Oxidative Phosphorylation Components Research Articles

Are Escherichia coli OXPHOS complexes concentrated in specialized zones within the plasma membrane?

Most organisms are able to synthesize ATP by OXPHOS (oxidative phosphorylation). Mitochondria in eukaryotes perform OXPHOS in the inner mitochondrial membrane, whereas the plasma membrane is used by prokaryotes. However, whereas OXPHOS is a well-understood process at the biochemical level, relatively little is known about its operation at the level of the whole-organelle/cell. We observed that a fluorescently labelled terminal oxidase, the cytochrome bd complex, is heterogeneously distributed in the Escherichia coli plasma membrane. This observation forms the basis of a working hypothesis that patches of the E. coli plasma membrane ('respirazones') are dedicated to respiratory function by the high concentration of OXPHOS components in these zones relative to the adjacent membrane. The formulation and physiological significance of this hypothesis are discussed in this paper.

Actuality of Warburg’s views in our understanding of renal cancer metabolism

More than 50 years ago, Warburg proposed that the shift in glucose metabolism from oxidative phosphorylation (OXPHOS) to glycolysis occurring in spite of an adequate oxygen supply was at the root of cancer. This hypothesis often disregarded over the following years has recently stirred up much interest due to progress made in cancer genetics and proteomics. Studies related to renal cancers have been particularly informative to understand how abnormal use of glucose and decrease in OXPHOS are linked to cell proliferation in tumors. Indeed, in aggressive tumors such as clear cell renal carcinoma, the von Hippel-Lindau factor invalidation stabilizes the hypoxia-inducible factor (HIF) in the presence of oxygen. HIF stimulating glycolytic gene expression increases the glycolytic flux. Deficiencies in genes involved in oxidative phosphorylation that can explain the down-regulation of OXPHOS components also begin to be identified. These findings are important in the search for novel therapeutic approaches to cancer treatment.

Lithium increases PGC‐1α expression and mitochondrial biogenesis in primary bovine aortic endothelial cells

Lithium is a therapeutic agent commonly used to treat bipolar disorder and its beneficial effects are thought to be due to a combination of activation of the Wnt/beta-catenin pathway via inhibition of glycogen synthase kinase-3beta and depletion of the inositol pool via inhibition of the inositol monophosphatase-1. We demonstrated that lithium in primary endothelial cells induced an increase in mitochondrial mass leading to an increase in ATP production without any significant change in mitochondrial efficiency. This increase in mitochondrial mass was associated with an increase in the mRNA levels of mitochondrial biogenesis transcription factors: nuclear respiratory factor-1 and -2beta, as well as mitochondrial transcription factors A and B2, which lead to the coordinated upregulation of oxidative phosphorylation components encoded by either the nuclear or mitochondrial genome. These effects of lithium on mitochondrial biogenesis were independent of the inhibition of glycogen synthase kinase-3beta and independent of inositol depletion. Also, expression of the coactivator PGC-1alpha was increased, whereas expression of the coactivator PRC was not affected. Lithium treatment rapidly induced a decrease in activating Akt-Ser473 phosphorylation and inhibitory Forkhead box class O (FOXO1)-Thr24 phosphorylation, as well as an increase in activating c-AMP responsive element binding (CREB)-Ser133 phosphorylation, two mechanisms known to control PGC-1alpha expression. Together, our results show that lithium induces mitochondrial biogenesis via CREB/PGC-1alpha and FOXO1/PGC-1alpha cascades, which highlight the pleiotropic effects of lithium and reveal also novel beneficial effects via preservation of mitochondrial functions.

Reduced TOR Signaling Extends Chronological Life Span via Increased Respiration and Upregulation of Mitochondrial Gene Expression

The relationships between mitochondrial respiration, reactive oxygen species (ROS), and life span are complex and remain controversial. Inhibition of the target of rapamycin (TOR) signaling pathway extends life span in several model organisms. We show here that deletion of the TOR1 gene extends chronological life span in Saccharomyces cerevisiae, primarily by increasing mitochondrial respiration via enhanced translation of mtDNA-encoded oxidative phosphorylation complex subunits. Unlike previously reported pathways regulating chronological life span, we demonstrate that deletion of TOR1 delays aging independently of the antioxidant gene SOD2. Furthermore, wild-type and tor1 null strains differ in life span only when respiration competent and grown in normoxia in the presence of glucose. We propose that inhibition of TOR signaling causes derepression of respiration during growth in glucose and that the subsequent increase in mitochondrial oxygen consumption limits intracellular oxygen and ROS-mediated damage during glycolytic growth, leading to lower cellular ROS and extension of chronological life span.

Role of Oxidative Stress in Cardiac Hypertrophy and Remodeling

Cardiac adaptation in response to intrinsic or external stress involves a complex process of chamber remodeling and myocyte molecular modifications. A fundamental response to increased biomechanical stress is cardiomyocyte and chamber hypertrophy. Although this may provide initial salutary compensation to the stress, sustained hypertrophic stimulation becomes maladaptive, worsening morbidity and mortality risks because of congestive heart failure and sudden death.1 Growing evidence highlights oxidative and nitrosative stresses as important mechanisms for this maladaptation.2–9 Oxidative stress occurs when excess reactive oxygen species (ROS) are generated that cannot be adequately countered by intrinsic antioxidant systems. Superoxide anion (O2−) can further combine with NO, forming reactive compounds such as peroxynitrite, generating nitroso-redox imbalance.4 ROS generation is a normal component of oxidative phosphorylation and plays a role in normal redox control of physiological signaling pathways.5,8,9 However, excessive ROS generation triggers cell dysfunction, lipid peroxidation, and DNA mutagenesis and can lead to irreversible cell damage or death.5,8,9 In this review, we discuss recent experimental evidence for the role of oxidant stress on cardiac remodeling, focusing on pressure- overload–induced hypertrophy and dilation. ROS include free radicals such as superoxide (O2−) and hydroxyl radical and compounds such as hydrogen peroxide (H2O2) that can be converted to radicals, and they participate in both normal and pathologic biochemical reactions.9 O2− is formed intracellularly (Figure 1) by activation of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase or xanthine oxidase (XO), uncoupling of NO synthase (NOS), and electron transport and “leakage” during oxidative phosphorylation in the mitochondria.5,8,9 H2O2 can generate the highly reactive hydroxyl radical via Fenton chemistry under pathological conditions.9 Figure 1. General schematic of generation pathways for ROS and antioxidant systems in the heart. Low levels of ROS are thought to …

Axonal neuropathy with optic atrophy is caused by mutations in mitofusin 2

Charcot-Marie-Tooth (CMT) neuropathy with visual impairment due to optic atrophy has been designated as hereditary motor and sensory neuropathy type VI (HMSN VI). Reports of affected families have indicated autosomal dominant and recessive forms, but the genetic cause of this disease has remained elusive. Here, we describe six HMSN VI families with a subacute onset of optic atrophy and subsequent slow recovery of visual acuity in 60% of the patients. Detailed clinical and genetic studies were performed. In each pedigree, we identified a unique mutation in the gene mitofusin 2 (MFN2). In three families, the MFN2 mutation occurred de novo; in two families the mutation was subsequently transmitted from father to son indicating autosomal dominant inheritance. MFN2 is a mitochondrial membrane protein that was recently reported to cause axonal CMT type 2A. It is intriguing that MFN2 shows functional overlap with optic atrophy 1 (OPA1), the protein underlying the most common form of autosomal dominant optic atrophy, and mitochondrial encoded oxidative phosphorylation components as seen in Leber's hereditary optic atrophy. We conclude that autosomal dominant HMSN VI is caused by mutations in MFN2, emphasizing the important role of mitochondrial function for both optic atrophies and peripheral neuropathies.

The Rhodomonas salina mitochondrial genome: bacteria-like operons, compact gene arrangement and complex repeat region

To gain insight into the mitochondrial genome structure and gene content of a putatively ancestral group of eukaryotes, the cryptophytes, we sequenced the complete mitochondrial DNA of Rhodomonas salina. The 48 063 bp circular-mapping molecule codes for 2 rRNAs, 27 tRNAs and 40 proteins including 23 components of oxidative phosphorylation, 15 ribosomal proteins and two subunits of tat translocase. One potential protein (ORF161) is without assigned function. Only two introns occur in the genome; both are present within cox1 belong to group II and contain RT open reading frames. Primitive genome features include bacteria-like rRNAs and tRNAs, ribosomal protein genes organized in large clusters resembling bacterial operons and the presence of the otherwise rare genes such as rps1 and tatA. The highly compact gene organization contrasts with the presence of a 4.7 kb long, repeat-containing intergenic region. Repeat motifs ∼40–700 bp long occur up to 31 times, forming a complex repeat structure. Tandem repeats are the major arrangement but the region also includes a large, ∼3 kb, inverted repeat and several potentially stable ∼40–80 bp long hairpin structures. We provide evidence that the large repeat region is involved in replication and transcription initiation, predict a promoter motif that occurs in three locations and discuss two likely scenarios of how this highly structured repeat region might have evolved.

Modular Kinetic Analysis of the Adenine Nucleotide Translocator–Mediated Effects of Palmitoyl-CoA on the Oxidative Phosphorylation in Isolated Rat Liver Mitochondria

To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial adenine nucleotide translocator, we applied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential (Deltapsi) and the fraction of matrix ATP as intermediates. We found that 5 mumol/l palmitoyl-CoA inhibited adenine nucleotide translocator, without direct effect on other components of oxidative phosphorylation. Indirect effects depended on how oxidative phosphorylation was regulated. When the electron donor and phosphate acceptor were in excess, and the mitochondrial "work" flux was allowed to vary, palmitoyl-CoA decreased phosphorylation flux by 38% and the fraction of ATP in the medium by 39%. Deltapsi increased by 15 mV, and the fraction of matrix ATP increased by 46%. Palmitoyl-CoA had a stronger effect when the flux through the mitochondrial electron transfer chain was maintained constant: Deltapsi increased by 27 mV, and the fraction of matrix ATP increased 2.6 times. When oxidative phosphorylation flux was kept constant by adjusting the rate using hexokinase, Deltapsi and the fraction of ATP were not affected. Palmitoyl-CoA increased the extramitochondrial AMP concentration significantly. The effects of palmitoyl-CoA in our model system support the proposed mechanism linking obesity and type 2 diabetes through an effect on adenine nucleotide translocator.

Modification of the Mitochondrial Proteome in Response to the Stress of Ethanol-dependent Hepatotoxicity

Mitochondria are particularly susceptible to increased formation of reactive oxygen and nitrogen species in the cell that can occur in response to pathological and xenobiotic stimuli. Proteomics can give insights into both mechanism of pathology and adaptation to stress. Herein we report the use of proteomics to evaluate alterations in the levels of mitochondrial proteins following chronic ethanol exposure in an animal model. Forty-three proteins showed differential expression, 13 increased and 30 decreased, as a consequence of chronic ethanol. Of these proteins, 25 were not previously known to be affected by chronic ethanol emphasizing the power of proteomic approaches in revealing global responses to stress. Both nuclear and mitochondrially encoded gene products of the oxidative phosphorylation complexes in mitochondria from ethanol-fed rats were decreased suggesting an assembly defect in this integrated metabolic pathway. Moreover mtDNA damage was increased by ethanol demonstrating that the effects of ethanol consumption extend beyond the proteome to encompass mtDNA. Taken together, we have demonstrated that chronic ethanol consumption extends to a modification of the mitochondrial proteome far broader than realized previously. These data also suggest that the response of mitochondria to stress may not involve non-discriminate changes in the proteome but is restricted to those metabolic pathways that have a direct role in a specific pathology.

Profiling gene transcription reveals a deficiency of mitochondrial oxidative phosphorylation in Trypanosoma cruzi-infected murine hearts: implications in chagasic myocarditis development

In this study, we report the host genetic responses that characterize Trypanosoma cruzi-induced myocarditis in a murine model of infection and disease development. The mRNA species from the myocardium of infected mice were assessed using cDNA microarray technology at immediate early, acute, and chronic stages of infection. The immediate early reaction of the host to T. cruzi infection was marked by up-regulation of transcripts indicative of proinflammatory and interferon-induced immune responses. Following acute infection, overexpression of transcripts for extracellular matrix (ECM) proteins, possibly initiated in response to myocardial injuries by invading and replicating parasites, was suggestive of active reparative and remodeling reactions. Surprisingly, progression to the cardiac disease phase was associated with coordinated down-regulation of a majority (>70%) of the differentially expressed genes. Among the most repressed genes were the troponins, essential for contractile function of the myofibrils, and the genes encoding components of oxidative phosphorylation (OXPHOS) pathways. Reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and biochemical assays confirmed the microarray results and provided evidence for the deficiency of OXPHOS complex IV in the chagasic murine heart. We discuss the apparent role of OXPHOS dysfunction in the cardiac hypertrophic and remodeling processes with the development of chagasic cardiomyopathy (CCM).

Microarray profiling of Erwinia chrysanthemi 3937 genes that are regulated during plant infection.

Microarray technology was used to identify genes in Erwinia chrysanthemi 3937 that are specifically up- or down-regulated in a plant host compared with growth in laboratory culture medium. Several genes were plant down-regulated, and almost all of them were homologues of well-known housekeeping genes, such as those encoding metabolic functions, oxidative phosphorylation components, and transcription or translation processes. On the other hand, almost all of the plant up-regulated genes were involved with specialized functions, including already known or new putative virulence factors, anaerobiosis, iron uptake, transporters or permeases, xenobiotic resistance, chemotaxis, and stress responses to reactive oxygen species and heat. A substantial number of the plant up-regulated genes do not appear to be directly involved in damaging the host, but are probably important in adapting the pathogen to the host environment. We constructed insertion mutations in several of the plant up-regulated E. chrysanthemi 3937 genes. Among these, mutations of Bacillus subtilis pps1, Escherichia coli purU, and Pseudomonas aeruginosa pheC homologues reduced virulence on African violet leaves. Thus, new insights were obtained into genes important in bacterial virulence.

Dual mutations reveal interactions between components of oxidative phosphorylation in Kluyveromyces lactis.

Loss of mtDNA or mitochondrial protein synthesis cannot be tolerated by wild-type Kluyveromyces lactis. The mitochondrial function responsible for rho(0)-lethality has been identified by disruption of nuclear genes encoding electron transport and F(0)-ATP synthase components of oxidative phosphorylation. Sporulation of diploid strains heterozygous for disruptions in genes for the two components of oxidative phosphorylation results in the formation of nonviable spores inferred to contain both disruptions. Lethality of spores is thought to result from absence of a transmembrane potential, Delta Psi, across the mitochondrial inner membrane due to lack of proton pumping by the electron transport chain or reversal of F(1)F(0)-ATP synthase. Synergistic lethality, caused by disruption of nuclear genes, or rho(0)-lethality can be suppressed by the atp2.1 mutation in the beta-subunit of F(1)-ATPase. Suppression is viewed as occurring by an increased hydrolysis of ATP by mutant F(1), allowing sufficient electrogenic exchange by the translocase of ADP in the matrix for ATP in the cytosol to maintain Delta Psi. In addition, lethality of haploid strains with a disruption of AAC encoding the ADP/ATP translocase can be suppressed by atp2.1. In this case suppression is considered to occur by mutant F(1) acting in the forward direction to partially uncouple ATP production, thereby stimulating respiration and relieving detrimental hyperpolarization of the inner membrane. Participation of the ADP/ATP translocase in suppression of rho(0)-lethality is supported by the observation that disruption of AAC abolishes suppressor activity of atp2.1.

Biology of Giardia lamblia.

Giardia lamblia is a common cause of diarrhea in humans and other mammals throughout the world. It can be distinguished from other Giardia species by light or electron microscopy. The two major genotypes of G. lamblia that infect humans are so different genetically and biologically that they may warrant separate species or subspecies designations. Trophozoites have nuclei and a well-developed cytoskeleton but lack mitochondria, peroxisomes, and the components of oxidative phosphorylation. They have an endomembrane system with at least some characteristics of the Golgi complex and encoplasmic reticulum, which becomes more extensive in encysting organisms. The primitive nature of the organelles and metabolism, as well as small-subunit rRNA phylogeny, has led to the proposal that Giardia spp. are among the most primitive eukaryotes. G. lamblia probably has a ploidy of 4 and a genome size of approximately 10 to 12 Mb divided among five chromosomes. Most genes have short 5' and 3' untranslated regions and promoter regions that are near the initiation codon. Trophozoites exhibit antigenic variation of an extensive repertoire of cysteine-rich variant-specific surface proteins. Expression is allele specific, and changes in expression from one vsp gene to another have not been associated with sequence alterations or gene rearrangements. The Giardia genome project promises to greatly increase our understanding of this interesting and enigmatic organism.

Up-regulation of Nuclear and Mitochondrial Genes in the Skeletal Muscle of Mice Lacking the Heart/Muscle Isoform of the Adenine Nucleotide Translocator

Mice deficient in the heart/muscle specific isoform of the adenine nucleotide translocator (ANT1) exhibit many of the hallmarks of human oxidative phosphorylation (OXPHOS) disease, including a dramatic proliferation of skeletal muscle mitochondria. Because many of the genes necessary for mitochondrial biosynthesis, OXPHOS function, and response to OXPHOS disease might be expected to be up-regulated in the Ant1(-/-) mouse, we used differential display reverse transcription-polymerase chain reaction techniques in an effort to identify these genes. 17 genes were identified as up-regulated in Ant1-deficient mice, and they fall into four categories: 1) nuclear and mitochondrial genes encoding OXPHOS components, 2) mitochondrial tRNA and rRNA genes, 3) genes involved in intermediary metabolism, and 4) an eclectic group of other genes. Among the latter genes, we identified the gene encoding anti-apoptotic Mcl-1, the Skd3 gene, and the WS-3 gene, which were previously unknown to be related to mitochondrial function. These results indicate that identification of genes up-regulated in the skeletal muscle of the Ant1-deficient mouse provides a novel method for identifying mammalian genes required for mitochondrial biogenesis.

Cloning, Expression, and Chromosomal Assignment of the Human Mitochondrial Intermediate Peptidase Gene (MIPEP)

The mitochondrial intermediate peptidase ofSaccharomyces cerevisiae(YMIP) is a component of the yeast mitochondrial protein import machinery critically involved in the biogenesis of the oxidative phosphorylation (OXPHOS) system. This leader peptidase removes specific octapeptides from the amino terminus of nuclear-encoded OXPHOS subunits and components of the mitochondrial genetic apparatus. To address the biologic role of the human peptidase [MIPEP gene, HMIP polypeptide], we have initiated its molecular and functional characterization. A full-length cDNA was isolated by screening a human liver library using a rat MIP (RMIP) cDNA as a probe. The encoded protein contained a typical mitochondrial leader peptide and showed 92 and 54% homology to RMIP and YMIP, respectively. A survey of human mitochondrial protein precursors revealed that, similar to YMIP, HMIP is primarily involved in the maturation of OXPHOS-related proteins. Northern analysis showed that the MIPEP gene is differentially expressed in human tissues, with the highest levels of expression in the heart, skeletal muscle, and pancreas, three organ systems that are frequently affected in OXPHOS disorders. Using fluorescencein situhybridization, the MIPEP locus was assigned to 13q12. This information offers the possibility of testing the potential involvement of HMIP in the pathophysiology of nuclear-driven OXPHOS disorders.

Ca2+ stimulates both the respiratory and phosphorylation subsystems in rat heart mitochondria.

Stimulation of mitochondrial respiration by physiological concentrations of Ca2+ was studied to determine which components of oxidative phosphorylation are affected by Ca2+. The kinetic dependence of the respiratory chain, phosphorylation subsystem and proton leak on the mitochondrial membrane potential in isolated rat heart mitochondria respiring on 2-oxoglutarate or succinate was measured at two different concentrations of external free Ca2+. The results show that proton leak is not directly affected by Ca2+, but that both the respiratory and phosphorylation systems can be directly stimulated by Ca2+ depending on conditions. Although Ca2+ directly stimulates the phosphorylation system, this has relatively little effect on respiration rate with 2-oxoglutarate in States 3 and 4 because the subsystem has little control over respiration. However, in intermediate states, the phosphorylation system has greater control and Ca2+ stimulation of this system contributes substantially to the stimulation of respiration and phosphorylation. In the case of succinate oxidation neither the respiratory subsystem nor the phosphorylation system is stimulated by Ca2+.

Myocardial Ca(2+)- and ATP-cycling imbalances in end-stage dilated and ischemic cardiomyopathies.

We have previously demonstrated deficiencies in myocardial cycling of Ca2+, and ATP turnover, in animals with heart failure (HF). The objective of this study was to determine the relevance of these changes to human HF. We used the Ca2+ dye, indo-1, and the Ca(2+)-channel modulator ryanodine to examine Ca(2+)-cycling in homogenates containing 2.5% myocardium from 12 patients undergoing cardiac transplantations because of ischemic or idiopathic dilated cardiomyopathies (ISCM, DCM), and compared them to homogenates from 11 organ donors who died from noncardiac causes. Key enzymes of ATP production and utilization were also assayed. In HF due to either ISCM or DCM, compared to nonfailing myocardium, rate constants (x 10(-3) s-1) for sarcoplasmic reticulum Ca(2+)-pumping (41.6 +/- 16.0 versus 15.1 +/- 5.9) and Ca(2+)-channel (25.1 +/- 8.3 versus 6.2 +/- 4.1) activities were decreased by 64 and 75%, respectively. These changes in rate constants were associated with a three-fold increase in ionized Ca2+ concentration. Compared to nonfailing myocardium, activities (IU/g) of ATP turnover were also decreased in ISCM and DCM HF by 39%, 30%, and 34%, respectively, for ATP production capacity of creatine kinase (1830 +/- 130 versus 1110 +/- 411) and oxidative phosphorylation (20.0 +/- 3.3 and 14.1 +/- 4.8), and for ATP utilization (28.2 +/- 18.7 versus 18.7 +/- 4.0). Myoglobin, a key component of oxidative phosphorylation, was approximately 50% lower with HF (1.72 +/- 0.30 versus 0.97 +/- 0.20 mg/g). As in animal models, cycling of Ca2+ and ATP turnover were markedly impaired in human heart failure. There were no consistent biochemical differences attributable to difference in etiology, excepting that myoglobin deficiency was 33% greater in ISCM than DCM. We conclude that ATP and Ca2+ cycling are significantly impaired in human HF due to DCM and ISCM.

Effects of β-adrenoceptor blockers on mitochondrial ATPase activity in guinea pig heart preparations

The effects of three β-adrenoceptor blockers atenolol, indenolol and nadolol on myocardial mitochondrial ATPase (ATP: phosphohydrolase EC 3.6.1.3) activity were evaluated and compared with that of propranolol in guinea pig heart preparations. Propranolol and indenolol inhibited ATPase activity with IC 50 values of 4.4 ± 0.5 and 5.3 ± 0.4 mM, respectively. In contrast, however, nadolol and atenolol markedly enhanced mitochondrial ATPase activity. Atenolol increased the enzyme activity by approximately 5,240 and 950%, while nadolol enhanced it by 13,280 and 2800% at 100 μM, 1.0 mM and 10.0 mM, respectively. The results indicate that these drugs exhibit two modes of interaction with the mitochondrial ATPase: inhibition by propranolol and indenolol and stimulation by atenolol and nadolol. The inhibitory actions are probably related to the membrane-stabilizing effects and therefore antiarrhythmic actions of the two drugs, while the stimulatory effects of atenolol and nadolol are probably a result of interactions with some component of oxidative phosphorylation or the respiratory chain.

Kinetics of Pi-Pi exchange in rat liver mitochondria. Rapid filtration experiments in the millisecond time range.

Phosphate-phosphate exchange through the inorganic phosphate (Pi) carrier of rat liver mitochondria was investigated by a new rapid filtration technique, which does not require the use of transport inhibitors to stop the reaction and offers high time resolution (starting from 10 ms), thus allowing kinetic measurements on a fine time scale even at room temperature. At approximately 22 degrees C, isotopic equilibrium of [32P]Pi is achieved within 0.8-2.5 s--depending on the Pi concentration--and an initial linear phase, lasting for 400-500 ms, is observed. Complete inhibition of Pi exchange by an excess (33 nmol/mg) of mersalyl, a well-known organomercurial inhibitor, required 200 ms, pointing to the insufficiency of this reagent for effective inhibitor stop. On the other hand, investigation of the effect of mersalyl (allowed to react with mitochondria for at least 20 s) on the initial rate of Pi exchange supports earlier observations on the protective effect of this inhibitor; i.e., up to 3 nmol of mersalyl/mg of protein does not decrease the transport rate whereas these low concentrations protect approximately 50% of the transport capacity from irreversible inactivation by N-ethylmaleimide. In nonrespiring mitochondria, at pH 7.3, Pi exchange exhibited a Km of 1.6 mM and a Vmax of 3.0 mumol min-1 (mg of mitochondrial protein)-1. The increase of the membrane potential without any concomitant change of delta pH had no significant influence on the kinetic parameters. The maximal velocity of Pi transport is significantly higher than the maximal velocity of all the other components of oxidative phosphorylation at comparable temperatures. The possible physiological significance of this excess capacity is discussed.

Aminoglycoside-resistant mutants of Pseudomonas aeruginosa deficient in cytochrome d, nitrite reductase, and aerobic transport.

Two gentamicin-resistant mutants of Pseudomonas aeruginosa PAO 503 were selected after ethyl methane sulfonate mutagenesis. Mutant PAO 2403 had significantly increased resistance to aminoglycoside but not to other antibiotics. Mutant PAO 2402 showed a similar spectrum of resistance but of lower magnitude. Both mutants showed no detectable cytochrome d and had a high frequency of reversion to a fully wild-type phenotype. PAO 2403 had a marked decrease and PAO 2402 had a moderate decrease in nitrite reductase activity. Both mutants had reduced uptake of gentamicin and dihydrostreptomycin. Mutant PAO 2403 showed a general decrease in transport rate of cationic compounds, whereas mutant PAO 2402 had only deficient glucose transport. Both mutants showed enhanced rates of glutamine transport and no change in glutamic acid transport. Other components of electron transport and oxidative phosphorylation were normal. These mutants involve ferrocytochrome C551 oxidoreductase formed only on anaerobic growth but illustrate transport defects in aerobically grown cells.