Pyruvate Decarboxylase Activity Research Articles

RESPONSES OF PAPAYA TO SHORT-TERM EXPOSURE OF INSECTICIDAL O2 ATMOSPHERE

Papaya fruits were exposed to a continuous flow of an atmosphere containing less than 0.5% O2 (the balance is N2) for 0 to 5 days at 20°C. Fruits were evaluated every day after exposure to low O2 atmosphere, and simultaneously after 3 days in air at 20°C. During every evaluation period part of the fruits were promptly frozen for the analysis of the activity of the enzymes alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lactate dehydrogenase (LDH), and pyruvic acid. The rest of the fruits were evaluated for flesh firmness, external and internal appearance, incidence of decay, and presence of off–flavors. Fruits had major decay problems and presented some off–flavors after 3 days in low O2 and 3 days in air at 20°C. The intolerance of the fruit to low O2 is, correlated with an increase, after 3 days, in the activity of ADH and PDC but not with the activity of LDH. On the basis of these results, it is concluded that insecticidal O2 atmosphere can be used as a quarantine insect control treatment in papaya for up to 3 days without the risk of significant fruit injury.

Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDC1 and PDC5

Recently we deleted the pyruvate decarboxylase structural gene PDC1 from the genome of the yeast Saccharomyces cerevisiae. The pdc1 deletion mutants had pyruvate decarboxylase activity due to the presence of a second structural gene [Schaaff, I., Green, J. B. A., Gozalbo, D. & Hohmann, S. (1989) Curr. Genet. 15, 75-81]. We cloned and sequenced this gene which we call PDC5. The predicted amino acid sequences of PDC1 and PDC5 are 88% identical. Deletion of PDC5 did not cause any decrease in the specific pyruvate decarboxylase activity while pdc1 deletion mutants had 80% of the wild-type activity. Deletion mutants lacking both PDC1 and PDC5 did not show any detectable pyruvate decarboxylase activity in vitro and were unable to ferment glucose. This indicates that PDC1 and PDC5 are the only structural genes for pyruvate decarboxylase in yeast. The PDC5 isoenzyme showed a slightly higher Km value for its substrate pyruvate than the PDC1 product (PDC5: Km = 8 mM; PDC1: Km = 5 mM), as measured in crude extract of pdc1 and pdc5 deletion mutants, respectively. PDC5 is only expressed in pdc1 deletion mutants. No mRNA transcribed from PDC5 could be detected in wild-type cells. Thus, in addition to the control by glucose induction, pyruvate decarboxylase activity seems to be subject to autoregulation. Similar phenomena have been described previously for tubulin, histones and a ribosomal protein but not for metabolic enzymes.

Transient-State Analysis of Metabolic Fluxes in Crabtree-Positive and Crabtree-Negative Yeasts

In bakers' yeast, an immediate alcoholic fermentation begins when a glucose pulse is added to glucose-limited, aerobically grown cells. The mechanism of this short-term Crabtree effect was investigated via a comparative enzymic analysis of eight yeast species. It was established that the fermentation rate of the organisms upon transition from glucose limitation to glucose excess is positively correlated with the level of pyruvate decarboxylase (EC 4.1.1.1). In the Crabtree-negative yeasts, the pyruvate decarboxylase activity was low and did not increase when excess glucose was added. In contrast, in the Crabtree-positive yeasts, the activity of this enzyme was on the average sixfold higher and increased after exposure to glucose excess. In Crabtree-negative species, relatively high activities of acetaldehyde dehydrogenases (EC 1.2.1.4 and EC 1.2.1.5) and acetyl coenzyme A synthetase (EC 6.2.1.1), in addition to low pyruvate decarboxylase activities, were present. Thus, in these yeasts, acetaldehyde can be effectively oxidized via a bypass that circumvents the reduction of acetaldehyde to ethanol. Growth rates of most Crabtree-positive yeasts did not increase upon transition from glucose limitation to glucose excess. In contrast, the Crabtree-negative yeasts exhibited enhanced rates of biomass production which in most cases could be ascribed to the intracellular accumulation of reserve carbohydrates. Generally, the glucose consumption rate after a glucose pulse was higher in the Crabtree-positive yeasts than in the Crabtree-negative yeasts. However, the respiratory capacities of steady-state cultures of Crabtree-positive yeasts were not significantly different from those of Crabtree-negative yeasts. Thus, a limited respiratory capacity is not the primary cause of the Crabtree effect in yeasts. Instead, the difference between Crabtree-positive and Crabtree-negative yeasts is attributed to differences in the kinetics of glucose uptake, synthesis of reserve carbohydrates, and pyruvate metabolism.

Expression of Zymomonas mobilis adhB (encoding alcohol dehydrogenase II) and adhB-lacZ operon fusions in recombinant Z. mobilis

The Zymomonas mobilis alcohol dehydrogenase II gene (adhB) was overexpressed 7- to 14-fold on a recombinant plasmid, accompanied by a small decrease in growth rate. A fragment containing the truncated gene with promoter reduced expression from the chromosomal gene as measured immunologically and enzymatically, consistent with the presence of a trans-active regulatory factor and positive regulatory control. Both the complete gene and the promoter fragment increased pyruvate decarboxylase and glucokinase activities, with no effect on alcohol dehydrogenase I or eight glycolytic enzymes. Tandem promoters from adhB expressed beta-galactosidase at higher levels than did either promoter alone in operon fusions. Addition of 50 microM zinc sulfate in minimal medium reduced the expression of adhB and of the operon fusions. Abundant but inactive alcohol dehydrogenase II was produced in iron-limited cells. This inactive enzyme did not form intracellular aggregates, and no morphological changes were apparent by transmission electron microscopy.

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

The role of pyruvate metabolism in the triggering of aerobic, alcoholic fermentation in Saccharomyces cerevisiae has been studied. Since Candida utilis does not exhibit a Crabtree effect, this yeast was used as a reference organism. The localization, activity and kinetic properties of pyruvate carboxylase (EC 6.4.1), the pyruvate dehydrogenase complex and pyruvate decarboxylase (EC 4.1.1.1) in cells of glucose-limited chemostat cultures of the two yeast were compared. In contrast to the general situation in fungi, plants and animals, pyruvate carboxylase was found to be a cytosolic enzyme in both yeasts. This implies that for anabolic processes, transport of C 4-dicarboxylic acids into the mitochondria is required. Isolated mitochondria from both yeast exhibited the same kinetics with respect to oxidation of malate. Also, the affinity of isolated mitochondria for pyruvate oxidation and the in situ activity of the pyruvate dehydrogenase complex was similar in both types of mitochondria. The activity of the cytosolic enzyme pyruvate decarboxylase in S. cerevisiae from glucose-limited chemostat cultures was 8-fold that in C. utilis. The enzyme was purified from both organisms, and its kinetic properties were determined. Pyruvate decarboxylase of both yeast was competitively inhibited by inorganic phosphate. The enzyme of S. cerevisiae was more sensitive to this inhibitor than the enzyme of C. utilis. The in vivo role of phosphate inhibition of pyruvate decarboxylase upon transition of cells from glucose limitation to glucose excess and the associated triggering of alcoholic fermentation was investigated with 31P-NMR. In both yeasts this transition resulted in a rapid drop of the cytosolic inorganic phosphate concentration. It is concluded that the relief from phosphate inhibition does stimulate alcoholic fermentation, but it is not a prerequisite for pyruvate decarboxylase to become active in vivo. Rather, a high glycolytic flux and a high level of this enzymes are decisive for the occurrence of alcoholic fermentation after transfer of cells from glucose limitation to glucose excess.

Neonatal pyruvate dehydrogenase deficiency with lipoate responsive lactic acidaemia and hyperammonaemia

A 2-day-old girl developed a severe lactic acidosis with a normal lactate/pyruvate ratio and hyperammonaemia. Plasma arginine and citrulline levels were below the limit of detection. In muscle total pyruvate dehydrogenase complex (PDHC) and pyruvate decarboxylase (E1) activities were reduced to a fraction of lower control values. The acute neonatal period was bridged with peritoneal dialysis, dichloroacetate therapy, supplements of arginine and branched chain amino acids, a complete vitamin B complex and lipoic acid. Lactate homeostasis responded to pharmacological supplements of lipoic acid. At age 1 year the child was hypotonic, showed severe developmental retardation, optic atrophy and cranial dysmorphism. She died aged 1 year 8 months with signs of respiratory paralysis but with normal lactate levels under assisted breathing. Pathological findings at autopsy were suggestive of Leigh syndrome, interstitial pneumonia and extensive fatty infiltration of hepatocytes. Regression analysis of data from 187 plasma amino acid determinations from the patient over a period of 1 year 8 months revealed a persistent-imbalance involving alanine, glutamic acid, glutamine, proline, citrulline and branched chain amino acids. Aspects of acute and long-term therapy in this patient and some implications of the imbalances in plasma amino acids are discussed.

Identification, cloning and characterisation of a new gene required for full pyruvate decarboxylase activity in Saccharomyces cerevisiae.

Biochemical evidence that pyruvate decarboxylase in S. cerevisiae might be constituted from two independently encoded subunits led us to question genetic evidence for a single structural gene. The main evidence for this was that three "structural" mutations appeared to be alleles of the same gene, PDC1 (Schmitt and Zimmermann 1982). We report that one of these mutations (pdc1-30) is not allelic either to other pdc1 alleles or to pdc2 mutations and therefore is has been renamed pdc3-30 thus identifying a new gene, PDC3. We have cloned the PDC3 gene, it represents a unique sequence in the genome and targeted integration shows tight linkage to the PDC3 locus. However, the size, abundance and regulation of the PDC3 transcript suggest that it does not encode a second structural gene. Possible functions for the PDC3 gene product are discussed.

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)

A deletion of the PDC1 gene for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations

We deleted most of the pyruvate decarboxylase structural gene PDC1 from the genome of Saccharomyces cerevisiae. Surprisingly, mutants carrying this deletion allele showed a completely different phenotype than previously described point mutations. They were able to ferment glucose and their specific pyruvate decarboxylase activity was only reduced to 45% of the wild type level. Northern blot analysis revealed that a sequence in the yeast genome homologous to PDC1 and formerly designated as a possible pseudogene is expressed and may code for a different but closely related pyruvate decarboxylase. The products of the two PDC genes seem to form hybrid oligomers, however both homooligomers have enzyme activity. Thus, the product of the PDC1 gene is not absolutely necessary for glucose fermentation in yeast.

Cholinergic system and memory in the rat: Effects of chronic ethanol, embryonic basal forebrain brain transplants and excitotoxic lesions of cholinergic basal forebrain projection system

Oral administration of ethanol (20% v/v) to male Sprague-Dawley rats for different periods of time up to 28 weeks resulted in profound reductions of acetylcholine content, in vitro synthesis and release of acetylcholine, choline uptake, activities of choline acetyltransferase, acetylcholinesterase and pyruvate decarboxylase, content of noradrenaline, serotonin and, to a lesser extent, dopamine throughout the brain. Changes were fully and partially reversible by a 4 weeks' ethanol-free period following a treatment of 8 and 18 weeks, respectively. They remained persistent, however, after 28 weeks of treatment. Performance in an eight arm-radial maze revealed a severe impairment in both spatial and non-spatial reference and working memory. A similar pattern of memory impairment was obtained after ibotenate lesion of the cholinergic basal forebrain projection system. In order to test whether this memory impairment depends on cholinergic deafferentation of the cortex, cholinergic-rich fetal basal forebrain cell suspensions were transplanted into cortex, hippocampus or both these sites in ethanol treated rats. Cholinergic-rich transplants, but not cholinergic-poor transplants, were effective in ameliorating impaired memory function and measures of cholinergic activity in the basal forebrain projection system. The behavioural efficacy of the basal forebrain grafts was well correlated with measures of both transplant volume and the degree to which they restored acetylcholine content at the transplant site; these transplants had no effect, however, on brain monoamine levels. The effects of the cholinergic-rich transplants into cortical and hippocampal sites were additive in their amelioration of performance in the radial maze. Similarly, ibotenate lesions of the sites of origin of the cholinergic projections to neocortex (in the region of the nucleus basalis magnocellularis) and hippocampus (the medial septal areas and nucleus of the diagonal band), respectively, were additive in their deleterious effects on maze performance. There were no qualitative differences in the susceptibility of the four different types of memory performance measured (spatial and non-spatial reference and working memory) to the effects of ethanol, ibotenate lesions of the cholinergic projection system, or cholinergic-rich brain tissue transplants. Thus, overall, the results indicate that the forebrain cholinergic system acts as a whole, without major functional differences between the projections originating in the medial septal area/diagonal band complex and the basal nucleus, and that it discharges a very general function in cognitive processes. It is suggested that chronic ethanol treatment in rat, known to induce similar changes in Man, might represent a model suitable to investigate the relationship of chronically induced degeneration in the cholinergic basal forebrain projection system and impaired cognitive function.

Evidence Does Not Support Ethylene as a Cue for Synthesis of Alcohol Dehydrogenase and Pyruvate Decarboxylase During Exposure to Hypoxia

Low O2 concentrations increased the activity of alcohol dehydrogenase (ADH; EC 1.1.1.1) and pyruvate decarboxylase (PDC; EC 4.1.1.1), but exogenously applied ethylene did not reproduce this effect in either maize roots or rice coleoptiles. However, ethylene was clearly exerting its known effects on the maize roots, since it inhibited root extension, and promoted aerenchyma formation. Furthermore, treatment with silver nitrate, an antagonist of ethylene action, failed to inhibit the rise in ADH activity in O2-deficient maize roots. We conclude that ethylene causes morphological adaptations to O2-deficiency, but is not involved in concurrent increases in activities of ADH and PDC. Alternative causes for the increases in enzyme activity are proposed.

Thiamin diphosphate catalysis. Mechanistic divergence as a probe of substrate activation of pyruvate decarboxylase

Thiamin diphosphate catalysis. Mechanistic divergence as a probe of substrate activation of pyruvate decarboxylase

Alcohol Dehydrogenase and Ethanol in the Stems of Trees

Anaerobic fermentation in plants is usually thought to be a transient phenomenon, brought about by environmental limitations to oxygen availability, or by structural constraints to oxygen transport. The vascular cambium of trees is separated from the air by the outer bark and secondary phloem, and we hypothesized that the cambium may experience sufficient hypoxia to induce anaerobic fermentation. We found high alcohol dehydrogenase activity in the cambium of several tree species. Mean activity of alcohol dehydrogenase in Populus deltoides was 165 micromoles NADH oxidized per minute per gram fresh weight in May. Pyruvate decarboxylase activity was also present in the cambium of P. deltoides, with mean activity of 26 micromoles NADH oxidized per minute per gram fresh weight in May. Lactate dehydrogenase activity was not present in any tree species we examined. Contrary to our expectation, alcohol dehydrogenase activity was inversely related to bark thickness in Acer saccharum and unrelated to bark thickness in two Populus species. Bark thickness may be less important in limiting oxygen availability to the cambium than is oxygen consumption by rapidly respiring phloem and cambium in actively growing trees. Ethanol was present in the vascular cambium of all species examined, with mean concentrations of 35 to 143 nanomoles per gram fresh weight, depending on species. Ethanol was also present in xylem sap and may have been released from the cambium into the transpiration stream. The presence in the cambium of the enzymes necessary for fermentation as well as the products of fermentation is evidence that respiration in the vascular cambium of trees may be oxygen-limited, but other biosynthetic origins of ethanol have not been ruled out.

Acetoin and Phenylacetylcarbinol Formation by the Pyruvate Decarboxylases ofZymomonas MobilisandSaccharomyces CarlsbergensisStephanie Bringer-Meyer

Cell suspensions of Zymomonas mobilis and Saccharomyces carlsbergensis and the pyruvate decarboxylases from the two organisms were compared with respect to their efficiencies of acyloin formation. Although Z. mobilis contained five times more pyruvate decarboxylase activity than yeast, sugar-fermenting suspensions of Z. mobilis produced, in the presence of benzaldehyde, 4–5 times less phenylacetylcarbinol than the yeast. The pyruvate decarboxylases of both organisms catalyzed acetoin and phenylacetylcarbinol synthesis from pyruvate and acetaldehyde or benzaldehyde, but the affinity of the Z. mobilis pyruvate decarboxylase towards the aldehyde reactants was lower than that of the yeast enzyme. Because of the limited solubility of benzaldehyde, neither enzyme could be saturated with this substrate for phenyl-acetylcarbinol synthesis. Studies with 2-toluidinonaphthalene-6-sulfonate and substrate analogues showed that the catalytic sites of pyruvate decarboxylase from Z. mobilis were less lipophilic than thos...

Alcohol Dehydrogenase and Pyruvate Decarboxylase Activity in Leaves and Roots of Eastern Cottonwood (Populus deltoides Bartr.) and Soybean (Glycine max L.)

Pyruvate decarboxylase (PDC, EC 4.1.1.1) and alcohol dehydrogenase (ADH, EC 1.1.1.1) are responsible for the anaerobic production of acetaldehyde and ethanol in higher plants. In developing soybean embryos, ADH activity increased upon imbibition and then declined exponentially with development, and was undetectable in leaves by 30 days after imbibition. PDC was not detectable in soybean leaves. In contrast, ADH activity remained high in developing cottonwood seedlings, with no decline in activity during development. ADH activity in the first fully expanded leaf of cottonwood was 230 micromoles NADH oxidized per minute per gram dry weight, and increased with leaf age. Maximal PDC activity of cottonwood leaves was 10 micromoles NADH oxidized per minute per gram dry weight. ADH activity in cottonwood roots was induced by anaerobic stress, increasing from 58 to 205 micromoles NADH oxidized per minute per gram dry weight in intact plants in 48 hours, and from 38 to 246 micromoles NADH oxidized per minute per gram dry weight in detached roots in 48 hours. Leaf ADH activity increased by 10 to 20% on exposure to anaerobic conditions. Crude leaf enzyme extracts with high ADH activity reduced little or no NADH when other aldehydes, such as trans-2-hexenal, were provided as substrate. ADH and PDC are constitutive enzyme in cottonwood leaves, but their metabolic role is not known.

Alcohol Dehydrogenase and Pyruvate Decarboxylase Activities in Flood‐Tolerant and Susceptible Corn Seeds during Flooding1

AbstractEarly planted corn (Zea maysL.) is frequently subjected to flooding stress, especially in poorly drained soils. This study characterizes the role of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) enzymes in the flooding susceptibility of corn seeds; PDC and ADH activities were assayed in a flood‐tolerant (A632) and a flood‐susceptible (Mo17) inbred during flooding at 10 and 25 °C. Each inbred consisted of two seed lots possessing different germination levels (95 and 75% germination). Flooding induced an increase in the activity of both enzymes. However, the increase was greater and sustained longer for ADH than PDC. The maximum level of ADH activity achieved was similar in both inbreds, but was greater in the high‐quality than in the low‐quality seeds. Since the highquality seeds survived flooding longer than the lowquality seeds, no correlation between flooding susceptibility and maximum ADH activity was observed. The two inbreds also exhibited similar ADH isoenzyme patterns. These data indicate that flooding susceptibility is not associated with PDC or ADH activities in corn seeds.

Anaerobic Stress in Germinating Castor Bean, Ethanol Metabolism, and Effects on Subcellular Organelles

Endosperms from castor beans (Ricinus communis) germinated for 0 to 6 days were exposed to anoxia for 0 to 15 hours. Ethanol, the only alcohol detected by gas chromatography in the tissue, accumulates to a concentration of 15 millimolar during the first 2 to 4 hours of anoxia and subsequently decreases. The absolute amount of ethanol varies from 10 micromoles per 5-day endosperm after 4 hours anoxia to less than 1 micromole in 2-day endosperm after 4 hours. Lactate content is 2 micromoles or less per endosperm. Alcohol dehydrogenase and pyruvate decarboxylase activities, which are localized in cytosolic fractions, are not greatly affected by anoxia. The recoveries of the marker enzymes and protein in endoplasmic reticulum (ER) and mitochondrial fractions decrease during anoxia. After 15 hours, the recovery of NADPH cytochrome c reductase is 15% of that in controls, fumarase is 50%, and catalase is 75%.Glyoxysomes and ER are capable of converting ethanol to acetaldehyde which was measured using the fluorogenic reagent, 5,5-dimethyl-1,3-cyclohexanedione. The glyoxysomal activity is dependent on a hydrogen peroxide-generating substrate and the ER is dependent on NADPH. However, these activities are less than 3% of the alcohol dehydrogenase activity.

Pyruvate decarboxylase and alcohol dehydrogenase in the insect trypanosomatid, Crithidia fasciculata

1. 1. Cell-free extracts of the insect trypanosomatid Crithidia fasciculata, obtained by grinding with glass powder in a mortar, contained activity of pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1). Both enzymes were found in the soluble fraction (105,000 g supernatant) and were partially purified by ammonium sulphate fractionation and gel filtration on Sephadex G-200. 2. 2. Pyruvate decarboxylase, free of lactate dehydrogenase and with little endogenous alcohol dehydrogenase, was assayed spectrophotometrically with commercial yeast alcohol dehydrogenase as coupled enzyme. The activity was increased nearly 2-fold by the addition of thiamine pyrophosphate (0.1 mM) and MgCl 2 (1 mM). The apparent K m for pyruvate was 2 mM, and the optimal pH was 6.0. 3. 3. Alcohol dehydrogenase was nearly 14-fold more active in the direction of acetaldehyde reduction than in the direction of ethanol oxidation. The optimal pH was about 7.0, and the apparent K m values were 35 μM for acetaldehyde (at 0.15 mM NADH) and 14 μM for NADH (at 60 μM acetaldehyde). 4. 4. Both enzymes were strongly inhibited by thiol reagents such as p-chloromercuribenzoate and phenolphthalein mercuric acetate; pyruvate decarboxylase was the more sensitive. 5. 5. The properties of both enzymes suggest that the enzyme system for ethanol production by C. fasciculata is very similar to that present in baker's yeast.

Acetaldehyde formation by mitochondria from the free-living nematode Panagrellus redivivus.

The mitochondrial fraction from the free-living nematode Panagrellus redivivus decarboxylates pyruvate to produce significant amounts of acetaldehyde. Acetaldehyde formation is stimulated by thiamin pyrophosphate, shows a sharp optimum at pH 6.8 and is greater under anaerobic than aerobic conditions. The pyruvate decarboxylase activity cofractionates with, and is probably a partial reaction of, the pyruvate dehydrogenase complex. Acetaldehyde production is modulated by NAD+, ATP and acetyl-CoA and is greatly stimulated by lipoic acid. The pyruvate decarboxylase system is extremely sensitive to thiol-group inhibitors and is inhibited by oxygen in the presence of pyruvate.

Effect of Nitrogen Source and Vitamins on Ethanol and Pyruvate Production byBipolaris MaydisRace T

The dry weight of Bipolaris maydis race T incubated three da in a glucose-mineral salts liquid medium containing asparagine as the nitrogen source was approximately twice that obtained with either glutamate or NH4C1. Thiamine at 0.1 or 1 Mg/rnl was inhibitory to growth at pH 5 in asparagine medium, was stimulatory at pH 6.5 in glutamate medium, and had no effect in NH4C1 medium at any pH tested. The concentration of ethanol in the culture medium was greatest with glutamate as the nitrogen source and least with NH4C1. When asparagine was the nitrogen source, caused an increase in extracellular ethanol levels and a decrease in the levels of both intra- and extracellular pyruvate. This appeared to be due to a thiamine-mediated increase in pyruvate decarboxylase but not alcohol dehydrogenase activity. No similar trends were evident in either NH4C1 or glutamate media. The intracellular pyruvate level appeared to be a more important regulator of ethanol production than the activities of pyruvate decarboxylase and alcohol dehydrogenase. Of six other vitamins tested on glucose-asparagine pH 5 medium, only folic acid stimulated growth; biotin was inhibitory and the others had no effect. The inhibitory effect of biotin could be nullified by the presence of thiamine. Although each vitamin could stimulate ethanol production, only folic acid and pantothenate were as effective as thiamine. Thiamine inhibits sporulation in Bipolaris maydis (Nisikado) Shoemaker [Drechslera maydis (Nisikado) Subram. & Jain; Helminthosporium maydis Ni? sikado] race T, the southern corn leaf blight pathogen, when it is incubated on an agar medium containing L-asparagine, ammonium citrate, or sodium nitrate as the nitrogen source (Garraway, 1973; Garraway and Evans, 1977). Although B. maydis does not sporulate in liquid media, we have used tissue grown in this manner as a model system to investigate biochemical effects of under defined nutritional conditions (Evans and Garraway, 1976). When B. maydis is incubated in shake culture in asparagine medium, inhibits growth and stimulates ethanol production. We have previously shown that this latter event is due to a thiamine-mediated increase in the synthesis of pyruvate decarboxylase (PD), an enzyme in the alcoholic fermentation pathway that requires pyrophosphate as a cofactor. In the absence of added thiamine, PD activity is low, and pyruvate accumulates intracellularly. Apparently a portion of the intracellular pyruvate pool leaks into the medium where it can be detected in relatively high concentrations. As the concentration of the medium is increased, PD activity also increases and the pyruvate level drops as it is converted to acetaldehyde. Acetaldehyde is then converted to ethanol via alcohol dehydrogenase (ADH), an enzyme whose activity is not affected by thia? mine (Evans and Garraway, 1976). Because ethanol production may be a mechanism by which inhibits growth and sporulation in B. maydis, we wished to determine if the thiamine