[Theoretical analysis of the possibility of existence of oscillations in photosynthesis].

A mathematical model for carbohydrate and carbon dioxide metabolism in a chloroplast has been constructed. If it is taken into consideration that the rate of the efflux of sugars is restricted as their concentration increases, then even with a simplified representation of the work of the Calvin cycle, it can be shown that there is a set of the parameters of the model at which stable oscillations can appear in the system.

[Role of transamination in the mobilization of respiratory substrates in germinating seeds of castor oil plants].

The metabolism of 1,4-14C-succinate and 2,3-14C-succinate and the activity of succinic semialdehyde dehydrogenase (EC 1.2.1.16) were studied in germinating seeds of castor oil plants (Ricinus communis L.). Succinate metabolism involved succinate dehydrogenase and was sensitive to metabolites of the gamma-aminobutyric acid shunt. Considerable accumulation of the label in amino acids reflected the progression of transamination reactions. Succinic semialdehyde dehydrogenase was purified from the endosperm of castor oil plants. Kinetic characteristics of the enzyme were evaluated. Our study indicates that the mobilization of respiratory substrates during germination of castor oil plants is related to active transamination of ketoacids in the Krebs cycle and involves the gamma-aminobutyric acid shunt.

Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments.

On the basis of the equilibrium of adenylate kinase (AK; EC 2.7.4.3), which interconverts MgATP and free AMP with MgADP and free ADP, an approach has been worked out to calculate concentrations of free magnesium (Mg(2+)), based on concentrations of total ATP, ADP and AMP in plant tissues and in individual subcellular compartments. Based on reported total adenylate contents, [Mg(2+)] in plant tissues and organelles varies significantly depending on light and dark regimes, plant age and developmental stage. In steady-state conditions, [Mg(2+)] in chloroplasts is similar in light and darkness (in the millimolar range), whereas in the cytosol it is very low in the light and increases to about 0.4 mM in darkness. During the dark-to-light transition (photosynthetic induction), the [Mg(2+)] in chloroplasts falls to low values (0.2 mM or less), corresponding to a delay in photosynthetic oxygen evolution. This delay is considered to result from lower activities of Mg-dependent enzymes in the Calvin cycle. In mitochondria, the changes in [Mg(2+)] are similar but smoother. On the other hand, when the transition from light to darkness is considered, an initial increase in [Mg(2+)] occurs in both chloroplasts and mitochondria, which may be of importance for the control of key regulatory enzymes (e.g. mitochondrial malic enzyme and pyruvate dehydrogenase complex) and for processes connected with light-enhanced dark respiration. A rationale is presented for a possible role of [MgATP]/[MgADP] ratio (rather than [ATP(total)]/[ADP(total)]) as an important component of metabolic cellular control. It is postulated that assays of total adenylates may provide an accurate measure of [Mg(2+)] in plant tissues/cells and subcellular compartments, given that the adenylates are equilibrated by AK.

Decarboxylation of glycine contributes to carbon isotope fractionation in photosynthetic organisms.

Carbon isotope effects were investigated for the reaction catalyzed by the glycine decarboxylase complex (GDC; EC 2.1.2.10). Mitochondria isolated from leaves of pea (Pisum sativum L.) and spinach (Spinacia oleracea L.) were incubated with glycine, and the CO(2) evolved was analyzed for the carbon isotope ratio (delta(13)C). Within the range of parameters tested (temperature, pH, combination of cofactors NAD(+), ADP, pyridoxal 5-phosphate), carbon isotope shifts of CO(2) relative to the C(1)-carboxyl carbon of glycine varied from +14 per thousand to -7 per thousand. The maximum effect of cofactors was observed for NAD(+), the removal of which resulted in a strong (12)C enrichment of the CO(2) evolved. This indicates the possibility of isotope effects with both positive and negative signs in the GDC reaction. The measurement of delta(13)C in the leaves of the GDC-deficient barley (Hordeum vulgare L.) mutant (LaPr 87/30) plants indicated that photorespiratory carbon isotope fractionation, opposite in sign when compared to the carbon isotope effect during CO(2) photoassimilation, takes place in vivo. Thus the key reaction of photorespiration catalyzed by GDC, together with the key reaction of CO(2) fixation catalyzed by ribulose-1,5-bisphosphate carboxylase, both contribute to carbon isotope fractionation in photosynthesis.

[Effect of salt stress on respiration metabolism in higher plants]. / Vliianie solevogo stressa na dykhatel'nyi metabolism vysshikh rastenii.

We studied the activity of NADP-dependent isocitrate dehydrogenase, malate dehydrogenase, succinate dehydrogenase, catalase, and peroxidase as well as the rate of 14CO2 release after introduction of labeled substrates for glycolysis and citrate acid cycle within 24 h after salt stress (1% NaCl) in 10-14 days old germinants of wheat (Triticum aestivum L.) and maize (Zea mays L.) as well as thallus of small duckweed (Wolffia arrhiza (L.) Hork ex Wimmer). Oscillations in the enzymes activity with 4-6 h period have been revealed under stress conditions. Activity of glycolysis decreased in wheat and maize and increased in duckweed under the influence of stress stimulus. Six hours after NaCl action decarboxylation of exogenous citrate and succinate was enhanced in all three plants while the rate of exogenous malate decarboxylation was decreased. We conclude that adaptation of higher plans to salinization is accompanied by rearrangements in oxidative metabolism reflected by oscillations in activity of the enzymes involved in oxidative metabolism.

[Light-dependent changes in the enzyme activity of the ascorbate-glutathione cycle and ascorbate oxidase in the leaves of pea]. / Svetozavisimye izmeneniia aktivnosti fermentov askorbat-glutationovogo tsikla i askorbatoksidazy v list'iakh gorokha.

Light-determined activation of ferments of ascorbate-glutation cycle, ascorbate-oxidase in chloroplasts and cytosol is demonstrated as well as ascorbate-peroxidase, monodehydroascorbate-reductase, glutation-reductase and ascorbate-oxydase in mitochondria. On the other hands activity of mitochondrial dehydroascorbate-reductase increased on reduction of light most likely due to function of electron transport from glutation to dehydroascorbate in mitochondria. Glutation metabolism is proved to be endogenic catalytic process where the amount reconstructed glutation changes slowly with a delay and gradually follow light changes. Light dependable changes of glutation content in chloroplasts ensure resistance of ferment system again hydrogen peroxide and superoxide radicals that generate intensively at light.

[Induction of glyoxylate cycle enzymes in various tissues from starving rats]. / Induktsiia fermentov glioksilatnogo tsikla v razlichnykh tkaniakh golodaiushchikh krys.

The induction of glyoxylate cycle enzyme activities was revealed in the liver and other organs of starving rats. A five day deprivation of food was followed by the appearance of isocitrate lyase (ICL) and malate synthase activities and the increase of malate dehydrogenase (MDH) and citrate synthase activities. The induction of MDH was associated with the appearance of its new isoform with Rf 0.52. ICL activity was revealed in the liver, blood, pancreas, kidney, lungs, heart, and skeletal muscles of starving rats, reaching a peak on day 5 of food deprivation. No significant changes of blood glucose level in starving rats were revealed until day 9. A homogeneous ICL preparation with a specific activity of 12.4 IU per mg protein was obtained as the results of five-stage purification procedure.

Two separate transhydrogenase activities are present in plant mitochondria.

Inside-out submitochondrial particles from both potato tubers and pea leaves catalyze the transfer of hydride equivalents from NADPH to NAD(+) as monitored with a substrate-regenerating system. The NAD(+) analogue acetylpyridine adenine dinucleotide is also reduced by NADPH and incomplete inhibition by the complex I inhibitor diphenyleneiodonium (DPI) indicates that two enzymes are involved in this reaction. Gel-filtration chromatography of solubilized mitochondrial membrane complexes confirms that the DPI-sensitive TH activity is due to NADH-ubiquinone oxidoreductase (EC 1.6.5.3, complex I), whereas the DPI-insensitive activity is due to a separate enzyme eluting around 220 kDa. The DPI-insensitive TH activity is specific for the 4B proton on NADH, whereas there is no indication of a 4A-specific activity characteristic of a mammalian-type energy-linked TH. The DPI-insensitive TH may be similar to the soluble type of transhydrogenase found in, e.g., Pseudomonas. The presence of non-energy-linked TH activities directly coupling the matrix NAD(H) and NADP(H) pools will have important consequences for the regulation of NADP-linked processes in plant mitochondria.

Foundations of metabolic organization: coherence as a basis of computational properties in metabolic networks.

Biological organization is based on the coherent energy transfer allowing for macromolecules to operate with high efficiency and realize computation. Computation is executed with virtually 100% efficiency via the coherent operation of molecular machines in which low-energy recognitions trigger energy-driven non-equilibrium dynamic processes. The recognition process is of quantum mechanical nature being a non-demolition measurement. It underlies the enzymatic conversion of a substrate into the product (an elementary metabolic phenomenon); the switching via separation of the direct and reverse routes in futile cycles provides the generation and complication of metabolic networks (coherence within cycles is maintained by the supramolecular organization of enzymes); the genetic level corresponding to the appearance of digital information is based on reflective arrows (catalysts realize their own self-reproduction) and operation of hypercycles. Every metabolic cycle via reciprocal regulation of both its halves can generate rhythms and spatial structures (resulting from the temporally organized depositions from the cycles). Via coherent events which percolate from the elementary submolecular level to organismic entities, self-assembly based on the molecular complementarity is realized and the dynamic informational field operating within the metabolic network is generated.

Glyoxylate cycle enzymes are present in liver peroxisomes of alloxan-treated rats.

Key enzymes of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), have been detected in the liver of alloxan-treated rats. The activity of ICL in rat liver was 0.040 micromol/min/mg protein and the activity of MS was 0.022 micromol/min/mg protein. These enzymes were associated with the peroxisomal fraction. The activities of citrate synthase, malate synthase and malate dehydrogenase detected in the peroxisomal fraction were also increased by alloxan treatment. Isocitrate lyase was partially purified and displayed catalytic and regulatory properties similar to those of the enzyme isolated from the liver of starved rats (Popov, V.N. et al. (1996) FEBS Lett. 391, 87-90).

Time, reflectivity and information processing in living systems: a sketch for the unified information paradigm in biology.

The recognition activity of biomacromolecules based on quantum non-demolition measurements is regarded as the basis of information processing. Reflective arrows in the set of mappings appearing from quantum measurements correspond to the Gödel numbers created inside a system overcoming its incompleteness. Temporal evolution is a consequence of contradictory statements about the whole system in which a reflective arrow is both an element of the system and its signification. It results from the solution of a paradox in which the system generates new descriptions non-deducible from its previous states. The active combinatorial process of self modification of information, being an internalized language game, allows a system to create Gödel numbers. The whole system is constructed according to percolating coherent events, providing 'vertical' self-assembly that is predetermined by the encoding and internal language games.

Involvement of cyanide-resistant and rotenone-insensitive pathways of mitochondrial electron transport during oxidation of glycine in higher plants.

Metabolism of glycine in isolated mitochondria and protoplasts was investigated in photosynthetic, etiolated (barley and pea leaves) and fat-storing (maize scutellum) tissues using methods of [1-(14)C]glycine incorporation and counting of 14CO2 evolved, oxymetric measurement of glycine oxidation and rapid fractionation of protoplasts incubated in photorespiratory conditions with consequent determination of ATP/ADP ratios in different cell compartments. The involvement of different paths of electron transport in mitochondria during operation of glycine decarboxylase complex (GDC) was tested in different conditions, using aminoacetonitrile (AAN), the inhibitor of glycine oxidation in mitochondria, rotenone, the inhibitor of Complex I of mitochondrial electron transport, and inhibitors of cytochrome oxidase and alternative oxidase. It was shown that glycine has a preference to other substrates oxidized in mitochondria only in photosynthetic tissue where succinate and malate even stimulated its oxidation. Rotenone had no or small effect on glycine oxidation, whereas the role of cyanide-resistant path increased in the presence of ATP. Glycine oxidation increased ATP/ADP ratio in cytosol of barley protoplasts incubated in the presence of CO2, but not in the CO2-free medium indicating that in conditions of high photorespiratory flux oxidation of NADH formed in the GDC reaction passes via the non-coupled paths. Activity of GDC in fat-storing tissue correlated with the activity of glyoxylate-cycle enzymes, glycine oxidation did not reveal preference to other substrates and the involvement of paths non-connected with proton translocation was not pronounced. It is suggested that the preference of glycine to other substrates oxidized in mitochondria is achieved in photosynthetic tissue by switching to rotenone-insensitive intramitochrondrial NADH oxidation and by increasing of alternative oxidase involvement in the presence of glycine.

[Purification and properties of isocitrate lyase and malate synthase from fasting rat liver]. / Ochistka i svoistva izotsitratlizay i malatsintazy iz pecheni golodaiushchikh krys.

Key enzymes of glyoxylate cycle, isocitrate lyase and malate synthase, are active in the fasting rat liver. The enzymes were synthesized on day 3 after food deprivation and their activities were maximal on day 5 of fasting. Specific activity of isocitrate lyase was 0.06 units/mg protein and specific activity of malate synthase was 0.03 units/mg protein. Isocitrate lyase was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose chromatography and Toyopearl HW-65 gel filtration. Enzyme was purified to specific activity of 9.0 units/mg protein with 8.2% yield. Molecular mass of isocitrate lyase was 145 kD according to gel filtration. Catalytic characteristics of isocitrate lyase indicate that the enzyme follows Michaelis-Menten kinetics (Km for isocitrate is 0.07 mM), is competitively inhibited by glucose-I-phosphate (Ki = 1.1 mM) and glucose-6-phosphate (Ki = 1.9 mM), and is activate by ADP; optimal pH is 7.4. Malate synthase was partially purified by ammonium sulfate fractionation and Sephadex G-25 gel filtration. Enzyme was purified to specific activity of 0.15 units/mg protein with 45% yield. Km of malate synthase for acetyl-CoA was 0.2 mM and Km for glyoxylate was 0.3 mM; optimal pH was 7.6.

Induction of glyoxylate cycle enzymes in rat liver upon food starvation.

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of food-starved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The K(m)(isocitrate) was 68 microM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a K(m)(acetyl-CoA) of 0.2 microM, a K(m)(glyoxylate) of 3 mM and a pH optimum of 7.6.

Fractionation of carbon (13C/12C) isotopes in glycine decarboxylase reaction.

Fractionation of carbon isotopes (13C/12C) by glycine decarboxylase (GDC) was investigated in mitochondrial preparations isolated from photosynthetic tissues of different plants (Pisum, Medicago, Triticum, Hordeum, Spinacia, Brassica, Wolffia). 20 mM glycine was supplied to mitochondria, and the CO2 formed was absorbed and analyzed for isotopic content. CO2 evolved by mitochondria of Pisum was enriched up to 8% in 12C compared to the carboxylic atom of glycine. CO2 evolved by mitochondria of the other plants investigated was enriched by 5-16% in 13C. Carbon isotope effects were sensitive to reaction conditions (pH and the presence of GDC cofactors). Theoretical treatment of the reaction mechanism enabled us to conclude that the value and even the sign of the carbon isotope effect in glycine decarboxylation depend on the contribution of the enzyme-substrate binding step and of the decarboxylation step itself to the overall reaction rate. Therefore, the fractionation of carbon isotopes in GDC reaction was revealed which provides essential isotopic effects in plants in addition to the well-known effect of carbon isotope fractionation by the central photosynthetic enzyme, ribulose-1,5-biphosphate carboxylase.

Alternative system of succinate oxidation in glyoxysomes of higher plants.

Succinate oxidation in scutella of germinating seeds of wheat and maize was investigated. Besides oxidation via succinate dehydrogenase (SDH; EC 1.3.99.1), an alternative path of succinate oxidation insensitive to SDH inhibitors--malonate and thenoyltrifluoroacetone (TTFA)--was revealed. Using isopicnic sucrose gradient it was shown that this path is localized in glyoxysomal membranes. Glyoxysomal succinate oxidase (GSO) converts succinate directly into malate with the production of hydrogen peroxide identified using auxiliary enzymes malate dehydrogenase and peroxidase. GSO is most active during the intensive operation of the glyoxylate cycle (3-5 days of germination). Quinacrine, the inhibitor of flavine-containing oxidases, strongly suppressed the activity of GSO. Km for succinate is 18 mM for GSO from maize scutellum. It is concluded that in scutella of cereal seeds the glyoxysomal succinate oxidation non-linked with ATP synthesis operates.

The role of metabolic transformations in generation of biological order.

Transformations of metabolic organization are considered as a ground of structure generation during ontogenesis and evolution. The emergence of non-linearity in metabolic pathways which leads to the formation of biochemical cycles is caused by the separation between the direct and reverse enzymatic reactions, one of them being coupled with the pool of macroergs, whereas the other--non-coupled. The absence of couplings in one of the halves of such primary cycle can lead to the increase of metabolic flow and to the generation of bifurcations resulting in the formation of new metabolic pathway. For their internalization the genetic reconstructions are necessary. Deposition of compounds from the cycles leads to the generation of internal rhythms and to morphogenetic processes. Morphology is formed by stable trajectories of the formation and deposition of certain compounds according to the principles of curvilinear symmetry.

Quantum mechanical properties of biosystems: a framework for complexity, structural stability, and transformations.

Internal quantum non-demolition measurements are inherent for biological organization and determine the essential features of living systems. Low energy dissipation in these measurements provided by slow conformational relaxation of biomacromolecular complexes (regarded as measuring devices) is the main precondition of enzyme operation and information transfer determining the steady non-equilibrium state of biosystems. The presence of an internal formal description inside a biosystem, expressed in genetic structures (developmental program), is a consequence of its quantum properties. Incompleteness of this formal description provides the possibility of the generation of new functional relations and interconnections inside the system. This is a logical precondition of an evolutionary process. The quantum mechanical uncertainty that underlies the appearance of bifurcations is considered to be the main physical foundation of complication and irreversible transformation of biosystems.