Succinate Uptake Research Articles

Aspartate transport by the Dct system in Rhizobium leguminosarum negatively affects nitrogen-regulated operons

Amino acid uptake by the general amino acid permease (Aap) of Rhizobium leguminosarum strain 3841 was severely reduced by the presence of aspartate in the growth medium when glucose was the carbon source. The reduction in transport by the Aap appeared to be caused by inhibition of uptake and not by transcriptional repression. However, as measured with lacZ fusions, the Ntr-regulated gene glnII was repressed by aspartate. The negative regulatory effect on both the Aap and glnII was prevented by mutation of any component of the dicarboxylate transport (Dct) system or by the inclusion of a C4- dicarboxylate in the growth medium, including the non-metabolizable analogue 2-methylsuccinate. As measured by total uptake and with a dctA-lacZ fusion, aspartate was an efficient inducer of the Dct system, but slightly less so than succinate alone or succinate and aspartate together. Thus, aspartate does not cause overexpression of DctA leading to improper regulation of other operons. Transport measurements revealed that the Dct system has an apparent Km for succinate of 5 microM and an apparent Ki for aspartate inhibition of succinate uptake of 5 mM. These data imply that the Dct-mediated accumulation of aspartate causes an unregulated build-up of aspartate or a metabolic product of it in the cell. This accumulation of aspartate is prevented either by mutation of the dct system or by the presence of a higher affinity substrate that will reduce access of aspartate to the carrier protein. Elevation or disruption of the intracellular aspartate pool is predicted to disrupt N-regulated operons and nitrogen fixation.

The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids.

The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein in similar to models proposed for other integral membrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1.5 kb. The mea1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5' to the MFml gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid.

Metabolic characterization ofAcetobacter diazotrophicus

Carbon and nitrogen metabolism were investigated in Acetobacter diazotrophicus Pal 3, a N2-fixing bacterium able to grow at low pH and at high sugar concentration. Enzymatic, respiratory, and uptake studies were performed. The main active pathway for the catabolism of phosphorylated glucose was the pentose phosphate pathway. In addition, A. diazotrophicus directly oxidized glucose, gluconate, and ketogluconates through respiratory chain-linked enzymes. Soluble enzymes for the oxidation of glucose and gluconate were also found. Acetobacter diazotrophicus had a complete tricarboxylic acid cycle with a respiratory chain-linked malate dehydrogenase. The ability to grow on two- and three-carbon substrates would be explained by the presence of gluconeogenesis. Lack of bacterial growth on dicarboxylates was explained by the absence of a transport system. Ammonium assimilation proceeded mainly through glutamate dehydrogenase under ammonium excess but also through energy-demanding glutamine synthetase and glutamate synthase under N2-fixing conditions. Acetobacter diazotrophicus was not able to transport sucrose and its ability to grow on this disaccharide was explained by the presence of an extracellular enzyme with saccharolytic activity.Key words: Acetobacter diazotrophicus, carbon–nitrogen metabolism, extracellular saccharolytic activity, sucrose–succinate uptake.

Effect of renal ischaemia on organic compound transport in rabbit kidney proximal tubule.

This study was carried out to determine the effect of renal ischaemia on transport systems for organic compounds in the rabbit kidney proximal tubule. Ischaemia for 30 or 60 min. induced glucosuria and phosphaturia, which was accompanied by polyuria and natriuresis. The Na(+)-dependent uptake of glucose, succinate and L-glutamate by brush-border membrane vesicles was not altered by 30 or 60 min. of ischaemia, while the H+/tetraethylammonium antiport was significantly inhibited after 30 min. of ischaemia. When the duration of ischaemia was extended to 120 min. the uptake of glucose and succinate by brush-border membrane vesicles was also significantly attenuated, but the L-glutamate uptake was not altered. The uptake of glucose, succinate and L-glutamate by basolateral membrane vesicles was not impaired even with 120 min. of ischaemia, suggesting that transport systems for organic compounds in the brush-border membrane are more sensitive to ischaemia than those in the basolateral membrane. Ouabain-sensitive oxygen consumption in renal cortical slices was not depressed by 60 min. of ischaemia. When kidneys were reperfused for 60 min. following 60 min. of ischaemia, the Na(+)-glucose and Na(+)-succinate cotransport and the H+/tetraethylammonium antiport were not different from the control, but the recovery of alkaline phosphatase was significantly reduced. When kidneys were subjected to ischaemia for 60 min., a loss of brush-border microvilli and plasma membrane was observed after 5 or 60 min. of reflow in the proximal convoluted tubule. After 3 hr of reflow, focal necrosis appeared although the microvilli were partially regenerated.(ABSTRACT TRUNCATED AT 250 WORDS)

Carrier-mediated transport is involved in mucosal succinate uptake by rat large intestine.

Mucosal uptake of succinate across the luminal epithelial border in various segments of the large intestine of the rat (proximal and distal colon, caecum) was investigated using an in vitro mucosal uptake technique. For comparison, some experiments with jejunal preparations were also performed. Additionally, disappearance of succinate from the lumen of ligated loops of the proximal and distal colon was measured in vivo. In the ligated loop experiments, the succinate concentration of the instilled solution significantly decreased during a period of 30 min in both colonic segments, indicating absorption of succinate. The in vitro experiments revealed a distinct Na+ dependence of mucosal succinate uptake in all intestinal segments investigated. Because succinate uptake by the colonic mucosa of germ-free rats was similarly enhanced in the presence of Na+ compared with Na(+)-free conditions, measured uptake appears to be due to mucosal uptake rather than to Na(+)-dependent uptake of succinate by bacteria associated with the mucosa. The evaluation of the kinetics of succinate transport in the proximal colon and jejunum, respectively, revealed a single, Na(+)-dependent saturable component and a diffusive component in both intestinal segments. With respect to the apparent kinetic parameters of the carrier-mediated transport component, the transport capacity (Vmax) of the jejunal carrier appears to be substantially higher compared with the colonic mechanism (80 and 11 nmol cm-2 (5 min)-1, respectively) whereas the affinity constants (Km) did not differ markedly in both segments (0.67 and 0.41 mmol l-1, respectively). Mucosal uptake of 14C-labelled succinate in the proximal colon was inhibited by unlabelled succinate, fumarate, citrate and tricarballylate, but not by glutamate, L-leucine or butyrate. Na(+)-dependent uptake of the tricarboxylate tricarballylate but not of the dicarboxylate succinate was significantly enhanced by lowering the pH from 7.0 to 5.5, indicating preferential transport of the protonated forms of tricarboxylates. It is concluded from the present results that a Na(+)-dependent, saturable carrier is involved in the uptake of structurally related tri- and dicarboxylates by the colonic mucosa of the rat.

Expression of a rat renal sodium-dependent dicarboxylate transporter in Xenopus oocytes.

Microinjection of mRNA isolated from rat kidney cortex into Xenopus laevis oocytes resulted in the expression of a Na(+)-dependent dicarboxylate transporter, as detected by uptake measurements with [14C]succinate as substrate. The expressed transporter showed an S-shaped Na(+)-dependence with half-maximal activation at 19-21 mM Na+ and a Hill coefficient between 2 and 3. Endogenous succinate uptake was not Na(+)-dependent. Na(+)-stimulated succinate uptake in mRNA-injected oocytes exhibited a maximum at pH 7.5, whereas endogenous Na(+)-independent transporter was fastest at pH 8.5. The expressed dicarboxylate transporter also differed from the endogenous transporter in its sensitivity to citrate as well as dicarboxylates in trans and cis configurations. The expressed transporter resembled the renal basolateral transporter, especially with respect to affinity for succinate (Km 28 microM), activation by Na+, pH-dependence and substrate specificity. After injection of size-fractionated mRNA, succinate uptake was expressed by mRNA of 2-3 kb. Our results suggest expression of the basolateral Na(+)-dependent dicarboxylate transporter after injection of mRNA from rat kidney into Xenopus oocytes.

A metabolic study of Buchnera, the intracellular bacterial symbionts of the pea aphid Acyrthosiphon pisum

Cells of the bacterium Buchnera were isolated from embryos of the pea aphid Acyrthosiphon pisum, with an intact perisymbiont membrane (the insect membrane which surrounds each bacterial cell inside the aphid). The bacterial preparations respired aerobically, consuming oxygen at an average rate of 24 nmol (mg protein)−1 min−1. The bacteria took up a range of carboxylic acids and the amino acids glutamate and aspartate from an external concentration of 0·5 mm at rates of 1–10 nmol (mg protein)−1 h−1; glucose was taken up at 0·17 nmol (mg protein)−1 h−1. Glutamate uptake was proportional to its external concentration, at all concentrations tested between 15 μm and 10 mm. Saturable systems for the uptake of succinate and aspartate were identified. The kinetic constants were: K m 0·79 mm, V max 12·6 nmol (mg protein)−1 min−1 for succinate; and K m 0·22 mm, V max 3·3 nmol (mg protein)−1 min−1 for aspartate. Succinate uptake was not inhibited by the uncoupler CCCP and was markedly stimulated by ATP, suggesting that its transport is not linked to a proton-motive force but is dependent on an energized membrane and possibly mediated by a co-transport system involving another ion.

Identification of sodium-dependent and sodium-independent dicarboxylate transport systems in rat liver basolateral membrane vesicles.

The mechanisms involved in the hepatocellular uptake of Krebs-cycle intermediates were investigated in isolated basolateral (sinusoidal and lateral) rat liver plasma membrane (blLPM) vesicles. An inwardly directed Na+ gradient markedly stimulated uptake of 2-oxoglutarate and succinate into voltage- and pH-clamped blLPM vesicles. This Na(+)-dependent portion of the dicarboxylate uptake was characterized by (a) saturability with increasing substrate concentrations (Km = 6.4-10 mM; Vmax approximately 0.2 nmol min-1 mg protein-1), (b) cis-inhibition by lithium (10 mM), other Krebs-cycle dicarboxylates (1 mM) and DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; 1 mM) but not by sulphate, monocarboxylates, oxalate, acidic amino acids, bile salts and probenecid, (c) stimulation by an intravesicular negative K(+)-diffusion potential indicating electrogenic [(Na+)n greater than 2-succinate] cotransport, and (d) a pH optimum for transport between 7.0 and 7.5. In the absence of Na+, an inside alkaline pH gradient also markedly stimulated 2-oxoglutarate uptake. This pH-gradient-driven 2-oxoglutarate uptake was insensitive to lithium, but could also be inhibited by DIDS and succinate. Furthermore, saturation kinetics demonstrated Km (approximately 34 mM) and Vmax (approximately 0.8 nmol min-1 mg protein-1) values that were clearly different from those of the Na(+)-dependent uptake system. These results indicate the occurrence of two separate dicarboxylate transport systems along the sinusoidal border of hepatocytes, one being a Na(+)-dicarboxylate symporter and the other representing an anion-exchange system.

A succinate transport mutant of Bradyrhizobium japonicum forms ineffective nodules on soybeans.

Biochemical evidence has shown that dicarboxylic acids actively support symbiotic nitrogen fixation by both fast- and slow-growing Rhizobium. Mutants defective in the active uptake of succinate have been previously described only in species of the fast-growing rhizobium. This article is a report on the isolation of mutants defective in dicarboxylate transport in a slow-growing species of rhizobium, Bradyrhizobium japonicum. One of these presumptive dicarboxylate transport mutants, GTS, was characterized further. Cultured GTS was unable to accumulate [14C]succinate above background levels but possessed normal rates of malate dehydrogenase, fumarase, and hydroxybutyrate dehydrogenase activities. When inoculated onto soybeans, GTS produced a Nod+, Fix- phenotype. The bacteroids isolated from these nodules failed to accumulate labelled succinate. Electron micrographs of nodules formed by inoculation with GTS appeared normal with the exceptions of more prominent peribacteroid spaces in the infected cells and the appearance of starch granules in the noninfected cells. The phenotypical and morphological changes observed for B. japonicum are similar to those previously reported for the fast-growing species.

Dicarboxylate transport in renal basolateral and brush-border membrane vesicles.

Characteristics of succinate transport were determined in basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated in parallel from rabbit renal cortex. The uptake of succinate was markedly stimulated by the imposition of an inwardly directed Na+ gradient, showing an "overshoot" phenomenon in both membrane preparations. The stimulation of succinate uptake by an inwardly directed Na+ gradient was not significantly affected by pH clamp or inhibition of Na(+)-H+ exchange. The Na(+)-dependent and -independent succinate uptakes were not stimulated by an outwardly directed pH gradient. The Na dependence of succinate uptake exhibited sigmoidal kinetics, with Hill coefficients of 2.17 and 2.38 in BLMV and BBMV, respectively. The Na(+)-dependent succinate uptake by BLMV and BBMV was stimulated by a valinomycin-induced inside-negative potential. The Na(+)-dependent succinate uptake by BLMV and BBMV followed a simple Michaelis-Menten kinetics, with an apparent Km of 22.20 +/- 4.08 and 71.52 +/- 0.14 microM and a Vmax of 39.0 +/- 3.72 and 70.20 +/- 0.96 nmol/(mg.min), respectively. The substrate specificity and the inhibitor sensitivity of the succinate transport system appeared to be very similar in both membranes. These results indicate that both the renal brush-border and basolateral membranes possess the Na(+)-dependent dicarboxylate transport system with very similar properties but with different substrate affinity and transport capacity.

Hypochlorous acid and myeloperoxidase-catalyzed oxidation of iron-sulfur clusters in bacterial respiratory dehydrogenases.

Hypochlorous acid and related oxidants derived from myeloperoxidase-catalyzed reactions contribute to the microbicidal activities of phagocytosing neutrophils and monocytes. Microbial iron-sulfur (Fe/S) clusters have been suggested as general targets of myeloperoxidase-derived oxidations, but no susceptible Fe/S site has yet been identified. In this study, the effects of HOCl and myeloperoxidase-catalyzed peroxidation of chloride ion upon EPR-detectable Fe/S clusters in Escherichia coli and Pseudomonas aeruginosa were examined. Increasing amounts of oxidant produced progressive loss of signal amplitudes from the S-1 and S-3 Fe/S clusters of succinate:ubiquinone oxidoreductase in respiring membrane fragments. These changes were compared to loss of microbial viability, succinate uptake rates, succinate dehydrogenase activity and succinate-dependent respiration. The amounts of oxidant required to destroy Fe/S clusters exceeded the amounts required to kill organisms or inhibit respiratory function by factors of four or five. Power saturation characteristics of the S-1 signal indicated that the S-2 signal was also resistant to modification, even in highly oxidized membranes. Loss of succinate-dependent respiration was closely associated with HOCl and myeloperoxidase-mediated microbicidal activity against P. aeruginosa and was also an early event in the oxidant-mediated metabolic dysfunctions of E. coli. However, these effects were not caused by the destruction of the Fe/S clusters within the succinate:ubiquinone oxidoreductase. Rather, the major respiration-inhibiting lesion(s) appeared to reside at points in the respiratory chain between the Fe/S clusters and the ubiquinone reductase site.

Inactivation of taurine transporter by calcium in purified human placental brush border membrane vesicles

We investigated the effects of calcium on the activity of the taurine transporter in purified human placental brush border membrane vesicles. Treatment of the membrane vesicles with calcium markedly inhibited taurine uptake. The magnitude of inhibition was dependent on the calcium concentration and the treatment time. Free ionized Ca 2+ was responsible for this effect because EGTA, a Ca 2+ chelator, totally abolished the calcium-induced effect. Uptake of succinate, which occurs via a Na +-dependent process as does the uptake of taurine, was reduced only to a small extent by the calcium treatment. This result indicates that the effect of Ca 2+ on taurine uptake was not due to an accelerated dissipation of the Na + gradient as a result of an increased Na + permeability of the membrane. Preloading the vesicles with phospholipase inhibitors such as neomycin and quinacrine significantly protected the taurine transporter from the Ca 2+ effect, raising the possibility that Ca 2+-activated phospholipases may mediate the Ca 2+ effect. Kinetic analysis revealed that Ca 2+ decreased the affinity of the transporter for taurine as well as the translocation rate of the taurine-loaded transporter complex.

Deficient utilization of succinate in a sterol 14α-demethylation mutant ofCandida albicans

Growth of a wild-type strain (KD14) started after a lag of approximately 15 h when glucose-grown cells of Candida albicans were incubated at 25 degrees C, with succinate as a sole energy/carbon source. No growth was observed, however, for a sterol 14 alpha-demethylation mutant (KD4900). During the lag period, the wild-type cells retained their respiratory activity, as measured with glucose as an added substrate, and developed the capacity to incorporate labelled succinate. In contrast, incubation with succinate for 15 h resulted in diminished respiratory activity and failed to induce the succinate uptake capacity of the mutant cells. These two differences from the wild-type seem to account for the inability of the mutant to grow in the presence of a non-fermentable substrate.

Succinate transport by a ruminal selenomonad and its regulation by carbohydrate availability and osmotic strength

Washed cells of strain H18, a newly isolated ruminal selenomonad, decarboxylated succinate 25-fold faster than Selenomonas ruminantium HD4 (130 versus 5 nmol min-1 mg of protein-1, respectively). Batch cultures of strain H18 which were fermenting glucose did not utilize succinate, and glucose-limited continuous cultures were only able to decarboxylate significant amounts of succinate at slow (less than 0.1 h-1) dilution rates. Strain H18 grew more slowly on lactate than glucose (0.2 versus 0.4 h-1, respectively), and more than half of the lactate was initially converted to succinate. Succinate was only utilized after growth on lactate had ceased. Although nonenergized and glucose-energized cells had similar proton motive forces and ATP levels, glucose-energized cells were unable to transport succinate. Transport by nonenergized cells was decreased by small increases in osmotic strength, and it is possible that energy-dependent inhibition of succinate transport was related to changes in cell turgor. Since cells which were deenergized with 2-deoxyglucose or iodoacetate did not transport succinate, it appeared that glycogen metabolism was providing the driving force for succinate uptake. An artificial delta pH drove succinate transport in deenergized cells, but an artificial membrane potential (delta psi) could not serve as a driving force. Because succinate is nearly fully dissociated at pH 7.0 and the transport process was electroneutral, it appeared that succinate was taken up in symport with two protons. An Eadie-Hofstee plot indicated that the rate of uptake was unusually rapid at high substrate concentrations, but the low-velocity, high-affinity component could account for succinate utilization by stationary cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

The renal mitochondrial toxicity of beta-lactam antibiotics

The nephrotoxic beta-lactam antibiotics cephaloridine, cephaloglycin, and imipenem produce irreversible injury to renal mitochondrial anionic substrate uptake and respiration after 1 to 2 h of in vivo exposure. Toxicity during in vitro exposure is nearly identical but is immediate in onset and is reversed by the mitochondria being washed or the substrate concentrations being increased. A model of injury that accounts for these findings proposes that the beta-lactams fit carriers for mitochondrial substrate uptake, causing inhibition that is initially reversible and becomes irreversible as the antibiotics acylate the transporters. These studies were designed to create an environment of prolonged in vitro exposure, first, to determine whether toxicity becomes irreversible with time and, second, to study the molecular properties of toxicity. Respiration with and the uptake of succinate and ADP were measured in rabbit renal cortical mitochondria exposed for 2 to 6 h to 300 to 3,000 micrograms of cephalexin (nontoxic) or cephaloglycin or imipenem (nephrotoxic) per mL and then washed to remove the antibiotic. In vitro cephalexin reduced respiration only slightly and was therefore not studied further. Cephaloglycin and imipenem irreversibly reduced both respiration and succinate uptake. ADP uptake was unaffected by cephaloglycin and was slightly reduced by imipenem. Finally, cilastatin, which prevents the tubular necrosis produced by imipenem in vivo, reduced its mitochondrial toxicity in vitro. It is concluded that the pattern of in vitro injury of the nephrotoxic beta-lactams to mitochondrial substrate uptake and respiration evolves in a time-dependent and concentration-dependent manner, consistent with the proposed model of acylation and inactivation of substrate transporters, and that the protective action of cilastatin against imipenem occurs at least partly at a subcellular level.

Specificity and regulation of the dicarboxylate carrier on the peribacteroid membrane of soybean nodules

Malate and succinate were taken up rapidly by isolated, intact peribacteroid units (PBUs) from soybean (Glycine max (L.) Merr.) root nodules and inhibited each other in a competitive manner. Malonate uptake was slower and was severely inhibited by equimolar malate in the reaction medium. The apparent Km for malonate uptake was higher than that for malate and succinate uptake. Malate uptake by PBUs was inhibited by (in diminishing order of severity) oxaloacetate, fumarate, succinate, phthalonate and oxoglutarate. Malonate and butylmalonate inhibited only slightly and pyruvate,isocitrate and glutamate not at all. Of these compounds, only oxaloacetate, fumarate and succinate inhibited malate uptake by free bacteroids. Malate uptake by PBUs was inhibited severely by the uncoupler carbonylcyanidem-chlorophenyl hydrazone and the respiratory poison KCN, and was stimulated by ATP. We conclude that the peribacteroid membrane contains a dicarboxylate transport system which is distinct from that on the bacteroid membrane and other plant membranes. This system can catalyse the rapid uptake of a range of dicarboxylates into PBUs, with malate and succinate preferred substrates, and is likely to play an important role in symbiotic nitrogen fixation. Energization of both the bacteroid and peribacteroid membranes controls the rate of dicarboxylate transport into peribacteroid units.

Generation of a membrane potential by sodium-dependent succinate efflux in Selenomonas ruminantium

When Selenomonas ruminantium HD4 was grown in a chemostat, maximal succinate production and the highest molar growth yield values were both observed at a dilution rate of roughly 0.2 h-1. To determine the possible relationship between succinate efflux and high molar growth yields, the generation of a membrane potential by succinate efflux was studied in whole cells and vesicles (inside-out and right-side-out) prepared from S. ruminantium. Washed whole cells took up succinate in the absence of an exogenous energy supply; uptake was completely abolished by brief treatment with dinitrophenol or with nigericin and valinomycin. High levels of sodium ions (with respect to the intracellular sodium concentration in the assay buffer had a stimulatory effect on succinate uptake. When succinate was added to inside-out vesicles, a membrane potential (inside positive) was generated, as indicated by fluorescence quenching of the anionic lipophilic dye Oxonol V. Fluorescence quenching was sensitive to uncoupling by gramicidin D but only partially sensitive to the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In right-side-out vesicles, succinate uptake could be driven by an artificially imposed sodium gradient but not by a potassium diffusion potential; imposition of both a sodium gradient and potassium diffusion potential resulted in improved succinate uptake. The generation of a membrane potential (inside negative) upon succinate efflux was demonstrated directly in right-side-out vesicles when succinate-loaded vesicles were diluted into succinate-free buffer, and the lipophilic cationic probe tetraphenylphosphonium accumulated in the vesicles. Results indicate that an electrogenic succinate-sodium symporter is present in S. ruminantium. Transport of succinate out of the cell via the symporter might be responsible for the high molar growth yields obtained by this organism when it is grown at dilution rates where maximal succinate production occurs.

Thienamycin nephrotoxicity: Mitochondrial injury and oxidative effects of imipenem in the rabbit kidney

The nephrotoxic cephalosoprins cephaloridine and cephaloglycin both produce mitochondrial respiratory toxicity in renal cortex. Recent work has provided evidence that this respiratory toxicity is caused acylation and inactivation of mitochondrial anionic substrate transporters. While cephaloridine also causes significant lipid peroxidative injury in cortical mitochondria and microsomes, cephaloglycin causes little or no oxidative damage under identical conditions. The recently released thienamycin antibiotic, imipenem, like the toxic cephalosporins, produces acute proximal tubular necrosis which can be prevented completely by prior administration of probenecid. The ability of imipenem to block mitochondrial substrate uptake and respiration and produce oxidative changes has not been examined. We therefore evaluated the effects of imipenem in rabbit renal cortex on the following: (1) mitochondrial function [respiration with and uptake of succinate, and uptake of ADP]; and (2) evidence of oxidative change [depletion of reduced glutathione (GSH), production of oxidized glutathione (GSSG), and production of lipid peroxidative injury, as reflected in microsomal conjugated dienes (CDs)]. The mitochondrial effects of 300 mg/kg body wt of imipenem, given i.v. 1 and 2 hr before killing the animals, were comparable to those of the nephrotoxic cephalosporins. There was singnificant reduction of respiration with, and unidirectional uptake of, succinate at both times, while mitochondrial ADP transport was comparatively unaffected. Imipenem also depleted GSH and increased GSSG and CDs at 1 hr. These effects, however, were considerably smaller than those of a comparably nephrotoxic dose of cephaloridine, and this evidence of oxidative stress had resolved by 2 hr. We conclude that imipenem and the nephrotoxic cephalosporins have similar effects on mitochondrial substrate uptake and respiration, but differ significantly in their production of oxidative injury.

Delayed Nodule Development in a Succinate Transport Mutant of Bradyrhizobium japonicum

Summary Soybeans infected with Bradyrhizobium japonicum 29ITn5, a mutant of strain 61-A-101 with a 88%, respectively 44 %, lower V max for the two succinate uptake systems, revealed a delayed onset of nitrogenase activity, a chlorotic phenotype, reduced dry weights and low nitrogen content of the plant tops. Nitrogenase activity of free-living cells of strain 29ITn5 was reduced to 4–7 % of the wild type. Electron micrographs of nodule sections indicated normally shaped bacteroids with only a few PHBA granules. Peripheral amyloplast accumulation and internal vacuolation were especially noticeable in the infected cells.

Dicarboxylate transport in human placental brush-border membrane vesicles

Pathways for transport of dicarboxylic acid metabolites by human placental epithelia were investigated using apical membrane vesicles isolated by divalent cation precipitation. The presence of Na +/dicarboxylate cotransport was assessed directly by [ 14C]succinate tracer flux measurements and indirectly by fluorescence determinations of voltage sensitive dye responses. The imposition of an inwardly directed Na + gradient stimulated vesicle uptake of succinate achieving levels approximately 5-fold greater than those observed at equilibrium. The increased succinate uptake was specific for Na + as no stimulation was observed in the presence of Li +, K + or choline + gradients. In addition to concentrative accumulation of succinate, a direct coupling of Na +/succinate cotransport was suggested by the absence of a sizeable conductive pathway for succinate uptake and decreased succinate uptake levels associated with a more rapid decay of an imposed Na + gradient. Na + gradient-driven succinate uptake was not the result of parallel Na +/H + and succinate/OH − exchange activities but was reduced by the Na +-coupled transport inhibitor harmaline. The voltage sensitivity of Na + gradient-driven succinate uptake suggests Na +/succinate cotransport is electrogenic occurring with net transfer of positive charge. Substrate-specificity studies suggest the tricarboxylic acid cycle intermediates as candidates for transport by the Na +-coupled pathway. Decreasing pH increased the citrate-induced inhibition of succinate uptake suggesting divalent citrate as the preferred substrate for transport. Initial rate determinations of succinate uptake indicate succinate interacts with a single saturable site ( K m 33 μM) with a maximal transport rate of 0.5 nmol/mg per min.