Absence Of Citrate Research Articles

Preparative induction and characterization of L-antithrombin: a structural homologue of latent plasminogen activator inhibitor-1.

The inhibitory mechanism of the serpin family of serine protease inhibitors is characterized by a remarkable degree of conformational flexibility. Various conformational states have been elucidated by X-ray crystallography and indicate that the inhibitory loop, the central A-beta-sheet, and the outside edge of the C-beta-sheet are particularly mobile. However, no crystal structure of a serpin-enzyme complex is yet available, and the likely nature of the protease-complexed serpin remains for biochemical and biophysical researchers to examine. Here, we show that the biochemical induction of the latent state of antithrombin is slow relative to polymer formation, and infer that this may reflect structural features that are important for the regulation of the initial docking and subsequent locking of serpins with cognate proteases. L-Antithrombin was induced by incubation of native antithrombin at 60 degrees C for 10 h in the presence of citrate to prevent polymerization. L-Antithrombin was more stable to denaturation by both heat and urea than native antithrombin. Whereas native antithrombin formed binary complexes with synthetic peptide homologues of the inhibitory loop, biochemically induced L-antithrombin did not, indicating that the inhibitory loop of L-antithrombin is probably fully inserted into the A-beta-sheet as in the crystal structure. This was confirmed by limited proteolysis studies which demonstrated that the inhibitory loop of L-antithrombin could not be cleaved by five proteases which do cleave the loop of native antithrombin. The limited proteolysis studies also indicated that the "gate" region (residues 236-248) of the biochemically induced L-antithrombin was in a conformation substantially different from that of the native antithrombin. This again is similar to L-antithrombin in the crystal structure in which the gate has "opened" away from the body of the molecule by a rotation of 24 degrees to facilitate the relocation of strand 1C from its ordered position in the C-beta-sheet to a disordered surface loop. At 60 degrees C in the absence of citrate, antithrombin (and other serpins) rapidly polymerizes. In the presence of citrate, the formation of L-antithrombin is slow and increases with time, indicating that the inhibition of polymer formation by citrate allows the time necessary for the much slower formation of the L form. We therefore suggest that L-antithrombin formation is a two-step process: an initial rapid conformational change, probably including partial incorporation of the reactive loop into the A-sheet (as in the active molecule in the crystal structure) and displacement of s1C from the C-beta-sheet which supports polymer formation, and a much slower transition to complete loop insertion within the A-beta-sheet. It is likely that both the first rapid transitional step and the structural features that impose resistance to the second more extensive conformational change reflect the optimization of the unique inhibitory function in the serpins.

Effect of citrate on growth of Lactococcus lactis subsp. lactis in milk.

The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly. Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains. Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains. At 26 degrees C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show that citrate metabolism slightly stimulates the growth of lactococci in milk.

An EPR Investigation of the Products of the Reaction of Cytosolic and Mitochondrial Aconitases with Nitric Oxide

Cellular studies have indicated that some Fe-S proteins, and the aconitases in particular, are targets for nitric oxide. Specifically, NO has been implicated in the intracellular process of the conversion of active cytosolic aconitase containing a [4Fe-4S] cluster, to its apo-form which functions as an iron-regulatory protein. We have undertaken the in vitro study of the reaction of NO with purified forms of both mitochondrial and cytosolic aconitases by following enzyme activity and by observing the formation of EPR signals not shown by the original reactants. Inactivation by either NO solutions or NO-producing NONOates under anaerobic conditions is seen for both enzyme isoforms. This inactivation, which occurs in the presence or absence of substrate, is accompanied by the appearance of the g = 2.02 signals of the [3Fe-4S] clusters and the g approximately 2.04 signal of a protein-bound dinitrosyl-iron-dithiol complex in the d7 state. In addition, in the reaction of cytosolic aconitase, the transient formation of a thiyl radical, g parallel = 2.11 and g perpendicular = 2.03, is observed. Disassembly of the [3Fe-4S] clusters of the inactive forms of the enzymes upon the anaerobic addition of NO is also accompanied by the formation of the g approximately 2.04 species and in the case of mitochondrial aconitase, a transient signal at g approximately 2. 032 appeared. This signal is tentatively assigned to the d9 form of an iron-nitrosyl-histidyl complex of the mitochondrial protein. Inactivation of the [4Fe-4S] forms of both aconitases by either superoxide anion or peroxynitrite produces the g = 2.02 [3Fe-4S] proteins.

Gastrointestinal absorption, tissue retention, and urinary excretion of dietary aluminum in rats determined by using 26Al

We used accelerator mass spectrometry (AMS) and 26AI to study the plasma concentration, urinary excretion, and retention in bone, brain, and liver of a single dose of a dietary concentration of aluminum ingested either with or without citrate by 2-month-old Wistar rats. In the absence of citrate, cumulative urinary excretion and skeleton retention were each approximately 0.05% of the total 26AI dose ingested. 26AI retention in brain and liver were approximately 4 x 10(-8) and 2 x 10(-6), respectively. Concomitant citrate intake increased these median values by about two- to fivefold, although this factor was highly variable in individual rats. Independent of citrate administration, 90% of the 26AI excreted in urine (measured cumulatively over 30 days) was excreted within the first 48 h. Uptake by bone was rapid (approximately 1 h) and permanent over the 30-day duration of the experiment.

Effect of Citrate on Production of Diacetyl and Acetoin by Lactococcus lactis ssp. lactis CNRZ 483 Cultivated in the Presence of Oxygen

The effect was studied of trisodium citrate addition on the growth and formation of diacetyl and acetoin by Lactococcus lactis ssp. lactis CNRZ 483 in a whey-based medium under an oxygen pressure of 2atm. Growth increased, as shown by the final absorbance of the culture, which was 1.2 in the absence of citrate and 2.8 in the presence of 26mM citrate. In the absence of citrate, maximal concentrations of diacetyl and acetoin were 0.09 and 0.29mM, respectively. When citrate was added at 6.2, 11.5, and 26mM, the maximal diacetyl concentrations were 0.45, 0.53, and 0.85mM, and those of acetoin were 3.6, 4.5, and 8.5mM, respectively. However, under these conditions, the yield of citrate bioconversion to diacetyl and acetoin decreased as the initial concentration of citrate increased. Yields were 121, 83, and 70% in the presence of 6.2, 11.5, and 26mM citrate, respectively.

Acetyl Coenzyme A Carboxylase Activity in Neonatal Rat Cardiac Myocytes in Culture: Citrate Dependence and Effects of Hypoxia

The presence of a 280,000Mrisoform of acetyl-CoA carboxylase (ACC-280) in the cardiac myocyte suggests that heart muscle is capable of malonyl-CoA synthesis. Cellular factors which regulate activity of ACC-280 are unknown. We have employed a neonatal rat cardiac myocyte culture (where the majority of ACC is present as ACC-280) to examine the effects of hypoxia and decreased cellular ATP on the activity of ACC in the cells. The myocyte culture has the following advantages over similar studies in the intact rat heart: the presence of a pure population of myocytes and the ability to measure cytosolic ACC free from contamination by mitochondrial carboxylases. ACC activity in cultured cardiac myocytes is completely dependent on the presence of citrate (A0.5= 3.8 mM). Under control conditions, the cytosolic citrate concentrationin situis determined to be less than 1 mM. With 5 h of hypoxia, cytosolic ATP decreases from 9.85 ± 0.23 to 2.83 ± 0.25 mMand cytosolic AMP increases from undetectable levels to 40 ± 0.4 μM. With hypoxia, a significant portion of the total ACC activity is now expressed in the absence of citrate and the amount of activity which is stimulated by 10 mMcitrate is significantly less (1.268 ± 0.106 nmol/4 × 105cells) than is seen under control conditions (3.042 ± 0.048). There are no significant changes in the total amount of cellular protein on the plates after 5 h of hypoxia. Consistent with net ACC activation in hypoxia, malonyl-CoA levels increase in the cells by 7 h of hypoxia. Decreased radioactive phosphate content of immunopurified ACC-280 after 5 h of hypoxia is consistent with net dephosphorylation of ACC-280 and increased citrate-independent activity.

Functional properties of the purified Na(+)-dependent citrate carrier of Klebsiella pneumoniae: evidence for asymmetric orientation of the carrier protein in proteoliposomes.

The sodium-ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) was purified and reconstituted into liposomes to investigate the properties of this transport system without interference from other proteins. Citrate uptake was an electroneutral process, where delta pNa+ and/or delta pH are driving forces. Delta psi was unable to stimulate citrate transport, either alone or in addition to the other driving forces. Sodium ions on the inside of the proteoliposomes stimulated the uptake of citrate, indicating that Na+ ions recycle during the transport of citrate. CitS also performed Na+ counterflow in the absence of citrate. The citrate carrier performed citrate/citrate counterflow but no heterologous antiport of citrate with one of the end products arising from the anaerobic citrate fermentation pathway (acetate, formate, or bicarbonate) in K. pneumoniae. Citrate counterflow kinetics revealed that CitS transports citrate according to a simultaneous type of mechanism. The Km and Ki values revealed two binding sites for citrate: one with low and one with high affinity. This transport mode is in accord with an asymmetric organization of the carrier protein in proteoliposomes.

Artificial simulation of the early stages of renal stone formation.

To study the initial steps of calcium oxalate monohydrate (COM) stone formation and the influence of the inhibitor substances citrate and phytic acid, using an in vitro model. Experiments were performed using a device to simulate renal stone formation in conditions similar to those found in humans. COM crystal formation took place directly on the solid substrate in contact with the urine but no previous formation occurs in the liquid with subsequent attachment. The first COM crystals appeared after 3 h, in the presence of citrate at a physiological concentration of 1.7 x 10(-3) M. A decrease in the total amount of COM was observed during the next 9 h. After 24 h the quantity of COM crystals was the same irrespective of the presence or absence of citrate. In the presence of phytic acid (6 x 10(-3) M) the appearance of the first COM crystals was delayed by > 24 h. At 78 h a few small COM crystals were detected. A fixed solid surface, irrespective of its composition, in contact with normal urine sooner or later develops stone-forming compounds on its surface. The composition of these compounds depends on the supersaturated salts that are present. Under the experimental conditions citrate had only a limited capacity to inhibit COM crystal development. In contrast, phytic acid exhibited a much higher capacity for inhibition even at a considerably lower concentration than that of citric acid.

The physiology of Lactococcus lactis subsp. lactis biovar. diacetylactis immobilized in hollow-fibre bioreactors: glucose, lactose and citrate metabolism at high cell densities

Lactococcus lactis subsp. lactis biovar. diacetylactis was selected to study the physiological influences of immobilization and growth to high cell densities. Cells were cultivated on glucose or lactose medium in the presence and absence of citrate. With excess glucose the cells produced mainly lactate as the fermentation product (homofermentative) providing that not all of the substrate was consumed. The population so cultivated was exposed to extreme gradients of pH and lactate concentrations. When the glucose concentration was reduced the population showed a mixed product profile with half of the glucose being fermented to lactate, the remainder to formate, acetate, ethanol and 2,3-butanediol. Inclusion of citrate in the medium shifted the population to homofermentation, with respect to the amount of glucose or lactose consumed. The citrate was metabolized via the pyruvate-formate lyase and α-acetolactate synthase routes. The pH of the medium was shown to strongly influence the product profile from citrate, presumably by affecting the activity of the key enzymes of pyruvate metabolism. The lactococci immobilized at high cell densities show product profiles typical of carbohydrate limitation at low dilution rates.

The Effect of Citrate and pH on Zinc Uptake by Wheat

AbstractZinc uptake by plants may be influenced by its reaction with organic ligands in the rhizosphere. Therefore, four experiments were conducted to examine the effects of an organic ligand (citrate) and pH on the uptake of Zn by wheat (Triticum aestivum L. emend. Theil). Plants were grown for 21 to 28 d in a nutrient solution (containing 0–0.05 μmol L−1 Zn) in a temperature controlled tank, either in the absence or presence of citrate and at constant or variable pH (3.7–7.1). Dry matter weights of plant parts and Zn content in the shoots were determined. The activities of Zn in the nutrient solution were estimated. Shoot dry matter of the wheat plants in each experiment correlated well with either ZnT/[H+] or Σ(moles Zn1charge)/[H+] (ZnT = total Zn, Zn1 = each zinc species). Both parameters could explain the relative shoot dry matter of the plants from all experiments in one equation (y = A + BE−cx; r2 = 0.79 and 0.77, respectively). In the absence of citrate, shoot dry matter as well as Zn content increased with increasing pH and increasing total Zn concentrations in solution. In the presence of citrate, the shoot dry matter of wheat plants that were grown in nutrient solution with constant pH increased with the total Zn concentrations. However, the effect of the total Zn concentrations in the solution containing citrate with variable pH were less important than the effect of pH.

Partial purification of the cyanide-resistant alternative oxidase of skunk cabbage (Symplocarpus foetidus) mitochondria.

A partial purification of the cyanide-resistant, alternative oxidase from skunk cabbage (Symplocarpus foetidus L.) spadix mitochondria is described. Skunk cabbage mitochondria were solubilized in N,N-bis-(3-D-glucon-amido-propyl)deoxycholamide and the alternative oxidase was purified using a batch DEAE-cellulose treatment, followed by precipitation with Extracti-Gel and chromatography on Sephadex G-200. Following pooling and concentrating of the most active fractions from the gel filtration column, a 20- to 30-fold purification of the alternative oxidase was obtained, with no evidence of contamination by cytochrome c oxidase (complex IV) or cytochrome c reductase (complex III). Polyacrylamide gel electrophoresis of the partially purified oxidase showed major polypeptides at 36 and 29 kD, both of which react with monoclonal antibodies raised against the Sauromatum guttatum alternative oxidase. The purified oxidase fraction showed no absorbance in the visible spectral region, and addition of sodium borohydride induced no absorbance changes in the ultraviolet region. The purified alternative oxidase catalyzed the four-electron reduction of oxygen to water in the absence of citrate, but catalyzed an apparent two-electron reduction of oxygen to hydrogen peroxide in the presence of 0.7 M citrate.

Calcium oxalate monohydrate crystallization: citrate inhibition of nucleation and growth steps

The inhibitory action of citrate on calcium oxalate monohydrate (COM) crystallization has been examined in terms of nucleation and crystal growth kinetic properties. Lag-time data for the appearance of crystals and [ 14C] oxalate incorporation under crystal growth conditions allowed us to investigate the influence of citrate at physiological levels (3.5mM). Moreover, through the use of the EQUIL software, we formulated our solutions based on calculations of solute composition such that free calcium concentrations were the same in the absence and presence of this tricarboxylic acid. The presence of citrate had little effect on the apparent interfacial free energy as determined by nucleation kinetic studies, but total particle production was greater in the absence of citrate; this was evident from electron microscopy and was also indicated by corresponding values of pre-exponential terms of the Gibbs-Thomson equation. Crystal growth rates were lowered in the presence of citrate to 30% of the uninhibited value, and distinctive morphological habit modifications were also observed by scanning electron microscopy. Together, these findings suggest that citrate may influence COM crystallization at several stages, and we present a model for face-specific growth inhibition by citrate acting on the (010) COM crystal face.

Effect of varying citrate levels on C4 compound formation and on enzyme levels in Leuconostoc mesenteroides subsp. cremoris grown in continuous culture

The effects of citrate on diacetyl, acetoin and 2,3-butylene glycol (2,3-BG) production by Leuconostoc mesenteroides subsp. cremoris grown in continuous culture at pH 5.2 were studied. In glucose alone end-product production agreed with the theoretical stoichiometry. In the presence of citrate, lactate and acetate production was higher than the theoretical stoichiometry from glucose. Lactate production was constant when the initial citrate concentration was increased whereas ethanol production strongly decreased. In the absence of citrate, citrate lyase (CL) exhibited weak activity. Diacetyl reductase (DR) and acetoin reductase (AR) exhibited basal activity. When varying citrate concentrations ranging from 10 to 75 mm were added to glucose broth, DR, AR, lactate dehydrogenase, NADH oxidase and alcohol dehydrogenase decreased as the initial citrate concentration increased suggesting that they were partly repressed by citrate. In contrast, CL increased and the specific citrate utilization rate also increased in the same way, indicating no saturation of the first step of citrate metabolism. Acetate kinase (AK) was slightly higher in the presence of citrate and increased when the initial citrate concentration increased. This result was correlated with an increase of acetate from the acetyl phosphate pathway. More ATP was produced in the presence of citrate, which could explain the increase in biomass formation. Citrate bioconversion into diacetyl, acetoin and 2,3-BG increased as the initial citrate increased.

Kinetics of cadmium hydroxide precipitation

The kinetics of cadmium hydroxide precipitation in the presence and in the absence of citrate was investigated. Precipitation experiments were conducted in a continuous stirred tank reactor. Experiments were performed with initial cadmium concentrations ranging from 2.0 to 4.0 × 10 −4 M; a range commonly occurring in electroplating rinsewater. A population balance model was used to compute the nucleation and particle growth rates and relate these to the supersaturation ratio. Particle growth rates found in the presence of citrate were much lower than that in the absence of citrate. The examination of growth inhibition suggested that the mechanism was surface reaction controlled.

Purification, characterization, and ontogeny of acetyl-CoA carboxylase isozyme of chick embryo brain.

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of fatty acids. Because fatty acids are required during myelination in the developing brain, it was proposed that the level of acetyl-CoA carboxylase may be highest in embryonic brain. The presence of acetyl-CoA carboxylase activity was detected in chick embryo brain. Its activity varied with age, showing a peak in the 17-18-day-old embryo and decreasing thereafter. The enzyme, affinity-purified from 18-day-old chick embryo brain, appeared as a major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (Mr 265,000), indistinguishable from the 265 kDa isozyme of liver acetyl-CoA carboxylase. It had significant activity (Sp act = 1.1 mumol/min per mg protein) in the absence of citrate. There was a maximum stimulation of only 25% in the presence of citrate. Dephosphorylation using [acetyl-CoA carboxylase] phosphatase 2 did not result in activation of the enzyme. Palmitoyl-CoA (0.1 mM) and malonyl-CoA (1 mM) inhibited the activity to 95% and 71%, respectively. Palmitoylcarnitine, however, did not show significant inhibition. The enzyme was inhibited (greater than 95%) by avidin; however, avidin did not show significant inhibition in the presence of excess biotin. The enzyme was also inhibited (greater than 90%) by antibodies against liver acetyl-CoA carboxylase. An immunoblot or avidin-blot detected only one protein band (Mr 265,000) in preparations from chick embryo brain or adult liver. These observations suggest that acetyl-CoA carboxylase is present in embryonic brain and that the enzyme appears to be similar to the 265 kDa isozyme of liver.

Voltammetric determination of Al(III) with adsorptive preconcentration of the pyrocatechol violet complex

A method is described for the determination of aluminium in aqueous samples by linear scan voltammetry after adsorptive accumulation of the pyrocatechol violet (PCV) complex. A pH 6.5 triethanolamine/perchloric acid electrolyte is used for the determination with adsorption onto a mercury drop electrode at −0.50 V (vs a Ag/AgCl/satd KCl reference). Scanning cathodically after adsorption gave a current peak at −0.70 V for reduction of adsorbed PCV and a peak at −0.90 V for reduction of the adsorbed Al-PCV complex. The peaks were well resolved. Using 1.0 μM PCV and a 60 s adsorption period gave linear calibration curves for Al in the range 1 to 25 ng mL−1 Al. The detection limit was 0.1 ng mL−1 Al. For analysis, calibration by the method of standard additions is required. Most other metal species do not interfere, but vanadium (V(IV) and V(V)) forms a complex with PCV that is adsorbed and reduced at the same potential as Al–PCV. The interference may be eliminated by complexation of the V with citrate. Al and V present together may be determined by analysis in the absence of citrate (total: Al + V) and in the presence of citrate (Al); V is determined as the difference in the two values.The analysis of a range of samples with Al concentrations from 6 ng mL−1 to 3 μg mL−1 gave results which were in good agreement with those obtained by other methods. Key words: aluminium, pyrocatechol violet, adsorption, voltammetry, environmental.

Influence of Citrate on the Kinetics of Fe(II) Oxidation and the Formation of Iron Oxyhydroxides

AbstractThe rate of Fe(II) oxidation at a constant rate of oxygen supply in the presence of citrate was measured at pH 6.0 at various citrate/Fe(II) molar ratios at 23.5°C in 0.01 M ferrous Perchlorate system. The kinetics followed a first-order reaction with respect to Fe(II) concentration at constant pH (6.0) and aeration (5 ml/min). The rate constant decreased exponentially from 41.3 × 10-4 to 7.6 × 10-4/min with an increase in the citrate/Fe(II) molar ratio from 0 to 0.1.The nature of the hydrolytic products formed after 120 min of oxidation was arrived at by X-ray powder diffraction (XRD), infrared spectrometry (IR), and transmission electron microscopic (TEM) analyses. In the absence of citrate, goethite (α-FeOOH) and poorly crystalline lepidocrocite (γ-FeOOH) were the oxidation products formed at pH 6.0. The formation of lepidocrocite was promoted at the expense of goethite at citrate/Fe(II) molar ratios of 0.0005 to 0.005. The formation of lepidocrocite was especially pronounced at a citrate/Fe(II) molar ratio of 0.001, as observed from the width at half height (WHH) and the area of the 020 XRD peak of lepidocrocite. At a citrate/Fe(II) molar ratio of 0.01, however, the crystallization was perturbed resulting in the formation of noncrystalline Fe oxides, and no precipitate was observed at a citrate/Fe molar ratio of 0.1. The strong complexation of Fe(II) with citrate retarded the kinetics of Fe(II) oxidation and the formation and hydrolysis of Fe(III). The complexation, electrostatic, and steric effects of the coexisting citrate anions in solution apparently influenced the oxygen coordination and the way by which the double rows of edge-sharing Fe(O,OH)6 octahedra linked during crystallization, resulting in the formation of lepidocrocite.

Inhibition of protein synthesis results in super-induction of procyclin (PARP) RNA levels

Procyclin is an abundant surface antigen found exclusively on the procyclic forms of African trypanosomes. We are interested in the induction of procyclin gene expression during differentiation from bloodstream forms. We find that increased levels of procyclin RNA are evident as early as 15 min after triggering differentiation. The increase in procyclin RNA levels requires the temperature shift from 37 degrees C to 27 degrees C and is aided by addition of the tricarboxylic acid cycle intermediate cis-aconitate. Maximal induction is observed with a combination of three triggers of differentiation: citrate, cis-aconitate and the temperature shift. Protein synthesis does not appear to be required for induction of procyclin RNA during differentiation. In fact, addition of protein synthesis inhibitors results in super-induction of procyclin RNA levels, even under conditions where no induction is normally observed (i.e., at 37 degrees C in the absence of citrate and cis-aconitate). This super-induction was observed with four different protein synthesis inhibitors that affect different stages of translation. Thus, the accumulation of procyclin transcripts may be under the control of a negative regulator whose effective levels are reduced during differentiation from bloodstream to procyclic forms.

Nature of enhanced mitochondrial oxidative metabolism by a calf blood extract

The effect of calf blood extract (Solcoseryl, SS) on mitochondrial oxidative function in various states was studied polarographically in vitro. 1) Mitochondrial respiration in all 4 conventional study states (Estabrook, 1967) was enhanced by the addition of SS, including states 1 and 2 (endogenous substrates only). 2) The effect of SS on mitochondrial oxygen consumption was concentration dependent, while ADP/O ratio remained constant. The effect of added respiratory substrates varied with the particular substrate at optimally active concentrations. With suboptimal substrate levels, ADP/O ratios were concentration dependent, in contrast to the SS effect. Under oligomycin ATPase inhibition, SS was no longer active, in contrast to DNP, which remained active. 3) In states 3 (added ADP) and 4 (ADP exhausted), oxygen consumption and oxidative phosphorylation were enhanced by SS in the presence or absence of citrate, glutamate, pyruvate, lactate, or ascorbate. However, in the presence of succinate, SS had no effect. 4) ADP/O ratio was decreased by SS in the presence of added substrate, suggesting that SS activation of H(+)-ATPase enhances ATP hydrolysis as well as oxidative phosphorylation and ATP synthesis. 5) The enhancing effect of SS on mitochondrial function is due to hydrophilic components of SS. The lipidic components obtained by Folch fraction of SS have no effect. It is concluded that the effects of SS respiratory substrates and uncouplers on mitochondrial function are essentially different. SS enhances both ATP synthesis and oxygen consumption by mitochondria.

Novel two-component transmembrane transcription control: regulation of iron dicitrate transport in Escherichia coli K-12

Citrate and iron have to enter only the periplasmic space in order to induce the citrate-dependent iron(III) transport system of Escherichia coli. The five transport genes fecABCDE form an operon and are transcribed from fecA to fecE. Two genes, termed fecI and fecR, that mediate induction by iron(III) dicitrate have been identified upstream of fecA. The fecI gene encodes a protein of 173 amino acids (molecular weight, 19,478); the fecR gene encodes a protein of 317 amino acids (molecular weight, 35,529). Chromosomal fecI::Mu d1 mutants were unable to grow with iron(III) dicitrate as the sole iron source and synthesized no FecA outer membrane receptor protein. Growth was restored by transformation with plasmids encoding fecI or fecI and fecR. FecA and beta-galactosidase syntheses under transcription control of the fecB gene (fecB::Mu d1) were constitutive in fecI transformants and were regulated by iron(III) dicitrate in fecI fecR transformants. The amino acid sequence of the FecI protein contains a region close to the carboxy-terminal end for which a helix-turn-helix motif is predicted, which is typical for DNA-binding regulatory proteins. The FecI protein was found in the membrane, and the FecR protein was found in the periplasmic fraction. It is proposed that the FecR protein is the sensor that recognizes iron(III) dicitrate in the periplasm. The FecI protein activates fec gene expression by binding to the fec operator region. In the absence of citrate, FecR inactivates FecI. The lack of sequence homologies to other transmembrane signaling proteins and the location of the two proteins suggest a new type of transmembrane control mechanism.