrogenase, if the cells were grown anaerobically with aromatic compounds. With non-aromatic substrates or after aerobic growth the coenzyme specificity of 2-oxoglutarate dehydrogenase changed to NAD⁺-specificity. The reaction stoichiometry and the apparent K _m-values of the enriched enzymes were determined: pyruvate 0.5 mM, coenzyme A 0.05 mM, NAD⁺ 0.25 mM; 2-oxoglutarate 0.6 mM, coenzyme A 0.05 mM, NADP⁺ 0.03 mM. Isocitrate dehydrogenase was NADP⁺-specific. The findings suggest that these strains contained at least two lipoamide dehydrogenases, one NAD⁺-specific, the other NADP⁺-specific.     

Regeneration of NAD(H) by alcohol dehydrogenase in gel-entrapped yeast cells

Anaerobically grown cells of Saccharomyces cerevisiae entrapped in polyacrylamide gel have been shown to provide a stable source of alcohol dehydrogenase [(ADH) alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1] for effective regeneration of NAD(H). This system was able to provide the coenzyme required for the operation of other dehydrogenases, such as lactate dehydrogenase [(LDH) l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27] and malate dehydrogenase [(MDH) l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37]. Yeast cells coimmobilized with a dehydrogenase are capable of the reversible regeneration of the reduced or oxidized coenzyme, depending on the additions made. A two-cell system can also be constituted using the same strain of yeast, adapted differently. Cells grown anaerobically and aerobically as sources of ADH and MDH, respectively, can operate efficiently on coimmobilization. The system can be used repeatedly without measurable loss of efficiency.     

Peculiarities of Ethanol Metabolism in an Acinetobacter sp. Mutant Strain Defective in Exopolysaccharide Synthesis

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD⁺-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD⁺ and NADP⁺ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C₄-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C₂-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.     

Effects of ethacrynic acid on ion transport and energy metabolism in slices of avian salt gland and of mammalian liver and kidney cortex

Summary Ethacrynic acid greatly inhibited net transport of ions and aerobic, energyconserving metabolism in slices of avian salt gland, rat liver, and rat and guinea-pig kidney cortex. The effects of increasing concentrations of ethacrynic acid on the transport of Na⁺, K⁺ and Cl^– ran closely parallel to its effects on tissue ATP levels and respiration. The concentration needed for maximal inhibition of transport reduced ATP levels by 80–90%. Respiration was reduced by 80–90% in salt gland and kidney cortex, and by a maximum of 30% in liver slices. The effects of low concentrations of ethacrynic acid required time to become fully manifest in some tissues, and the development of transport inhibition followed a similar course to decline of respiration and ATP levels. Ca²⁺ extrusion by liver cells was inhibited by ethacrynic acid. The concentration dependence of the inhibition was similar to that shown by the other transport systems inhibited. There was no distinction evident between the sensitivity of Na⁺ extrusion and of K⁺ accumulation to the diuretic. Lactate production increased as respiration decreased in the presence of increasing concentrations of ethacrynic acid. We conclude that ethacrynic acid acted primarily as an inhibitor of mitochondrial respiration and ATP synthesis in the tissue slices, and that inhibition of ion transport was a nonspecific consequence of the failure of the energy supply.     

Tumor growth of neurofibromin-deficient cells is driven by decreased respiration and hampered by NAD+ and SIRT3

Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion, decreasing both respiration and intracellular NAD⁺. Expression of the alternative NADH dehydrogenase NDI1 raises NAD⁺/NADH ratio, enhances the activity of the NAD⁺-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. The antineoplastic effect of NDI1 is mimicked by administration of NAD⁺ precursors or by rising expression of the NAD⁺ deacetylase SIRT3 and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes. These findings shed light on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD⁺/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.Subject terms: Cancer metabolism, Cell biology     

Studies on marine occurring yeasts: Respiration,fermentation and salt tolerance

Summary The effect of various NaCl concentrations on respiration and fermentation rates in cells with or without added glucose as exogenous substrate as well as on respiratory quotients was determined for Debaryomyces hansenii, Saccharomyces cerevisiae, Cryptococcus albidus, and Candida zeylanoides, all yeasts isolated from marine environment. A given strain had about the same respiratory and fermentatory intensity at 0% and 4% NaCl (w/v). A further increase considerably reduced the oxygen uptake or CO₂-evolution. D. hansenii was the most NaCl tolerant yeast tested, giving about 10% activity still at a concentration of 24% NaCl, whether the activities of whole cells or cell homogenates were determined. For S. cerevisiae or Cr. albidus the respiratory activity was reduced to about the same degree at 16% NaCl for whole cells, at 12% NaCl for homogenates of Cr. albidus. A somewhat higher NaCl concentration was evidently tolerated for respiration and fermentation than for growth, very obvious in the case of C. zeylanoides.The minimum values for water activity (a _w) permitting 10% respiration activity were higher when produced by electrolytes (NaCl, KCl, or Na₂SO₄), lower when due to sugars (metabolizable glucose or non-metabolizable lactose) and lowest when due to glycerol. The a _w per se was evidently not solely decisive for the limitation of respiration activity.Attempts were made to assess an effect of high NaCl concentrations on the glucose uptake.The potassium content was higher in cells of the highly halotolerant D. hansenii than in those of the other yeasts and decreased with the increase in external, consequently in internal, Na⁺ concentration. The decrease in K⁺ content can presumably only proceed to a certain extent, below which the ability for growth and respiration was lost.     

Molecular basis of negative co-operativity in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

The interactions of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase with NAD⁺ and with its fluorescent derivative 1, N⁶-etheno-adenine dinucleotide were investigated using a variety of spectroscopic methods. These techniques included: difference spectroscopy, circular dichroism, fluorescence and circular polarized luminescence. It was found that the greatest structural change in the protein tetramer occurs upon binding of the first mole of coenzyme. We have also demonstrated that progressive structural changes occur at the adenine subsite in the NAD⁺ binding site as a function of coenzyme saturation. These conformational changes are probably responsible for the progressive decrease in the affinity towards the coenzyme. It was also found that every NAD⁺ molecule induces the same conformational change of the nicotinamide subsite. These results offer a molecular explanation for the negative co-operativity in the binding of the coenzyme, without a change in the catalytic power of the NAD⁺ site as a function of coenzyme saturation. These results also offer a new explanation for the fact that enzyme exhibits half-of-the-sites reactivity towards certain ligands and full-site reactivity towards others. It is suggested that those ligands interacting at the adenine subsite of the NAD⁺ binding site induce the half-of-the-sites reactivity.Our results support the view that both the negative co-operativity in coenzyme binding and half-of-the-sites reactivity are due to ligand-induced conformational changes on an a priori symmetric glyceraldehyde-3-phosphate dehydrogenase molecule.     

Helminthosporium maydis Race T Toxin Induces Leakage of NAD from T Cytoplasm Corn Mitochondria

The mechanism by which Helminthosporium maydis race T toxin inhibits respiration dependent on NAD⁺-linked substrates in T cytoplasm corn mitochondria was investigated. The toxin did not cause leakage of the soluble matrix enzyme malate dehydrogenase from the mitochondria or inhibit malate dehydrogenase or isocitrate dehydrogenase directly. The toxin did increase the permeability of the inner membranes of T cytoplasm, but not N cytoplasm, mitochondria to NAD⁺. Added NAD⁺ partially or fully restored toxin-inhibited electron transport in T cytoplasm mitochondria. Thiamin pyrophosphate had a similar effect when malate was the substrate. It was concluded that the inhibition of respiration of NAD⁺-linked substrates by the toxin is due to depletion of the intramitochondrial pool of NAD⁺ and other coenzymes.     

Specific inactivation of glucose metabolism from eucaryotic cells by pentalenolactone

Pentalenolactone, an antibiotic related to the class of the sesquiterpene-lactones and produced by the strain Streptomyces arenae Tü-469, inhibits specifically the glucose metabolism by inactivation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD⁺ oxidoreductase (phosphorylating) EC 1.2.1.12). The sensitivity of several eucaryotic cell-systems for pentalenolactone was shown under in vivo conditions. The glycolytic as well as the gluconeogenetic pathway of mammalian cells can be completely inhibited with low concentration of the antibiotic. In all cases, the minimum inhibitory concentration is dependent on cell density. The inhibitory effect in vivo and in vitro does not seem to be species-specific. In erythrocytes from rats, in Ehrlich-ascites tumor cells and in Plasmodium vinckei infected erythrocytes from mice glycolysis can be inhibited with concentrations of 18–90 μM pentalenolactone. In hepatocytes, glycolysis as well as gluconeogenesis is prevented by the same concentrations. In contract to these results, in yeast the inhibition depends on growth conditions. The inhibition in glucose medium is cancelled by precultivation on acetate-containing medium.