Comparative studies on glutamate metabolism in synpatic and non-synaptic rat brain mitochondria.

1. The apparent Michaelis constants of the glutamate dehydrogenase (EC 1.4.1.3), the glutamate-oxaloacetate transaminase (EC 2.6.1.1) and the glutaminase (EC 3.5.1.2) of rat brain mitochondria derived from non-synaptic (M) and synaptic (SM2) sources were studied. 2. The kinetics of oxygen uptake of both populations of mitochondria in the presence of a fixed concentration of malate and various concentrations of glutamate or glutamine were investigated. 3. In both mitochondrial populations, glutamate-supported respiration in the presence of 2.5 mM-malate appears to be biphasic, one system (B) having an apparent Km for glutamate of 0.25 +/- 0.04 mM (n=7) and the other (A) of 1.64 +/- 0.5 mM (n=7) [when corrected for low-Km process, Km=2.4 +/- 0.75 mM (n=7)]. Aspartate production in these experiments followed kinetics of a single process with an apparent Km for glutamate of 1.8-2 mM, approximating to the high-Km process. 4. Oxygen-uptake measurement with both mitochondrial populations in the presence of malate and various glutamate concentrations in which amino-oxyacetate was present showed kinetics approximating only to the low-Km process (apparent Km for glutamate approximately 0.2 mM). Similar experiments in the presence of glutamate alone showed kinetics approximating only to the high-Km process (apparent Km for glutamate approximately 1-1.3 mM). 5. Oxygen uptake supported by glutamine (0-3 mM) and malate (2.5 mM) by the free (M) mitochondrial population, however, showed single-phase kinetics with an apparent Km for glutamine of 0.28 mM. 6. Aspartate and 2-oxoglutarate accumulation was measured in 'free' nonsynaptic (M) brain mitochondria oxidizing various concentrations of glutamate at a fixed malate concentration. Over a 30-fold increase in glutamate concentration, the flux through the glutamate-oxaloacetate transaminase increased 7--8-fold, whereas the flux through 2-oxoglutarate dehydrogenase increased about 2.5-fold. 7. The biphasic kinetics of glutamate-supported respiration by brain mitochondria in the presence of malate are interpreted as reflecting this change in the relative fluxes through transamination and 2-oxoglutarate metabolism.     

Ethanol-metabolizing pathways in deermice. Estimation of flux calculated from isotope effects

The apparent deuterium isotope effects on Vmax/Km (D(V/K] of ethanol oxidation in two deermouse strains (one having and one lacking hepatic alcohol dehydrogenase (ADH] were used to calculate flux through the ADH, microsomal ethanol-oxidizing system (MEOS), and catalase pathways. In vitro, D(V/K) values were 3.22 for ADH, 1.13 for MEOS, and 1.83 for catalase under physiological conditions of pH, temperature, and ionic strength. In vivo, in deermice lacking ADH (ADH-), D(V/K) was 1.20 +/- 0.09 (mean +/- S.E.) at 7.0 +/- 0.5 mM blood ethanol and 1.08 +/- 0.10 at 57.8 +/- 10.2 mM blood ethanol, consistent with ethanol oxidation principally by MEOS. Pretreatment of ADH- animals with the catalase inhibitor 3-amino-1,2,4-triazole did not significantly change D(V/K). ADH+ deermice exhibited D(V/K) values of 1.87 +/- 0.06 (untreated), 1.71 +/- 0.13 (pretreated with 3-amino-1,2,4-triazole), and 1.24 +/- 0.13 (after the ADH inhibitor, 4-methylpyrazole) at 5-7 mM blood ethanol levels. At elevated blood ethanol concentrations (58.1 +/- 2.4 mM), a D(V/K) of 1.37 +/- 0.21 was measured in the ADH+ strain. For measured D(V/K) values to accurately reflect pathway contributions, initial reaction conditions are essential. These were shown to exist by the following criteria: negligible fractional conversion of substrate to product and no measurable back reaction in deermice having a reversible enzyme (ADH). Thus, calculations from D(V/K) indicate that, even when ADH is present, non-ADH pathways (mostly MEOS) participate significantly in ethanol metabolism at all concentrations tested and play a major role at high levels.     

Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol dehydrogenases and the inhibition by ethanol and 4-methylpyrazole

Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1M sodium phosphate buffer, at pH 7.5 and 25°C, containing 0.5 mM NAD(+) and varied concentrations of substrate. K(M) values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12-57 mM, for methanol to be 2.0-3500 mM, for ethylene glycol to be 4.3-2600mM, and for isopropanol to be 0.73-3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for V(max) for the toxic alcohols were much less than that of the K(M) across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (K(is)) for the whole family being 0.062-960 μM and the intercept inhibition constants (K(ii)), 33-3000 μM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50mM are largely inhibited by 20 mM ethanol or by 50 μM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives.     

Ferroxidase kinetics of horse spleen apoferritin.

Protein ferroxidase site(s), which catalyze the reaction between ferrous ion and dioxygen, have long been thought to play a role in core formation in ferritin; however, the mechanism of the reaction has never been studied in detail. In the present work, the enzymatic activity of ferritin was examined using oximetry, the net Fe2+ oxidation reaction being as follows. [formula: see text] The reaction exhibits saturation kinetics with respect to both Fe2+ and O2 (apparent Michaelis constants: Km,Fe = 0.35 +/- 0.01 mM and Km,O2 = 0.14 +/- 0.03 mM). The enzyme has a turnover number kcat = 80 +/- 3 min-1 at 20 degrees C with maximal activity at pH 7. The kinetics are discussed in terms of two mechanisms, one involving monomeric and the other dimeric iron protein complexes. In both instances Fe(II) oxidation occurs in 1-electron steps. Zinc(II) is a competitive inhibitor of iron(II) oxidation at Zn2+/apoprotein ratios > or = 6 (inhibitor constant KI,Zn = 0.067 +/- 0.011 mM) but appears to be a noncompetitive inhibitor at lower ratios (< or = 2), indicating the presence of more than one type of zinc binding site on the protein. At increments of 50 Fe2+/protein or less, all of the iron is oxidized via the protein ferroxidase site(s), independent of the amount of core already present. However, when larger increments are employed, some iron oxidation appears to occur on the surface of the mineral core. The results of these studies emphasize the role of the protein shell in all phases of core growth and confirm the presence of a functionally important catalytic site in ferritin in addition to other binding sites on the protein for iron.     

Effect of intersubunit interaction in horse liver alcohol dehydrogenase on the kinetics of ethanol oxidation]

The kinetics of enzymatic oxidation of ethanol in the presence of alcohol dehydrogenase within a wide range of ethanol and NAD concentrations (pH 6.0--11.5) were studied. It was shown that high concentrations of ethanol (greater than 0.7--5 mM, depending on pH) and NAD (greater than 0.4--0.8 mM) activate alcohol dehydrogenase from horse liver within the pH range of 6.0--7.9. A mechanism of activation based on negative cooperativity of ADH subunits for binding of ethanol and NAD was proposed. The catalytic and Michaelis constants for alcohol dehydrogenase were calculated from ethanol and NAD at all pH values studied. The changes resulting from the subunit cooperativity were revealed. The nature of ionogenic groups of alcohol dehydrogenase, which affect the formation of complexes between the enzyme and NAD and ethanol, and the rate constants for catalytic oxidation of ethanol was assumed. The biological significance of the enzyme capacity for activation by high concentrations of ethanol within the physiological range of pH in the blood under excessive use of alcohol is discussed.     

The metabolic role of human ADH3 functioning as ethanol dehydrogenase

Human class III alcohol dehydrogenase (ADH3), also known as glutathione-dependent formaldehyde dehydrogenase, exhibited non-hyperbolic kinetics with ethanol at a near physiological pH 7.5. The S(0.5) and k(cat) were determined to be 3.4+/-0.3 M and 33+/-3 min(-1), and the Hill coefficient (h) 2.21+/-0.09, indicating positive cooperativity. Strikingly, the S(0.5) for ethanol was found to be 5.4 x 10(6)-fold higher than the K(m) for S-(hydroxymethyl)glutathione, a classic substrate for the enzyme, whereas the k(cat) for the former was 41% lower than that for the latter. Isotope effects on enzyme activity suggest that hydride transfer may be rate-limiting in the oxidation of ethanol. Kinetic simulations using the experimentally determined Hill constant suggest that gastric ADH3 may highly effectively contribute to the first-pass metabolism at 0.5-3 M ethanol, an attainable range in the gastric lumen during alcohol consumption. The positive cooperativity mainly accounts for this metabolic role of ADH3.     

Rhodobacter capsulatus 1-deoxy-D-xylulose 5-phosphate synthase: steady-state kinetics and substrate binding

1-Deoxy-d-xylulose 5-phosphate synthase (DXP synthase) catalyzes the thiamine diphosphate (TPP)-dependent condensation of pyruvate and d-glyceraldehyde 3-phosphate (GAP) to yield DXP in the first step of the methylerythritol phosphate pathway for isoprenoid biosynthesis. Steady-state kinetic constants for DXP synthase calculated from the initial velocities measured at varying concentrations of substrates were as follows: k(cat) = 1.9 +/- 0.1 s(-1), K(m)(GAP) = 0.068 +/- 0.001 mM, and K(m)(pyruvate) = 0.44 +/- 0.05 mM for pyruvate and GAP; k(cat) = 1.7 +/- 0.1 s(-1), K(m)(d-glyceraldehyde) = 33 +/- 3 mM, and K(m)(pyruvate) = 1.9 +/- 0.5 mM for d-glyceraldehyde and pyruvate. beta-Fluoropyruvate was investigated as a dead-end inhibitor for pyruvate. Double-reciprocal plots showed a competitive inhibition pattern with respect to pyruvate and noncompetitive inhibition with respect to GAP/d-glyceraldehyde. (14)CO(2) trapping experiments demonstrated that the binding of both substrates (pyruvate and GAP/d-glyceraldehyde) is required for the formation of a catalytically competent enzyme-substrate complex. These results are consistent with an ordered mechanism for DXP synthase where pyruvate binds before GAP/d-glyceraldehyde.     

Distinction between the two basic mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system

In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.     

Kinetics of inhibition of ethanol metabolism in rats and the rate-limiting role of alcohol dehydrogenase

If liver alcohol dehydrogenase were rate-limiting in ethanol metabolism, inhibitors of the enzyme should inhibit the metabolism with the same type of kinetics and the same kinetic constants in vitro and in vivo. Against varied concentrations of ethanol, 4-methylpyrazole is a competitive inhibitor of purified rat liver alcohol dehydrogenase (Kis = 0.11 microM, in 83 mM potassium phosphate and 40 mM KCl buffer, pH 7.3, 37 degrees C) and is competitive in rats (with Kis = 1.4 mumol/kg). Isobutyramide is essentially an uncompetitive inhibitor of purified enzyme (Kii = 0.33 mM) and of metabolism in vivo (Kii = 1.0 mmol/kg). Low concentrations of both inhibitors decreased the rate of metabolism as a direct function of their concentrations. Qualitatively, therefore, alcohol dehydrogenase activity appears to be a major rate-limiting factor in ethanol metabolism. Quantitatively, however, the constants may not agree because of distribution in the animal or metabolism of the inhibitors. At saturating concentrations of inhibitors, ethanol is eliminated by inhibitor-insensitive pathways, at about 10% of the total rate at a dose of ethanol of 10 mmol/kg. Uncompetitive inhibitors of alcohol dehydrogenase should be especially useful for inhibiting the metabolism of alcohols since they are effective even at saturating levels of alcohol, in contrast to competitive inhibitors, whose action is overcome by saturation with alcohol.     

Mouse alcohol dehydrogenase 4: kinetic mechanism, substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37 degrees C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD(+) is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K(m) for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

Evidence for a free radical chain mechanism in the reaction between peroxidase and indole-3-acetic acid at neutral pH

The oxidation of indole-3-acetic acid (IAA) catalyzed by horseradish peroxidase (HRP) in the absence of added H2O2 was studied at pH 7.4 using spectral and kinetic approaches. Upon addition of a hundred-fold excess of IAA to HRP the native enzyme was rapidly transformed to compound II (HRP-II). HRP-II was the predominant catalytic enzyme species during the steady state. No compound III was observed. HRP-II was slowly transformed to the stable inactive verdohemo-protein, P-670. A precursor of P-670, so-called P-940 was not detected. After the cessation of IAA oxidation there was neither oxygen consumption nor P-670 formation; the remaining HRP-II was spontaneously reduced to native enzyme. Single exponential kinetics were observed in the steady state for IAA oxidation, oxygen consumption and P-670 formation yielding identical first order rate constants of about 6 . 10(4) s(-1). A comparison of the rate of IAA oxidation by HRP-II in the steady state and in the transient state indicated that more than 1 3 of the IAA was oxidized non-enzymatically during the steady state, confirming that a free radical chain reaction is involved in the peroxidase-catalyzed oxidation of IAA. IAA oxidation stopped before IAA was completely consumed, which cannot be ascribed to enzyme inactivation because 30-50% of the enzyme was still active after the end of the reaction. Instead, incomplete IAA oxidation is explained in terms of termination of the free radical chain reaction. Bimolecular rate constants of IAA oxidation by HRP-I and HRP-II determined under transient state conditions were (2.2 +/- 0.1) x 10(3) M(-1) s(-1) and (2.3 +/- 0.2) x 10(2) M(-1) s(-1).     

Purification and characterization of an alcohol dehydrogenase from Lithospermum erythrorhizon cell cultures

An NAD-dependent alcohol dehydrogenase has been purified to apparent homogeneity from cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), using protamine sulphate and ammonium sulphate precipitation and chromatography on DEAE-Sephacel, Superdex 200, hydroxyapatite and HiTrap blue. The enzyme is a homodimer with a M_r of ca. 77,000. Each subunit with a M_r of 40,000 contains two zinc atoms. Its isoelectric point was found at pH 5.0. The best alcohol substrate of the enzyme is ethanol. The pH optimum for ethanol oxidation is at pH 8.7 and for acetaldehyde reduction at pH 4.6. The Michaelis constants for ethanol and NAD are 2.49 and 0.05 (pH 8.7), and for acetaldehyde and NADH 2.2 and 0.078 mM (pH 4.6), respectively. Partial amino acid sequences of the purified enzyme showed high homology to alcohol dehydrogenases from other plants.Abbreviations ADH alcohol dehydrogenase - DTT dithiothreitol - PMSF dephenylmethylsulfonyl fluoride - PVPP polyvinylpolypyrrolidone - IAA indole-3-acetic acid - TFA trifluoroacetic acid     

Mouse alcohol dehydrogenase 4: kinetic mechanism,substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37°C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD⁺ is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K_m for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

Investigation of the effects of some sulfonamide derivatives on the activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase from human erythrocytes

In this study, the in vitro effects of some sulfonamide derivatives, which are carbonic anhydrase inhibitors, on the enzymes activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase were investigated. For this purpose, these three enzymes were purified from human erythrocytes. Purification procedure composed of four steps; preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and gel filtration chromatography on Sephadex G-200. 5-(3alpha-Hydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3alpha,12alpha-Dihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3alpha,7alpha,12alpha-Trihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3), 5-(3alpha,Acetoxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (4), 5-(3alpha,7alpha,12alpha-Triacetoxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (5), 5-(3,7,12-Trioxo-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (6), acetazolamide, and dorzolamide were tested in this experiment. Compounds 3, 5, and dorzolamide showed inhibitory effects on the activity of 6-phosphogluconate dehydrogenase, and I(50) values and K(i) constants were calculated as 0.0601 mM, 0.00253 mM, and 1.41 mM and 0.0878 +/- 0.0274 mM, 0.0042 +/- 0.0009 mM, and 3.1446 +/- 0.2081 mM, respectively. Glutathione reductase was also inhibited by 1 and 2. I(50) values and K(i) constants were 0.0471 mM and 0.0723 +/- 0.0388 mM for 1 and 0.0045 mM and 0.0061 +/- 0.0014 mM, for 2. If these sulfonamide derivatives are proposed as drugs, some of which are being used in glaucoma treatment such as acetazolamide and dorzolamide, these results should be taken into consideration concerning via these enzymes.     

Competitive binding of acetate and chloride in photosystem II.

The binding of chloride and acetate to photosystem II (PSII) was examined to elucidate the mechanism of acetate inhibition. The mode of inhibition was studied, and individual binding sites were assigned by steady-state O2 evolution measurements in correlation with electron paramagnetic resonance (EPR) results. Two binding sites were found for acetate, one chloride-sensitive on the electron donor side and one chloride-insensitive on the electron acceptor side. The respective binding constants were as follows: KCl = 0.5 +/- 0.2 mM (chloride binding to the donor side), KI = 16 +/- 5 mM (acetate binding to the donor side), and KI' = 130 +/- 40 mM (acetate binding to the acceptor side). When acetate was bound to the acceptor side of PSII, 200 K illumination induced a narrowed form of the QA-FeII EPR signal, the yield of which was independent of the chloride concentration. When acetate was bound to the donor side, room-temperature illumination produced the S2YZ* state. EPR measurements showed that both the yield and formation rate of this state increased with acetate concentration. Increasing chloride concentrations slowed the rate of formation of the S2YZ* state, but did not affect the steady-state yield of the S2YZ* state. These findings indicate that the light-induced reactions in acetate-inhibited PSII are modulated by both donor side and acceptor side binding of acetate, while the steady-state yield of the S2YZ* state at the high PSII concentrations used for EPR measurements depends primarily on acceptor side turnover. Our data further support a close proximity of chloride to YZ*, indicating a possible role for chloride in the electron-transfer mechanism at the O2-evolving complex.     

Colloidal bismuth subcitrate and omeprazole inhibit alcohol dehydrogenase mediated acetaldehyde production by Helicobacter pylori

We have recently shown that Helicobacter pylori possesses marked alcohol dehydrogenase (ADH) activity and is capable - when incubated with an ethanol containing solution in vitro - of producing large amounts of acetaldehyde. In the present study we report that some drugs commonly used for the eradication of H. pylori and for the treatment of gastroduodenal diseases are potent ADH inhibitors and, consequently, effectively prevent bacterial oxidation of ethanol to acetaldehyde. Colloidal bismuth subcitrate (CBS), already at a concentration of 0.01 mM, inhibited H. pylori ADH by 93% at 0.5 M ethanol and decreased oxidation of 22 mM ethanol to acetaldehyde to 82% of control. At concentrations above 5 mM, CBS almost totally inhibited acetaldehyde formation. Omeprazole, a drug also known to suppress growth of H. pylori, also inhibited H. pylori ADH and suppressed bacterial acetaldehyde formation significantly to 69% of control at a drug concentration of 0.1 mM. By contrast, the H₂-receptor antagonists ranitidine and famotidine showed only modest effect on bacterial ADH and acetaldehyde production. We suggest that inhibition of bacterial ADH and a consequent suppression of acetaldehyde production from endogenous or exogenous ethanol may be a novel mechanism by which CBS and omeprazole exert their effect both on the growth of H. pylori as well as on H. pylori associated gastric injury.     

Kinetics of bicarbonate and chloride transport in human red cell membranes

Unidirectional [14C]HCO3- and 36Cl- efflux from human red cells and ghosts was studied under self-exchange conditions at pH 7.8 and 0 degrees C by means of the Millipore-Swinnex filtering technique. Control bicarbonate experiments showed that 14CO2 loss from the cells to the efflux medium was insignificant. The anion flux was determined under (a) symmetric variations of the anion concentration (C(i) = C(o) = 5-700 mM), and (b) asymmetric conditions with CAn constant on one side and varied on the other side of the membrane. Simple Michaelis-Menten-like kinetics (MM fit: J(eff) = J(eff)max.C/(K1/2 + C)) was used to describe anion flux dependence on C for (a) C(i) = C(o) = 5-100 mM, (b) C(i) = 6-100 mM, C(o) = constant, and (c) C(i) = constant, C(o) = 1-25 mM. At higher cellular concentrations noncompetitive self-inhibition by anion binding (inhibition constant Ki mM) to an intracellular site was included in the model (MS fit): J(eff) = J(eff)max.C(i)/[(K1/2 + C(i)).(1 + C(i)/Ki)]. The MM fits show that the external half-saturation constant, Ko1/2 ( = C(o)An for J(eff,o) = 1/2.j(eff,o)max) at C(o) = 1-25 mM is 1.5-2.4 mM (HCO3-) and 1.8-2.6 mM (Cl-). At C(o) = 1-260 mM Ko1/2 is 1.2-1.5 mM (HCO3-) and 1.4-1.8 mM (Cl-). The respective maximum flux, J(eff,o)max (nmol/[cm2.s]), for C(o) = 1-25 mM is 0.41-0.51 (HCO3-) and 0.28-0.38 (Cl-), and for C(o) = 1-260 mM 0.39-0.44 (HCO3-) and 0.27-0.31 (Cl-). The internal half-saturation constant, Ki1/2 mM is: MM fit (C(i) = 6-100 mM, C(o) = 50 mM), 18.0 mM (HCO3-) and 23.8 mM (Cl-); MS fit (C(i) = 6-920 mM, C(o) = 50 mM), 32.0 mM (HCO3-) and 45.1 mM (Cl-). The maximum flux, J(eff,i)max (nmol/[cm2.s]) is: MM fit; 0.50 (HCO3-) and 0.34 (Cl-); MS fit, 0.70 (HCO-3) and 0.50 (Cl-). The half-inhibition constants of the MS fit, Ki, are 393 mM (HCO3-) and 544 mM (Cl-). The MM fit shows that the symmetric half-saturation constant, Ks1/2, is 20.2 (HCO-3) and 23.9 (Cl-) mM, and J(eff,s)max is 0.51 (HCO3-) and 0.32 (Cl-) nmol/(cm2.s). The MS fit shows that for C = 5-700 mM Ks1/2 is 30.4 nM (HCO3-) and 50.1 mM (Cl-), and Ki is 541 mM (HCO3-) and 392 mM (Cl-).(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphofructokinase from muscle of the marine giant barnacle Austromegabalanus psittacus: kinetic characterization and effect of in vitro phosphorylation

The kinetic properties of phosphofructokinase from muscle of the giant cirripede Austromegabalanus psittacus were characterized, after partial purification by ion exchange chromatography on DEAE-cellulose. This enzyme showed differences regarding PFKs from other marine invertebrates: the affinity for fructose 6-phosphate (Fru 6-P) was very low, with an S(0.5) of 22.6+/-1.4 mM (mean+/-S.D., n=3), and a high cooperativity (n(H) of 2.90+/-0.21; mean+/-S.D., n=3). The barnacle PFK showed hyperbolic saturation kinetics for ATP (apparent K(m ATP)=70 microM, at 5 mM Fru 6-P, in the presence of 2 mM ammonium sulfate). ATP concentrations higher than 1 mM inhibited the enzyme. Ammonium sulfate activated the PFK several folds, increasing the affinity of the enzyme for Fru 6-P and V(max). 5'-AMP (0.2 mM) increased the affinity for Fru 6-P (S(0.5) of 6.2 mM). Fructose 2,6-bisphosphate activated the PFK, with a maximal activation at concentrations higher than 2 microM. Citrate reverted the activation of PFK produced by 0.2 mM 5'-AMP (IC(50 citrate)=2.0 mM), producing a higher inhibition than that exerted on other invertebrate PFKs. Barnacle muscular PFK was activated in vitro after exposure to exogenous cyclic-AMP (0.1 mM) as well as by phosphatidylserine (50 microg/ml), indicating a possible control by protein kinase A and a phospholipid dependent protein kinase (PKC). The results suggest a highly regulated enzyme in vivo, by allosteric mechanisms and also by protein phosphorylation.     

Ethanol production and toxicity in suspension-cultured carrot cells and embryos

The process of carrot (Daucus carota L.) somatic embryogenesis is highly sensitive to exogenously added ethanol, since 5 mM ethanol inhibits this process by 50%, whereas the growth of proliferating carrot cells is inhibited to the same extent by 20 mM ethanol. This is consistent with the fact that proliferating cultures produce ethanol and release it into the medium at concentrations up to 20 mM, whereas embryogenic culture medium contains less than 1 mM ethanol. Data are presented showing the influence of cell density and 2,4-dichlorophenoxyacetic acid on ethanol production and on the presence of an alcohol-dehydrogenase (EC 1.1.1.1.) inactivator in carrot embryos.Abbreviations ADH alcohol dehydrogenase - 6-BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - FW fresh weight     

Apparent affinity of the Na/K pump for ouabain in cultured chick cardiac myocytes. Effects of Nai and Ko

The measured apparent affinity (K0.5) of the Na/K pump for ouabain has been reported to vary over a wide range. In a previous report we found that changing Nai could alter apparent affinity by at least an order of magnitude and that the model presented predicted this variability. To increase our understanding of this variability, isolated cells or two- to three-cell clusters of cardiac myocytes from 11-d embryonic chick were used to measure the effects of Nai and Ko on the K0.5 of the Na/K pump for ouabain. Myocytes were whole-cell patch clamped and Na/K pump current (Ip) was measured in preparations exposed to a Ca-free modified Hank's solution (HBSS) that contained 1 mM Ba, 10 mM Cs, and 0.1 mM Cd. Under these conditions there are no Ko-sensitive currents other than Ip because removal of Ko in the presence of ouabain had no effect on the current-voltage (I-V) relation. The I-V relation for Ip showed that in the presence of 5.4 mM Ko and 51 mM Nai, Ip has a slight voltage dependence, decreasing approximately 30% from 0 to -130 mV. Increasing Nai in the patch pipette from 6 to 51 mM (Ko = 5.4 mM) caused Ip to increase from 0.46 +/- 0.07 (n = 5) to 1.34 +/- 0.08 microA/cm2 (n = 13) with a K0.5 for Nai of 17.4 mM and decreased the K0.5 for ouabain from 18.5 +/- 1.8 (n = 4) to 3.1 +/- 0.4 microM (n = 3). Similarly, varying Ko between 0.3 and 10.8 mM (Nai = 24 mM) increased Ip from 0.13 +/- 0.01 (n = 5) to 0.90 +/- 0.05 microA/cm2 (n = 5) with a K0.5 for Ko of 1.94 mM and increased K0.5 for ouabain from 0.56 +/- 0.14 (n = 3-6) to 10.0 +/- 1.1 microM (n = 6). All of these changes are predicted by the model presented. A qualitative explanation of these results is that Nai and Ko interact with the Na/K pump to shift the steady-state distribution of the Na/K pump molecules among the kinetic states. This shift in state distribution alters the probability that the Na/K pump will be in the conformation that binds ouabain with high affinity, thus altering the apparent affinity. In intact cells, the measured apparent affinity represents a combination of all the rate constants in the model and does not equate to simple first-order binding kinetics.(ABSTRACT TRUNCATED AT 400 WORDS)