Compartmentation of metabolism within mitochondria and plastids

An important component of metabolic regulation is compartmentation and specialization. Subcellular compartmentation and the role of individual organelles is well studied, though less consideration has been given to the extent to which organelles differ between cells. Organelles extracted from whole tissue homogenates will have generally originated from a range of cell types. This review describes and assesses the regulation of metabolic activity in plants at both the cellular and subcellular level by considering specialization of mitochondria and plastids.     

31P-NMR studies of metabolite compartmentation in Fasciola hepatica

Fasciola hepatica, the common liver fluke, is an anaerobic parasitic worm. Possible compartmentation of metabolites between different cell types, metabolic compartments, and free and macromolecule-bound species was investigated using 31P-NMR. A spectrum of the intact worm shows unusual metabolic features, among which are large amounts of glycerolphosphorylcholine, phospholipids mobile on the NMR time-scale, and free cytosolic ADP. Spectra from cells as different as those in oral sucker tissue and eggs showed similar features. Acidosis after serotonin administration was associated with parallel changes in chemical shifts of intracellular Pi and glucose 6-phosphate, suggesting that they are in the same metabolic compartment. Although 13.4 +/- 1.1 mumol/g wet wt. (n = 3) Mg2+ is present in fluke tissue, a considerable fraction is sequestered out of the cytosol. The intracellular free [Mg2+] was independently estimated from the chemical shifts of ATP and ADP as 1.6 +/- 0.5 mM and 2.9 +/- 0.7 mM, respectively. Quantitation of observable phosphate-containing metabolites in whole tissue and in perchlorate extracts demonstrated that 60% of the total ADP and 50% of the total Pi are 'NMR-invisible' in the intact fluke and therefore probably bound to macromolecules in the cells. The apparent ATP/ADP X Pi free concentration ratio is much lower in this anaerobic tissue than in mammalian oxidative tissues.     

The effect of water stress on the vacuole-extravacuole compartmentation of proline in potato cell suspension cultures

Solute compartmentation in cells is an important component of metabolic regulation. There is only little information on how stress treatment of cells effects this component. Therefore, the effect of water stress [10% (w/v) PEG 6000] on the vacuolar-extravacuolar proline compartmentation was studied in a cell suspension culture of Svlanum tuberosum L, cv, HH258, In non-stressed cells 34% of the total cellular proline was located in the vacuole. After 20 h of water stress the proline pool of the cells was increased 4-6 fold and only t6% of it was found in the vacuole. A negative correlation between the total cellular proline content and its percentage in the vacuole was observed, irrespective of the culture method (stress or non-stress culture). The stress-induced changes in proline compartmentation are discussed.     

Intracellular metabolic compartmentation assessed by 13C magnetic resonance spectroscopy

Our understanding of the brain has developed from the theory that it is one continuous cell to the knowledge that there are many brain cells originally termed neurons and, furthermore to the discovery of glial cells and their multiple functions. Thus, an increasing complexity was unraveled and we have not reached a complete understanding of the phenomenon which comprises the compartmentation of metabolic pathways and metabolites. This is an important principle needed to fully understand the metabolic processes of the brain. At the cellular level this concept is well established whereas intracellular compartmentation has yet to be explored. Using magnetic resonance spectroscopy (MRS) for analysis of isotopomer composition combined with quantification of amino acid contents it is possible to construct models that describe intracellular compartmentation. Results of studies of cultures of astrocytes and neurons incubated in media containing [U- 13C]glutamate in the presence or absence of thiopental may be used to propose an intracellular three compartment model of mitochondrial function. Due to the experimental paradigm only certain aspects of metabolism can be described. The present model consists of compartments assigned to CO(2) production, glutamate synthesis from ketoglutarate and finally synthesis of a four-carbon metabolite which is shuttled between compartments. It is likely that metabolism may be far more complex than this and we are only beginning to glimpse some aspects of compartmentation at the cellular level.     

Design of glycolysis

The design of the glycolytic pathway resulting from the continuous refinement of evolution is discussed with regard to three aspects. 1. Functional and structural properties of individual enzymes. The catalytic constants of the glycolytic enzymes are remarkably optimized; the turnover numbers are within one order of magnitude. The same is true for the molarities of catalytic centres in the cytosol, as is noted for yeast. Functional properties of the enzymes are reflected in their tertiary and quaternary structures. 2. Regulatory mechanisms of single enzymes. A classification of the various types of enzymic control mechanisms operating in the glycolytic pathway is given. In addition to the usual Michaelis-Menten saturation kinetics and the various types of inhibition there is control by positive and negative effectors based on oligomeric structures (fast acting, fine control) as well as regulation by chemical interconversion structures (fast acting, fine control) as well as regulation by chemical based on enzymes cascades (slow acting, very effective). 3. Functional and regulatory mechanisms of the whole glycolytic reaction pathway. A prominent feature is the high enzyme:substrate ratio, which guarantees fast response times. However, a quantitative treatment of the overall kinetics is limited by an incomplete knowledge of the enzymes' dynamic and chemical compartmentation as well as some of their control properties. From an analysis of the oscillatory state, certain control points in the glycolytic chain can be located that coincide with major branching points to other metabolic pathways. These points are controlled by fast-acting cooperative enzymes that operate in a flip-flop mechanism together with the respective antagonistic enzymes, preventing futile cycles. The gating enzymes leading to the glycogen store and the citric acid cycle are of the slow-acting but very effective interconvertible type. The combination of all the complex and intricate features of design yields a glycolytic network that enables the cell to respond to its various metabolic needs quickly, effectively and economically.     

SYNAPTIC AND NON-SYNAPTIC MITOCHONDRIA FROM RAT BRAIN: ISOLATION AND CHARACTERIZATION

Abstract— A method has been developed where by three distinct populations of metabolically active, well coupled and relatively pure mitochondria from rat brain may be prepared. Two mitochondrial populations are derived from synaptozomes and the third consists of 'free' (i.e. non-synaptic) mitochondria. These mitochondrial populations have been characterized with respect to both enzyme content and ability to oxidize substrates. The results indicate that these mitochondrial populations are heterogeneous with respect to maximal activities of certain enzymes concerned with the citric acid cycle and glutamate and 4-aminobutyrate metabolism as well as their ability to utilize various substrates. The data reported here also confirm that brain mitochondria are very heterogeneous and suggest that synaptic mitochondria may contain at least two sub-populations. The relations between the heterogeneity of brain mitochondria and the metabolic compartmentation of the citric acid cycle and related metabolites such as glutamate, aspartate and 4-aminobutyrate are briefly discussed in the light of two proposed models of metabolic compartmentation in the mammalian brain.     

Detection of circular excised DNA deletion elements in Tetrahymena thermophila during development.

Extensive programmed DNA deletion occurs in ciliates during development. In this study we examine the excised forms of two previously characterized deletion elements, the R- and M-element, in Tetrahymena. Using divergently oriented primers in polymerase chain reactions we have detected the junctions formed by joining the two ends of these elements, providing evidence for the presence of circular excised forms. These circular forms were detected in developing macronuclear DNA from 12-24 h after mating began, but not in micronuclear or whole cell DNA of vegetative cells. They are present at very low abundance, detectable after PCR only through hybridization with specific probes. Sequence analysis shows that the circle junctions occur at or very near the known ends of the elements. There is sequence microheterogeneity in these junctions, which does not support a simple reciprocal exchange model for DNA deletion. A model involving staggered cuts and variable mismatch repair is proposed to explain these results. This model also explains the sequence microheterogeneity previously detected among the junction sequences retained in the macronuclear chromosome.     

Frontiers in metabolic reconstruction and modeling of plant genomes

A major goal of post-genomic biology is to reconstruct and model in silico the metabolic networks of entire organisms. Work on bacteria is well advanced, and is now under way for plants and other eukaryotes. Genome-scale modelling in plants is much more challenging than in bacteria. The challenges come from features characteristic of higher organisms (subcellular compartmentation, tissue differentiation) and also from the particular severity in plants of a general problem: genome content whose functions remain undiscovered. This problem results in thousands of genes for which no function is known ('undiscovered genome content') and hundreds of enzymatic and transport functions for which no gene is yet identified. The severity of the undiscovered genome content problem in plants reflects their genome size and complexity. To bring the challenges of plant genome-scale modelling into focus, we first summarize the current status of plant genome-scale models. We then highlight the challenges - and ways to address them - in three areas: identifying genes for missing processes, modelling tissues as opposed to single cells, and finding metabolic functions encoded by undiscovered genome content. We also discuss the emerging view that a significant fraction of undiscovered genome content encodes functions that counter damage to metabolites inflicted by spontaneous chemical reactions or enzymatic mistakes.     

Compartmentation in plant metabolism

Cell fractionation and immunohistochemical studies in the last 40 years have revealed the extensive compartmentation of plant metabolism. In recent years, new protein mass spectrometry and fluorescent-protein tagging technologies have accelerated the flow of information, especially for Arabidopsis thaliana, but the intracellular locations of the majority of proteins in the plant proteome are still not known. Prediction programs that search for targeting information within protein sequences can be applied to whole proteomes, but predictions from different programs often do not agree with each other or, indeed, with experimentally determined results. The compartmentation of most pathways of primary metabolism is generally covered in plant physiology textbooks, so the focus here is mainly on newly discovered metabolic pathways in plants or pathways that have recently been revised. Ultimately, all of the pathways of plant metabolism are interconnected, and a major challenge facing plant biochemists is to understand the regulation and control of metabolic networks. One of the best-characterized networks links sucrose synthesis in the cytosol with photosynthetic CO(2) fixation and starch synthesis in the chloroplasts. One of the key features of this network is how the transport of pathway intermediates and signal metabolites across the chloroplast envelope conveys information between the two compartments, influencing the regulation of several enzymes to co-ordinate fluxes through the different pathways. It is widely accepted that chloroplasts and mitochondria originated from prokaryotic endosymbionts, and that new transporters and regulatory networks evolved to integrate metabolism in these organelles with the rest of the cell. Curiously, the present-day locations of many metabolic pathways within the cell often do not reflect their evolutionary origin, and there is evidence of extensive shuffling of enzymes and whole pathways between compartments during the evolution of plants.     

Sequence and characterization of the sperm-specific protein phi 3 from Mytilus californianus

We have shown that the sperm-specific protein phi 3 from Mytilus californianus (Conrad) exhibits compositional microheterogeneity. For the first time, we have isolated and characterized the three major components of this protein. These fractions display different electrophoretic mobilities on Triton/urea/acetic acid polycrylamide gels. However, they have a very similar molecular mass of 5 +/- 0.1 kDa as measured by sedimentation equilibrium in the analytical ultracentrifuge. All of them show a marked trend toward aggregation. We have also established the sequence for each of these three fractions. The sequencing data suggest an even greater extent of microheterogeneity for this protein. The predicted secondary structure from the sequences, as well as infrared analyzes carried out on the native protein, suggest a structure organization into an alpha helix.     

Combined noninvasive imaging and modeling approaches reveal metabolic compartmentation in the barley endosperm

The starchy endosperm of cereals is a priori taken as a metabolically uniform tissue. By applying a noninvasive assay based on (13)C/(1)H-magnetic resonance imaging (MRI) to barley (Hordeum vulgare) grains, we uncovered metabolic compartmentation in the endosperm. (13)C-Suc feeding during grain filling showed that the primary site of Ala synthesis was the central region of the endosperm, the part of the caryopsis experiencing the highest level of hypoxia. Region-specific metabolism in the endosperm was characterized by flux balance analysis (FBA) and metabolite profiling. FBA predicts that in the central region of the endosperm, the tricarboxylic acid cycle shifts to a noncyclic mode, accompanied by elevated glycolytic flux and the accumulation of Ala. The metabolic compartmentation within the endosperm is advantageous for the grain's carbon and energy economy, with a prominent role being played by Ala aminotransferase. An investigation of caryopses with a genetically perturbed tissue pattern demonstrated that Ala accumulation is a consequence of oxygen status, rather than being either tissue specific or dependent on the supply of Suc. Hence the (13)C-Ala gradient can be used as an in vivo marker for hypoxia. The combination of MRI and metabolic modeling offers opportunities for the noninvasive analysis of metabolic compartmentation in plants.     

Quantitative studies of enzyme-substrate compartmentation,functional coupling and metabolic channelling in muscle cells

Some historical aspects of development of the concepts of functional coupling, metabolic channelling, compartmentation and energy transfer networks are reviewed. Different quantitative approaches, including kinetic and mathematical modeling of energy metabolism, intracellular energy transfer and metabolic regulation of energy production and fluxes in the cells in vivo are analyzed. As an example of the system with metabolic channelling, thermodynamic aspects of the functioning the mitochondrial creatine kinase functionally coupled to the oxidative phosphorylation are considered. The internal thermodynamics of the mitochondrial creatine kinase reaction is similar to that for other isoenzymes of creatine kinase, and the oxidative phosphorylation process specifically influences steps of association and dissociation of MgATP with the enzyme due to channelling of ATP from adenine nucleotide translocase. A new paradigm of muscle bioenergetics - the paradigm of energy transfer and feedback signaling networks based on analysis of compartmentation phenomena and structural and functional interactions in the cell is described. Analysis of the results of mathematical modeling of the compartmentalized energy transfer leads to conclusion that both calcium and ADP, which concentration changes synchronously in contraction cycle, may simultaneously activate oxidative phosphorylation in the muscle cells in vivo. The importance of the phosphocreatine circuit among other pathways of intracellular energy transfer network is discussed on the basis of the recent data published in the literature, with some experimental demonstration. The results of studies of perfused rat hearts with completely inhibited creatine kinase show significantly decreased work capacity and respectively, energy fluxes, in these hearts in spite of significant activation of adenylate kinase system (Dzeja et al. this volume). These results, combined with those of mathematical analysis of the energy metabolism of hearts of transgenic mice with switched off creatine kinase isoenzymes confirm the importance of phosphocreatine pathway for energy transfer for cell function and energetics in mature heart and many other types of cells, as one of major parts of intracellular energy transfer network and metabolic regulation.     

Changes in structural organization of surface membrane during erythrocyte maturation

The effect of concanavalin A and its succinylated derivative on cell agglutination and potassium compartmentation of mature and immature erythrocytes was observed. The binding of tetravalent concanavalin A to the surface glycoproteins of rabbit erythrocytes leads to a change in the properties of the surface membrane, which results in an induction of cell agglutination and concomitant release of potassium from the cells. Both of the phenomena induced by concanavalin A are temperature dependent, and observed at above 15°C.Divalent succinylated concanavalin A, lacking the inducing activity of surface glycoprotein cross-linking into patches and caps, caused neither cell agglutination nor change in the potassium compartmentation of erythrocytes and reticulocytes.In the case of immature reticulocytes, however, remarkable agglutination of the cells was induced without a change in the potassium compartmentation after treatment with tetravalent concanavalin A.It is suggested that changes in the molecular organization of the surface membrane occur in which potassium compartmentation of the reticulocytes becomes more susceptible to surface glycoprotein cross-linking during cellular maturation.     

Strategies for metabolic flux analysis in plants using isotope labelling

Flux measurements through metabolic pathways generate insights into the integration of metabolism, and there is increasing interest in using such measurements to quantify the metabolic effects of mutation and genetic manipulation. Isotope labelling provides a powerful approach for measuring metabolic fluxes, and it gives rise to several distinct methods based on either dynamic or steady-state experiments. We discuss the application of these methods to photosynthetic and non-photosynthetic plant tissues, and we illustrate the different approaches with an analysis of the pathways interconverting hexose phosphates and triose phosphates. The complicating effects of the pentose phosphate pathway and the problems arising from the extensive compartmentation of plant cell metabolism are considered. The non-trivial nature of the analysis is emphasised by reference to invalid deductions in earlier work. It is concluded that steady-state isotopic labelling experiments can provide important information on the fluxes through primary metabolism in plants, and that the combination of stable isotope labelling with detection by nuclear magnetic resonance is particularly informative.     

The influence of pretreatment with different cations on anaerobic nitrite production by excisedPisum sativum roots

The influence of pretreatment with some cations on anaerobic nitrite production (in an assay medium lacking nitrate) by excised primary roots of pea (Pisum sativum L., ov. Raman), detached from six-day-old seedlings germinated in distilled water, was investigated. When the excised roots were precultivated in one-salt-solutions of KNO3, then these roots produced at 9 mM and 15 mM NO3- concentrations under anaerobic conditions significantly more NO2-, than those precultivated in a nutrient solution containing besides K+ ions also Ca2+ and Mg2+ ions, and they produced nitrite for a longer time. The KNO3 dependent increase in anaerobic NO2- production was counteracted most by Ca2+ and to a lesser extent by Mg2+; Na+ was without effect. NH4+ at higher concentrations (12 and 15 mM) significantly depressed nitrite production both by roots precultivated in a solution containing besides NH4+ only K+, and by roots precultivated in a full nutrient solution containing K+, Ca2+ and Mg2+, however at lower NH4+ concentrations (0.6 and 2mMNH4+; 15mMNO3-) the decrease was more conspicuous in the KNO3 solution than in the full nutrient solution. Nitrate reductase level was not influenced by this pretreatment. When 6% and 7.5% n-propanol, which increases membrane permeability and causes mixing of storage and metabolic nitrate pools in the cells, was added to the assay medium lacking nitrate, anaerobic nitrite production increased and the differences caused by the precultivation disappeared. These results show that higher K+ concentrations in unbalanced one-salt-solutions of KNO3 can cause higher membrane permeability by accentuating Ca8+ deficiency, which results in a faster penetration of NO3- from the storage pool to the sites of its reduction and in an easier penetration of NO2- out of the roots, and that higher NH4+ concentrations can change nitrate compartmentation and diminish the metabolic NO3- pool which results in a slower nitrate reduction. Besides that, lower NH4+ concentrations in KNO3 solutions (15mMNO3-) probably partially counteract the K+ dependent increase in membrane permeability. The results obtained show that there is no simple, direct relationship between the so-called metabolic pool of nitrate (i.e. anaerobic nitrite production) and the level of nitrate reductase, but that the velocity of nitrate reduction can be influenced by nitrate compartmentation in the cell.     

One- and two-dimensional 90.5-MHz 13C-NMR spectroscopy of the N-linked triantennary oligosaccharide units of calf fetuin

Complete assignments of all anomeric resonances in the proton and carbon spectra of the N-linked oligosaccharide units of fetuin were made using one- and two-dimensional NMR spectroscopy. We are able to confirm the presence of microheterogeneity in the N-acetylneuraminic acid linkages to the galactose residues and the presence of a unique triantennary structure which carries a side chain: NeuAc alpha(1-3)Gal beta(1-3)GlcNac beta(1-4)-. Anomeric carbon chemical shifts changes resulting from long-range conformational effects were observed.     

Insights into plant metabolic networks from steady-state metabolic flux analysis

Steady-state metabolic flux analysis (MFA) is an experimental approach that allows the measurement of multiple fluxes in the core network of primary carbon metabolism. It is based on isotopic labelling experiments, and although well established in the analysis of micro-organisms, and some mammalian systems, the extension of the method to plant cells has been challenging because of the extensive subcellular compartmentation of the metabolic network. Despite this difficulty there has been substantial progress in developing robust protocols for the analysis of heterotrophic plant metabolism by steady-state MFA, and flux maps have now been published that reflect the metabolic phenotypes of excised root tips, developing embryos and cotyledons, hairy root cultures, and cell suspensions under a variety of physiological conditions. There has been a steady improvement in the quality, extent and statistical reliability of these analyses, and new information is emerging on the performance of the plant metabolic network and the contributions of specific pathways.     

Metabolic flux analysis in plants: coping with complexity

Theory and experience in metabolic engineering both show that metabolism operates at the network level. In plants, this complexity is compounded by a high degree of compartmentation and the synthesis of a very wide array of secondary metabolic products. A further challenge to understanding and predicting plant metabolic function is posed by our ignorance about the structure of metabolic networks even in well-studied systems. Metabolic flux analysis (MFA) provides tools to measure and model the functioning of metabolism, and is making significant contributions to coping with their complexity.
This review gives an overview of different MFA approaches, the measurements required to implement them and the information they yield. The application of MFA methods to plant systems is then illustrated by several examples from the recent literature. Next, the challenges that plant metabolism poses for MFA are discussed together with ways that these can be addressed. Lastly, new developments in MFA are described that can be expected to improve the range and reliability of plant MFA in the coming years.     

Alterations in the sialylation and sulfation of secreted mouse thyrotropin in primary hypothyroidism

We investigated the effect of in vivo hypothyroidism on the sialylation and sulfation of thyroid-stimulating hormone (TSH) secreted by mouse pituitary explants. Oligosaccharides from secreted thyroid-stimulating hormone from hypothyroid animals contained greater sialic acid relative to sulfate in both alpha and beta subunits. Aging per se had little effect on thyroid-stimulating hormone sialylation or sulfation. Variable sialylation and sulfation demonstrates a mechanism for charge microheterogeneity of thyroid-stimulating hormone, and the increasing sialylation observed with hypothyroidism may functionally mediate the prolonged metabolic clearance that has been noted previously.     

Regulation of cytokinin content in plant cells

Cytokinin levels in plant cells are dependent on cytokinin biosynthesis and/or uptake from extracellular sources, metabolic interconversions, inactivation and degradation. Cytokinin conversion to compounds differing in polarity seems to be decisive for their entrapment within the cell and intracellular compartmentation, which affects their metabolic stability. Increase in cytokinin levels, resulting either from their uptake or intracellular biosynthesis, may promote further autoinductive accumulation of cytokinins which may function in the induction of cytokinin-initiated physiological processes. Accumulated cytokinins are capable of inducing cytokinin oxidase which consequently decreases cytokinin levels. This seems to be the mechanism of re-establishment and maintenance of cytokinin homeostasis required for further development of physiological events induced by transient cytokinin accumulation. Auxin may influence cytokinin levels by down regulation of cytokinin biosynthesis and/or by promotion of cytokinin degradation. A model of the regulation of cytokinin levels in plant cells based on these phenomena is presented and its physiological role(s) is discussed.