Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from $\text{Escherichia coli}$ K12 was found to be partially resistant to 50 μM carbonyl cyanide $\text{m}$-chlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCP completely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mM KCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additional studies of transport in a mutant (CBT302) deficient in membranebound Ca²⁺-, Mg²⁺-ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions.     

Effect of uncouplers on the bioenergetic properties of a carbonyl cyanide m-chlorophenylhydrazone-resistant mutant Escherichia Coli UV6

The effects of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and tri-n-butyltin chloride (Bu₃SnCl) on proline transport, proton uptake and the transmembrane pH gradient in intact cells have been compared in a CCCP-resistant mutant strain Escherichia coli UV6, and its parent strain, AN180. CCCP and Bu₃SnCl inhibited proline uptake in AN180 but the pH gradient was affected only by CCCP. Neither uncoupler affected the pH gradient in UV6 although inhibition of proline uptake occurred at high concentrations. CCCP caused efflux of accumulated proline in both strains but Bu₃SnCl was ineffective. Bu₃SnCl did not prevent the efflux of proline induced by CCCP, indicating that Bu₃SnCl had not inactivated the transport carrier. In contrast with the parent strain, CCCP failed to reverse the oxidation-dependent inhibition of the phosphotransferase system in UV6 even at concentrations causing inhibition of proline uptake. The phosphorylation potential of UV6 with succinate as substrate was lower than in AN180. This was associated with a 10-fold higher concentration of phosphate in succinate-grown UV6 than in AN180. These results suggest that CCCP and Bu₃SnCl have different sites of action on the membrane energization system of intact cells of E. coli. A possible explanation of the differences between AN180 and UV6 is that the energization system is altered in the CCCP-resistant mutant. Both uncouplers stimulated the uptake of protons by intact cells to the same extent in UV6 as in AN180. In UV6, and in AN180 with Bu₃SnCl, this was not accompanied by effects on the transmembrane pH gradient. The extent of proton uptake appeared to be related to the level of the highly anionic membrane-associated oligosaccharides in the periplasmic space. It is proposed the outer membrane acts as a partial barrier to protons and that the uncouplers can equilibrate protons between the extracellular medium and the periplasmic space in intact cells.     

Detection of a sodium pump in the terminal segment of the bacterial respiratory chain

An alkalo- and halotolerant aerobic microorganism has been isolated which, according to microbiological data and the ribosomal 5S-RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism termed as Bacillus FTU proved to be resistant to the protonophorous uncoupler CCCP. The fast growth of Bacillus FTU in the presence of CCCP was shown to require high Na+ concentrations in the medium. A procedure has been developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only upon addition of an exogenous respiratory substrate. The exhausted cells were found to oxidize ascorbate in the presence of TMPD in a cyanide-sensitive fashion. Ascorbate oxidation was coupled to the uphill Na+ extrusion stimulated by CCCP and a penetrating weak base, diethylamine (DEA), as well as by valinomycin with or without DEA. The operation of the Bacillus FTU terminal oxidase resulted in the generation of delta psi which, in a Na+ medium, was slightly decreased by CCCP and strongly by CCCP + DEA. In a K+ medium CCCP discharged delta psi even without DEA. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium. CCCP + DEA were inhibitory in both media. The data obtained indicate that there is a Na+-motive terminal oxidase in Bacillus FTU. It is suggested that delta microNa formed by the oxidase can be utilized by an Na+-driven ATP-synthase.     

The effect of carbonyl cyanide m-chlorophenylhydrazone on steroid transport in membrane vesicles of Pseudomonas testosteroni

The uncoupler carbonyl cyanide chlorophenylhydrazone (CCCP) was an effective inhibitor of steroid transport in membrane vesicles of Pseudomonas testosteroni between 10 microM and 1 microM CCCP. At these concentrations the inhibition of steroid transport was not due to an inhibition of the 3 beta and 17 beta-hydroxysteroid dehydrogenase enzyme. CCCP also affected testosterone-dependent oxygen consumption at concentrations up to 100 microM and inhibited respiration at 0.5 and 1 microM. The effect of CCCP on testosterone-dependent oxygen consumption indicated that CCCP was acting as an uncoupler. The concurrent inhibition of testosterone transport and stimulation of testosterone-dependent oxygen consumption at 10-100 microM CCCP supported the conclusion that transport and metabolism were tightly coupled processes. When membrane vesicles were pre-incubated with CCCP for 15 min, CCCP did inhibit transport and the 3 beta and 17 beta-hydroxysteroid dehydrogenase activity. However, both transport and enzyme inhibition could be prevented by the addition of NAD+ to the incubation mixture. This indicated that CCCP exhibits the properties of a sulfhydryl reagent under pre-incubated conditions.     

Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet

Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells.     

Energy coupling in the active transport of amino acids by bacteriohodopsin-containing cells of Halobacterium holobium.

Growth of Halobacterium halobium under illumination with limiting aeration induces bacteriorhodopsin formation and renders the cells capable of photophosphorylation. Cells depleted of endogenous reserves by a starvation treatment were used to investigate the means by which energy is coupled to the active transport of [14C]proline, -leucine, and -histidine. Proline was readily accumulated by irradiated cells under anaerobiosis even when the photophosphorylation was abolished by the adenosine triphosphatase inhibitor N,N'-dicyclohexylcarbodimiide (DCCD). The uptake of proline in the dark was limited except when the cells were allowed to accumulate adenosine 5'-triphosphate (ATP) by prior light exposure or by the oxidation of glycerol. DCCD inhibited this dark uptake. These findings essentially support Mitchell's chemiosmotic theory of active transport. The driving force is apparently the proton-motive force developed when protons are extruded from irradiated bacteriorhodopsin or by the dydrolysis of ATP by membrane adenosine triphosphatase. Carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton permeant known to abolish membrane potential, was a strong inhibitor of proline uptake. Leucine transport was also apparently driven by proton-motive force, although its kinetic properties differed from the proline system. Histidine transport is apparently not a chemiosmotic system. Dark- or light-exposed cells show comparable initial rats of histidine uptake, and these processes were only partially inhibited by DCCD or CCCP. The histidine system apparently does not utilize ATP per se since comparable rates of uptake were exhibited by cells of differing intracellular ATP levels. Irradiated cells did effect a greater total accumulation of histidine than dark-exposed cells. These findings suggest that ATP is needed for sustained transport.     

Counter-transport mediated by the lactose permease of Escherichia coli.

When the two main energy yielding pathways, respiration and the membrane ATPase of Escherichia coli are poisoned, the lactose permease is unable to accomplish accumulative transport of thiogalactosides, but the efflux of preloaded substrate can be coupled to a transiently uphill transport of exogenous substrate. This transient uphill transport, called overshoot has been reexamined with the possibility of an obligate H+ cotransport in mind. Overshoot can be diminished but not suppressed by a proton-conducting uncoupler, carbonyl cyanide m chlorophenylhydrazone, (CCCP) and by a liposoluble cation, triphenyl-methyl phosphonium (TPMP+). The effect of other factors, such as temperature, amount of permease and pH were also explored. The overshoot was found to decrease with increasing pH, until at pH 8 it became negligible. This is in sharp contrast with the relatively flat pH dependence of uphill and downhill transport in unpoisoned cells. CCCP and TPMP+ had no inhibitory effect on the overshoot at pH 6 and below.     

The Na+-motive terminal oxidase activity in an alkalo- and halo-tolerant Bacillus

An alkalo- and halo-tolerant aerobic microorganism has been isolated which, according to microbiological analysis data and the ribosomal 5S RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism, called Bacillus FTU, proved to be resistant to the protonophorous uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). The fast growth of Bacillus FTU in the presence of CCCP was shown to require a high Na+ concentration in the medium. A procedure was developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only when an exogenous respiratory substrate was added. The exhausted cells were found to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) in a cyanide-sensitive fashion. The ascorbate oxidation was coupled to the uphill Na+ extrusion which was stimulated by CCCP and a penetrating weak base, diethylamine, as well as by valinomycin with or without diethylamine. Operation of the Bacillus FTU terminal oxidase resulted in the generation of a delta psi which, in the Na+ medium, was slightly decreased by CCCP and strongly decreased by CCCP + diethylamine. In the K+ medium, CCCP discharged delta psi even without diethylamine. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium as if Na+- and H+-coupled oxidative phosphorylations were operative in the Na+ and K+ media, respectively. Inside-out subcellular vesicles of Bacillus FTU were found to be competent in the Na+ uptake supported by oxidation of ascorbate + TMPD or diaminodurene. CCCP or valinomycin + K+ increased the Na+ uptake very strongly. The process was completely inhibited by cyanide or monensin, the former, but not the latter, being inhibitory for respiration. The data obtained indicate that in Bacillus FTU there is not only H+-motive but also Na+-motive terminal oxidase activity.     

Uptake of glycine betaine and its analogues by bacteroids of Rhizobium meliloti

Bacteroids isolated from alfalfa nodules induced by Rhizobium meliloti 102F34 transported glycine betaine at a constant rate for up to 30 min. Addition of sodium salts greatly increased the uptake activity, whereas other salts or non-electrolytes had less effect. The apparent Km for glycine betaine uptake was 8.3 microM and V was about 0.84 nmol min-1 (mg protein)-1 in the presence of 200 mM-NaCl which gave maximum stimulation of the transport. Supplementing bacteroid suspensions with various energy-yielding substrates, or ATP, did not increase glycine betaine uptake rates. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the respiratory inhibitor potassium cyanide strongly inhibited glycine betaine uptake, but arsenate was totally inactive. Glycine betaine transport showed considerable structural specificity: choline, proline betaine, gamma-butyrobetaine and trigonelline did not competitively inhibit the system, although choline and proline betaine were transported by bacteroids. Both a high-affinity activity and a low-affinity activity were found for choline uptake. These osmoprotective compounds might have a significant role in the maintenance of nitrogenase activity in bacteroids subjected to salt stress.     

Multidrug resistance protein 4 (MRP4/ABCC4) mediates efflux of bimane-glutathione

Multidrug resistance proteins (MRPs) are ATP-dependent export pumps that mediate the export of organic anions. ABCC1 (MRP1), ABCC2 (MRP2) and ABCC3 (MRP3) are all able to facilitate the efflux of anionic conjugates including glutathione (GSH), glucuronide and sulfate conjugates of xenobiotics and endogenous molecules. Earlier studies showed that ABCC4 functions as an ATP-driven export pump for cyclic AMP and cyclic GMP, as well as estradiol-17-beta-D-glucuronide. However, it was unclear if other conjugated metabolites can be transported by ABCC4. Hence in this study, a fluorescent substrate, bimane-glutathione (bimane-GS) was used to further examine the transport activity of ABCC4. Using cells stably overexpressing ABCC4, this study shows that ABCC4 can facilitate the efflux of the glutathione conjugate, bimane-glutathione. Bimane-glutathione efflux increased with time and >85% of the conjugate was exported after 15min. This transport was abolished in the presence of 2.5microM carbonylcyanide m-chlorophenylhydrasone (CCCP), an uncoupler of oxidative phosphorylation. Inhibition was also observed with known inhibitors of MRP transporters including benzbromarone, verapamil and indomethacin. In addition, 100microM methotrexate, an ABCC4 substrate or 100microM 6-thioguanine (6-TG), a compound whose monophosphate metabolite is an ABCC4 substrate, reduced efflux by >40%. A concentration-dependent inhibition of bimane-glutathione efflux was observed with 1-chloro-2,4-dinitrobenzene (CDNB) which is metabolized intracellularly to the glutathione conjugate, 2,4-dinitrophenyl-glutathione (DNP-GS). The determination that ABCC4 can mediate the transport of glucuronide and glutathione conjugates indicates that ABCC4 may play a role in the cellular extrusion of Phase II detoxification metabolites.     

Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate.     

Synaptosomal bioenergetics. The role of glycolysis, pyruvate oxidation and responses to hypoglycaemia

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.     

Electron spin resonance studies of lipid fluidity changes in membranes of an uncoupler-resistant mutant of Escherichia coli

The fluidity of the lipids in membrane preparations from a mutant of Escherichia coli resistant to the uncoupler CCCP, grown at different temperatures with and without CCCP, was examined by electron spin resonance using the spin probe 5-doxyl stearic acid. The fluidity of the membrane lipids at the growth temperature, as estimated using electron spin resonance, was less in cells grown at lower temperatures. Precise homeoviscous adaptation was not observed. Growth in the presence of CCCP resulted in a decrease in membrane lipid fluidity, particularly in the inner (cytoplasmic) membrane. There was no change in the proportion of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in the cell envelope. However, there was an increase in the proportion of unsaturated fatty acids in membranes from cells grown with uncoupler. This was reflected in the increased fluidity of the lipids extracted from these membranes. This result is contrary to that expected from measurements of the fluidity of the lipid in these membranes. The decreased fluidity of the lipid in these membranes may be a consequence of the observed increase in the ratio of protein to phospholipid.     

Transport and catabolism of proline betaine in salt-stressed Rhizobium meliloti

Exogenous proline betaine (N,N-dimethylproline or stachydrine) highly stimulated the growth rate of Rhizobium meliloti, in media of inhibitory concentration of NaCl whereas proline was ineffective. High levels of proline betaine uptake occurred in cells grown in media of elevated osmotic strength; on the contrary, only low activity was found in cells grown in minimal medium. The apparent K _m was 10 M with a maximal transport rate of 25 nmol min^-1 mg^-1 of protein in 0.3 M NaCl-grown cells. The concentrative transport was totally abolished by KCN (2 mM), 2,4-dinitrophenol (2 mM), and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP 10 M) but was insensitive to arsenate (5 mM). Glycine betaine was a very potent inhibitor of proline betaine uptake while proline was not. Proline betaine transport was not reduced in osmotically shocked cells and no proline betaine binding activity was detected in the crude periplasmic shock fluid. In the absence of salt stress, Rhizobium meliloti actively catabolized proline betaine but this catabolism was blocked by increasing the osmotic strength of the medium. The osmolarity in the growth medium regulates the use of proline betaine either as a carbon and nitrogen source or as an osmoprotectant.Abbreviations LAS lactate-aspartate-salts - MSY mannitol-salts-yeast - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DCCD dicyclohexylcarbodiimide - KCN potassium cyanide - Hepes 4-(2-hydroxyethyl)-1-piperzine-ethanesulphonic acid     

Mechanism of stimulation of endogenous fermentation in yeast by carbonyl cyanide m-chlorophenylhydrazone

Addition of the uncoupler and protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to starved yeast cells starts endogenous alcoholic fermentation lasting about 20 min. Hexose 6-phosphates, fructose 2,6-bisphosphate, and pyruvate accumulate in less than 2 min after addition of CCCP from almost zero concentration to concentrations which correspond to 1/5-1/10 of the steady-state concentrations during fermentation of glucose. CCCP immediately causes a decrease of the intracellular cytosolic pH from 6.9 to 6.4. This change activates adenylate cyclase (Purwin, C., Nicolay, K., Scheffers, W.A., and Holzer, H. (1986) J. Biol. Chem. 261, 8744-8749) and leads to the previously observed transient increase of cyclic AMP. It is shown here that the following enzymes known from in vitro experiments to be activated by cyclic AMP-dependent phosphorylation are activated in the CCCP-treated starved yeast cells in vivo: glycogen phosphorylase, trehalase (pH 7), 6-phosphofructo-2-kinase. The activation of 6-phosphofructo-2-kinase leads to an accumulation of fructose 2,6-bisphosphate, which is known from in vitro experiments to activate 6-phosphofructo-1-kinase and to inhibit fructose-1,6-bisphosphatase. All effects observed in the intact yeast cells fit with the idea that the CCCP-initiated activation of adenylate cyclase leads to a sequence of events which by protein phosphorylation and allosteric effects initiates endogenous alcoholic fermentation.     

31P nuclear magnetic resonance study of the effect of azide on xylose fermentation by Candida tropicalis

Maximal ethanol production by Candida tropicalis grown on xylose was obtained at an oxygen transfer rate of 5 to 7 mmol/liter per h. Addition of 0.2 mM azide increased the ethanol yield by a factor of 3 to 4, based on the cell mass produced, and decreased the formation of the by-product xylitol by 80%. In the presence of azide, ethanol was reassimilated before the carbon source was depleted. At all oxygenation levels studied, azide caused 25 to 60% of the carbon to be lost, most probably as carbon dioxide. Identical spectra were obtained with 31P nuclear magnetic resonance spectroscopy performed on extracts of C. tropicalis grown on xylose in the absence and presence of azide. Azide lowered the levels of sugar phosphates. Enzymatic analysis showed extremely low levels of fructose 1,6-diphosphate compared with the levels obtained in the absence of azide, while the level of malate, a citric acid cycle intermediate, was not influenced by azide. 31P nuclear magnetic resonance spectroscopy performed on xylose-grown whole cells of C. tropicalis showed that azide lowered the intracellular pH, inhibited the uptake of external Pi, and decreased the buildup of polyphosphate in relation to results with untreated cells. Similar results were obtained with the uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), except that CCCP treatment led to extremely high levels of internal Pi. The dual effect of azide as a respiratory inhibitor and as an uncoupler is discussed with respect to the metabolism and product formation in xylose-assimilating C. tropicalis.     

Energy generation from the CO oxidation-hydrogen production pathway in Rubrivivax gelatinosus

When incubated in the presence of CO gas, Rubrivivax gelatinosus CBS induces a CO oxidation-H2 production pathway according to the stoichiometry CO + H2O --> CO2 + H2. Once induced, this pathway proceeds equally well in both light and darkness. When light is not present, CO can serve as the sole carbon source, supporting cell growth anaerobically with a cell doubling time of nearly 2 days. This observation suggests that the CO oxidation reaction yields energy. Indeed, new ATP synthesis was detected in darkness following CO additions to the gas phase of the culture, in contrast to the case for a control that received an inert gas such as argon. When the CO-to-H2 activity was determined in the presence of the electron transport uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), the rate of H2 production from CO oxidation was enhanced nearly 40% compared to that of the control. Upon the addition of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), we observed an inhibition of H2 production from CO oxidation which could be reversed upon the addition of CCCP. Collectively, these data strongly suggest that the CO-to-H2 reaction yields ATP driven by a transmembrane proton gradient, but the detailed mechanism of this reaction is not yet known. These findings encourage additional research aimed at long-term H2 production from gas streams containing CO.     

Stimulation of menaquinone-dependent electron transfer in the respiratory chain of Bacillus subtilis by membrane energization

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Increased proline transport resulting from growth of normal and Kirsten sarcoma virus-transformed BALB 3T3 cells in the presence of N(6), O(2')-dibutyryl cyclic adenosine 3',5'-monophosphate.

Proline transport in Kirsten sarcoma virus-transformed BALB 3T3 (Ki-3T3) cells was increased approximately twofold by 0.5 mm dibutyryl cAMP (dbcAMP), and the increase was observed whether transport was assayed in the presence or absence of cycloheximide. Two days of exposure to the analog was required for maximum stimulation. Increased proline transport contributed almost entirely to the increased incorporation of [¹⁴C]proline into noncollagen protein but for only 13% of the increased incorporation into collagen of dbcAMP-treated Ki-3T3 cells. Proline transport was further characterized using an assay system containing 0.1 mm cycloheximide, which did not affect transport over a 30-min period. The K_m for proline was decreased from 6.5 to 3.4 mm by dbcAMP treatment of Ki-3T3. Proline transport in Ki-3T3 proceeds almost entirely via the A system, and the effect of dbcAMP appears to be on this system specifically since glycine and glutamine transport, which are heterogeneous, were not affected but transport of N-methylaminoisobutyrate, a specific A system substrate, was increased by dbcAMP treatment. Although 0.5 mm butyrate increased proline transport in Ki-3T3 cells to a similar degree as dbcAMP, the effect of the latter appeared related to its action as a cAMP analog since N⁶-monobutyryl cAMP, having a stable butyryl group, and 8-bromo-cAMP also increased proline transport while dbcGMP did not. The rate of proline transport in normal BALB 3T3 cells was only 30–40% lower than that of Ki-3T3 cells at various growth stages, and dbcAMP and 8-bromo-cAMP treatment also increased proline transport in the normal cells. The results of these studies suggest that dbcAMP and other cAMP analogs induce the synthesis of an altered component of the A system for amino acid transport and that the effect of these compounds is unrelated to the effect of transformation on proline transport.     

Overexpression of UCP3 in cultured human muscle lowers mitochondrial membrane potential, raises ATP/ADP ratio, and favors fatty acid vs. glucose oxidation.

The skeletal muscle mitochondrial uncoupling protein-3 (UCP3) promotes substrate oxidation, but direct evidence for its metabolic role is lacking. Here, we show that UCP3 overexpression in cultured human muscle cells decreased mitochondrial membrane potential (DYm). Despite this, the ATP content was not significantly decreased compared with control cells, whereas ADP content was reduced and thus the ATP/ADP ratio raised. This finding was contrasts with the effect caused by the chemical protonophoric uncoupler, CCCP, which lowered DYm, ATP, and the ATP/ADP ratio. UCP3-overexpression enhanced oxidation of oleate, regardless of the presence of glucose, whereas etomoxir, which blocks fatty acid entry to mitochondria, suppressed the UCP3 effect. Glucose oxidation was stimulated in UCP3-overexpressing cells, but this effect was inhibited by oleate. UCP3 caused weak increase of both 2-Deoxyglucose uptake and glycolytic rate, which differed from the marked stimulation by CCCP. We concluded that UCP3 promoted nutrient oxidation by lowering DYm and enhanced fatty acid-dependent inhibition of glucose oxidation. Unlike the uncoupler CCCP, however, UCP3 raised the ATP/ADP ratio and modestly increased glucose uptake and glycolysis. We propose that this differential effect provides a biological significance to UCP3, which is up-regulated in metabolic stress situations where it could be involved in nutrient partitioning.