Mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate.

The mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate has been probed using initial velocity studies, deuterium isotope effects, and isotope partitioning of the E:Mg:malate complex. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:NAD:Mg:malate complexes. Fumarate is a positive heterotropic effector of the NAD-malic enzyme at low concentrations (K act approximately 0.05 mM) and an inhibitor competitive against malate (Ki approximately 25 mM). The activation by fumarate results in a decrease in the Ki malate and an increase in V/K malate of about 2-fold, while the maximum velocity remains constant. Isotope partitioning studies of E:Mg:[14C]malate indicate that the presence of fumarate results in a decrease in the malate off-rate constant by about 2.2-fold. The deuterium isotope effects on V and V/K malate are both 1.6 +/- 0.1 in the absence of fumarate, while in the presence of 0.5 mM fumarate DV is 1.6 +/- 0.1 and D(V/K malate) is 1.1 +/- 0.1. These data are also consistent with a decrease in the off-rate for malate from E:NAD:Mg:malate, resulting in an increase in the forward commitment factor for malate and manifested as a lower value for D(V/K malate). There is a discrimination between active and activator sites for the binding of dicarboxylic acids, with the activator site preferring the extended configuration of 4-carbon dicarboxylic acids, while the active site prefers a configuration in which the 4-carboxyl is twisted out of the C1-C3 plane. The physiologic importance and regulatory properties of fumarate in the parasite are also discussed.     

Channeling of TCA cycle intermediates in cultured Saccharomyces cerevisiae

Oxidation of [3-13C]propionate was studied in cultured yeast cells, and the distribution of label in the 2- and 3-positions of alanine was detected by 13C NMR. [3-13C]Propionate forms [2-13C]succinyl-CoA in the mitochondria which then enters the citric acid cycle and forms malate through two symmetrical intermediates, succinate and fumarate. If these symmetrical intermediates randomly diffuse from one enzyme to the next in mitochondria as is normally assumed, then 13C labeling in malate C2 and C3 must be equal. However, any direct transfer of metabolites from site to site between succinate thiokinase, succinate dehydrogenase, and fumarase would result in an uneven distribution of 13C in malate C2 and C3 and any molecules derived from malate. Since pyruvate may be derived from malate via the malic enzyme and subsequently converted into alanine by transamination, any 13C asymmetry in alanine C2 and C3 must directly reflect the 13C distribution in the malate pool. During oxidation of [3-13C]propionate, we detect a significant quantity of labeled alanine, where 13C enrichment in C3 is significantly higher than that in C2. Inhibition of succinate dehydrogenase with malonate or creating conditions that increase the chances of a back-reaction (from malate to fumarate) result in a significant decrease in the asymmetric labeling of alanine. Ubiquinone-deficient yeast cells (having only 10% of the oxidative capacity of wild-type cells) could slowly oxidize propionate, but in this case the 13C labeling was equal in the C2 and C3 of alanine, showing that isotope randomization had occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aconitase: its source of catalytic protons

An ordinary isotope partition experiment was performed to determine the rate of dissociation of the proton from the donor site for the hydration of cis-aconitate. Aconitase in [3H]water was efficiently diluted into well-mixed solutions of cis-aconitate. Citrate and isocitrate that were formed within 2 s were more heavily labeled than could be explained by consideration of an isotope effect in the processing of one proton per enzyme equivalent. Control experiments indicate that mixing was much more rapid than catalytic turnover, ruling out incompletely diluted [3H]water as a significant isotope source. Therefore, it appears that significantly more than one enzyme-bound tritium atom (protons) must have been used in the course of the multiple turnover of the enzyme after the dilution was complete. Isotope incorporation reached values in excess of four proton equivalents as a limit with simple Michaelis dependence on cis-aconitate. From the half-saturation concentration value for trapping, 0.15 mM, the t 1/2 for exchange of each of these protons with solvent appears to be approximately 0.1 s at 0 degrees C. The large number of protons trapped seems to suggest the existence of a structurally stabilized pool of protons, or water, that communicates between the active site base and the medium in the hydration of cis-aconitate. The proton abstracted in the dehydration of [3H]citrate is transferred directly to undissociated cis-aconitate to form isocitrate without dilution, or cis-aconitate having dissociated, the tritium passes to the medium, presumably through the pool of bound protons indicated above. All of the citrate-derived protons can be found in isocitrate if cis-aconitate is added in sufficient concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

A gamma-aminobutyric acid/benzodiazepine receptor complex from bovine cerebral cortex. Improved purification with preservation of regulatory sites and their interactions

A gamma-aminobutyrate/benzodiazepine receptor complex has been purified from bovine cerebral cortex by an improved procedure using a zwitterionic detergent. A high affinity binding site for the chloride ion channel-blocking ligand [35S]t-butyl bicyclophosphorothionate ( TBPS ) was co-purified with the high affinity binding sites for gamma-aminobutyrate and benzodiazepines. The latter two have previously been shown to reside on the same physical structure ( Sigel , E., Stephenson , F.A., Mamalaki , C., and Barnard , E. A. (1983) J. Biol. Chem. 258, 6965-6971). The dissociation constants, as measured in assay medium containing zwitterionic detergent were 90 +/- 20 nM for TBPS and 11 +/- 4 nM for [3H]flunitrazepam, whereas the binding of [3H]muscimol, a gamma-aminobutyrate agonist, showed a more complex binding behavior with more than one site. If the same preparation was assayed in a medium containing instead Triton X-100 as the detergent, the binding of TBPS was strongly inhibited, [3H]flunitrazepam binding was unaffected, and [3H]muscimol bound to a single class of sites with a dissociation constant of 33 +/- 3 nM. Regulatory interactions were retained in the complex isolated by the improved method: [3H]flunitrazepam binding was stimulated by gamma-aminobutyrate or by pentobarbital, and in a dose-dependent manner. The same two subunit types of Mr = 53,000 and 57,000 are present in the purified receptor complex as previously reported.     

Identification of the catalytic mechanism and estimation of kinetic parameters for fumarase

The enzyme fumarase catalyzes the reversible hydration of fumarate to malate. The reaction catalyzed by fumarase is critical for cellular energetics as a part of the tricarboxylic acid cycle, which produces reducing equivalents to drive oxidative ATP synthesis. A catalytic mechanism for the fumarase reaction that can account for the kinetic behavior of the enzyme observed in both isotope exchange studies and initial velocity studies has not yet been identified. In the present study, we develop an 11-state kinetic model of the enzyme based on the current consensus on its catalytic mechanism and design a series of experiments to estimate the model parameters and identify the major flux routes through the mechanism. The 11-state mechanism accounts for competitive binding of inhibitors and activation by different anions, including phosphate and fumarate. The model is identified from experimental time courses of the hydration of fumarate to malate obtained over a wide range of buffer and substrate concentrations. Further, the 11-state model is found to effectively reduce to a five-state model by lumping certain successive steps together to yield a mathematically less complex representation that is able to match the data. Analysis suggests the primary reaction route of the catalytic mechanism, with fumarate binding to the free unprotonated enzyme and a proton addition prior to malate release in the fumarate hydration reaction. In the reverse direction (malate dehydration), malate binds the protonated form of the enzyme, and a proton is generated before fumarate is released from the active site.     

Proton diffusion in the active site of triosephosphate isomerase

The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect. Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product. The transfer is believed to be due to the action of a unique basic residue in the active site. Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product. Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium. A case in point is triose-P isomerase (TIM), where kcat is approximately 10(4) s-1. T-Labeled substrate is found to lose approximately 95% of its T to the medium when totally converted to product. Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10. The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope. The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E.enediol state internal exchange. Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isotope partitioning for NAD-malic enzyme from Ascaris suum confirms a steady-state random kinetic mechanism

Isotope partitioning studies beginning with E.[14C]NAD, E.[14C]malate, E.[14C]NAD.Mg2+, and E.Mg.[14C]malate suggest a steady-state random mechanism for the NAD-malic enzyme. Isotope trapping beginning with E.[14C]NAD and with varying concentrations of Mg2+ and malate in the chase solution indicates that Mg2+ is added in rapid equilibrium and must be added prior to malate for productive ternary complex formation. Equal percentage trapping from E.[14C]NAD.Mg and E.Mg.[14C]malate indicates the mechanism is steady-state random with equal off-rates for NAD and malate from E.NAD.Mg.malate. The off-rates for both do not change significantly in the ternary E.Mg.malate and E.NAD.Mg complexes, nor does the off-rate change for NAD from E.NAD. No trapping of malate was obtained from E.[14C]malate, suggesting that this complex is nonproductive. A quantitative analysis of the data allows an estimation of values for a number of the rate constants along the reaction pathway.     

The tricarboxylic acid cycle in Dictyostelium discoideum. Metabolite concentrations, oxygen uptake and 14c-labelled amino acid labelling patterns.

Some aspects of tricarboxylic acid-cycle activity during differentiation and aging in Dictyostelium discoideum were examined. The concentrations of glutamate, aspartate, alanine, citrate, 2-oxoglutarate, succinate, fumarate, malate, oxaloacetate, pyruvate and acetyl-CoA were determined at four stages over the course of differentiation. The rate of O2 utilization was also determined over differentiation. In addition, experiments are described in which the specific radioactivities of citrate, 2-oxoglutarate, succinate, fumarate and malate were determined during a 30 min labelling of cells from the preculmination stage of development with [14C]glutamate, [14C]aspartate or [14C]alanine. A similar experiment was also performed with cells from the aggregation stage of development using [14C]glutamate.     

Cerebral dicarboxylate transport and metabolism studied with isotopically labelled fumarate,malate and malonate

Transport and metabolism of dicarboxylates may be important in the glial-neuronal metabolic interplay. Further, exogenous dicarboxylates have been suggested as cerebral energy substrates. After intrastriatal injection of [(14) C]fumarate or [(14) C]malate, glutamine attained a specific activity 4.1 and 2.6 times higher than that of glutamate, respectively, indicating predominantly glial uptake of these four-carbon dicarboxylates. In contrast, the three-carbon dicarboxylate [(14) C]malonate gave a specific activity in glutamate which was approximately five times higher than that of glutamine, indicating neuronal uptake of malonate. Therefore, neurones and glia take up different types of dicarboxylates, probably by different transport mechanisms. Labelling of alanine from [(14) C]fumarate and [(14) C]malate demonstrated extensive malate decarboxylation, presumably in glia. Intravenous injection of 75 micromol [U-(13) C]fumarate rapidly led to high concentrations of [U-(13) C]fumarate and [U-(13) C]malate in serum, but neither substrate labelled cerebral metabolites as determined by (13) C NMR spectroscopy. Only after conversion of [U-(13) C]fumarate into serum glucose was there (13) C-labelling of cerebral metabolites, and only at <10% of that obtained with 75 micromol [3-(13) C]lactate or [2-(13) C]acetate. These findings suggest a very low transport capacity for four-carbon dicarboxylates across the blood-brain barrier and rule out a role for exogenous fumarate as a cerebral energy substrate.     

Kinetics and regulation of hepatoma mitochondrial NAD(P) malic enzyme.

Kinetic studies of Morris 7777 hepatoma mitochondrial NAD(P) malic enzyme were consistent with an ordered mechanism where NAD adds to the enzyme before malate and dissociation of NADH from the enzyme is rate-limiting. In addition to its active site, malate apparently also associates with a lower affinity with an activator site. The activator fumarate competes with malate at the activator site and facilitates dissociation of NADH from the enzyme. The ratio of NAD(P) malic enzyme to malate dehydrogenase activity in the hepatoma mitochondrial extract was found to be too low, even in the presence of known inhibitors of malate dehydrogenase, to account for the known ability of NAD(P) malic enzyme to intercept exogenous malate from malate dehydrogenase in intact tumor mitochondria (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221). However, NAD(P) malic enzyme may be able to intercept exogenous malate because according to the present results, it can associate with the pyruvate dehydrogenase complex, which could localize NAD(P) malic enzyme in the vicinity of the inner mitochondrial membrane. The activity levels of some key metabolic enzymes were found to be different in Morris 7777 mitochondria than in liver or mitochondria of other rapidly dividing tumors. These results are discussed in terms of differences among tumors in their ability to utilize malate, glutamate, and citrate as respiratory fuels.     

Engineering water to act as an active site acid catalyst in a soluble fumarate reductase

The ability of an arginine residue to function as the active site acid catalyst in the fumarate reductase family of enzymes is now well-established. Recently, a dual role for the arginine during fumarate reduction has been proposed [Mowat, C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 12292-12298] in which it acts both as a Lewis acid in transition-state stabilization and as a Br?nsted acid in proton delivery. This proposal has led to the prediction that, if appropriately positioned, a water molecule would be capable of functioning as the active site Br?nsted acid. In this paper, we describe the construction and kinetic and crystallographic analysis of the Q363F single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the soluble fumarate reductase from Shewanella frigidimarina. Although replacement of the active site acid, Arg402, with alanine has been shown to eliminate fumarate reductase activity, this phenomenon is partially reversed by the additional substitution of Gln363 with phenylalanine. This Gln --> Phe substitution in the inactive R402A mutant enzyme was designed to "push" a water molecule close enough to the substrate C3 atom to allow it to act as a Br?nsted acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme does indeed reveal the introduction of a water molecule at the correct position in the active site to allow it to act as the catalytic proton donor. The 1.8 A resolution crystal structure of the Q363F mutant enzyme shows a water molecule similarly positioned, which can account for its measured fumarate reductase activity. However, in this mutant enzyme Michaelis complex formation is impaired due to significant and unpredicted structural changes at the active site.     

Substrate activation by malate induced by oxalate in the Ascaris suum NAD-malic enzyme reaction

Substrate activation of the rate of the NAD-malic enzyme reaction by malate is obtained in the presence but not in the absence of oxalate. The substrate activation is a result of competition between malate and oxalate for the E.NADH complex, with malate binding to the form of the complex unprotonated at an enzyme group with a pK of 4.9 and oxalate binding preferentially to the protonated form. The off-rate for NADH from the E.NADH complex is completely rate limiting when the group with a pK of 4.9 is protonated but is only one of several rate-limiting steps when it is unprotonated [Kiick, D.M., Harris, B.G., & Cook, P.F. (1986) Biochemistry 25, 227]. The competition by malate with oxalate thus results in an overall increase in the off-rate for NADH as a result of binding to the unprotonated form of E.NADH. Consistent with the proposed mechanism, the deuterium isotope effect on V for the nonsubstrate-activating malate concentration range decreases from 1.6 in the absence of oxalate to 1.3 in the presence of a concentration of oxalate equal to its Kii. The rate equation for the oxalate-induced substrate activation by malate is derived and presented in the Appendix. Data are discussed in terms of the overall mechanism of the NAD-malic enzyme.     

Physicochemical characteristics of the interaction of the glucocorticoid antagonist RU38486 with glucocorticoid receptors in vitro, and the role of molybdate

The physicochemical properties of complexes formed between the glucocorticoid antagonist, RU38486, and the glucocorticoid receptor in rat thymus cytosol were investigated and compared with those of complexes formed with the potent agonist, triamcinolone acetonide. The equilibrium dissociation constant for the interaction of [3H]RU38486 with the molybdate-stabilized glucocorticoid receptor was lower than that for [1,2,4-3H]triamcinolone acetonide at 0 degree C but higher at 25 degrees C, suggesting that hydrophobic interactions play a major role in the binding of RU38486. Differences in equilibrium constants were reflected in corresponding differences in dissociation rate constants; association rate constants for the two steroids were similar. The rate of dissociation of [3H]RU38486 from the glucocorticoid receptor was higher in the absence of molybdate than in its presence both at 0 degree C and at 25 degrees C, suggesting that molybdate modifies the physical state of the antagonist-receptor complex, but other physical properties were similar both in the presence and in the absence of molybdate. The rate of inactivation of the unoccupied glucocorticoid receptor at 25 degrees C in the absence of molybdate was lower in phosphate buffer than in Tris-HCl buffer but the rate of dissociation of [3H]RU38486 was the same in both buffers. The binding of RU38486 afforded little, if any, protection against inactivation in either buffer; [3H]RU38486 dissociated irreversibly from the inactivated receptor at the same rate as from the non-inactivated complex but molybdate had no effect on the dissociation kinetics of the inactivated complex. It is concluded that RU38486 interacts with the ground state of the glucocorticoid receptor in a manner which neither promotes receptor transformation nor prevents receptor inactivation.     

Partial purification and some properties of oxalacetase from Aspergillus niger.

1. Oxalacetase from Asperigillus niger was found to be an inducible enzyme, the induction being dependent not only on neutralisation of the acidic growth medium but also on the presence of carbonate. An explanation is proposed. 2. Three methods were established for the quantitative determination of oxalacetase activity. These are based on the determination of the product acetate, on the absorbance of oxaloacetate and on coupling the hydrolysis of oxaloacetate to the oxidation of malate by NAD in the presence of malate dehydrogenase. 3. Oxalacetase was purified about 50-fold from cell-free extracts of A. niger and used to determine some of its properties such as kinetic constants. 4. 2S-[U-14C, 3-2H2] Malate in the presence of oxalacetase, NAD and malate dehydrogenase was partially converted to acetate and oxalate. The 3H/14C ratio of the isolated acetate was nearly twice as high as that of the malate used initially. The result demonstrates that the keto form of oxaloacetate, not the enol, is the substrate of the enzyme. 5. Equimolecular mixtures of 2S, 3S-[3-2H1] malate + 2S-[2-2H1] malate (mixture 1) and 2S, 3R-[3-2H1, 3H1] malate + 2S, 3R-[2-2H1, 3-3H1] malate (mixture 2) were prepared from 2S-[3-3H2] malate by incubation with fumarase in normal and tritiated water, respectively. The isolated mixture 1, in the presence of oxalacetase, NAD and malate dehydrogenase was incubated in tritiated water for formation of acetate and oxalate; the isolated mixture 2 was treated likewise in normal water. 6. The mixtures of symmetrically labelled [3H1] acetate and chiral acetates thus produced were isolated and the configuration of the [3H1, 3H1] acetate specimens was determined in the sequence acetate leads to malate leads to fumarate, as usual. The [2H1, 3H1] acetate derived from 2S, 3S-[3-2H1] malate (present in mixture 1( yielded a malate which on incubation with fumarase retained 65.0% of its total tritium content. This chiral acetate, therefore, had the R configuration. The [2H1, 3H1] acetate derived from 2S, 3R-[2-2H1, 3-3H1] malate produced a malate which retained 35% of its total tritium content, and therefore had the S configuration. 7. It was concluded that the detachment of the oxaloyl residue from oxaloacetate and its replacement by a proton proceed with inversion of configuration at the methylene group which becomes methyl during the hydrolysis.     

Ascaris suum NAD-malic enzyme is activated by L-malate and fumarate binding to separate allosteric sites

The kinetic mechanism of activation of the mitochondrial NAD-malic enzyme from the parasitic roundworm Ascaris suum has been studied using a steady-state kinetic approach. The following conclusions are suggested. First, malate and fumarate increase the activity of the enzyme in both reaction directions as a result of binding to separate allosteric sites, i.e., sites that exist in addition to the active site. The binding of malate and fumarate is synergistic with the K(act) decreasing by >or=10-fold at saturating concentrations of the other activator. Second, the presence of the activators decreases the K(m) for pyruvate 3-4-fold, and the K(i) (Mn) >or=20-fold in the direction of reductive carboxylation; similar effects are obtained with fumarate in the direction of oxidative decarboxylation. The greatest effect of the activators is thus expressed at low reactant concentrations, i.e., physiologic concentrations of reactant, where activation of >or=15-fold is observed. A recent crystallographic structure of the human mitochondrial NAD malic enzyme [13] shows fumarate bound to an allosteric site. Site-directed mutagenesis was used to change R105, homologous to R91 in the fumarate activator site of the human enzyme, to alanine. The R105A mutant enzyme exhibits the same maximum rate and V/K(NAD) as does the wild-type enzyme, but 7-8-fold decrease in both V/K(malate) and V/K(Mg), indicating the importance of this residue in the activator site. In addition, neither fumarate nor malate activates the enzyme in either reaction direction. Finally, a change in K143 (a residue in a positive pocket adjacent to that which contains R105), to alanine results in an increase in the K(act) for malate by about an order of magnitude such that it is now of the same magnitude as the K(m) for malate. The K143A mutant enzyme also exhibits an increase in the K(act) for fumarate (in the absence of malate) from 200 microM to about 25 mM.     

Evidence against the incorporation into protein of amino acids directly from the membrane transport system in rat heart

The possibility suggested recently [Hider, R.C., Fern, E.B. and London, D.R. (1969) Biochem. J. 114, 171-178; Hider, R.C., Fern, E.B. and London, D.R. (1971) Biochem. J. 121, 817-827; van Venrooij, W.J., Poort, C., Kramer, M.F. and Jansen, M.T. (1972) Eur. J. Biochem. 30, 427-433; and Adamson, L.F., Herington, A.C. and Bornstein, J. (1972) Biochim. Biophys. Acta 282, 352-365] that protein synthesis takes place using amino acids directly from the membrane transport system and not from an intracellular pool has been investigated in rat heart. The tissue was perfused first for 30 min with either [14C]glycine or [14C]leucine and then for a further 30 min with identical medium containing [3H]glycine or [3H]leucine, respectively. After an initial lag, [14C]glycine was incorporated into protein at a linear rate up to 60 min. The [3H]glycine was accumulated into tissue water and incorporated just as readily as the [14C]glycine had been. The rate of total protein synthesis agrees with literature values only if intracellular and not extracellular specific activity values are used in the calculation. Some glycine was converted to serine or threonine. Leucine influx and efflux were very rapid in contrast to the relatively slow exchange reported for incubated tissues [Hider, R.C., Fern, E.B. and London, D.R. (1969) Biochem. J. 114, 171-178; Hider, R.C., Fern, E.B. and London, D.R. (1971) Biochem. J. 121, 817-827; van Venrooij, W.J., Poort, C., Kramer, M.F. and Jansen, M.T. (1972) Eur. J. Biochem. 30, 427-433]. The results are consistent with the existence of an intracellular precursor pool for glycine. Some possible reasons for the discrepancies between this and the other studies are discussed.     

Intracellular proton regulation of ClC-0

Some CLC proteins function as passive Cl(-) ion channels whereas others are secondary active chloride/proton antiporters. Voltage-dependent gating of the model Torpedo channel ClC-0 is modulated by intracellular and extracellular pH, possibly reflecting a mechanistic relationship with the chloride/proton coupling of CLC antiporters. We used inside-out patch clamp measurements and mutagenesis to explore the dependence of the fast gating mechanism of ClC-0 on intracellular pH and to identify the putative intracellular proton acceptor(s). Among the tested residues (S123, K129, R133, K149, E166, F214L, S224, E226, V227, C229, R305, R312, C415, H472, F418, V419, P420, and Y512) only mutants of E166, F214, and F418 qualitatively changed the pH(int) dependence. No tested amino acid emerged as a valid candidate for being a pH sensor. A detailed kinetic analysis of the dependence of fast gate relaxations on pH(int) and [Cl(-)](int) provided quantitative constraints on possible mechanistic models of gating. In one particular model, a proton is generated by the dissociation of a water molecule in an intrapore chloride ion binding site. The proton is delivered to the side chain of E166 leading to the opening of the channel, while the hydroxyl ion is stabilized in the internal/central anion binding site. Deuterium isotope effects confirm that proton transfer is rate limiting for fast gate opening and that channel closure depends mostly on the concentration of OH(-) ions. The gating model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on pH(int) and [Cl(-)](int).     

Disequilibrium in the malate dehydrogenase reaction in rat liver mitochondria in vivo

1. When [2-(14)C]pyruvate is injected into rats the C3-position of liver glutamate becomes more heavily labelled than the C2-position, thus establishing that oxaloacetate and fumarate are not in equilibrium in rat liver mitochondria in vivo. The amount of disequilibrium was shown to be simply related to the value that the C3-label/C2-label ratio would have were no label recycled. This ratio, z, was calculated for post-absorptive rats in environmental temperatures of 20 degrees and 30 degrees C from determinations of the distribution of label within glutamate 1, 3 and 10min after intravenous injection of [2-(14)C]pyruvate. The values of z (best estimate and range) were 1.65 (1.60-1.69) in rats at 20 degrees C and 2.43 (2.23-2.63) in rats at 30 degrees C. These values of z imply the following rates of interconversion in mitochondria of fumarate and oxaloacetate (in terms of the oxaloacetate-->citrate flux, R) in rats at 20 degrees C: [Formula: see text] and in rats at 30 degrees C: [Formula: see text] 2. The kinetic parameters of malate dehydrogenase and fumarate hydratase and the intramitochondrial concentrations of NAD(+) and NADH under (as far as could be judged) conditions in vivo were collated. From them and the best estimates of R now available were calculated the rates of interconversion of fumarate, malate and oxaloacetate required to give the found values of z. These rates showed that the fumarate hydratase reaction was nearly in equilibrium, but that the malate dehydrogenase reaction was considerably out of equilibrium. The calculations also led to the following conclusions. 3. In livers of rats at 20 degrees and 30 degrees C mitochondrial malate concentrations were respectively about 5 and 1.5 times mean cellular concentrations. 4. Mitochondrial oxaloacetate concentrations were less than 0.2 of the mean cellular concentrations. They were also only 0.65 and 0.55 of the equilibrium concentrations for the malate dehydrogenase reaction in rats at 20 degrees and 30 degrees C respectively. 5. Malate dehydrogenase activity was low because of the very low oxaloacetate concentrations in the mitochondria and the very small fraction of the enzyme complexed with NAD(+), i.e. in each direction one substrate concentration was very sub-optimal.     

A 1H NMR study of succinate synthesis from exogenous precursors in oxygen-deprived rat heart mitochondria

Succinate synthesis from exogenous malate, alpha-ketoglutarate, oxaloacetate and L-glutamate in isolated oxygen-deprived rat heart mitochondria was studied using 1H NMR. The highest rate of succinate synthesis was observed during incubation of mitochondria with a mixture of L-glutamate and oxaloacetate. When mitochondria were incubated with [U-13C] glutamate and oxaloacetate the [U-13C] succinate/succinate and aspartate/succinate ratios were equal to 2. This suggests that the succinate produced from [U-13C] alpha-keto-glutarate formed via transamination of [U-13C] glutamate with oxaloacetate by aspartate aminotransferase exceeds twofold that synthesized via oxaloacetate reduction. It may thus be expected that GTP yield in a reaction catalyzed by the succinic thiokinase will be 2 times higher that of ATP production coupled with NADH-dependent fumarate reduction.