Pool Sizes Of Intermediates Research Articles

CO2 -responsiveness of leaf isoprene emission: Why do speciesdiffer?

Leaf isoprene emission rate, I, decreases with increasing atmospheric CO2 concentration with major implications for global change. There is a significant interspecific variability in [CO2 ]-responsiveness of I, but the extent of this variation is unknown and its reasons are not understood. We hypothesized that the magnitude of emission reduction reflects the size and changeability of precursor pools responsible for isoprene emission (dimethylallyl diphosphate, DMADP and 2-methyl-erythritol 2,4-cyclodiphosphate, MEcDP). Changes in I and intermediate pool sizes upon increase of [CO2 ] from 400 to 1500 μmol/mol were studied in nine woody species spanning boreal to tropical ecosystems. I varied 10-fold, total substrate pool size 37-fold and the ratio of DMADP/MEcDP pool sizes 57-fold. At higher [CO2 ], I was reduced on average by 65%, but [CO2 ]-responsiveness varied an order of magnitude across species. The increase in [CO2 ] resulted in concomitant reductions in both substrate pools. The variation in [CO2 ]-responsiveness across species scaled with the reduction in pool sizes, the substrate pool size supported and the share of DMADP in total substrate pool. This study highlights a major interspecific variation in [CO2 ]-responsiveness of isoprene emission and conclusively links this variation to interspecific variability in [CO2 ] effects on substrate availability and intermediate pool size.

768-P: MSDC-0602 Is a Direct Mitochondrial Pyruvate Carrier Inhibitor That Modulates Central Carbon Metabolism in Mice and Humans

Mitochondrial pyruvate carrier (MPC) disruption attenuates type 2 diabetes NAFLD in mice. MSDC-0602, a PPARγ-sparing, TZD-like molecule that inhibits the MPC, was recently tested in a phase 2B trial for treating NASH, which reported beneficial effects on glucose and insulin homeostasis. Yet, the broad, in vivo metabolomic effects of MSDC-0602 treatment have remained undefined. Here, we tested MSDC-062 metabolic mechanisms of action in vitro and in vivo, spanning from mitochondrial pyruvate uptake assays in isolated mouse mitochondria, through cultured mouse liver cells, in vivo in mice, and in humans participating in the EMMINENCE trial. First, we examined MSDC-0602’s effect in isolated mouse liver mitochondria: Pyruvate-oxidation was decreased by MSDC-0602, and 2.5 µM MSDC-0602 was sufficient to decrease mitochondrial pyruvate uptake by >50%. We extended these findings to AML12 mouse liver cells and mouse liver in vivo. In AML12 cells, MSDC-0602 treatment increased pyruvate and lactate and decreased alanine. These results were comparable but not identical to results from parallel testing of the specific MPC inhibitor UK5099. 14-day treatment of NCD and HFD fed mice with MSDC-0602 decreased liver alanine and branched chain amino acids. As we previously observed with MPC liver-specific knockout mice, the HFD-induced liver TCA intermediate pool size expansion was mitigated by MSDC-0602. Accordingly, MSDC-0602-dependent metabolomic changes were more pronounced in HFD- versus NCD-fed mice. Human EMMINENCE trial participants receiving MSDC-0602 showed decreased circulating alanine, ketone bodies, and glucose. Similarly, circulating and liver glucose were decreased in HFD mice treated with MSDC-0602. Together, these data are consistent with MSDC-0602 modulating in vivo metabolism in a partially MPC-dependent manner and by additional mechanisms that remain to be understood. Disclosure A. J. Rauckhorst: None. D. J. Pape: None. J. R. Colca: Board Member; Self; Metabolic Solutions Development Company, LLC, Employee; Self; Cirius Therapeutics. E. B. Taylor: None. Funding American Diabetes Association (1-18-PDF-060 to A.J.R.); National Institute of Diabetes and Digestive and Kidney Diseases (DK104998); Cirius Therapeutics

Remodeling of substrate consumption in the murine sTAC model of heart failure

BackgroundEnergy metabolism and substrate selection are key aspects of correct myocardial mechanical function. Myocardial preference for oxidizable substrates changes in both hypertrophy and in overt failure. Previous work has shown that glucose oxidation is upregulated in overpressure hypertrophy, but its fate in overt failure is less clear. Anaplerotic flux of pyruvate into the tricarboxylic acid cycle (TCA) has been posited as a secondary fate of glycolysis, aside from pyruvate oxidation or lactate production. Methods and resultsA model of heart failure that emulates both valvular and hypertensive heart disease, the severe transaortic constriction (sTAC) mouse, was assayed for changes in substrate preference using metabolomic and carbon-13 flux measurements. Quantitative measures of O2 consumption in the Langendorff perfused mouse heart were paired with 13C isotopomer analysis to assess TCA cycle turnover. Since the heart accommodates oxidation of all physiological energy sources, the utilization of carbohydrates, fatty acids, and ketones were measured simultaneously using a triple-tracer NMR method. The fractional contribution of glucose to acetyl-CoA production was upregulated in heart failure, while other sources were not significantly different. A model that includes both pyruvate carboxylation and anaplerosis through succinyl-CoA produced superior fits to the data compared to a model using only pyruvate carboxylation. In the sTAC heart, anaplerosis through succinyl-CoA is elevated, while pyruvate carboxylation was not. Metabolomic data showed depleted TCA cycle intermediate pool sizes versus the control, in agreement with previous results. ConclusionIn the sTAC heart failure model, the glucose contribution to acetyl-CoA production was significantly higher, with compensatory changes in fatty acid and ketone oxidation not reaching a significant level. Anaplerosis through succinyl-CoA is also upregulated, and is likely used to preserve TCA cycle intermediate pool sizes. The triple tracer method used here is new, and can be used to assess sources of acetyl-CoA production in any oxidative tissue.

The Effect of Switchback Parameters on Root Pass Formation of Butt Welds with Variable Gap

Root pass manufacturing in automated welding is still a challenge when the backing plate is not feasible. Using the concept of bead formation in an original way, the GMAW (Gas Metal Arc Welding) switchback technique was assessed against linear movement as a means of facing this challenge. Experimental work was applied, keeping the process parametrization and joint configuration, so that only the switchback parameters were modified, i.e., the stroke lengths and speeds. Thermography was used to estimate the effect of the switchback parameters on bead formation. The results showed the potential of the switchback technique as a means of favoring weld pool control. Surprisingly, the operational gap range is not necessarily larger when switchback is applied. The strong influence of stroke lengths and speeds on the process performance was characterized. In general, the results showed that linear movement leads to larger pools and deeper penetrations, more adequate for gaps with no clearances. Shorter stroke lengths and slower stroke speeds (intermediate pool size) better suit root gaps that are not too wide, while longer stroke lengths and faster stroke speeds (smaller pool size, more easily sustainable) are applicable to larger root gaps.

The Precision Control of Autophagic Flux and Vesicle Dynamics-A Micropattern Approach.

Autophagy failure is implicated in age-related human disease. A decrease in the rate of protein degradation through the entire autophagy pathway, i.e., autophagic flux, has been associated with the onset of cellular proteotoxity and cell death. Although the precision control of autophagy as a pharmacological intervention has received major attention, mammalian model systems that enable a dissection of the relationship between autophagic flux and pathway intermediate pool sizes remain largely underexplored. Here, we make use of a micropattern-based fluorescence life cell imaging approach, allowing a high degree of experimental control and cellular geometry constraints. By assessing two autophagy modulators in a system that achieves a similarly raised autophagic flux, we measure their impact on the pathway intermediate pool size, autophagosome velocity, and motion. Our results reveal a differential effect of autophagic flux enhancement on pathway intermediate pool sizes, velocities, and directionality of autophagosome motion, suggesting distinct control over autophagy function. These findings may be of importance for better understanding the fine-tuning autophagic activity and protein degradation proficiency in different cell and tissue types of age-associated pathologies.

Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence

Synechocystis sp. strain PCC 6803 has been widely used as a photo-biorefinery chassis. Based on its genome annotation, this species contains a complete TCA cycle, an Embden-Meyerhof-Parnas pathway (EMPP), an oxidative pentose phosphate pathway (OPPP), and an Entner-Doudoroff pathway (EDP). To evaluate how Synechocystis 6803 catabolizes glucose under heterotrophic conditions, we performed 13 C metabolic flux analysis, metabolite pool size analysis, gene knockouts, and heterologous expressions. The results revealed a cyclic mode of flux through the OPPP. Small, but non-zero, fluxes were observed through the TCA cycle and the malic shunt. Independent knockouts of 6-phosphogluconate dehydrogenase (gnd) and malic enzyme (me) corroborated these results, as neither mutant could grow under dark heterotrophic conditions. Our data also indicate that Synechocystis 6803 metabolism relies upon oxidative phosphorylation to generate ATP from NADPH under dark or insufficient light conditions. The pool sizes of intermediates in the TCA cycle, particularly acetyl-CoA, were found to be several fold lower in Synechocystis 6803 (compared to E. coli metabolite pool sizes), while its sugar phosphate intermediates were several-fold higher. Moreover, negligible flux was detected through the native, or heterologous, EDP in the wild type or Δgnd strains under heterotrophic conditions. Comparing photoautotrophic, photomixotrophic, and heterotrophic conditions, the Calvin cycle, OPPP, and EMPP in Synechocystis 6803 possess the ability to regulate their fluxes under various growth conditions (plastic), whereas its TCA cycle always maintains at low levels (rigid). This work also demonstrates how genetic profiles do not always reflect actual metabolic flux through native or heterologous pathways. Biotechnol. Bioeng. 2017;114: 1593-1602. © 2017 Wiley Periodicals, Inc.

Metabolic analysis reveals changes in the mevalonate and juvenile hormone synthesis pathways linked to the mosquito reproductive physiology

Juvenile hormone (JH) regulates reproductive maturation in insects; therefore interruption of JH biosynthesis has been considered as a strategy for the development of target-specific insecticides. The corpora allata (CA) from mosquitoes is highly specialized to supply variable levels of JH, which are linked to ovarian developmental stages and influenced by nutritional signals. However, very little is known about how changes in JH synthesis relate to reproductive physiology and how JH synthesis regulation is translated into changes in the CA machinery. With the advent of new methods that facilitate the analysis of transcripts, enzymes and metabolites in the minuscule CA, we were able to provide comprehensive descriptions of the mevalonic (MVA) and JH synthesis pathways by integrating information on changes in the basic components of those pathways. Our results revealed remarkable dynamic changes in JH synthesis and exposed part of a complex mechanism that regulates CA activity. Principal component (PC) analyses validated that both pathways (MVAP and JH-branch) are transcriptionally co-regulated as a single unit, and catalytic activities for the enzymes of the MVAP and JH-branch also changed in a coordinate fashion. Metabolite studies showed that global fluctuations in the intermediate pool sizes in the MVAP and JH-branch were often inversely related. PC analyses suggest that in female mosquitoes, there are at least 4 developmental switches that alter JH synthesis by modulating the flux at distinctive points in both pathways.

No effect of glutamine supplementation and hyperoxia on oxidative metabolism and performance during high-intensity exercise

Glutamine enhances the exercise-induced expansion of the tricarboxylic acid intermediate pool. The aim of the present study was to determine whether oral glutamine, alone or in combination with hyperoxia, influenced oxidative metabolism and cycle time-trial performance. Eight participants consumed either placebo or 0.125 g · kg body mass−1 of glutamine in 5 ml · kg body mass−1 placebo 1 h before exercise in normoxic (control and glutamine respectively) or hyperoxic (FiO2 = 50%; hyperoxia and hyperoxia + glutamine respectively) conditions. Participants then cycled for 6 min at 70% maximal oxygen uptake ([Vdot]O2max) immediately before completing a brief high-intensity time-trial (∼4 min) during which a pre-determined volume of work was completed as fast as possible. The increment in pulmonary oxygen uptake during the performance test (Δ[Vdot]O2max, P = 0.02) and exercise performance (control: 243 s, s x = 7; glutamine: 242 s, s x = 3; hyperoxia: 231 s, s x = 3; hyperoxia + glutamine: 228 s, s x = 5; P < 0.01) were significantly improved in hyperoxic conditions. There was some evidence that glutamine ingestion increased Δ[Vdot]O2max in normoxia, but not hyperoxia (interaction drink/FiO2, P = 0.04), but there was no main effect or impact on performance. Overall, the data show no effect of glutamine ingestion either alone or in combination with hyperoxia, and thus no limiting effect of the tricarboxylic acid intermediate pool size, on oxidative metabolism and performance during maximal exercise.

Tricarboxylic Acid Cycle Intermediate Pool Size

The tricarboxylic acid (TCA) cycle is the major final common pathway for oxidation of carbohydrates, lipids and some amino acids, which produces reducing equivalents in the form of nicotinamide adenine dinucleotide and flavin adenine dinucleotide that result in production of large amounts of adenosine triphosphate (ATP) via oxidative phosphorylation. Although regulated primarily by the products of ATP hydrolysis, in particular adenosine diphosphate, the rate of delivery of reducing equivalents to the electron transport chain is also a potential regulatory step of oxidative phosphorylation. The TCA cycle is responsible for the generation of approximately 67% of all reducing equivalents per molecule of glucose, hence factors that influence TCA cycle flux will be of critical importance for oxidative phosphorylation. TCA cycle flux is dependent upon the supply of acetyl units, activation of the three non-equilibrium reactions within the TCA cycle, and it has been suggested that an increase in the total concentration of the TCA cycle intermediates (TCAi) is also necessary to augment and maintain TCA cycle flux during exercise. This article reviews the evidence of the functional importance of the TCAi pool size for oxidative metabolism in exercising human skeletal muscle. In parallel with increased oxidative metabolism and TCA cycle flux during exercise, there is an exercise intensity-dependent 4- to 5-fold increase in the concentration of the TCAi. TCAi concentration reaches a peak after 10-15 minutes of exercise, and thereafter tends to decline. This seems to support the suggestion that the concentration of TCAi may be of functional importance for oxidative phosphorylation. However, researchers have been able to induce dissociations between TCAi pool size and oxidative energy provision using a variety of nutritional, pharmacological and exercise interventions. Brief periods of endurance training (5 days or 7 weeks) have been found to result in reduced TCAi pool expansion at the start of exercise (same absolute work intensity) in parallel with either equivalent or increased oxidative energy provision. Cycloserine inhibits alanine aminotransferase, which catalyses the predominant anaplerotic reaction in exercising human muscle. When infused into contracting rat hindlimb muscle, TCAi pool expansion was reduced by 25% with no significant change in oxidative energy provision or power output. Glutamine supplementation has been shown to enhance TCAi pool expansion at the start of exercise with no increase in oxidative energy provision. In summary, there is a consistent dissociation between the extent of TCAi pool expansion at the onset of exercise and oxidative energy provision. At the other end of the spectrum, the parallel loss of TCAi, glycogen and adenine nucleotides and accumulation of inosine monophosphate during prolonged exercise has led to the suggestion that there is a link between muscle glycogen depletion, reduced TCA cycle flux and the development of fatigue. However, analysis of serial biopsies during prolonged exercise demonstrated dissociation between muscle TCAi content and both muscle glycogen content and muscle oxygen uptake. In addition, the delay in fatigue development achieved through increased carbohydrate availability does not attenuate TCAi reduction during prolonged exercise. Therefore, TCAi concentration in whole muscle homogenate does not seem to be of functional importance. However, TCAi content can currently only be measured in whole muscle homogenate rather than the mitochondrial subfraction where TCA cycle reactions occur. In addition, anaplerotic flux rather than TCAi content per se is likely to be of greater importance in determining TCA cycle flux, since TCAi content is probably merely reflective of anaplerotic substrate concentration. Methodological advances are required to allow researchers to address the questions of whether oxidative phosphorylation is limited by mitochondrial TCAi content and/or anaplerotic flux.

Metabolic alterations beyond fatty acid oxidation defects in PPARα null mice hearts

Subjects with fatty acid oxidation (FAO) defects develop a cardiomyopathy, yet the underlying mechanism is unclear. Using our established working mouse heart model and 13C-methodology, we compared the metabolic and functional response of hearts from PPARα null mice, a model of FAO defect, and control C57BL/6 mice at two workloads. Compared to controls, perfused hearts from PPARα null mice depicted functional parameters similar to controls at 12 mmHg preload, but displayed an impaired response to a raise in preload as reflected by a 20% decline in aortic flow and cardiac efficiency, and enhanced MVO2 (20%) and lactate dehydrogenase release (2-fold) (p<0.05). At the metabolic level, these hearts showed the expected shift from FA (4-fold down) to carbohydrate (CHO: 2-fold up; P<0.001), yet flux through anaplerotic pyruvate carboxylation (PC) expressed relative to citric acid cycle (CAC) or pyruvate decarboxylation (PDC), as well as CAC intermediate levels, were similar to controls at both preloads. However, glycolytic rates, expressed as absolute values or relative to PDC were differentially affected by preloads, suggesting a potential mismatch between cytosolic and mitochondrial CHO metabolism in PPARα null mice hearts. Interestingly, when clamped in vivo, these hearts showed higher levels of citrate and malate, suggesting additional, yet-to-be identified, factor(s) determining CAC intermediate pool size in vivo. Gene expression analysis revealed no change for PC, but 20% lower propionyl-CoA carboxylase mRNA levels. Collectively, our data highlight metabolic alterations in PPARα null mice hearts, beyond their lower FAO, which may determine their response to increased energy demand. (Supported by NIH & CIHR)

Multienzyme mevalonate pathway bioreactor.

The five-carbon metabolic intermediate isopentenyl diphosphate constitutes the basic building block for the biosynthesis of all isoprenoids in all forms of life. Two distinct pathways lead from amphibolic intermediates to isopentenyl diphosphate. The Gram-positive cocci and certain other pathogenic bacteria employ exclusively the mevalonate pathway, a set of six enzyme-catalyzed reactions that convert 3 mol of acetyl-CoA to 1 mol each of carbon dioxide and isopentenyl diphosphate. The survival of the Gram-positive cocci requires a fully functional set of mevalonate pathway enzymes. These enzymes therefore represent potential targets of inhibitors that might be employed as antibiotics directed against multidrug-resistant strains of certain bacterial pathogens. A rapid throughput, bioreactor-based assay to assess the effects of potential inhibitors on several enzymes simultaneously should prove useful for the survey of candidate inhibitors. To approach this goal, and as a proof of concept, we employed enzymes from the Gram-positive pathogen Enterococcus faecalis. Purified recombinant enzymes that catalyze the first three reactions of the mevalonate pathway were immobilized in two kinds of continuous flow enzyme bioreactors: a classical hollow fiber bioreactor and an immobilized plug flow bioreactor that exploited a novel method of enzyme immobilization. Both bioreactor types employed recombinant acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase from E. faecalis to convert acetyl-CoA to mevalonate, the central intermediate of the mevalonate pathway. Reactor performance was monitored continuously by spectrophotometric measurement of the concentration of NADPH in the reactor effluent. Additional potential applications of an Ni(++) affinity support bioreactor include using recombinant enzymes from extremophiles for biosynthetic applications. Finally, linking a Ni(++) affinity support bioreactor to an HPLC-mass spectrometer would provide an experimental and pedagogical tool for study of metabolite flux and pool sizes of intermediates to model regulation in intact cells.

A mathematical model of C(4) photosynthesis: The mechanism of concentrating CO(2) in NADP-malic enzyme type species.

A computer model comprising light reactions in PS II and PS I, electron-proton transport reactions in mesophyll and bundle sheath chloroplasts, all enzymatic reactions and most of the known regulatory functions of NADP-ME type C(4) photosynthesis has been developed as a system of differential budget equations for intermediate compounds. Rate-equations were designed on principles of multisubstrate-multiproduct enzyme kinetics. Some of the 275 constants needed (DeltaG(0)' and K (m) values) were available from literature and others (V (m)) were estimated from reported rates and pool sizes. The model provided good simulations for rates of photosynthesis and pool sizes of intermediates under varying light, CO(2) and O(2). A basic novelty of the model is coupling of NADPH production via NADP-ME with ATP production and regulation of the C(3) cycle in bundle sheath chloroplasts. The functional range of the ATP/NADPH ratio in bundle sheath chloroplasts extends from 1.5 to 2.1, being energetically most efficient around 2. In the presence of such stoichiometry, the CO(2) concentrating function can be explained on the basis of two processes: (a) extra ATP consumption for starch and protein synthesis in bundle sheath leads to a faster NADPH and CO(2) import compared with CO(2) fixation in bundle sheath, and (b) the residual photorespiratory activity consumes RuBP by oxygenation, NADPH and ATP and causes the imported CO(2) to accumulate in bundle sheath cells. As a wider application, the model may be used for predicting results of genetic engineering of plants.

Tricarboxylic acid cycle intermediate pool size and estimated cycle flux in human muscle during exercise.

We examined the relationship between tricarboxylic acid (TCA) cycle intermediate (TCAI) pool size, TCA cycle flux (calculated from leg O2 uptake), and pyruvate dehydrogenase activity (PDHa) in human skeletal muscle. Six males performed moderate leg extensor exercise for 10 min, followed immediately by intense exercise until exhaustion (3.8 +/- 0.5 min). The sum of seven measured TCAI (SigmaTCAI) increased (P </= 0.05) from 1.39 +/- 0.11 at rest to 2. 88 +/- 0.31 after 10 min and to 5.38 +/- 0.31 mmol/kg dry wt at exhaustion. TCA cycle flux increased approximately 70-fold during submaximal exercise and was approximately 100-fold higher than rest at exhaustion. PDHa corresponded to 77 and 90% of TCA cycle flux during submaximal and maximal exercise, respectively. The present data demonstrate that a tremendous increase in TCA cycle flux can occur in skeletal muscle despite a relatively small change in TCAI pool size. It is suggested that the increase in SigmaTCAI during exercise may primarily reflect an imbalance between the rate of pyruvate production and its rate of oxidation in the TCA cycle.

Cytoplasmic domains of the common beta-chain of the GM-CSF/IL-3/IL-5 receptors that are required for inducing differentiation or clonal suppression in myeloid leukaemic cell lines.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that controls the production and function of myeloid cells by interaction with a cell surface receptor composed of a specific ligand-binding alpha-chain (hGMRalpha) and a shared signal-transducing beta-chain (beta c). Co-expression of human GMR alpha-chain and wild-type human beta c in two murine leukaemic cell lines (M1 and WEHI-3B D+) conferred the ability to terminally differentiate into macrophages when stimulated with human GM-CSF. Analysis of cytoplasmic truncation mutants of beta c showed that residues to amino acid 783 (numbering from the first amino acid of the leader sequence) were sufficient for the GM-CSF-dependent induction of all aspects of differentiation in both cell types. However, shorter truncations selectively lost, in a cell-specific manner, first the capacity to induce macrophage migration in agar and then cell surface differentiation antigens and clonal suppression of proliferative potential. The data suggest that different aspects of the differentiated phenotype can be dissociated with the required signalling pathways originating from distinct regions of the receptor cytoplasmic domain and cooperating to produce a fully differentiated macrophage. The cooperativity of these pathways and limiting cell signalling intermediate pool sizes could explain the observed cell line differences and may have implications for normal haemopoiesis.

Why and when channelling can decrease pool size at constant net flux in a simple dynamic channel

Cornish-Bowden and Cárdenas (Cornish-Bowden, A. and Cárdenas M.L. (1993) Eur. J. Biochem. 213, 87–92) have suggested that simulation results previously published by us (Mendes, P., Kell, D.B. and Westerhoff, H.V. (1992) Eur. J. Biochem. 204, 255–266) which had demonstrated that large reductions of intermediate pool sizes could be accompanied by increasing channel flux in a model metabolic pathway, were an artefact of changes in the pathway's on erall flux of the order of 0.0075%, or of inappropriate alterations of enzyme activities. They also asserted to prove that “channelling of an intermediate cannot affect its free concentration at constant net flux”. We consider the co-response of the intermediate metabolite concentration (‘pool’) and the channel flux to changes in kinetic (or thermodynamic) parameters. Both by analytical proofs and by numerical examples we show that this co-response can be positive, negative or null, depending on the parameter change. In particular, we prove that there is always a number of ways of changing parameters such that the intermediate metabolite concentration decreases with increasing channel flux, whether the total flux varies or is constant. We also show that increased stability of the (dynamic) enzyme-intermediate-enzyme complex, as well as a single parameter change that similarly displays no cross-over effects, car, lead to decreased to decreased intermediate metabolite concentration and increased channel flux at constant total flux. In general, a non-zero co-response of the intermediate metabolite concentration (‘pool’) and the channel flux to changes in kinetic (or other) parameters is the rule rather than the exception. More specifically: (i) The algebraic analysis (‘general proof’) given in Cornish-Bowden and Cárdenas (1993) contains the constraint that the elasticities of various steps to the modulation parameters which were used to vary the channel flux at constant net flux were unity. This is an unfortunate and unnecessary constraint which, when lifted, means that the concentration of the pool in the general case can indeed change at constant net flux. A ‘simplified proof’ given in Cornish-Bowden and Cárdenas (1993) also fails, due in addition to the consequent failure to include mass conservation relations for some of the enzymes. (ii) In the systems studied by Cornish-Bowden and Cárdenas (1993), flux is properly to be considered as a variable (since it varies during the transition to the steady state), and not a parameter, and as such cannot per se affect the magnitude of other variables in the steady state. (iii) By relaxing the constraint referred to in (i), and by making dual modulations (i.e., of more than one parameter at once) which are different from those carried out in Cornish-Bowden and Cárdenas (1993) we find many instances in which channelling (described by a parameter p) does significantly affect the concentration of the pool intermediate C at constant total flux. (iv) In the same pathways, but in which the flux is held constant by setting it via a zero-order flux-generating reaction, the addition of a channel is also able significantly to modulate the size of the pool at constant total flux. Our results show that the effectiveness of channelling in decreasing a pool, even at constant flux, is very much a reality.

The carbon flux to triacylglycerol in maturing oilseed rape embryos.

Oilseed rape is an important agricultural crop with the seed oil having many industrial as well as nutritional uses [l]. Considerable work has been carried out on the enzymes leading to the formation of storage TAGS by the Kennedy pathway [2]. We have examined this pathway for possible points which might show significant flux control and previous data have indicated that DAGAT (the final enzyme) may do so when TAG accumulation is high [3]. We have found that TAG synthesis was higher in the light compared with dark periods and have used these conditions to manipulate carbon flux. A low-erucate spring variety of oilseed rape (Brmsica napus L. cv Shiralee) was grown [4] and seeds harvested at 30 DAP, either towards the end of the dark period or 4 hr into the light period. For lipid analysis the seeds were steam-killed and the embryos rapidly dissected into [so-propanol, heated for 30 min at 70°C and analyzed as previously [4]. For n.m.r. studies and enzyme preparations the seeds were stored at -70°C until needed. Embryos were dissected out a few at a time and again stored at -70°C prior to use. In the P-n.m.r. analyses for G3P, neutralized perchloric acid extracts were prepared !?om 4 g frozen embryos [5]. For G3PAT, LPAAT and DAGAT assays radiolabelled substrates were used and the lipid products extracted, separated and analyzed [4]. PAP activity was determined by measuring the production of Pi, by the method of Chen et al. [6] as modified by Ichihara et al. [7]. The rate of lipid synthesis in the light was higher than in the dark as deduced from radiolabelling experiments involving [Clacetate and ['Hlglycerol precursors [S]. When we compared pool sizes of intermediates in the Kennedy pathway, there were significant changes in their amounts on light treatment (Table 1). Thus, under conditions of increased carbon flux through the pathway to TAG, there was a significant build-up of material in the PA and DAG pools. There was also an apparent increase in the level of G3P, although because of the amount of material needed for n.m.r. we were only able to make a single determination. The increased levels of endogenous PA and DAG suggested that PAP and DAGAT could be limiting for fat accumulation during light treatment. Microsomal fractions from embryos are capable of incorporating radioactivity from precursors into TAG [S]. In addition, subcellular fractionation studies have shown that activity of Kennedy pathway enzymes are found in such membranes. Therefore, we measured the 'four enzymes in microsomes from embryos derived from rape plants subjected to light or dark treatment. In terms of either total or specific activity (Table 2), there were no significant changes for any of the enzymes assayed. Therefore, the increases in pool sizes induced by light treatment (Table 1) cannot be explained by decreases in particular enzyme activities. Three enzymes of the Kennedy pathway had relatively low activities (Table 2) and the substrate pool sizes for each rose when carbon flux was increased through the pathway (Table 1). 31

Levels of ethanolamine intermediates in the human and rat visual system structures: Comparison with neural tissues of a lower vertebrate ( Mustelus canis) and an invertebrate ( Loligo pealei)

Levels of ethanolamine intermediates in the retina and optic nerves of autopsied human donors and in the rat visual system (retina, optic nerve, lateral geniculate body, superior colliculus) were measured. Amounts were also obtained from the retina, optic nerve, and optic tectum of a primitive elasmobranch, the smooth dogfish Mustelus canis, and from the related nervous structures (retina, optic lobe, fin nerve, and stellate ganglia) of a marine invertebrate, the squid Loligo pealei. In all regions of the human and rat nervous system, the pool size of CDP-ethanolamine (values ranging between 10–31 nmol/g wet wt) was much smaller than that of free ethanolamine (values ranging between 197–395 nmol/g wet wt), whereas glycerophosphorylethanolamine was present in relatively high content (values ranging between 125–280 nmol/g wet wt). In nervous system regions of the dogfish and squid, the distribution of values followed the same general trend as observed for humans and rats, even if all regions had less ethanolamine intermediates compared to the mammalian counterpart. In dogfish and squid retina, glycerophosphorylethanolamine showed the highest pool size among the ethanolamine derivatives analyzed (16 and 44 nmol/g wet wt, respectively). The present study confirms the basic similarity of ethanolamine intermediate pool size patterns in the nervous system structures (with the exception of the retina) of animal species which have widely different phylogenetic positions. The data support the proposal that the levels reached by ethanolamine and its derivatives in the nervous tissue is the result of an ancient evolutionary development of metabolic pathways for the maintenance of phosphatidylethanolamine membraneous content.

Alterations in substrate utilization in the reperfused myocardium: a direct analysis by 13C NMR.

An alternative 13C NMR method which allows direct determination of substrate oxidation in tissue for up to three competing 13C-enriched substrates is presented. Oxidation of competing substrates can be measured by 13C NMR spectroscopy under non-steady-state conditions if the relative areas of the glutamate C3 and C4 resonances can be determined. The accuracy of this measurement is limited during brief exposure to 13C-enriched substrates because of the low enrichment in the C3 carbon. The glutamate C4 resonance from a tissue sample which has oxidized a combination of [1,2-13C]acetate (or a uniformly enriched fatty acid mixture) and [3-13C]lactate appears as a nine-line resonance consisting of four multiplet components: a singlet (C4S), two doublets with differing one-bond coupling constants (C4D34 and C4D45), and a quartet (C4Q). It is shown that the sum of the C4S + C4D34 resonance areas versus the C4D45 + C4Q resonance areas directly reports the relative utilization of [3-13C]lactate versus [1,2-13C]acetate, respectively, regardless of citric acid cycle intermediate pool sizes or carbon flux through anaplerotic reactions. We also show that homonuclear 13C decoupling of the glutamate C2 resonance collapses the C3 resonance multiplet into an apparent triplet (actually, a singlet plus a doublet); the relative area of the singlet component reflects the amount of unlabeled acetyl-CoA entering the cycle. The method has been used to determine the contribution of lactate/acetate/glucose to acetyl-CoA in normoxic and reperfused rat hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Levels of choline intermediates in the visual system structures and in peripheral nerve of the rat: Comparison with neural tissues of a lower vertebrate (Mustelus canis) and an invertebrate (Loligo pealei)

The levels of choline intermediate endogenous pools in structures of the visual system (retina, optic nerve, lateral geniculate body, superior colliculus) and in sciatic nerve of adult (4-month-old) and young (30-day-old) rats were measured. The amounts were also obtained from retina, optic nerve, optic tectum and cranio-spinal nerves of a primitive elasmobranch, the smooth dogfish Mustelus canis, and from related nervous structures (retina, optic lobe, fin nerve, stellar nerve and stellate ganglia) of a marine invertebrate, the squid Loligo pealei. In all regions of rat nervous system, the pool size of CDP-choline was much smaller than that of free choline, whereas GroPCho was present in a relatively higher content. The pool sizes of choline intermediates in 30- and 120-day-old rats were nearly the same. In nervous system regions of the dogfish and squid, the values followed the same general trend as observed for rat. Squid nervous tissues had the lowest choline and GroPCho contents. The rat retina showed the lowest glycerophosphorylcholine phosphodiesterase activity. The chemical studies described here confirm the basic similarity in the pattern of choline intermediate pool sizes among animal species widely different in phylogenetic position. The data highly reinforce the idea that the precursor role of choline and catabolic pathways for the maintenance of the PtdCho membraneous pool seem highly conserved during evolution.

The Effect of Sodium Nutrition on the Pool Sizes of Intermediates of the C Photosynthetic Pathway

Changes in C4 pathway intermediates in response to sodium nutrition, consistent with the hypothesis that there is a limitation in the conversion of pyruvate to phosphoenolpyruvate (PEP) in the mesophyll cells in sodium-deficiency, were observed in the C4 species, Kochia childsii, Chloris gayana, Amaranthus edulis, Amaranthus tricolor and Atriplex spongiosa. In the C3 species Lycopersicon esculentum (tomato), no differences were observed when grown with or without added sodium. Of the group 1 elements, only sodium, irrespective of the salt supplied to deficient cultures, effected these changes in the C4 species. In the light, concentrations of aspartate, PEP and 3-phosphoglycerate (3-PGA) were lower and those of pyruvate and alanine were greater in sodium-deficient than normal plants. In the dark, concentrations of aspartate, pyruvate, alanine, PEP and 3-PGA were similar in sodium-deficient and normal plants. In the C4 species Atriplex spongiosa, the concentration of sodium required to bring about these changes corresponded to that required for growth responses. Rapid increases in the concentrations of malate, PEP and 3-PGA and decreases in pyruvate and alanine were observed following the direct application of sodium to leaves.