External Substrate Concentration Research Articles

Continuous Production of DHA and EPA Ethyl Esters via Lipase-Catalyzed Transesterification in an Ultrasonic Packed-Bed Bioreactor

Ethyl esters of omega-3 fatty acids are active pharmaceutical ingredients used for the reduction in triglycerides in the treatment of hyperlipidemia. Herein, an ultrasonic packed-bed bioreactor was developed for continuous production of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) ethyl esters from DHA+EPA concentrate and ethyl acetate (EA) using an immobilized lipase, Novozym® 435, as a biocatalyst. A three-level–two-factor central composite design combined with a response surface methodology (RSM) was employed to evaluate the packed-bed bioreactor with or without ultrasonication on the conversion of DHA + EPA ethyl ester. The highest conversion of 99% was achieved with ultrasonication at the condition of 1 mL min−1 flow rate and 100 mM DHA + EPA concentration. Our results also showed that the ultrasonic packed-bed bioreactor has a higher external mass transfer coefficient and a lower external substrate concentration on the surface of the immobilized enzyme. The effect of ultrasound was also demonstrated by a kinetic model in the batch reaction that the specificity constant (V′max/K2) in the ultrasonic bath was 8.9 times higher than that of the shaking bath, indicating the ultrasonication increased the affinity between enzymes and substrates and, therefore, increasing reaction rate. An experiment performed under the highest conversion conditions showed that the enzyme in the bioreactor remained stable at least for 5 days and maintained a 98% conversion.

A systems-level model reveals that 1,2-Propanediol utilization microcompartments enhance pathway flux through intermediate sequestration

The spatial organization of metabolism is common to all domains of life. Enteric and other bacteria use subcellular organelles known as bacterial microcompartments to spatially organize the metabolism of pathogenicity-relevant carbon sources, such as 1,2-propanediol. The organelles are thought to sequester a private cofactor pool, minimize the effects of toxic intermediates, and enhance flux through the encapsulated metabolic pathways. We develop a mathematical model of the function of the 1,2-propanediol utilization microcompartment of Salmonella enterica and use it to analyze the function of the microcompartment organelles in detail. Our model makes accurate estimates of doubling times based on an optimized compartment shell permeability determined by maximizing metabolic flux in the model. The compartments function primarily to decouple cytosolic intermediate concentrations from the concentrations in the microcompartment, allowing significant enhancement in pathway flux by the generation of large concentration gradients across the microcompartment shell. We find that selective permeability of the microcompartment shell is not absolutely necessary, but is often beneficial in establishing this intermediate-trapping function. Our findings also implicate active transport of the 1,2-propanediol substrate under conditions of low external substrate concentration, and we present a mathematical bound, in terms of external 1,2-propanediol substrate concentration and diffusive rates, on when active transport of the substrate is advantageous. By allowing us to predict experimentally inaccessible aspects of microcompartment function, such as intra-microcompartment metabolite concentrations, our model presents avenues for future research and underscores the importance of carefully considering changes in external metabolite concentrations and other conditions during batch cultures. Our results also suggest that the encapsulation of heterologous pathways in bacterial microcompartments might yield significant benefits for pathway flux, as well as for toxicity mitigation.

Cellular and intracellular transport of vitamin C. The physiologic aspects

Vitamin C requirement is satisfied by natural sources and vitamin C supplements in the ordinary human diet. The two major forms of vitamin C in the diet are L-ascorbic acid and L-dehydroascorbic acid. Both ascorbate and dehydroascorbate are absorbed along the entire length of the human intestine. The reduced form, L-ascorbic acid is imported by an active mechanism, requiring two sodium-dependent vitamin C transporters (SVCT1 and SVCT2). The transport of the oxidized form, dehydroascorbate is mediated by glucose transporters GLUT1, GLUT3 and possibly GLUT4. Initial rate of uptake of both ascorbate and dehydroascorbate is saturable with increasing external substrate concentration. Vitamin C plasma concentrations are tightly controlled when the vitamin is taken orally. It has two simple reasons, on the one hand, the capacity of the transporters is limited, on the other hand the two Na+-dependent transporters can be down-regulated by an elevated level of ascorbate.

A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp(112)) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.

Relationship between phosphate affinities and cell size and shape in various bacteria and phytoplankton

Substrate affinity expresses the ability of an osmotroph organism to compete for a sub- strate at permanently low external concentrations and is thus a central parameter in conceptual and mathematical models of aquatic food webs. Assuming diffusion transport in the surrounding medium to be the limiting process at low external substrate concentrations, the theoretical maximum affinity (α max ) and its dependence on cell size and shape for a given osmotroph organism can be calculated from Fick's law of diffusion in combination with knowledge of the amount of substrate required to form a new cell. For a non-respired substrate, the actual affinity (α) can also be expressed as the bio- mass-specific turnover rate of the substrate, α = (TB) -1 . Combining a measure of biomass (B), with determination of substrate turnover time (T), the affinity can thus be determined experimentally. We used this approach to compare measured with theoretical maximum affinities for phosphate in labo- ratory cultures of osmotrophic microorganisms. For bacteria and autotrophic flagellates, we found relatively good agreement between experimental and theoretical maximum values, suggesting that diffusion limitation is actually approached in P-limited cultures. Assuming P-free vacuoles, the the- ory predicts diatom affinities to exceed that of similarly sized flagellates. This prediction is consistent with our experimental observations. Previous reports of diatoms being unsuccessful under P-limited conditions may therefore need a more complex explanation than lack of competitive ability in diatoms.

α-Isopropylmalate, a Leucine Biosynthesis Intermediate in Yeast, Is Transported by the Mitochondrial Oxalacetate Carrier

In Saccharomyces cerevisiae, alpha-isopropylmalate (alpha-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported alpha-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di- and tricarboxylate transporters as well as the previously proposed alpha-IPM transporter. Transport was saturable with a half-saturation constant of 75 +/- 4 microm for alpha-IPM and 0.31 +/- 0.04 mm for beta-IPM and was inhibited by the substrates of Oac1p. Though not transported, alpha-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, alpha-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an alpha-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with alpha-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant DeltaOAC1DeltaLEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of DeltaOAC1DeltaLEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates alpha-ketoisocaproate and alpha-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of alpha-IPM, probably in exchange for oxalacetate.

A unified phenomenological analysis of the experimental velocity curves in molecular motors

We present a unified phenomenological kinetic framework to analyze the experimental data of several motor proteins (either linear or rotatory). This formalism allows us to discriminate the characteristic times of most relevant subprocesses. Explicitly, internal mechanical as well as chemical times are taken into account and joined together in a full-cycle time where effusion, diffusion and chemical rates, viscoelastic friction, and overdamped motion are considered. This approach clarifies the most relevant mechanisms in a particular motor by using the available experimental data of velocity versus external load and substrate concentration. We apply our analysis to three real molecular motors for which enough experimental data are available: the bacterial flagellar motor [Yoshiyuki et al., J. Mol. Biol. 377, 1043 (2003)], conventional kinesin (kinesin-1) [Block et al., Proc. Natl. Acad. Sci. U.S.A. 100, 2351 (2003)], and a RAN polymerase [Abbondanzieril, Nature (London) 438, 460 (2003)]. Moreover, the mechanism of stalling a motor is revised and split into two different concepts (mechanical and chemical stalling) that shed light to the understanding of backstepping in kinesin-1.

Uptake kinetics of different arsenic species by Brassica carinata

The time- and concentration-dependent uptake kinetics for arsenate and arsenite were determined in 15-day-old excised roots. In both cases, arsenite showed a mono-phasic influx with the isotherm data fitting a linear model better than a non-linear one. The time- and the concentration-dependent uptake of arsenate displayed a hyperbolic kinetic. Greater uptake of arsenate, compared with arsenite, was found especially at lower external substrate concentrations. Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems because arsenate uptake was strongly inhibited in the presence of phosphate, whereas arsenite uptake was not affected.

Establishing a Definitive Stoichiometry for the Na +/Monocarboxylate Cotransporter SMCT1

Several different stoichiometries have been proposed for the Na +/monocarboxylate cotransporter SMCT1, including variable Na +/substrate stoichiometry. In this work, we have definitively established an invariant 2:1 cotransport stoichiometry for SMCT1. By using two independent means of assay, we first showed that SMCT1 exhibits a 2:1 stoichiometry for Na +/lactate cotransport. Radiolabel uptake experiments proved that, unlike lactate, propionic acid diffuses passively through oocyte membranes and, consequently, propionate is a poor candidate for stoichiometric determination by these methods. Although we previously determined SMCT1 stoichiometry by measuring reversal potentials, this technique produced erroneous values, because SMCT1 simultaneously mediates both an inwardly rectifying cotransport current and an outwardly rectifying anionic leak current; the leak current predominates in the range where reversal potentials are observed. We therefore employed a method that compared the effect of halving the external Na + concentration to the effect of halving the external substrate concentration on zero-current potentials. Both lactate and propionate were cotransported through SMCT1 using 2:1 stoichiometries. The leak current passing through the protein has a 1 osmolyte/charge stoichiometry. Identification of cotransporter stoichiometry is not always a trivial task and it can lead to a much better understanding of the transport activity mediated by the protein in question.

ScOPT1 and AtOPT4 function as proton-coupled oligopeptide transporters with broad but distinct substrate specificities

A group of OPTs (oligopeptide transporters) exclusively identified in plants and fungi are proposed to transport oligopeptides and derivatives of three to six amino acids in length, but their transport mechanisms and biological functions are poorly understood. We expressed the Saccharomyces cerevisiae (yeast) OPT ScOPT1 and five Arabidopsis thaliana AtOPTs in Xenopus laevis oocytes for two-electrode voltage-clamp studies. ScOPT1 produced inward currents in response to GSH or GSSG, the phytochelatin (PC) PC2 and oligopeptides including the tetrapeptide GGFL, but not KLGL. Inward currents were dependent on the external proton and substrate concentrations, with high affinity for both. This and the inward currents evoked by substrates with net negative charges showed that ScOPT1 is a proton-coupled transporter. ScOPT1 displayed highest apparent affinity for PC2, with small differences in the maximal current among substrates. Glutathione transport by any of the tested AtOPTs, including AtOPT6, was not detected in yeast growth complementation assays. With AtOPT4, initially only small KLGL-dependent currents were recorded in batches of oocytes showing high ScOPT1 expression. AtOPT4 expression was optimized by swapping the 5'-untranslated region with that of ScOPT1. AtOPT4 displayed a higher affinity for KLGL than ScOPT1 did for any peptide. AtOPT4-mediated KLGL transport was detectable at pH 5.0, but not at pH 6.0 or 7.0. Taken together, our results demonstrate that ScOPT1 and AtOPT4 are proton-coupled OPTs with broad but distinct substrate specificities and affinities.

Impact of apoplast volume on ionic relations in plant cells.

Ionic relations of plant cells with free-running transmembrane voltage, V, can be modelled by electrocoupling of known, V-relevant and V-gated transporters. For cells in vitro, with constant external substrate concentrations, alternating states of solute uptake and release can be predicted. Here the model is extended to treat cells with limited external substrates, e.g., parenchyma cells in situ with a small external space, the apoplast. This model accounts also for cytoplasmic H+ buffering and apoplastic buffering of H+, K+ and Ca2+ by fixed anions. In general, the model converges to thermodynamic equilibrium for K+ between apoplast and symplast, and to equal steady-state rates of uptake and release for Cl- and H+. Oscillations of this model are rare and very sensitive to the volume portion of the apoplast.

Citrin and aralar1 are Ca(2+)-stimulated aspartate/glutamate transporters in mitochondria.

The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF-hand Ca(2+)-binding motifs in their N-terminal domains. Both proteins and their C-terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H(+). Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca(2+) on the external side of the inner mitochondrial membrane, where the Ca(2+)-binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca(2+) through a mechanism independent of Ca(2+) entry into mitochondria, and suggest a novel mechanism of Ca(2+) regulation of the aspartate/malate shuttle.

Arg-425 of the Citrate Transporter CitP Is Responsible for High Affinity Binding of Di- and Tricarboxylates

The citrate transporter of Leuconostoc mesenteroides (CitP) catalyzes exchange of divalent anionic citrate from the medium for monovalent anionic lactate, which is an end product of citrate degradation. The exchange generates a membrane potential and thus metabolic energy for the cell. The mechanism by which CitP transports both a divalent and a monovalent substrate was the subject of this investigation. Previous studies indicated that CitP is specific for substrates containing a 2-hydroxycarboxylate motif, HO-CR(2)-COO(-). CitP has a high affinity for substrates that have a "second" carboxylate at one of the R groups, such as divalent citrate and (S)-malate (Bandell, M., and Lolkema, J. S. (1999) Biochemistry 38, 10352-10360). Monovalent anionic substrates that lack this second carboxylate were found to bind with a low affinity. In the present study we have constructed site-directed mutants, changing Arg-425 into a lysine or a cysteine residue. By using two substrates, i.e. (S)-malate and 2-hydroxyisobutyrate, the substrate specificity of the mutants was analyzed. In both mutants the affinity for divalent (S)-malate was strongly decreased, whereas the affinity for monovalent 2-hydroxyisobutyrate was not. The largest effect was seen when the arginine was changed into the neutral cysteine, which reduced the affinity for (S)-malate over 50-fold. Chemical modification of the R425C mutant with the sulfhydryl reagent 2-aminoethyl methanethiosulfonate, which restores the positive charge at position 425, dramatically reactivated the mutant transporter. The R425C and R425K mutants revealed a substrate protectable inhibition by other sulfhydryl reagents and the lysine reagent 2,4,6-trinitrobenzene sulfonate, respectively. It is concluded that Arg-425 complexes the charged carboxylate present in divalent substrates but that is absent in monovalent substrates, and thus plays an important role in the generation of the membrane potential.

Mathematical Treatment of Transport Data of Bacterial Transport Systems to Estimate Limitation in Diffusion through the Outer Membrane

Bacterial transport systems are traditionally treated as enzymes exhibiting a saturable binding site giving rise to an apparent Kmof transport, whereas the maximal rate of transport is regarded equivalent to the Vmaxof enzymatic reactions. Thus, the Michaelis–Menten theory is usually applied in the analysis of transport data and Kmand Vmaxare derived from the treatment of data obtained from the rate of transport at varying substrate concentrations. Such an analysis tacitly assumes that the substrate recognition site of the transport system is freely accessible to substrate. However, this is not always the case. In systems endowed with high affinity in the μ M range or those recognizing large substrates or those exhibiting high Vmax, the diffusion through the outer membrane may become rate determining, particularly at low external substrate concentrations. In such a situation the dependence of the overall rate of transport (from the medium into the cytoplasm) on the substrate concentration in the medium will no longer follow Michaelis–Menten kinetics. By analysing the deviation of transport data from the corresponding ideal Michaelis–Menten plot we developed a method that allows us to determine diffusion limitation through the outer membrane. The method allows us to find the correctKm of the transport system functioning at the inner membrane even under conditions of strong diffusion limitation through the outer membrane. The model was tested and validified with the Escherichia coli binding protein-dependent ABC transporter for maltose. The corresponding systems for sn -glycerol-3-phospate of Escherichia coli and the α -cyclodextrin transport of Klebsiella oxitoca were used as test systems.

A structured model of dual-limitation kinetics

A structured model of substrate-utilization kinetics that encompasses dual-limitation conditions, caused by simultaneously low concentrations of the electron donor and the electron acceptor, is developed by incorporating the internal cofactor responses into the kinetic variables. The structured model is based on an assumption that the maximum specific electron-donor-oxidation rate (q(md)) is not a constant, but is linearly controlled by the intracellular chemical potentials, log(NAD/NADH) and log(ATP/ADP . P(i)). Determination of the kinetic parameters for the dual-limitation model, using experimental data from the companion article, verifies that q(md) varies and demonstrates that the NAD/NADH ratio affects q(md) in a positive direction; thus, an increase of the ratio increases the rate of electron-donor utilization. Because the internal NAD/NADH ratio rises with an increase in S(ar) the specific electron-donor-utilization rate is accelerated by high S(a). Since the ratio also increases as the specific electron-donor-utilization rate falls, the specific rate is intrinsically accelerated by the cofactor response when it becomes low due to a depletion of electron donor. Because the cofactor responses upon changes of the external substrate concentrations are systematic, the dual-limitation model can be expressed as a function of only external concentrations of electron donor and electron acceptor, which results in a multiplicative (double-Monod) form. Thus, dual limitation by both substrates reduces the overall reaction rate below the rate expected from single limitation by only one, the most severely limiting, substrate. (c) 1996 John Wiley & Sons, Inc.

Modelling and Adaptive Control of Biochemical Processes

For a general dynamic biochemical process described by an ordinary differential equation, we introduce the concept of non-cyclic processes. This replaces the conservation of mass assumption and is sufficient to guarantee invariance of the positive orthant and boundedness of the trajectories. Non-cyclic processes are characterized in terms of the stoichiometric matrix and an algorithm is presented which decides in finitely many steps whether a process is non-cyclic or not. The concept of λ-tracking is applied to an example to regulate some external substrate concentration by the feedrate in the presence of noise corrupting the output and in the presence of input constraints.

Optimization and Control of Metabolic Systems

Evolutionary optimization principles are applied to explain the catalytic efficiency of single enzymes as well as the structural design of metabolic pathways. Special results concern (1) the optimal values of elementary rate constants of enzymatic mechanisms as functions of the external substrate and product concentrations; (2) the distribution of enzyme concentrations in metabolic pathways for states of maximal fluxes, and (3) the optimal distribution of ATP consuming and ATP producing reactions in glycolysis. Conclusions are drawn for the distribution of flux control coefficients resulting from optimization processes.

Evidence for a low-affinity, high-capacity uniport for amino acids in Bombyx mori larval midgut.

We investigated the kinetics of leucine influx as a function of external substrate concentration between 0.03 and 16 mM in brush-border membrane vesicles (BBMV) prepared from the middle region of Bombyx mori larval midgut. A detailed kinetic analysis of leucine uptake led to the identification, in parallel with the K(+)-dependent symporter for neutral amino acids, of a K(+)-independent, low-affinity, high-capacity system. The parameter values of the Michaelis constant (7.12 mM) and maximal rate of transport (4.48 nmol.7 s-1.mg protein-1) were not influenced by an external alkaline pH nor by a transmembrane electrical potential difference. The uniporter is poorly specific, as it displayed the following rank of preference: Leu, His, Val, Ile, Phe, Ser > Lys, Arg, Gln > Pro, 2-amino-2-norbornane-carboxylic acid, Ala, Gly. The kinetic analysis performed in BBMV prepared from the posterior midgut portion indicates that the low-affinity, high-capacity uniporter is present along the entire length of the silkworm larval midgut with similar expression and functional properties.

A Darwinian theory for the origin of cellular differentiation.

In this theory, cell differentiation is a two-step mechanism at each stage of development. In the first step, gene expression is unstable. It occurs stochastically and produces different cell types. In the second step gene expression is stabilized by means of cellular interactions. However, this stabilization cannot occur until the combination of cell phenotypes corresponding to the developmental stage is expressed. This selection mechanism prevents disorganizing consequences of stochasticity in gene expression and directs the embryo towards the adult stage. Instability and stochasticity in gene expression are caused by random displacement of regulators along DNA, whereas phosphorylation and/or dephosphorylation of transcriptional regulators triggered by signal transduction between cells are responsible for the stabilization of stochastic gene expression. The origin of cellular differentiation is explained as an adaptation of cells to metabolic gradients created by substrate diffusion inside growing cell populations. This mechanism provides cells with complementary metabolism, increasing their ability to use food resources. Because the metabolic gradients are dependent on external substrate concentrations, cellular differentiation can also be viewed as an extension of natural selection inside organisms.

Anion channel blockers inhibit swelling-activated anion, cation, and nonelectrolyte transport in HeLa cells.

The effect of osmotic cell swelling on the permeability of HeLa cells to a range of structurally unrelated solutes including taurine, sorbitol, thymidine, choline, and K+ (96Rb+) was investigated. For each solute tested, reduction in the osmolality of the medium from 300 to 200 mosmol/kgH2O caused a significant increase in the unidirectional influx rate. In each case, the osmotically activated transport component was nonsaturable up to external substrate concentrations of 50 mM. Inhibitors of the swelling-activated anion channel of HeLa cells [quinine, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, niflumate, 1,9-dideoxyforskolin, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and tamoxifen] blocked the osmotically activated influx of each of the different substrates tested, as well as the osmotically activated efflux of taurine and I-. Tamoxifen and NPPB were similarly effective at blocking the osmotically activated efflux of 96Rb+. The simplest of several hypotheses consistent with the data is that the osmotically activated transport of the different solutes tested here is via a swelling-activated anion-selective channel that has a significant cation permeability and a minimum pore diameter of 8-9 A.