The oxidation of choline by liver slices and mitochondria during liver development in the rat

Betaine is the major oxidation product of [Me-¹⁴C] choline produced by rat liver slices. Liver slices from adult rats rapidly oxidize [Me-¹⁴C] choline to betaine and the bulk of the betaine produced is recovered in the incubation medium. Considerably more choline is oxidized to betaine than is phosphorylated to phosphorylcholine. The rate of phosphorylation of choline appears to be independent of the rate of choline oxidation. Liver slices from fetal and young rats oxidize choline to betaine at a lower rate than adult liver slices.The ability of mitochondria to oxidize [Me-¹⁴C] choline to betaine aldehyde and betaine is considerably lower in fetal liver than in adult liver. The major product with both fetal and adult mitochondria is betaine aldehyde. Choline oxidation by mitochondria begins to increase 1 day prior to birth and increases progressively to adult levels by 18 days. The developmental pattern for choline oxidation is similar to the pattern for succinic dehydrogenase activity.     

Control of choline oxidation in rat kidney mitochondria

Choline is a quaternary amino cationic organic alcohol that is oxidized to betaine in liver and kidney mitochondria. Betaine acts as an intracellular organic osmolyte in the medulla of the kidney. Evidence is provided that kidney mitochondria have a choline transporter in their inner membrane. The transporter has a Km of 173 ± 64 μM and a Vmax of 0.4 ± 0.1 nmol/min/mg mitochondrial protein (at 10 °C). Uptake of choline is not coupled to betaine efflux. Transporter activity demonstrates a dependence on membrane potential and choline transport is inhibited by hemicholinium-3. Steady-state oxygen consumption due to choline oxidation in kidney mitochondria was measurable at 37 °C (125 ± 6 pmolO₂/min/mg mitochondrial protein), in the absence of other mitochondrial electron transport chain substrates and the choline transporter was shown to be the major site of control (96 ± 4%) over choline oxidation flux in isolated kidney mitochondria. We conclude that the choline transporter in rat kidney mitochondria is the major site of control over the production of the organic osmolyte, betaine.     

Choline and betaine aldehyde oxidation by rat liver mitochondria

1. The question of the ability or inability of rat liver mitochondria to oxidize externally added or internally generated betaine aldehyde has been reexamined. Well washed mitochondria were demonstrated to contain approx. 7% of the post-nuclear betaine aldehyde dehydrogenase as an integral component. The enzyme is approximately equally distributed between the inner membrane and the intermembrane plus matrix fractions. Significantly, none was found in the outer membrane fraction. The mitochondrial enzyme was shown to be functional under all the conditions tested; betaine aldehyde generated within the mitochondria by choline oxidation or added externally was oxidized to betaine in significant amounts.

2. The stoichiometry for the complete oxidation of choline or externally added betaine aldehyde was confirmed to be 2 and 1 moles, respectively, of O₂ utilized per mole of substrate added. Depending on the reaction conditions employed, considerable variation in the relative amount of choline oxidase and betaine aldehyde oxidase activities of mitochondria was observed when they were allowed to oxidize only a portion of the choline added. The necessity of measuring the contribution of betaine aldehyde oxidase in studies of choline oxidase is discussed.

3. Reasons for the discrepancies in the literature concerning the ability of mitochondria to oxidize betaine aldehyde are discussed.     

Measurement of the formation of betaine aldehyde and betaine in rat liver mitochondria by a high pressure liquid chromatography-radioenzymatic assay.

A new assay procedure for measurement of rat liver mitochondrial choline dehydrogenase was developed. Oxidation of [methyl-14C]choline to [methyl-14C]betaine aldehyde and [methyl-14C]betaine was measured after isolating these compounds using HPLC. We observed that NAD+ was required for conversion of betaine aldehyde to betaine in rat liver mitochondria. In the absence of this cofactor, oxidation of choline led to the accumulation of betaine aldehyde. The apparent Km of the mitochondrial choline dehydrogenase for choline was 0.14-0.27 mM, which is significantly lower than previously reported. A partially purified preparation of choline dehydrogenase catalyzed betaine aldehyde formation only in the presence of exogenous electron acceptors (e.g., phenazine methosulfate). This preparation failed to catalyze the formation of betaine even in the presence of NAD+, indicating that betaine aldehyde dehydrogenase may be a separate enzyme from choline dehydrogenase.     

The transport of sulphate and sulphite in rat liver mitochondria

1. The mechanism of sulphite and sulphate permeation into rat liver mitochondria was investigated. 2. Extramitochondrial sulphite and sulphate elicit efflux of intramitochondrial phosphate, malate, succinate and malonate. The sulphate-dependent effluxes and the sulphite-dependent efflux of dicarboxylate anions are inhibited by butylmalonate, phenylsuccinate and mersalyl. Inhibition of the phosphate efflux produced by sulphite is caused by mersalyl alone and by N-ethylmaleimide and butylmalonate when present together. 3. External sulphite and sulphate cause efflux of intramitochondrial sulphate, and this is inhibited by butylmalonate, phenylsuccinate and mersalyl. 4. External sulphite and sulphate do not cause efflux of oxoglutarate or citrate. 5. Mitochondria swell when suspended in an iso-osmotic solution of ammonium sulphite; this is not inhibited by N-ethylmaleimide or mersalyl. 6. Low concentrations of sulphite, but not sulphate, produce mitochondrial swelling in iso-osmotic solutions of ammonium malate, succinate, malonate, sulphate, or phosphate in the presence of N-ethylmaleimide. 7. It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO(2) or an exchange of the sulphite anion for hydroxyl ion(s).     

Permeability transition in rat liver mitochondria is modulated by the ATP-Mg/Pi carrier

Mitochondrial permeability transition, due to opening of the permeability transition pore (PTP), is triggered by Ca2+ in conjunction with an inducing agent such as phosphate. However, incubation of rat liver mitochondria in the presence of low micromolar concentrations of Ca2+ and millimolar concentrations of phosphate is known to also cause net efflux of matrix adenine nucleotides via the ATP-Mg/Pi carrier. This raises the possibility that adenine nucleotide depletion through this mechanism contributes to mitochondrial permeability transition. Results of this study show that phosphate-induced opening of the mitochondrial PTP is, at least in part, secondary to depletion of the intramitochondrial adenine nucleotide content via the ATP-Mg/Pi carrier. Delaying net adenine nucleotide efflux from mitochondria also delays the onset of phosphate-induced PTP opening. Moreover, mitochondria that are depleted of matrix adenine nucleotides via the ATP-Mg/Pi carrier show highly increased susceptibility to swelling induced by high Ca2+ concentration, atractyloside, and the prooxidant tert-butylhydroperoxide. Thus the ATPMg/Pi carrier, by regulating the matrix adenine nucleotide content, can modulate the sensitivity of rat liver mitochondria to undergo permeability transition. This has important implications for hepatocytes under cellular conditions in which the intramitochondrial adenine nucleotide pool size is depleted, such as in hypoxia or ischemia, or during reperfusion when the mitochondria are exposed to increased oxidative stress.     

Choline oxidation by intact spinach chloroplasts

Plants synthesize betaine by a two-step oxidation of choline (choline → betaine aldehyde → betaine). Protoplast-derived chloroplasts of spinach (Spinacia oleracea L.) carry out both reactions, more rapidly in light than in darkness (AD Hanson et al. 1985 Proc Natl Acad Sci USA 82: 3678-3682). We investigated the light-stimulated oxidation of choline, using spinach chloroplasts isolated directly from leaves. The rates of choline oxidation obtained (dark and light rates: 10-50 and 100-300 nanomoles per hour per milligram chlorophyll, respectively) were approximately 20-fold higher than for protoplast-derived chloroplasts. Betaine aldehyde was the main product. Choline oxidation in darkness and light was suppressed by hypoxia. Neither uncouplers nor the Calvin cycle inhibitor glyceraldehyde greatly affected choline oxidation in the light, and maximal choline oxidation was attained far below light saturation of CO₂ fixation. The light stimulation of choline oxidation was abolished by the PSII inhibitors DCMU and dibromothymoquinone, and was partially restored by adding reduced diaminodurene, an electron donor to PSI. Both methyl viologen and phenazine methosulfate prevented choline oxidation. Adding dihydroxyacetone phosphate, which can generate NADPH in organello, doubled the dark rate of choline oxidation. These results indicate that choline oxidation in chloroplasts requires oxygen, and reducing power generated from PSI. Enzymic reactions consistent with these requirements are discussed.     

Control of choline oxidation by rat-liver mitochondria

The steady-state concentrations of choline and its reaction products in intact rat-liver mitochondria were determined under different conditions. From these measurements, it is concluded that in a sucrose medium choline dehydrogenation and betaine aldehyde dehydrogenation are the rate-limiting steps in overall choline oxidation under “State-3” or uncoupled conditions, respectively.Ageing of the mitochondria leads to changes in the mitochondrial membrane, resulting in a markedly different pattern of oxidation products. This finding explains why rotenone inhibits oxygen uptake with choline as substrate in fresh but not in aged mitochondria.     

Control of choline oxidation by rat-liver mitochondria.

The steady-state concentrations of choline and its reaction products in intact rat-liver mitochondria were determined under different conditions. From these measurements, it is concluded that in a sucrose medium choline dehydrogenation and betaine aldehyde dehydrogenation are the rate-limiting steps in overall choline oxidation under "State-3" or uncoupled conditions, respectively. Ageing of the mitochondria leads to changes in the mitochondrial membrane, resulting in a markedly different pattern of oxidation products. This finding explains why rotenone inhibits oxygen uptake with choline as substrate in fresh but not in aged mitochondria.     

The effect of mersalyl on the oxidation of succinate by housefly mitochondria

1. Succinate is oxidized very rapidly by housefly flight-muscle mitochondria in the presence of rotenone.

2. Phosphate (or arsenate) is a strong inhibitor of this succinate oxidation.

3. The inhibitory effect of phosphate (or arsenate) can be overcome by mersalyl and other sulphydryl reagents.

4. Under conditions of rapid succinate oxidation the intramitochondrial phosphate concentration is low.

5. It is suggested that phosphate competes with succinate for entry into the mitochondria.     

Control of mitochondrial Mg++-efflux]

Inorganic phosphate stimulates the release of Mg++ from liver mitochondria, depending on concentration; a concentration as low as 0.1 mM phosphate is already effective. The process is dependent on the electron transfer of the respiratory chain, and its rate is highest under conditions of endogenous respiration and with ascorbate and TMPD as substrates, respectively. The phosphate stimulated release of Mg++ is followed, with a pronounced delay, by a Ca++ efflux and a swelling of mitochondria. Addition of EGTA strongly reduced the rate of Mg++ liberation in the presence and absence of inorganic phosphate. Exogenous Ca++ is able to abolish the EGTA effect. ADP and ATP inhibit the phosphate stimulated release of Mg++. Phosphoenol pyruvate and free fatty acids enhance the rate of Mg++ and Ca++ efflux from the mitochondria. The results permit the conclusion that inorganic phosphate, Ca++ and various metabolites of the cell metabolism influence the Mg++ distribution between the extra- and intramitochondrial space, thus controlling the permeability of the mitochondrial inner membrane for monovalent cations.     

Efflux of choline and glycine betaine from osmoregulating cells of Escherichia coli

We present evidence that glycine betaine (betaine) which was synthesized from choline was excreted and reaccumulated in osmoregulating cells of Escherichia coli. Choline which was accumulated in bet mutants defective in betaine synthesis was shown to be excreted in response to betaine uptake. Our data suggest that E. coli has efflux systems for betaine and choline which are independent of the uptake systems for these metabolites. The ProU system of E. coli, but not that of Salmonella typhimurium, can mediate low-affinity choline uptake.     

Characteristics of adenine nucleotide fluxes and transport in human tumor mitochondria

The efflux of adenine nucleotides from three human tumor mitochondria has been investigated with mitochondria prelabeled with radioactive ATP. Uncouplers induce a large efflux of adenine nucleotides from mitochondria from human hepatoma and oat cell carcinoma while efflux from astrocytoma mitochondria is less. This efflux does not require exchangeable anions, i.e., adenine nucleotides or pyrophosphate, in the extramitochondrial medium, and is not sensitive to atractyloside. The efflux is more extensive with dinitrophenol and CCCP than with valinomycin-K+, and may account for the differential effects of the two types of uncouplers on uncoupler-stimulated ATPase of tumor mitochondria previously reported by us. Dinitrophenol and CCCP do not elicit any efflux of adenine nucleotides from normal liver mitochondria. Efflux of orthophosphate from tumor mitochondria is also greater with dinitrophenol and CCCP; however, the more interesting finding is that the concentration of orthophosphate in these mitochondria is unusually high, i.e., 10-40-times greater than the intramitochondrial phosphate concentration of liver mitochondria. Atractyloside sensitive transport of ATP and ADP in human tumor mitochondria has also been determined. Vmax values for both ADP and ATP transport are lower than those obtained with liver mitochondria, especially with ADP transport. ATP transport in tumor mitochondria is not affected by CCCP in contrast to the 4-5-fold stimulation observed in liver mitochondria.     

Choline oxidation and choline dehydrogenase

The oxidation of choline by both freshly prepared and aged rat liver mitochondria is inhibited by amytal. Whereas rotenone inhibits choline-cytochromec reductase only in the case of freshly prepared mitochondria, the extent of inhibition is influenced by preincubation, but the inhibition is not secondary to the inhibited oxidation of betaine aldehyde, the product of choline oxidation. Evidence shows that rotenone is able to inhibit the swelling of rat liver mitochondria and the inhibition of choline-cytochromec reductase by rotenone is related to the inhibition of mitochondrial swelling. Nine inhibitors of choline dehydrogenase have been reported. Among those, some belong to the category of acetylcholine esterase inhibitor. In view of the structure of those inhibitors, it seems quite likely that there is an anionic site at the active center of choline dehydrogenase. Purification of choline dehydrogenase in its native form has been accomplished by solubilization with Lubrol WX, hydroxyapatite, and DEAE-Sepharose chromatography and sucrose gradient ultracentrifugation. The preparation is pure as judged by SDS-PAGE and Ultrogel AcA 34 column chromatography. The molecular weight determined by SDS-PAGE is approximately 61,000. There is 0.23 mg phospholipid/mg protein and the Stokes' radius of protein-Lubrol-phospholipid mixed micelles is about 59 A.     

Mechanistic studies of choline oxidase with betaine aldehyde and its isosteric analogue 3,3-dimethylbutyraldehyde

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine via two sequential FAD-dependent reactions in which betaine aldehyde is formed as an intermediate. The chemical mechanism for the oxidation of choline catalyzed by choline oxidase was recently elucidated by using kinetic isotope effects [Fan, F., and Gadda, G. (2005) J. Am. Chem. Soc. 127, 2067-2074]. In this study, the oxidation of betaine aldehyde has been investigated by using spectroscopic and kinetic analyses with betaine aldehyde and its isosteric analogue 3,3-dimethylbutyraldehyde. The pH dependence of the kcat/Km and kcat values with betaine aldehyde showed that a catalytic base with a pKa of approximately 6.7 is required for betaine aldehyde oxidation. Complete reduction of the enzyme-bound flavin was observed in a stopped-flow spectrophotometer upon anaerobic mixing with betaine aldehyde or choline at pH 8, with similar k(red) values > or = 48 s(-1). In contrast, only 10-26% of the enzyme-bound flavin was reduced by 3,3-dimethylbutyraldehyde between pH 6 and 10. Furthermore, this compound acted as a competitive inhibitor versus choline. NMR spectroscopic analyses indicated that betaine aldehyde exists predominantly (99%) as a diol form in aqueous solution. In contrast, the thermodynamic equilibrium for 3,3-dimethylbutyraldehyde favors the aldehyde (> or = 65%) over the hydrated form in the pH range from 6 to 10. The keto species of 3,3-dimethylbutyraldehyde is reactive toward enzymic nucleophiles, as suggested by the kinetic data with NAD+-dependent yeast aldehyde dehydrogenase. The data presented suggest that choline oxidase utilizes the hydrated species of the aldehyde as substrate in a mechanism for aldehyde oxidation in which hydride transfer is triggered by an active site base.     

Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria

1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl(2) during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H(+) ions, and inhibited by ATP, NADH and Ca(2+). 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a ;classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.     

The transport of L-cysteinesulfinate in rat liver mitochondria.

1. The mechanism of L-cysteinesulfinate permeation into rat liver mitochondria has been investigated. 2. Mitochondria do not swell in ammonium or potassium salts of L-cysteinesulfinate in all the conditions tested, including the presence of valinomycin and/or carbonylcyanide p-trifluoromethoxyphenylhydrazone. 3. The activation of malate oxidation by L-cysteinesulfinate is abolished by aminooxyacetate, an inhibitor of the intramitochondrial aspartate aminotransferase, it is not inhibited by high concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone (in contrast to the oxidation of malate plus glutamate) and it is decreased on lowering the pH of the medium. 4. All the aspartate formed during the oxidation of malate plus L-cysteinesulfinate is exported into the extramitochondrial space. 5. Homocysteinesulfinate, cysteate and homocysteate, which are all good substrates of the mitochondrial aspartate aminotransferase, are unable to activate the oxidation of malate. Homocysteinesulfinate and homocysteate have no inhibitory effect on the L-cysteinesulfinate-induced respiration, whereas cysteate inhibits it competitively with respect to L-cysteinesulfinate. 6. In contrast to D-aspartate, D-cysteinesulfinate and D-glutamate, L-aspartate inhibits the oxidation of malate plus L-cysteinesulfinate in a competitive way with respect to L-cysteinesulfinate. Vice versa, L-cysteinesulfinate inhibits the influx of L-aspartate. 7. Externally added L-cysteinesulfinate elicits efflux of intramitochondrial L-aspartate or L-glutamate. The cysteinesulfinate analogues homocysteinesulfinate, cysteate and homocysteate and the D-stereoisomers of cysteinesulfinate, aspartate and glutamate do not cause a significant release of internal glutamate or aspartate, indicating a high degree of specificity of the exchange reactions. External L-cysteinesulfinate does not cause efflux of intramitochondrial Pi, malate, malonate, citrate, oxoglutarate, pyruvate or ADP. The L-cysteinesulfinate-aspartate and L-cysteinesulfinate-glutamate exchanges are inhibited by glisoxepide and by known substrates of the glutamate-aspartate carrier. 8. The exchange between external L-cysteinesulfinate and intramitochondrial glutamate is accompanied by translocation of protons across the mitochondrial membrane in the same direction as glutamate. The L-cysteinesulfinate-aspartate exchange, on the other hand, is not accompanied by H+ translocation. 9. The ratios delta H+/delta glutamate, delta L-cysteinesulfinate/delta glutamate and delta L-cysteinesulfinate/delta aspartate are close to unity. 10. It is concluded that L-cysteinesulfinate is transported by the glutamate-aspartate carrier of rat liver mitochondria. The present data suggest that the dissociated form of L-cysteinesulfinate exchanges with H+-compensated glutamate or with negatively charged aspartate.     

The inhibition of mitochondrial dicarboxylate transport by inorganic phosphate, some phosphate esters and some phosphonate compounds

1. P(i) competitively inhibited succinate oxidation by intact uncoupled mitochondria in the presence of sufficient N-ethylmaleimide to block the phosphate carrier, with a K(i) of 2.5mm. 2. Of a large number of phosphate esters and phosphonate compounds, phenyl phosphate and phenylphosphonate were found to inhibit competitively uncoupled succinate oxidation by intact but not broken mitochondria. By comparison, benzoate was a relatively weak competitive inhibitor of succinate oxidation by intact mitochondria but a relatively potent inhibitor of succinate dehydrogenase. 3. Phenyl phosphate and phenylphosphonate were non-penetrant, and inhibited P(i)-dependent swelling of mitochondria suspended in isosmolar ammonium malate in a manner non-competitive with P(i). The inhibitors did not affect mitochondrial swelling when tested with P(i) alone. 4. It is concluded that: (i) phenyl phosphate and phenylphosphonate behaved as non-penetrant analogues of P(i), since their inhibitory properties were in strict contrast with those of benzoate; (ii) phenyl phosphate and phenylphosphonate interacted with the dicarboxylate carrier but not with the phosphate carrier; (iii) P(i) was effective as a competitive inhibitor of succinate oxidation because of its being either an alternative substrate for the dicarboxylate carrier or competitive with succinate for the intramitochondrial cations as proposed by Harris & Manger (1968).     

The choline dehydrogenase BetA of Acinetobacter baumannii: a flavoprotein responsible for osmotic stress protection

Acinetobacter baumannii is outstanding for its ability to cope with low water activities which significantly contributes to its persistence in hospital environments. The vast majority of bacteria are able to prevent loss of cellular water by amassing osmoactive compatible solutes or their precursors into the cytoplasm. One such precursor of an osmoprotectant is choline that is taken up from the environment and oxidized to the compatible solute glycine betaine. Here, we report the identification of the osmotic stress operon betIBA in A. baumannii. This operon encodes the choline oxidation pathway important for the production of the solute glycine betaine. The salt-sensitive phenotype of a betA deletion strain could not be rescued by addition of choline, which is consistent with the role of BetA in choline oxidation. We found that BetA is a choline dehydrogenase but also mediates in vitro the oxidation of glycine betaine aldehyde to glycine betaine. BetA was found to be associated with the membrane and to contain a flavin, indicative for BetA donating electrons into the respiratory chain. The choline dehydrogenase activity was not salt dependent but was stimulated by the compatible solute glutamate.     

Selection, mapping, and characterization of osmoregulatory mutants of Escherichia coli blocked in the choline-glycine betaine pathway.

Osmotically stressed Escherichia coli cells synthesize the osmoprotectant glycine betaine by oxidation of choline through glycine betaine aldehyde (choline----glycine betaine aldehyde----glycine betaine; B. Landfald and A.R. Str?m, J. Bacteriol. 165:849-855, 1986. Mutants blocked at the level of choline dehydrogenase were isolated by selection of strains which did not grow at elevated osmotic strength in the presence of choline but grew when supplemented with glycine betaine. A gene governing the choline dehydrogenase activity was named betA. Mapping by P1 transduction, F' complementation, and deletion mutagenesis showed the betA gene to be located at 7.5 min in the argF-codAB region of the chromosome. Mutants carrying deletions of this region also lacked glycine betaine aldehyde dehydrogenase activity and high-affinity uptake activity for choline; these deletions did not influence the activities of glycine betaine uptake or low-affinity choline uptake, both of which were osmotically regulated.