Studies on the mechanism of oxidative phosphorylation. Flow-force relationships in mitochondrial energy-linked reactions

The relationship between the steady-state level of membrane potential (delta psi) and the rates of energy production and consumption has been studied in mitochondria and submitochondrial particles. The energy-linked reactions investigated were oxidative phosphorylation (with NADH, succinate, and beta-hydroxybutyrate as respiratory substrates) and nucleoside triphosphate-driven transhydrogenation from NADH to NADP and uphill electron transfer from succinate to NAD. Results have shown the following. 1) Attenuation of the rates of the energy-producing reactions results in a parallel change in the rates of the energy-consuming reactions with little or no change in the magnitude of steady-state delta psi. 2) At low rates of energy production and consumption, steady-state delta psi decreases. However, this is due largely to the energy leak of the system which lowers static-head delta psi when the rate of energy production is slow. 3) When the rate of energy production and static-head delta psi are held constant, and the rate of energy consumption is diminished by partial inhibition or the use of suboptimal conditions (e.g. subsaturating substrate concentrations), then even a small decrease in the rate of energy consumption results in an upward adjustment of the level of steady-state delta psi. The lower the rate of energy input, the greater the upward adjustment of steady-state delta psi upon suppression of the rate of energy consumption. 4) The above results have been discussed with regard to the role of bulk-phase delta mu H+ or delta psi in the mitochondrial energy transfer reactions.     

Studies of energy-linked reactions. Inhibition of oxidative phosphorylation by DL-8-methyldihydrolipoate.

1. DL-8-Methyldihydrolipoate was shown to be a potent inhibitor of mitochondrial oxidative phosphorylation and ATP-driven energy-linked reactions. 2. ADP-stimulated respiration utilizing pyruvate + malate and succinate in both ox heart and rat liver mitochondria is inhibited; oxidative phosphorylation using pyruvate + malate, succinate and ascorbate + NNN'N'-tetramethyl-p-phenylenediamine as substrates is also inhibited; uncoupler-stimulated respiration is unaffected regardless of the substrate used. 3. Mitochondrial oligomycin-sensitive adenosine triphosphatase is inhibited in both the membrane-bound form and the purified detergent-dispersed preparation. 4. ATP-driven transhydrogenase and the ATP-driven energy-linked reduction of NAD+ by succinate in ox heart submitochondrial particles are inhibited, whereas the respiratory-chain-driven transhydrogenase is unaffected. 5. DL-8-Methyl-lipoate has no immediate effect on the above reactions, demonstrating the requirement for the reduced form for inhibition. 6. The inhibitory properties of DL-8-methyldihydrolipoate are analogous to those of oligomycin and provide further evidence of a role for lipoic acid in oxidative phosphorylation.     

A comparison of the effects of NN′-dicyclohexylcarbodi-imide, oligomycin A and aurovertin on energy-linked reactions in mitochondria and submitochondrial particles

1. The effects of dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enzyme systems related to respiratory-chain phosphorylation were compared. Dicyclohexylcarbodi-imide and oligomycin A have very similar functional effects, giving 50% inhibition of ATP-utilizing and ATP-generating systems at concentrations below 0.8nmole/mg. of submitochondrial-particle protein. Aurovertin is a more potent inhibitor of ATP synthesis, giving 50% inhibition at 0.2nmole/mg. of protein. However, aurovertin is a less potent inhibitor of ATP-utilizing systems: the ATP-driven energy-linked nicotinamide nucleotide transhydrogenase is 50% inhibited at 3.0nmoles/mg. of protein and the ATP-driven reduction of NAD(+) by succinate is 50% inhibited at 0.95nmole/mg. of protein. 2. With EDTA-particles (prepared by subjecting mitochondria to ultrasonic radiation at pH9 in the presence of 2mm-EDTA) the maximum stimulation of the ATP-driven partial reactions is effected by similar concentrations of oligomycin A and dicylcohexylcarbodi-imide, but the latter is less effective. The stimulatory effects of suboptimum concentrations of dicyclohexylcarbodi-imide and oligomycin A are additive. Aurovertin does not stimulate these reactions or interfere with the stimulation by the other inhibitors. 3. Dicyclohexylcarbodi-imide and oligomycin A stimulate the aerobic energy-linked nicotinamide nucleotide transhydrogenase of EDTA-particles, but the optimum concentration is higher than that required for the ATP-driven partial reactions. Aurovertin has no effect on this reaction. 4. The site of action of dicyclohexylcarbodi-imide is in CF(0), the mitochondrial fraction that confers oligomycin sensitivity on F(1) mitochondrial adenosine triphosphatase.     

Control of cellular redox potential as measured in a steady-state, cell-free system

A cell-free system consisting of rat liver mitochondria, liver cytosol, lactate, and the substrates intrinsic to the malate-aspartate shuttle was reconstituted for studies of steady-state substrate fluxes and, more specifically, to evaluate further the mechanism of control of the intra- and extramitochondrial steady states of the free NAD+/NADH ratios. Soluble (F1) ATPase or 2,4-dinitrophenol (DNP) were added in varying amounts to alter substrate fluxes and the constant energy state of this 'open' metabolizing system. The steady-state redox segregation (1.36 log NAD+/NADH ratio out vs NAD+/NADH in the mitochondrial matrix) was maximally about 3 kcal, and declined together with the membrane potential (delta psi) and log ATP/ADP, which obtain on imposing an increasing energy load on the system. It is concluded that transmembrane movement of reducing equivalents is coupled to electron transfer through delta psi, mediated by the electrogenic exchange of glutamate and aspartate. When delta psi was high (near State 4), delta G redox was approximately the same as that generated without flux of reducing equivalents [E. J. Davis, J. Bremer, and K. E. Akerman (1980) J. Biol. Chem. 255, 2277-2283], suggesting that delta Gredox is in near thermodynamic equilibrium with delta psi. If the steady-state ATP/ADP ratio was altered with an energy load (F1-ATPase), delta Gredox decreased more steeply than delta psi (tetraphenyl phosphonium-sensitive electrode used to measure delta psi). At comparable ranges of ATP/ADP, both delta Gredox and delta psi decreased more steeply with uncoupler than with an external ADP-regenerating system.     

Evaluation of the kinetics of the intracellular reduction of 2,6-dichlorophenolindophenol in normal and transformed hepatocytes measured by amperometric methods

Amperometric methods were used to study the kinetics of intracellular reduction of 2,6-dichlorophenolindophenol (DCIP) in normal and transformed hepatocytes with glucose and succinate as substrates. The curves showing the formation of DCIPred as a function of time were biphasic, the first part obeying the equation of a pseudo-first-order reaction, the final part corresponding to Michaelis-Menten kinetics. A statistical method was used to estimate pseudo-first-order rate constants k as well as Km and Vmax values. At saturating glucose concentrations k, Km and Vmax values were higher in normal compared to transformed cells. Decreasing glucose concentrations revealed lowered saturation concentrations in tumour cells compared to normal cells. With succinate as substrate for hepatocytes, k values were higher than with glucose, while Km and Vmax were about the same. Hepatoma cells did not metabolize succinate. K values could be attributed to intracellular dehydrogenase activities including cytosolic and mitochondrial processes. Differences in pseudo-first-order rate constants between normal and tumour cells may therefore represent characteristic alterations associated with transformation.     

A mechanism of sulfite neurotoxicity: direct inhibition of glutamate dehydrogenase

Exposure of Neuro-2a and PC12 cells to micromolar concentrations of sulfite caused an increase in reactive oxygen species and a decrease in ATP. Likewise, the biosynthesis of ATP in intact rat brain mitochondria from the oxidation of glutamate was inhibited by micromolar sulfite. Glutamate-driven respiration increased the mitochondrial membrane potential (MMP), and this was abolished by sulfite but the MMP generated by oxidation of malate and succinate was not affected. The increased rate of production of NADH from exogenous NAD+ and glutamate added to rat brain mitochondrial extracts was inhibited by sulfite, and mitochondria preincubated with sulfite failed to reduce NAD+. Glutamate dehydrogenase (GDH) in rat brain mitochondrial extract was inhibited dose-dependently by sulfite as was the activity of a purified enzyme. An increase in the Km (glutamate) and a decrease in Vmax resulting in an attenuation in Vmax/Km (glutamate) at 100 microm sulfite suggest a mixed type of inhibition. However, uncompetitive inhibition was noted with decreases in both Km (NAD+) and Vmax, whereas Vmax/Km (NAD+) remained relatively constant. We propose that GDH is one target of action of sulfite, leading to a decrease in alpha-ketoglutarate and a diminished flux through the tricarboxylic acid cycle accompanied by a decrease in NADH through the mitochondrial electron transport chain, a decreased MMP, and a decrease in ATP synthesis. Because glutamate is a major metabolite in the brain, inhibition of GDH by sulfite could contribute to the severe phenotype of sulfite oxidase deficiency in human infants.     

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer in bovine submitochondrial particles provide evidence for direct interaction between ATPase and NADH:Q oxidoreductase

From the chemiosmotic hypothesis it follows that no change is expected in potency of an uncoupler to inhibit an energy-driven reaction in an energy-transducing membrane if the energy-requiring part of the reaction, the so-called secondary proton pump, is partially inhibited by a specific, tightly bound inhibitor. An increase in potency upon inhibition of the primary pump may be expected, due to a lower rate of the total proton flow that can be used by the secondary pump and dissipated by the uncoupler. Contrary to this prediction several uncouplers (S13, SF6847, 2,4-dinitrophenol, valinomycin + nigericin) show an increase in uncoupling efficiency in ATP-driven reverse electron transfer (reversal) upon inhibition of the secondary pump in this reaction, the NADH:Q oxidoreductase, by rotenone. The increase in uncoupling efficiency is proportional to the decrease in the rate of reversal, that is to the decrease in concentration of active secondary pump. Similarly, upon inhibition of the primary pump, the ATPase, with oligomycin, an increase in uncoupling efficiency was found, also proportional to the decrease in the rate of reversal. When the pore-forming uncoupler gramicidin was used, no change in uncoupling potency was found upon inhibition of NADH:Q oxidoreductase. Inhibition of the ATPase, however, resulted in a proportionally lower uncoupling titre for gramicidin, just as was found for S13 in the presence of oligomycin. A difference was also found in the relative concentrations of S13 and gramicidin required to stimulate ATP hydrolysis or to inhibit reversal. The amount of S13 needed to stimulate ATP hydrolysis was clearly higher than the amount needed to inhibit reversal. On the contrary, the titre of gramicidin for both actions was about the same. To explain these results we propose that gramicidin uncouples via dissipation of the bulk delta mu H+, whereas the carrier-type uncouplers preferentially interfere with the direct energy transduction between the ATPase and redox enzymes. This is in accordance with the recently developed collision hypothesis.     

Kinetics of human erythrocyte glucose-6-phosphate dehydrogenase dimers

The steady-state kinetics of human erythrocyte glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) dimers were studied by initial rate measurement. These experiments gave intersecting double-reciprocal plots suggesting a ternary complex mechanism with a Km for NADP and glucose 6-phosphate of 11 microM and 43 microM, respectively. These studies were combined with rate measurements in the presence of one product (NADPH), dead-end inhibitors, as well as alternative substrates. The inhibition by NADPH was found to be competitive with respect to both substrates. Alternate substrates experiments gave linear double-reciprocal plots over a wide range of substrate concentrations. The results suggest that the dimeric enzyme follows either a random or a Theorell-Chance mechanism.     

Biochemical comparison of the Neurospora crassa wild-type and the temperature-sensitive leucine-auxotroph mutant leu-5. Detailed kinetic comparison of the leucyl-tRNA synthetases

The cytoplasmic leucyl-tRNA synthetases were purified from a wild-type Neurospora crassa and from a temperature-sensitive leucine-auxotroph (leu-5) mutant. A detailed steady-state kinetic study of the aminoacylation of the tRNALeu from N. crassa by the purified synthetases was carried out. These enzymes need preincubation with dithioerythritol and spermine before the assay in order to become fully active. The Kappm value for leucine was lowered by high ATP concentrations and correspondingly the Kappm,ATP was lowered by high leucine concentrations. The Kappm,Leu was lowered by high pH, a pK value of 6.7 (at 30 degrees C) was calculated for the ionizable group affecting the Km. At the concentrations of 2 mM ATP, 20 microM leucine, 0.3 microM tRNALeu, and pH 7 the apparent Km values were Kappm,ATP = 1.3 mM, Kappm,Leu = 49 microM and Kappm,tRNA = 0.15 microM. No essentially altered cytoplasmic leucyl-tRNA synthetase was produced by the temperature-sensitive mutant strain when kept at 37 degrees C. In none of these experiments could we find any difference between the wild-type enzyme and the enzyme from the mutant strain (whether grown at permissive temperature, 28 degrees C, or grown at permissive temperature for 24 h followed by growth at 37 degrees C). We therefore think that the small difference in the Km value for leucine of the wild-type and mutant enzyme, established in some earlier investigations, is not due to a difference in the kinetic properties of the enzyme molecules but to an external influence. The almost total lack of the mitochondrial leucyl-tRNA synthetase in the mutant strain besides the leucine autotrophy remains the only difference between the wild-type and mutant strains.     

The permeability transition in heart mitochondria is regulated synergistically by ADP and cyclosporin A.

Heart mitochondria respiring in a sucrose medium containing P(i) show a permeability transition when challenged with Ca2+ and an oxidant such as cumene hydroperoxide. The transition results from the opening of a Ca(2+)-dependent pore and is evidenced by loss of membrane potential (delta psi) and osmotic swelling due to uptake of sucrose and other solutes. In the absence of oxidant, high concentrations of Ca2+ (100-150 microM) are necessary to induce loss of delta psi and initiate swelling. Cyclosporin A delays the loss of delta psi but enhances swelling under these conditions, apparently by promoting better retention of accumulated Ca2+. Cyclosporin A and ADP together restore delta psi in respiring mitochondria that have undergone the permeability transition at levels that are not effective when either is added alone. When the state of the Ca(2+)-dependent pore is assessed using passive osmotic contraction in response to polyethylene glycol (Haworth, R. A., and Hunter, D. R. (1979) Arch. Biochem. Biophys. 195, 460-467), cyclosporin A is found to be a partial inhibitor of solute flow through the open pore. Cyclosporin A decreases the Vmax of passive contraction and increases the Km for Ca2+ without affecting the Hill slope. ADP in the presence of carboxyatractyloside closes the pore almost completely even in the presence of 40 microM Ca2+. ADP shows mixed type inhibition of the Ca(2+)-dependent pore, and cyclosporin A increases the affinity of the pore for ADP. It is concluded that cyclosporin A and ADP act synergistically to close the Ca(2+)-dependent pore of the mitochondrion and that the pore is probably not formed directly from the adenine nucleotide transporter.     

The active transport of 2-keto-D-gluconate in vesicles prepared from Pseudomonas purida.

The transport of 2-keto-D-gluconate (alpha-D-arabino-2-hexulopyranosonic acid; 2KGA) in vesicles prepared from glucose-grown Pseudomonas putida occurs by a saturable process with a Km of 110.0 +/- 2.9 microM and a Vmax. of 0.55 +/- 0.04 nmol X min-1 X (mg of protein)-1. The provision of phenazine methosulphate/ascorbate or L-malate leads to an accumulation of intravescular 2KGA, a decrease in the Km value to 50 +/- 2.1 microM and 35 +/- 2.9 microM respectively and no change in the Vmax. In the presence of electron donors the transport of 2KGA is inhibited by the respiratory poisons antimycin A, rotenone and the uncoupler 2,4-dinitrophenol. 2KGA transport is also competitively inhibited by 4-deoxy-4-fluoro-2-keto- or 3-deoxy-3-fluoro-2-keto-D-gluconate with Ki values of 50 microM and 160 microM respectively. The carrier system for 2KGA is repressed in vesicles from cells grown on succinate. Such vesicles transport 2KGA by non-specific physical diffusion with a Km value of infinity in the absence or presence of electron donors. Vesicles from glucose or succinate grown cells, in the presence of phenazine methosulphate/ascorbate at pH 6.6, generate a proton-motive force (delta p) of approx. 140 mV. The delta p, composed of proton gradient (delta pH) and a membrane potential (delta psi), is collapsed in the presence of dinitrophenol. Based on the results obtained with valinomycin, nigericin and carbonyl cyanide m-chlorophenylhydrazone, the active transport of 2KGA at pH 6.6 is coupled predominately to the delta pH component of delta p.     

Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria.

The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are discussed.     

Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on the formation of the enzyme-substrate complex

The kinetic parameters of bovine prothrombin activation by factor Xa were determined in the absence and presence of factor Va as a function of the phospholipid concentration and composition. In the absence of factor Va, the Km for prothrombin increases proportionally with the phospholipid concentration and correlates well with the affinity of prothrombin for the different membranes. Phospholipid vesicles with a high affinity for prothrombin yield low Km values compared to membranes with less favorable binding parameters. At limited phospholipid concentrations, the Vmax of prothrombin activation correlates with the binding affinity of factor Xa for the various phospholipid vesicles. Membranes with a high affinity for factor Xa have high Vmax values, while for membranes with a low affinity a low Vmax is observed. Extrapolation of double-reciprocal plots of 1/Vmax vs. 1/[phospholipid] to infinite phospholipid concentrations, a condition at which all factor Xa would participate in prothrombin activation, yields a kcat of 2-4 min-1 independent of the type and amount of acidic phospholipid present in the vesicles. Also, in the presence of factor Va the Km for prothrombin varies proportionally with the phospholipid concentration. There is, however, no correlation between the binding parameters and the Km. Factor Va drastically lowers the Km for prothrombin for vesicles that have a low affinity for prothrombin. Vesicles composed of 20 mol % phosphatidylglycerol and 80 mol % phosphatidylcholine have a Km of 0.04 microM when factor Va is present, compared to 2.2 microM determined in the absence of factor Va.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem

We show that the structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. This conclusion is based on mutagenesis of the E. coli initiator tRNA gene followed by measurement of kinetic parameters for formylation of the mutant tRNAs in vitro and function in protein synthesis in vivo. The first base pair found at the end of the amino acid acceptor stem in all other tRNAs is replaced by a C.A. "mismatch" in E. coli initiator tRNA. Mutation of this C.A. to U:A, a weak base pair, or U.G., a mismatch, has little effect on formylation, whereas mutation to C:G, a strong base pair, has a dramatic effect lowering Vmax/Kappm by 495-fold. Mutation of the second basepair G2:C71 to U2:A71 lowers Vmax/Kappm by 236-fold. Replacement of the third base-pair C3:G70 by U3:A70, A3:U70, or G3:C70 lowers Vmax/Kappm by about 67-, 27-, and 30-fold, respectively. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and T psi C stem have little or no effect on formylation.     

Mechanism of proline transport in Escherichia coli K12. I. Effect of a membrane potential on the kinetics of 2H+/proline symport in cytoplasmic membrane vesicles

The stoichiometric coupling mechanism of the membrane potential (delta psi) in the reaction of H+/proline symport was investigated kinetically, using cytoplasmic membrane vesicles of the proline carrier-overproducing strain of Escherichia coli MinS/ pLC4 -45. When a delta psi was imposed across the cytoplasmic membrane by respiration, the Michaelis constant of transport (Kt) was lowered to about 1 microM, which was 2 orders of magnitude smaller than that of passive influx and efflux, and the maximum velocity (Vmax) was concomitantly enhanced as an exponential function of delta psi. Thermodynamically, the carrier translocated proline with a stoichiometry of 2 mol of protons versus 1 mol of substrate when driven by a delta psi at pH 8.0. Data on the delta psi dependence of Vmax of proline transport could be explained quantitatively by the Geck-Heinz hypothesis (Geck, P., and Heinz, E. (1976) Biochim, Biophys. Acta 443, 49-63). A symmetrical model of the 2H+/proline symport via formation of a carrier/H+/substrate (CH+H+S) intermediate is proposed. In this model, the effect of delta psi on the Kt was resolved as stimulation of formation of a transport intermediate, whereas the effect of delta psi on the Vmax was explained by enhancement of translocation of loaded carriers between the two sides of the membrane.     

Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase.

The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme--NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.     

A continuous spectrophotometric determination of hepatic microsomal azo reductase activity and its dependence on cytochrome P-450.

1. A continuous spectrophotometric determination of rat hepatic microsomal anaerobic azo reductase activity has been developed. 2. The addition of soluble flavins (riboflavin, FMN or FAD) greatly increased this NADPH-dependent activity towards a number of azo substrates. 3. Investigations with amaranth as substrate gave an apparent Km of 34 microM and Vmax. of 4 nmol/min per mg of microsomal protein. The inclusion of a fixed concentration of FMN increased Vmax. and greatly decreased Km, the magnitude of these changes reflecting the concentration of flavin present. 4. Investigations using a fixed amaranth concentration over a range of flavin concentrations gave biphasic double-reciprocal plots with two apparent Km and Vmax. values. 5. Pretreatment of animals with cobaltous chloride, 2-allyl-2-isopropylacetamide, carbon tetrachloride, phenobarbitone and 3-methylcholanthrene altered azo reductase activity in parallel with changes in cytochrome P-450 content. 6. The significance of these results is discussed in terms of the electron-transfer components present in the hepatic microsomal fraction.     

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.     

L-malate transport and proton symport in vesicles prepared from Pseudomonas putida

In vesicles from glucose-grown Pseudomonas putida, L-malate is transported by nonspecific physical diffusion. L-Malate also acts as an electron donor and generates a proton motive force (delta p) of 129 mV which is composed of a membrane potential (delta psi) of 60 mV and a delta pH of 69 mV. In contrast, vesicles from succinate-grown cells transport L-malate by a carrier-mediated system with a Km value of 14.3 mM and a Vmax of 313 nmol X mg protein-1 X min-1, generate no delta psi, delta pH, or delta p when L-malate is the electron donor, and produce an extravesicular alkaline pH during the transport of L-malate. A kinetic analysis of this L-malate-induced proton transport gives a Km value of 16 mM and a Vmax of 667 nmol H+ X mg protein-1 X min-1. This corresponds to a H+/L-malate ratio of 2.1. The failure to generate a delta p in these vesicles is considered, therefore, to be consistent with the induction in succinate-grown cells of an electrogenic proton symport L-malate transport system.     

Kinetic study of porcine kidney betaine aldehyde dehydrogenase

Porcine kidney betaine aldehyde dehydrogenase (EC 1.2.1.8) kinetic properties were determined at low substrate concentrations. The double-reciprocal plots of initial velocity versus substrate concentration are linear and intersect at the left of the 1/v axis and showed substrate inhibition with betaine aldehyde. Studies of inhibition by NADH and dead-end analogs showed that NADH is a mixed inhibitor against NAD(+) and betaine aldehyde. AMP is competitive with respect to NAD(+) and mixed with betaine aldehyde. Choline is competitive against betaine aldehyde and uncompetitive with respect to NAD(+). The kinetic behavior is consistent with an Iso-Ordered Bi-Bi Steady-State mechanism.