Generation Of Metabolic Energy Research Articles

Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy

The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated. In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generated by the metabolism of a glycolytic substrate. Malolactic fermentation results in the synthesis of ATP which is inhibited by the ionophore nigericin and the F0F1-ATPase inhibitor N,N-dicyclohexylcarbodiimide. Since substrate-level phosphorylation does not occur during malolactic fermentation, the generation of metabolic energy must originate from the uptake of L-malate and/or excretion of L-lactate. The initiation of malolactic fermentation is stimulated by the presence of L-lactate intracellularly, suggesting that L-malate is exchanged for L-lactate. Direct evidence for heterologous L-malate/L-lactate (and homologous L-malate/L-malate) antiport has been obtained with membrane vesicles of an L. lactis mutant deficient in malolactic enzyme. In membrane vesicles fused with liposomes, L-malate efflux and L-malate/L-lactate antiport are stimulated by a membrane potential (inside negative), indicating that net negative charge is moved to the outside in the efflux and antiport reaction. In membrane vesicles fused with liposomes in which cytochrome c oxidase was incorporated as a proton motive force-generating mechanism, transport of L-malate can be driven by a pH gradient alone, i.e., in the absence of L-lactate as countersubstrate. A membrane potential (inside negative) inhibits uptake of L-malate, indicating that L-malate is transported an an electronegative monoanionic species (or dianionic species together with a proton). The experiments described suggest that the generation of metabolic energy during malolactic fermentation arises from electrogenic malate/lactate antiport and electrogenic malate uptake (in combination with outward diffusion of lactic acid), together with proton consumption as result of decarboxylation of L-malate. The net energy gain would be equivalent to one proton translocated form the inside to the outside per L-malate metabolized.

Oxalate:formate exchange: The basis for energy coupling in Oxalobacter

In the Gram-negative anaerobe, Oxalobacter formigenes, the generation of metabolic energy depends on the transport and decarboxylation of oxalate. We have now used assays of reconstitution to study the movements of oxalate and to characterize the exchange of oxalate with formate, its immediate metabolic derivative. Membranes of O. formigenes were solubilized with octyl-beta-D-glucopyranoside in the presence of 20% glycerol and Escherichia coli phospholipid, and detergent extracts were reconstituted by detergent dilution. [14C]Oxalate was taken up by proteoliposomes loaded with unlabeled oxalate, but not by similarly loaded liposomes or by proteoliposomes containing sulfate in place of oxalate. Oxalate transport did not depend on the presence of sodium or potassium, nor was it affected by valinomycin (1 microM), nigericin (1 microM), or a proton conductor, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (5 microM) when potassium was at equal concentration on either side of the membrane. Such data suggest the presence of an overall neutral oxalate self-exchange, independent of common cations or anions. Kinetic analysis of the reaction in proteoliposomes gave a Michaelis constant (Kt) for oxalate transport of 0.24 mM and a maximal velocity (Vmax) of 99 mumol/min/mg of protein. A direct exchange of oxalate and formate was indicated by the observations that formate inhibited oxalate transport and that delayed addition of formate released [14C]oxalate accumulated during oxalate exchange. Moreover, [14C]formate was taken up by oxalate-loaded proteoliposomes (but not liposomes), and this heterologous reaction could be blocked by external oxalate. Further studies, using formate-loaded proteoliposomes, suggested that the heterologous exchange was electrogenic. Thus, for assays in which N-methylglucamine served as both internal and external cation, formate-loaded particles took up oxalate at a rate of 2.4 mumol/min/mg of protein. When external or internal N-methylglucamine was replaced by potassium in the presence of valinomycin, there was, respectively, a 7-fold stimulation or an 8-fold inhibition of oxalate accumulation, demonstrating that net negative charge moved in parallel with oxalate during the heterologous exchange. The work summarized here suggests the presence of an unusually rapid and electrogenic oxalate2-:formate1- antiport in membranes of O. formigenes. Since a proton is consumed during the intracellular decarboxylation that converts oxalate into formate plus CO2, antiport of oxalate and formate would play a central role in a biochemical cycle consisting of (a) oxalate influx, (b) oxalate decarboxylation, and (c) formate efflux.(ABSTRACT TRUNCATED AT 400 WORDS)

Bioenergetics and Solute Transport in Lactococci

During the last few years the studies about the physiology and bioenergetics of lactic acid bacteria during growth and starvation have evolved from a descriptive level to an analysis of the molecular events in the regulation of various processes. Considerable progress has been made in the understanding of the modes of metabolic energy generation, the mechanism of homeostasis of the internal pH, and the mechanism and regulatory processes of transport systems for sugars, amino acids, peptides, and ions. Detailed studies of these transport processes have been performed in cytoplasmic membrane vesicles of these organisms in which a foreign proton pump has been introduced to generate a high proton motive force.

37] Generation of a protonmotive force in anaerobic bacteria by end-product efflux

Publisher Summary This chapter deals with the generation of a Protonmotive force in anaerobic bacteria by end-product efflux. The protonmotive force acts as a driving force or regulatory parameter in a large number of different metabolic processes. Until recently only three primary proton pumps have been described in bacteria: (1) membranebound electron transfer chains; (2) the proton-translocating adenosine triphosphatase (ATPase) complex; and (3) in halobacteria, the light-driven proton pump bacteriorhodopsin. A number of experimental approaches can supply information about the generation of metabolic energy by end-product efflux: (1) uptake experiments, to demonstrate carrier activity and to obtain information on the H + /end-product stoichiometry; (2) efflux experiments from preloaded membranes, to demonstrate end-product efflux driven Ap generation (and subsequent ATP synthesis); (3) simultaneous measurements of the end-product gradient and Ap in growing or actively metabolizing cells, to obtain information on the H + / end-product stoichiometry and possible contributions of energy recycling to the overall production of metabolic energy; and (4) yield experiments under various conditions.

Energy recycling by lactate efflux in growing and nongrowing cells of Streptococcus cremoris.

Streptococcus cremoris was grown in pH-regulated batch and continuous cultures with lactose as the energy source. During growth the magnitude and composition of the electrochemical proton gradient and the lactate concentration gradient were determined. The upper limit of the number of protons translocated with a lactate molecule during lactate excretion (the proton-lactate stoichiometry) was calculated from the magnitudes of the membrane potential, the transmembrane pH difference, and the lactate concentration gradient. In cells growing in continuous culture, a low lactate concentration gradient (an internal lactate concentration of 35 to 45 mM at an external lactate concentration of 25 mM) existed. The cell yield (Ymax lactose) increased with increasing growth pH. In batch culture at pH 6.34, a considerable lactate gradient (more than 60 mV) was present during the early stages of growth. As growth continued, the electrochemical proton gradient did not change significantly (from -100 to -110 mV), but the lactate gradient decreased gradually. The H+-lactate stoichiometry of the excretion process decreased from 1.5 to about 0.9. In nongrowing cells, the magnitude and composition of the electrochemical proton gradient was dependent on the external pH but not on the external lactate concentration (up to 50 mM). The magnitude of the lactate gradient was independent of the external pH but decreased greatly with increasing external lactate concentrations. At very low lactate concentrations, a lactate gradient of 100 mV existed, which decreased to about 40 mV at 50 mM external lactate. As a consequence, the proton-lactate stoichiometry decreased with increasing external concentrations of protons and lactate at pH 7.0 from 1 mM lactate to 1.1 at 50 mM lactate and at pH 5.5 from 1.4 at l mM lactate to 0.7 at 50 mM lactate. The data presented in this paper suggest that a decrease in external pH and an increase in external lactate concentration both result in lower proton-lactate stoichiometry values and therefore in a decrease of the generation of metabolic energy by the end product efflux process.

The interaction between electron transfer, proton motive force and solute transport in bacteria.

The properties of proton solute symport have been studied in Streptococcus cremoris, Rhodopseudomonas sphaeroides and Escherichia coli. In the homolactic fermentative organism S. cremoris the efflux of lactate is a membrane protein-mediated process, which can lead to the generation of a proton motive force. These observations support the energy-recycling model that postulates the generation of metabolic energy by end-product efflux. Studies with oxidants and reductants and specific dithiol reagents in E. coli membrane vesicles demonstrated the presence of two redox-sensitive dithiol-disulphide groups in the transport proteins of proline and lactose. The redox state of these groups is controlled by the redox potential of the environment and by the proton motive force. One redox-sensitive group is located at the inner surface, the other at the outer surface of the membrane. In Rps. sphaeroides and E. coli the activity of several transport proteins depends on the activity of the electron transfer systems. On the basis of these results a redox model for proton solute transport coupled in parallel to the electron transfer system is postulated.

Trace Elements and Blood Pressure

Essential trace elements such as zinc, iron, and copper participate in various enzyme reactions directly related to the regulation of blood pressure and indirectly related to generation of oxidative metabolic energy, alterations in blood lipid levels, and alterations in taste acuity. The toxicological action of several heavy metal ions including cadmium, lead, mercury, and thallium can cause hypertension by affecting hormone metabolism, vasoconstriction, and renal tubular function. We conclude, however, that neither deficiencies of essential elements nor the presence of toxic heavy metals are primary causes of hypertension in our population.

Metabolism of One-Carbon Compounds by Chemotrophic Anaerobes

Publisher Summary This chapter focuses on the metabolism of one-carbon (C1) compounds chemotrophic anaerobes. C1-metabolizing species possess novel physiologies that are distinctive in many ways from the autotrophic and heterotrophic microorganisms, which seemingly are more widespread in the microbial world. C1 compounds refer to any oxidizable one-carbon substrate that contains carbon-bound electrons. A C1 substrate differs noticeably from the C1 compound carbon dioxide because, in addition to being able to be reduced or assimilated, it can also be oxidized and can provide electrons for use in energy metabolism or cell synthesis. The term “chemotrophic anaerobe” refers to any obligately non-oxygen-catabolizing microbial species whose growth is solely dependent on the generation of metabolic energy from chemical substrates. The chapter examines the microbial physiology that accounts for unicarbonotrophy in anaerobes. The C1 metabolites enter anaerobic ecosystems of the biosphere either as pollutants from aerobic environments, volcanic or deep subsurface emanations, or via chemical transformation reactions performed by anaerobic microorganisms. The formation of C1 metabolites often necessitates their removal in biological elemental cycles because they can accumulate and alters normal carbon and electron flow within a cell, or becomes toxic and result in cell death.

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose).

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.

Effects of inhibiting protein synthesis on the secretion of surfactant by type II cells in primary culture

Pulmonary surfactant is isolated from the alveolar lumen as a complex of lipid and protein (King R.J. (1974) Fed. Proc. 23, 2238–2247) and is probably secreted as such (King R.J., Martin H., Mitts D. and Holmstrom F.M. (1977) J. Appl. Physiol. 42, 483–491). We wished to determine whether the secretion of this complex was dependent upon cellular activities associated with the synthesis of protein, and whether the pre-formed lipids of surfactant would be released from the cell even though the synthesis of newly-formed protein was inhibited. Alveolar epithelial Type II cells were isolated from adult rat lung and grown to confluency in primary culture. The synthesis and secretion of the apolipoprotein of surfactant and its principal lipid, dipalmitoyl phosphatidyl-choline, were followed by isotopic precursor techniques. The synthesis of the apolipoprotein was reduced to 14% of control by 1 · 10 −4 M cycloheximide and to 2.5% of control by 4 · 10 −4 M cycloheximide. These concentrations of cycloheximide, however, had no effect on the rate of synthesis or release of DPPC. The secretion of the apolipoprotein which had been synthesized before the addition of 1 · 10 −4 M cycloheximide was not inhibited by this compound. Cells maintained at 5°C neither synthesized nor released surfactant. We conclude, therefore, that the synthesis of cellular protein is not required for the secretion of surfactant, but that the continuous generation of metabolic energy may be essential. These results, together with those of previous kinetic studies (see above references), suggest that the lipid and protein constituents of surfactant may be contained within lamellar bodies prior to their release into the extracellular environment.

Brain Hexokinase, the Prototype Ambiquitous Enzyme

Publisher Summary The brain is a voracious consumer of glucose—and of oxygen because the predominant route of glucose metabolism is aerobic oxidation via glycolysis and the tricarboxylic acid (TCA) cycle. Under special circumstances that lead to ketosis, ketone body metabolism might contribute to the generation of metabolic energy in the brain, but even under these conditions, an absolute requirement for a basal level of glucose metabolism remains. The critical dependence of normal brain function on an adequate supply of blood-borne glucose and oxygen has been demonstrated. Transport of glucose through the blood-brain barrier is a carrier-mediated process. The principal metabolic pathway involved is glycolysis. Under most conditions, regulation of glucose utilization equates with regulation of glycolysis. The principal control points for cerebral glycolysis are the hexokinase and phosphofructokinase reactions. Brain hexokinase offers a challenging opportunity for understanding how molecular structure and cellular structure might be inter-woven in regulating the in vivo activity of an enzyme of central importance for cerebral energy metabolism.

NEUTRON SCATTERING AND BIOLOGICAL STRUCTURES

The discovery of the helical structure of DNA, the determination of the three-dimensional structure of proteins, and the unraveling of the structure of viruses are among the outstang achievements of structural biology that have contributed much to our understanding of the molecular basis of life. These elucidations of the architectural details of biological structures provide insight into such diverse biological processes as the biosynthesis of macromolecules, the generation and storage of metabolic and photosynthetic energy, the expression and transmission of genetic information, and the control and recognition of physiological processes. The scattering of slow neutrons from condensed matter provides information about the structure and dynamical properties of such matter on a submicroscopic level. The type of information provided is related to, but qualitatively different from, that obtained by the scattering of x-rays. The neutron is an uncharged probe that interacts with the atoms of matter through two forces: the nuclear force, and a weaker, but longer-range, magnetic force between the neutron's magnetic moment and the magnetic moments of the electrons in the atoms. Although most present neutron studies in physics deal with magnetic and dynamical problems, in studies of structural chemistry neutrons are used to locate hydrogen atoms and tomore » analyze charge distribution in chemical bonds. Most biological neutron studies deal with structural problems, such as the position of atoms in a moleculelike protein or the relative position of proteins, lipids, drugs, and other entities in larger biological assemblies such as membranes.« less

Oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase in stationary-phase cultures of Bacillus subtilis

Glutamine phosphoribosylpyrophosphate amidotransferase (ATase) activity is rapidly inactivated in stationary-phase cells of Bacillus subtilis. The inactivation of APase requires both the cessation of rapid cell growth and the presence of oxygen. ATase is inactivated in two protease-deficient mutant strains at a rate similar to that seen in the wild type, and is stable in anaerobic cell-free extracts of the parent strain. These results suggest that the inactivation of ATase is not the result of general proteolysis. The inactivation of ATase in stationary-phase cultures can be inhibited by oxygen starvation. This oxygen requirement does not reflect a dependence on the generation of metabolic energy, but appears to be a direct requirement for molecular oxygen. ATase synthesis is repressed by the addition of adenosine, and is inactivated only after the cessation of exponential growth. Addition of chloramphenicol or rifampin to exponential- and stationary-phase cells does not inhibit ATase inactivation, suggesting that protein or ribonucleic acid synthesis is not required for inactivation. ATase is inactivated at the end of exponential growth in cells that have exhausted a required amino acid.

Inactivation of aspartic transcarbamylase in sporulating Bacillus subtilis: demonstration of a requirement for metabolic energy.

The aspartic transcarbamylase (ATCase) activity of Bacillus subtilis cells disappears rapidly from stationary-phase cells prior to sporulation. ATCase activity does not appear in the culture fluid during the stationary phase; hence the enzyme appears to be inactivated in the cells. The enzyme is inactivated normally in two different mutants lacking proteases; the activity is very stable in crude extracts of cells or in the culture fluid. These results suggest that ATCase is not inactivated by the general proteolysis that occurs in sporulating bacteria. The inactivation of ATCase can be completely inhibited after it has begun by oxygen starvation or addition of fluoroacetate. Inhibitors of oxidative phosphorylation and electron transport also interrupt the inactivation of ATCase. The inactivation of ATCase is very slow in two mutant strains that are deficient in enzymes of tricarboxylic acid cycle. Addition of gluconate to stationary cultures of the mutant strains, which is known to restore depleted adenosine 5'-triphosphate pools in these bacteria, also restores inactivation of ATCase. These experiments support the conclusion that the generation of metabolic energy is necessary for the inactivation of ATCase in stationary cells. ATCase activity is stable in growing cells in which ATCase synthesis is repressed by addition of uracil; the enzyme is inactivated normally, however, when such cells cease growing.

Increased transport of 2-aminoisobutyric acid in rat lymphocytes stimulated with concanavalin A

The transport of 2-aminoisobutyric acid has been studied in stimulated rat lymphocytes. The capacity to accumulate 2-aminoisobutyric acid increased twofold after a 5 min incubation with phytohaemagglutinin (PHA) and concanavalin A (conA). It reached a maximum level which was 6–8 times the level of the control culture after 24 h of incubation with PHA. The early increased transport induced by conA could not be attributed to a decreased efflux, increased exchange diffusion or passive diffusion but reflected an increased rate of active influx. Kinetic analysis showed an increase in the maximum velocity of uptake ( V max) while no effect was observed on the apparent affinity ( Km). The increased uptake was dependent on protein synthesis and only partly dependent on the generation of metabolic energy. It is concluded that concanavalin A has no direct effect on the binding of 2-aminoisobutyric acid to the carrier and on the mobility or the number of carriers.

Inhibition of membrane transport in Streptococcus faecalis by uncouplers of oxidative phosphorylation and its relationship to proton conduction.

We studied the effect of compounds that uncouple oxidative phosphorylation on membrane function in Streptoccocus faecalis, an organism which relies upon glycolysis for the generation of metabolic energy. At low concentrations (ranging from 10(-7) to 10(-4)m), tetrachlorosalicylanilide, tetramethyldipicrylamine, carbonylcyanide m-chlorophenylhydrazone, pentachlorophenol, and dicoumarol strongly inhibited energy-dependent transport of rubidium, phosphate, and certain amino acids. However, these compounds had little effect on the generation of adenosine triphosphate via glycolysis or on its utilization for the synthesis of macromolecules. They also did not seriously inhibit uptake of those monosaccharides and amino acids which do not require concurrent metabolism. It is proposed that the uncouplers interfere with the utilization of metabolic energy for membrane transport. The uncouplers accelerated the translocation of protons across the cytoplasmic membrane. It appears that a proton-impermeable membrane is required for transport, perhaps, because a proton gradient is involved in the coupling of metabolic energy to the translocation of substrates across the membrane.