Ammonia inhibits insulin stimulation of the Krebs cycle: Further insight into mechanism of hepatic coma

Oxidation of [2,3-¹⁴C]succinate in the intramitochondrial Krebs cycle was used as a probe to investigate the effect of ammonia on protein incorporation and Krebs cycle oxidation of succinate carbons in isolated rat hepatocytes. At low concentrations of ammonium chloride (0.1 to 0.5 mM) a slight increase in¹⁴CO₂ formation from [2,3-¹⁴C]succinate was observed, however, the stimulatory effect of insulin was significantly reduced. Insulin failed to cause any stimulation of succinate carbons incorporation into hepatocyte protein in the presence of ammonium chloride. Addition of ammonium chloride also depressed the movement of tracer carbons into the gluconeogenesis pathway. The activity of the amphibolic amino acid pool was significantly enhanced by ammonia. The data presented in this paper lend strong support to the Krebs-cycle depletion theory of hepatic coma. They also suggest that reduced mitochondrial Krebs cycle activity caused by increased amphibolic depletion of substrates results in loss of insulin sensitivity in ammonia toxicity.Special issue dedicated to Dr. Santiago Grisolia.     

The Mechanism of Acetate and Pyruvate Oxidation by Trypanosoma cruzi

The epimastigote or culture form of Trypanosoma cruzi oxidizes [3-¹⁴C] pyruvate and [2-¹⁴C] acetate to ¹⁴CO₂ without an apparent increase in overall respiration. This oxidation takes place through the tricarboxylic acid cycle as shown by (a) the incorporation of substrate ¹⁴C into cycle intermediates; (b) the earlier liberation of acetate carboxyl carbon as CO₂; and (c) the characteristic intramolecular distribution of pyruvate and acetate carbon atoms in the skeletal carbon of aspartic and glutamic acids. Upon oxidation of [3-¹⁴C] pyruvate and [2-¹⁴C] acetate, two of the products, alanine and glutamic acid, are found to account for more than 50% of incorporated ¹⁴C; labeling of alanine predominates with [3-¹⁴C] pyruvate while labeling of glutamic acid predominates with [2-¹⁴C] acetate. Using [1- or 6-¹⁴C] glucose as substrate, the pattern of ¹⁴C distribution in soluble metabolites closely resembles that obtained with [3-¹⁴C] pyruvate, in accordance with the joint operation of the Embden-Meyerhof pathway and Krebs cycle. The cycle operation depends on electron transport through the mitochondrial respiratory chain, since antimycin A, at a relatively low concentration, inhibits the oxidation of [2-¹⁴C] acetate to ¹⁴CO₂, to the same extent as the parasite respiration. Though functional in T. cruzi epimastigotes, the oxidative role of the Krebs’ cycle is apparently limited by the absence of an efficient oxidative apparatus. The cycle operation does, however, constitute an important source of skeletal carbon for the biosynthesis of amino acids and can contribute to the process of glycogenesis.     

Effect of tolbutamide on the metabolism of Tetrahymena

Tolbutamide partially inhibited the growth but increased the glycogen content of Tetrahymena pyriformis in logarithmically growing cultures. Tolbutamide slightly increased ¹⁴CO² production from [1-¹⁴C] and [6-¹⁴HC] glucose and [2-¹⁴C] pyruvate, but had little effect on the oxidation of [1-¹⁴C] acetate when any of these substrates were added to the proteose-peptone medium in which the cells had been grown. Measurement of ¹⁴CO₂ production from [1-¹⁴C] and [2-^I4C]-glyoxylate showed that this substrate was primarily oxidized via the glyoxylate cycle, with little if any oxidation occurring via the peroxisomal glyoxylate oxidase. Addition of tolbutamide inhibited the glyoxylate cycle as indicated by a marked reduction in label appearing in CO₂ and in glycogen from labeled acetate. In control cells, addition of acetate strongly inhibited the oxidation of [2-¹⁴C]-pyruvate whereas addition of pyruvate had little effect on the oxidation of [1-¹⁴C]-acetate. Acetate was more effective than pyruvate in preventing the growth inhibitory and glycogen-increasing effects of tolbutamide. The data suggest that one effect of tolbutamide may be to interfere with the transfer of isocitrate and acetyl CoA across mitochondrial membranes.     

Effect of insulin on the metabolic distribution of carbons 1, 2, and 3 of pyruvate

It has long been known that the carbons of pyruvate are converted to CO2 at different points in the metabolic process. This report deals with the observation that insulin affects the oxidation of carbons 2 and 3 primarily and has little effect on the oxidation of the carboxyl carbon. Oxidation of different carbons of pyruvate and their incorporation into various metabolic components was studied in isolated rat hepatocytes. Insulin stimulated the 14CO2 production from [2-14C]- and [3-14C]pyruvate and from [U-14C]alanine. However, it had little or no effect on the activity of the pyruvate dehydrogenase complex as measured by the evolution of 14CO2 from [1-14C]pyruvate or [1-14C] alanine. Insulin also stimulated the incorporation of carbons 2 and 3 of pyruvate into protein but had no effect on the incorporation of carbon 1. Incorporation of [1-14C]- and [U-14C]alanine into protein was differentially enhanced by insulin in a manner similar to that of the pyruvate carbons. The fact that insulin stimulates the incorporation of [1-14C]alanine into protein but not [1-14C]pyruvate suggests the possibility of a compartmentation of pyruvate metabolism in the isolated hepatocytes. These studies show that the stimulation of [2-14C]- and [3-14C]pyruvate incorporation into protein involves the stimulatory effect of insulin on the activity of the Krebs cycle which is evident from the fact that insulin did not stimulate the pyruvate carbons to enter protein via alanine but the incorporation via glutamate was increased by about 40%.     

Conditional lethal mutants of Chinese hamster cells: mutants requiring exogenous carbondioxide for growth

A series of Chinese hamster cell lines were tested and found to be able to proliferate in the absence of added bicarbonate and carbondioxide if hypoxanthine and uridine were present in the medium. Conversely, cells incapable of salvaging one of these precursors, such as hypoxanthine-guanine phosphoribosyltransferase (HGPRT^?) deficient cells did not multiply under these conditions. We describe another variant capable of utilizing hypoxanthine and uridine which has an absolute requirement for exogenous CO₂/NaHCO₃ for growth. These cells appear to be defective in the complete oxidation of pyruvate to carbondioxide, and indications are that the entry of pyruvate into the Krebs cycle is affected.     

Partial characterization of the pathway from propionate to acetate in the cabbage looper,Trichoplusia ni

Experiments were performed to characterize the metabolism of propionate to acetate in the cabbage looper Trichoplusia ni and correlate the results with vitamin B₁₂ levels. Fourth and fifth instar larvae contain 2–4 pg vitamin B₁₂/mg dry wt whereas pupae and adults do not contain detectable amounts. In vivo studies as a function of time in larvae, pupae and adults gave evidence that [2-¹⁴C]propionate was converted to 3-hydroxypropionate and then to acetate, which subsequently labeled Krebs cycle intermediates. Radioactivity from [1-¹⁴C]propionate was recovered only in the propionate and 3-hydroxypropionate fractions, and not in acetate or Krebs cycle intermediates, suggesting that carbon 1 of propionate was lost as carbon dioxide and that carbons 2 and 3 of propionate were retained during conversion to acetate. The enzymes of this pathway were located entirely in the mitochondrial fraction. Cyanide inhibited the metabolism of propionate to 3-hydroxypropionate and acetate in mitochondrial preparations, whereas carbon monoxide did not. [2,3-¹⁴C]Acrylic acid was metabolized to 3-hydroxypropionate, which is consistent with a dehydrogenase converting propionate to acrylate which is then hydrated to 3-hydroxypropionate and then oxidized and decarboxylated to acetate.     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.     

Conditionally lethal mutations in Chinese hamster cells. Characterization of a cell line with a possible defect in the krebs cycle

A variant Chinese hamster cell line has been isolated from a mutagenized population that has a markedly reduced ability to oxidize a variety of substrates via the Krebs cycle. The production of ¹⁴CO₂ from ¹⁴C-labeled compounds was measured using pyruvate, acetate, β-hydroxybutyrate, palmitate and glutamate, and in all cases it was negligible in the mutant. In contrast to this, significant amounts of ¹⁴CO₂ were produced from ¹⁴C-aspartate and ¹⁴C-succinate which suggests that some reactions of the Krebs cycle can take place and this conclusion is supported by tracer experiments with labeled compounds. The rate of respiration measured with a Clark oxygen electrode in the mutant was compared to several normal Chinese hamster cell lines and was found to be only 8%. Mitochondria appear to be present in normal numbers and with only minor differences in morphology. The measurement of difference spectra between oxidized and reduced states permits us to conclude that the cytochromes are all present and functional. These results lead us to believe that there may be a defect in the Krebs cycle between α-ketoglutarate and succinate. Alternatively a defect in a structural component of the mitochondria or in the electron-transport chain itself may be causing pleiotropic effects in the Krebs cycle and respiration.     

An estimation of pyruvate recycling during gluconeogenesis in the perfused rat liver

To estimate the degree of recycling of pyruvate during gluconeogenesis, an isotope tracer procedure was employed. Using the isolated, perfused rat liver with pyruvate-2-¹⁴C in the perfusion fluid, the 3-carbon acids lactate and pyruvate were isolated and the distribution of ¹⁴C in each carbon was assayed. It can be shown that the degree of recycling can be approximated as twice the sum of ¹⁴C in carbons 1 and 3. Glucose, acetoacetate, and β-hydroxybutyrate were also determined, and their ¹⁴C distribution estimated by appropriate degradation procedures. In livers from fasted rats, recycling of pyruvate during 1 hr incubation occurred at a rate of 0.21 μmoles ± 0.02 (SE)/min/g while gluconeogenesis occurred at a rate of 0.49 ± 0.11 μmoles pyruvate-2-¹⁴C/min/g. In livers from carbohydrate-fed rats, the ratio was reversed, with 0.35 ± 0.06 μmoles pyruvate-2-¹⁴C recycled and only 0.09 ± 0.03 μmoles converted to glucose. These patterns were not affected by the simultaneous presence of octanoate in the perfusion, during which ketone body production was greatly increased. Only about 20% of the ketone bodies formed were derived from pyruvate, much less with octanoate present, and over 95% of the total radioactivity was in carbons 1 and 3 of acetoacetate as anticipated from the degree of pyruvate recycling. The glucose invariably had 3–4% of its total activity in carbons 3 and 4 and the remainder distributed approximately equally in carbons 1, 2, 5, and 6. The radioactivity in respired CO₂ indicated that about 13–25% of the total O₂ uptake was due to pyruvate oxidation to CO₂.     

The role of the Krebs cycle in the generation of intramitochondrial reducing equivalents for the 11 beta-hydroxylation of deoxycorticosterone in isolated rat adrenal cells

Isolated rat adrenal cells were used to study the possible pathways of intramitochondrial NADPH generation for 11β-hydroxylation of 11-deoxycorticosterone. Pyruvate was efficiently utilized by the mitochondria as shown by evolution of ¹⁴CO₂ from [1-¹⁴C]- and [2-¹⁴C]pyruvate. Citrate, isocitrate, succinate, and malate were not utilized by intact cells due to their inability to permeate the plasma membrane. For every mole of corticosterone formed, 1.9 and 0.8 moles of ¹⁴CO₂ were formed from [1-¹⁴C]- and [2-¹⁴C]pyruvate, respectively, indicating that pyruvate dehydrogenase was quite active and supplied acetyl C?oA to the Krebs cycle. Fluorocitrate and 2,4-dinitrophenol inhibited 11β-hydroxylation of 11-deoxycorticosterone as well as the production of ¹⁴CO₂ from [2-¹⁴C]pyruvate. Comparison of data with the two inhibitors showed that for the same percentage of inhibition of ¹⁴CO₂ production, the inhibition of 11β-hydroxylation was greater with 2,4-dinitrophenol than with fluorocitrate. It is concluded that operation of the Krebs cycle may be essential for 11β-hydroxylation to occur primarily because NADH generated by the cycle provides ATP, via the respiratory chain, as well as the substrate for the energy-linked transhydrogenase that forms NADPH. The NADPH required for 11β-hydroxylation seems to be derived to a large extent via the energy-linked transhydrogenase.     

Both Forward and Reverse TCA Cycles Operate in Green Sulfur Bacteria

The anoxygenic green sulfur bacteria (GSBs) assimilate CO₂ autotrophically through the reductive (reverse) tricarboxylic acid (RTCA) cycle. Some organic carbon sources, such as acetate and pyruvate, can be assimilated during the phototrophic growth of the GSBs, in the presence of CO₂ or HCO₃⁻. It has not been established why the inorganic carbonis required for incorporating organic carbon for growth and how the organic carbons are assimilated. In this report, we probed carbon flux during autotrophic and mixotrophic growth of the GSB Chlorobaculum tepidum. Our data indicate the following: (a) the RTCA cycle is active during autotrophic and mixotrophic growth; (b) the flux from pyruvate to acetyl-CoA is very low and acetyl-CoA is synthesized through the RTCA cycle and acetate assimilation; (c) pyruvate is largely assimilated through the RTCA cycle; and (d) acetate can be assimilated via both of the RTCA as well as the oxidative (forward) TCA (OTCA) cycle. The OTCA cycle revealed herein may explain better cell growth during mixotrophic growth with acetate, as energy is generated through the OTCA cycle. Furthermore, the genes specific for the OTCA cycle are either absent or down-regulated during phototrophic growth, implying that the OTCA cycle is not complete, and CO₂ is required for the RTCA cycle to produce metabolites in the TCA cycle. Moreover, CO₂ is essential for assimilating acetate and pyruvate through the CO₂-anaplerotic pathway and pyruvate synthesis from acetyl-CoA.     

Assimilation and Metabolism of Exogenous Organic Compounds by the Strict Autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus

The assimilation and utilization of the individual carbon atoms of pyruvate and acetate by cells of Thiobacillus thioparus and T. neapolitanus, in the presence and absence of an energy source, were studied by use of radioactive substrates. Both organisms produced ¹⁴CO₂ from ¹⁴C-labeled pyruvate, but more came from carbon 1 than from carbons 2 or 3. The conversion of the carbons of acetate to CO₂ by both organisms was much less than that from any of the pyruvate carbons. When labeled pyruvate and acetate were incubated with these organisms, small amounts of radioactivity were found in the tricholoacetic acid-soluble material, nucleic acids, and lipids, and larger amounts were found in the protein fraction. The composition of the incubation medium affected the amount of utilization and incorporation of labeled substrates by both organisms. The presence of an exogenous energy source (Na₂S₂O₃) suppressed incorporation of the labeled substrates into various cellular components by T. thioparus, but enhanced incorporation by T. neapolitanus. When ¹⁴C-pyruvate was used as a substrate, as many as 12 radioactive compounds were found in the water-soluble fraction in the experiments with T. neapolitanus, whereas no more than three radioactive compounds were detected in this fraction in the experiments with T. thioparus. Of the total ¹⁴C activity found in the water-soluble fractions, malic acid contained the highest percentage. These findings are discussed in light of the overall metabolism of these two sulfur-oxidizing obligate chemoautotrophs, as well as in relation to the biochemical basis of chemoautotrophy.     

Alternative pathways of glucose utilization in brain. Changes in the pattern of glucose utilization in brain during development and the effect of phenazine methosulfate on the integration of metabolic routes.

The activities of alternative pathways of glucose metabolism in developing rat brain were evaluated by measurement of the yields of ¹⁴CO₂ from glucose labeled with ¹⁴C on carbons 1, 2, 3 + 4, 6 and uniformly labeled glucose, from the detritiation of [2-³H]glucose and from the incorporation of ¹⁴C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. The glycolytic route and tricarboxylic acid cycle (¹⁴CO₂ yield from carbons 3, 4, and 6 of glucose) increased during development. The flux through the glutamate-γ-aminobutyric route (¹⁴CO₂ yield from carbon 2-carbon 6 of glucose) also showed an increase with development. In contrast, the proportion of glucose metabolized via the pentose phosphate pathway was markedly decreased as development progressed. The artificial electron acceptor, phenazine methosulfate, was used as a probe to investigate the effect of alterations in the redox state of $\text{NADP}{}^{\mathrm{+}}\text{NADPH}$ couple on a number of NADP-linked systems in developing brain. Phenazine methosulfate produced a massive (20- to 50-fold) stimulation of the pentose phosphate pathway, in contrast, the incorporation of glucose carbon into fatty acids and flux through the glutamate-γ-aminobutyrate shunt were sharply decreased. The effects of phenazine methosulfate on the incorporation of glucose into glyceride glycerol, on the flux of glucose through the pyruvate dehydrogenase reaction and tricarboxylic acid cycle, all processes linked to the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ couple, appeared to be minimal in the brain at the stages of development studied, i.e., 1, 5, 10, 20 days, and in the adult rat. The significance of the massive reserve potential of the pentose phosphate pathway in the developing brain is discussed.     

The stimulus secretion coupling of glucose-induced insulin release.

In isolated rat pancreatic islets, exogenous l-lactate causes a dose-related enhancement of glucose-induced insulin release and shifts the sigmoidal curve relating insulin output to ambient glucose concentrations to the left. l-Lactate also enhances α-ketoisocaproate-induced insulin release and glucose-induced proinsulin biosynthesis. l-Lactate rapidly accumulates in the islet cells, is converted to pyruvate and CO₂, and raises the intracellular concentration of both ATP and NAD(P)H. On a molar basis, the insulinotropic capacity of nutrients ranges as follows d-glucose ? l-lactate > pyruvate = d/l-lactate > d-lactate and does not correlate with their respective oxidation rates. However, when allowance is made for the intracellular interconversion of these exogenous nutrients, for their reciprocal influence upon oxidation rates, and for their sparing action on the utilization of endogenous fuels, a close correlation is found between the aptitude of glucose, l-lactate, and pyruvate to generate reducing equivalents and to stimulate insulin release. It is proposed that the concentration of NAD(P)H in islet cells affects the ionophoretic fluxes of cations (K⁺, Ca²⁺) across membrane systems and, hence, regulates the net uptake of Ca²⁺ and subsequent release of insulin. The effect of l-lactate upon Ca²⁺ handling is sufficiently rapid to account for the immediate secretory response to this nutrient.     

Contribution of Extracellular Glutamine as An Anaplerotic Substrate to Neuronal Metabolism: A Re-Evaluation by Multinuclear NMR Spectroscopy in Primary Cultured Neurons

Multinuclear NMR spectroscopy is used to investigate the effect of glutamine on neuronal glucose metabolism. Primary neurons were incubated with [1-¹³C]glucose in the absence or presence of glutamine (2 mM) and/or NH₄Cl (5 mM). After ammonia-treatment, the concentrations of high-energy phosphates decreased up to 84% of control, which was aggravated in glutamine-containing medium (up to 42% of control). These effects could not be attributed to changes in mitochondrial glucose oxidation. Withdrawal of glutamine decreased amino acid concentrations, e.g. of glutamate to 53%, but also considerably lessened the ¹³C enrichment in [4-¹³C]glutamate to 8.3% of control, and decreased the ¹³C-enrichment in acetyl-CoA entering the Krebs cycle (P<0.001). Thus, although glutamine is potent in replenishing neuronal glutamate stores, glutamate formation is mainly attributed to its de novo synthesis from glucose. Furthermore, mitochondrial glucose metabolism strongly depends on the supply of carbons from glutamine, indicating that exogenous glutamine is a well-suited substrate to replenish neuronal Krebs cycle intermediates.     

Biodegradation of 14C-Labeled Model and Cornstalk Lignins, Phenols, Model Phenolase Humic Polymers, and Fungal Melanins as Influenced by a Readily Available Carbon Source and Soil

After 6 months of incubation in a fertile neutral sandy loam, about 48% of the ring carbons and 2-carbons and 60% of the OCH₃ carbons of specifically labeled coniferyl alcohol had evolved as CO₂. After 1 year, corresponding values were 55 and 65%. When coniferyl alcohol units were linked into model and cornstalk lignins, about 23% of the ring carbons and 2-carbons and 39% of the OCH₃ carbons had evolved as CO₂ after 6 months. After 1 year, corresponding values were about 28 and 46%. The addition of orange leaves (0.5%, wt/wt) after 6 months did not significantly increase the evolution of ¹⁴CO₂. Addition of orange leaves (0.5%, wt/wt) with specifically ¹⁴C-labeled pyrocatechol, coumaryl alcohol, model lignins, humic acid-type phenolic polymers and of uniformly ¹⁴C-labeled fungal melanins did not increase labeled C losses or C losses from the orange leaves. Decomposition of protein and pyrocatechol linked into model humic acid polymers, coniferyl alcohol C in model lignins, and Eurotium echinulatum melanin in six soils varied from 2 to 14%. Significant differences in C losses were related to soils and were not influenced by orange leaf applications.     

Sul Metabolismo Dell'Alcool Etilico in Tessuti Di Fusto Di « Pisum »

Abstract

On the metabolism of ethanol in the Pea stem tissues. — The average concentration of ethanol in the growing part of the etiolated pea internodes is of the order of 10^-3M. Previous work showed that auxin at growth promoting concentration markedly lowers this level in the excised internodes. This finding prompted a series of investigations on C¹⁴ labeled ethanol utilization in this material.

The capacity of the segments to metabolize ethanol is remarkable: with an external ethanol concentration 5X10^-3M the C¹⁴ labeled CO₂ originated from 1-C¹⁴ ethanol accounted for about 10% of total CO₂ produced during the first hour of treatment. Moreover, an amount of ethanol about 10 fold higher that that dissimilated to CO₂ was metabolized to various yet unidentified compounds. The ratio between the contribution of ethanol to CO₂ and that to other metabolites appeared maximal in the first period after feeding the labeled compound. This ratio was significantly higher then that found for 6-C¹⁴ glucose.

These preliminary results suggest the possibility that ethanol produced in glycolysis could represent an interesting metabolite in an anabolic pathway different from the one leading from pyruvate to the Krebs cycle acids.     

Anaerobic and aerobic energy metabolism in the larvae (tetrathyridia) of Mesocestoides corti

The tetrathyridia of Mesocestoides corti produce lactate, succinate, acetate, and CO₂ as major carbon-containing end products during in vitro incubation with glucose as the substrate. Differences in the rate of glucose consumption and lactate production under anaerobic or aerobic conditions were observed, but their significance could not be determined. However, succinate production was greatly decreased in the presence of oxygen.The relative activities and intracellular distribution of various enzymes involved in energy-supplying metabolism of the larvae appear to conform to the pathways observed in other parasitic helminths known to produce lactate, succinate, and volatile fatty acids as metabolic end products. Some common features found in this respect are the relatively low pyruvate kinase activity, the presence of a highly active cytoplasmic phosphoenolpyruvate carboxylase and the capability of mitochondrial membrane bound fumarate reductase to reduce fumarate by means of NADH. Although a stimulatory effect of fructose-1,6-diphosphate on the reaction velocity of pyruvate kinase occurred, the absolute activity of this enzyme is very low.Nearly all the enzymes required for Krebs cycle activity are available in the tetrathyridia. Under the assay conditions employed by us, only NAD-dependent isocitrate dehydrogenase could not be demonstrated. The small amounts of ¹⁴CO₂ liberated from 6-¹⁴C-glucose suggest that the cycle in its classical form probably only functions at a very low rate. The incorporation of ¹⁴C from labeled glucose into glycogen indicates the presence of enzymes capable of glycogenesis. The incorporation rate was found to be higher in the presence of oxygen than under anaerobic conditions. On account of the very low NAD-linked glycerol-3-phosphate dehydrogenase activity the glycerolphosphate cycle may be of minor importance for the tetrathyridia.As a result of these studies a scheme for the main carbohydrate dissimilating pathways in the tetrathyridia is proposed and the significance of oxygen with respect to energy-supplying metabolism is discussed.     

Oxidation of carbon sources via the tricarboxylic acid cycle during calcium-induced conidation of Penicillium notatum

The TCA cycle was examined during Ca²⁺-induced conidiation in Penicillium notatum over the 12-h period after addition of Ca²⁺ to vegetative cultures. Conidiation was independent of Ca²⁺ when certain intermediates and derivatives of the TCA cycle served as sole carbon sources. Arsenite and malonate augmented the effect of Ca²⁺ on conidiation but did not substitute for it. Mitochondria from vegetative cells had low rates of oxidation of TCA cycle intermediates and, with the exception of pyruvate, aconitate and glutamate, these were poorly linked to phosphorylation processes. Calcium ions affected mitochondrial function causing reduced oxidation of oxoglutarate, elimination of pyruvate oxidation and a decline in respiratory control of these substrates with increased oxidation of NADH and NADPH. Radiorespirometric studies and enzyme searches revealed a complete but weakly oxidative TCA cycle in vegetative cells. In Ca²⁺-induced cells oxoglutarate dehydrogenase activity was deleted within 6.5 h of Ca²⁺ addition and this was accompanied by establishment of an incomplete Krebs cycle. Calcium-induced conidiation was associated with increased capacity for acetate and glutamate metabolism involving an activated glyoxylate shunt which may be related to enhanced biosynthetic demand. The metabolic basis of the Ca²⁺ effect on conidiation is discussed in connection with previous findings.     

Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii

Chloroplastic respiration was monitored by measuring ¹⁴CO₂ from ¹⁴C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K_m for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of ¹⁴CO₂ was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO₂ evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO₂ evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH₄Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO₂ and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.