ATPase Specific Activity Research Articles

Changes in gill Na+, K+ ‐ATPase specific activity during seaward migration of wild juvenile chinook salmon

Migration of wild juvenile chinook salmon Oncorhynchus tshawytscha during the first 80 km of their 254 km migration through the Rogue River, Oregon, was significantly slower than that during the last 170 km. Gill Na+, K+ ‐ATPase specific activity did not increase significantly during the first 38 km of migration. Specific activities during the next 43 km did increase significantly. Specific activities continued to increase as the fish moved downstream, reaching a maximum within 44 km from the Pacific Ocean.

Changes in gill Na+, K+-ATPase specific activity during seaward migration of wild juvenile chinook salmon

Changes in gill Na+, K+-ATPase specific activity during seaward migration of wild juvenile chinook salmon

Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy.

The Escherichia coli protein DbpA is unique in its subclass of DEAD box RNA helicases, because it possesses ATPase-specific activity toward the peptidyl transferase center in 23S rRNA. Although its remarkable ATPase activity had been well defined toward various substrates, its RNA helicase activity remained to be characterized. Herein, we show by using biochemical assays and atomic force microscopy that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence. This work presents an attempt to investigate the action of DEAD box proteins by a single-molecule visualization methodology. Our atomic force microscopy images enabled us to observe directly the unwinding reaction of a DEAD box helicase on long stretches of double-stranded RNA. Specifically, we could differentiate between the binding of DbpA to RNA in the absence of ATP and the formation of a Y-shaped intermediate after its progression through double-stranded RNA in the presence of ATP. Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases. However, accumulated evidence and the results reported herein suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases.

Abolishment of Proton Pumping and Accumulation in the E1P Conformational State of a Plant Plasma Membrane H+-ATPase by Substitution of a Conserved Aspartyl Residue in Transmembrane Segment 6

The plasma membrane H(+)-ATPase AHA2 of Arabidopsis thaliana, which belongs to the P-type ATPase superfamily of cation-transporting ATPases, pumps protons out of the cell. To investigate the mechanism of ion transport by P-type ATPases we have mutagenized Asp(684), a residue in transmembrane segment M6 of AHA2 that is conserved in Ca(2+)-, Na(+)/K(+)-, H(+)/K(+)-, and H(+)-ATPases and which coordinates Ca(2+) ions in the SERCA1 Ca(2+)-ATPase. We describe the expression, purification, and biochemical analysis of the Asp(684) --> Asn mutant, and provide evidence that Asp(684) in the plasma membrane H(+)-ATPase is required for any coupling between ATP hydrolysis, enzyme conformational changes, and H(+)-transport. Proton pumping by the reconstituted mutant enzyme was completely abolished, whereas ATP was still hydrolyzed. The mutant was insensitive to the inhibitor vanadate, which preferentially binds to P-type ATPases in the E(2) conformation. During catalysis the Asp(684) --> Asn enzyme accumulated a phosphorylated intermediate whose stability was sensitive to addition of ADP. We conclude that the mutant enzyme is locked in the E(1) conformation and is unable to proceed through the E(1)P-E(2)P transition.

Characterisation of red and white muscle myosin heavy chain gene coding sequences from antarctic and tropical fish

To understand molecular adaptation for locomotion at different environmental temperatures, we have studied the myosin heavy chain genes as these encode the molecular motors involved. For this purpose, cDNA libraries from white (fast) and red (slow) myotomal muscle of an Antarctic and a tropical fish were constructed and from these different myosin heavy chain cDNAs were isolated. Northern and in situ hybridisation confirmed in which type of muscle these isoform genes are expressed. The cDNAs were sequenced and the structure of the ATPase sites compared. There was a marked similarity between the tropical fast myosin and the Antarctic slow myosin in the loop 1 region, which has similar amino acid side chains, charge distribution and conformation. These findings help to explain why the myofibrils isolated from white muscle of tropical fish show a lower specific ATPase activity than the white muscle of Antarctic fish but a similar activity to the Antarctic red (slow) muscle. It also provides insight into the way molecular motors in Antarctic fish have evolved to produce more power and thus ensure effective swimming at near zero temperatures by the substitution or addition of a few residues in strategic regions, which include the ATPase site.

Identification and Functional Expression of Four Isoforms of ATPase II, the Putative Aminophospholipid Translocase: EFFECT OF ISOFORM VARIATION ON THE ATPase ACTIVITY AND PHOSPHOLIPID SPECIFICITY

ATPase II, a vanadate-sensitive and phosphatidylserine-dependent Mg(2+)-ATPase, is a member of a subfamily of P-type ATPase and is presumably responsible for aminophospholipid translocation activity in eukaryotic cells. The aminophospholipid translocation activity plays an important physiological role in the maintenance of membrane phospholipid asymmetry that is observed in the plasma membrane as well as the membranes of certain cellular organelles. While the preparations of ATPase II from different sources share common fundamental properties, such as substrate specificity, inhibitor spectrum, and phospholipid dependence, they are divergent in several characteristics. These include specific ATPase activity and phospholipid selectivity. We report here the identification of four isoforms of ATPase II in bovine brain. These isoforms are formed by a combination of two major variations in their primary sequences and show that the structural variation of these isoforms has functional significance in both ATPase activity and phosholipid selectivity. Furthermore, studies with the phosphoenzyme intermediate of ATPase II and its recombinant isoforms revealed that phosphatidylserine is essential for the dephosphorylation of the intermediate. Without phosphatidylserine, ATPase II would be accumulated as phosphoenzyme in the presence of ATP, resulting in the interruption of its catalytic cycle.

A Plant Plasma Membrane H+-ATPase Expressed in Yeast Is Activated by Phosphorylation at Its Penultimate Residue and Binding of 14-3-3 Regulatory Proteins in the Absence of Fusicoccin

The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified. Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2. This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants. When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form. Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region. The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated. In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin. Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase. These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H(+) pumping activity. Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth. We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme.

NaCl Effects on Root Plasma Membrane ATPase of Salt Tolerant Wheat

Wheat seedlings of a salt tolerant cultivar were grown hydroponically in presence and absence of 100 mM NaCl. Roots were harvested, and the plasma membrane (PM) fraction was purified. PM ATPase required a divalent cations for activity (Mg > Mn > Ca > Co > Zn > Ni > Cu), and it was further stimulated by monovalent cations (K > Rb > NH4 > Li > choline > Cs). The pH optima were 6.0 and 5.6 in absence and presence of 25 mM KCl, respectively. The enzyme was sensitive to vanadate and DCCD but insensitive to azide, oligomycine and nitrate. The enzyme displayed a high preference for ATP but was also able to hydrolyze other nucleotide tri- and diphosphates. The enzyme activity showed a simple Michaelis-Menten kinetics for the substrate Mg2+-ATP in both control and salt exposed roots. The polypeptide patterns of control and salt stressed PM fractions, detected by SDS-PAGE, were very similar. NaCl substantially reduced the PM ATPase specific activity, whereas it had little effect on the apparent Km for Mg2+-ATP. Since the root PM ATPase of salt sensitive and resistant genotypes responded similarly to salinity stress, it seems unlikely that the mechanism of salt tolerance in wheat is primarily based on differences in PM ATPase characteristics.

Adenosine modulates the (Na(+)+K(+))ATPase activity in malpighian tubules isolated from Rhodnius prolixus.

The role of adenosine on regulation of the (Na(+)+K(+))ATPase activity present in the Malpighian tubules isolated from Rhodnius prolixus was investigated. Adenosine decreases the (Na(+)+K(+)) ATPase specific activity by 88%, in a dose-dependent manner, with maximal effect at a concentration of 10(-9) M. This effect was mimicked by N(6)-cyclohexyladenosine (CHA) at 10(-8) M, an agonist for A(1) adenosine receptor, and was reversed by 10(-9) M 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an antagonist for A(1) adenosine receptor. On the other hand, 5'-N-ethyl-carboxamide adenosine (NECA), an agonist for A(2) adenosine receptor, used in the range of 10(-9)-10(-5) M, did not change the (Na(+)+K(+))ATPase specific activity. In the same way, 10(-8) M 3, 7-dimethyl-1-propargylxanthine (DMPX), an antagonist for A(2) adenosine receptor, did not modify the inhibitory effect of adenosine. These data suggest that the inhibitory effect of adenosine on the (Na(+)+K(+))ATPase specific activity present in Malpighian tubules from Rhodnius prolixus is mediated by A(1) adenosine receptor activation. Arch.

Na(+)/K(+)-ATPase immunoreactivity in branchial chloride cells of Oreochromis mossambicus exposed to copper.

Chloride cells were identified by Na(+)/K(+)-ATPase immunocytochemistry at the light and electron microscope levels in gills of freshwater tilapia Oreochromis mossambicus. Turnover of chloride cells was enhanced by exposing the fish to waterborne copper (3.2 micromol l(-)(1)) for 14 days, as indicated by a 38 % increase in cells expressing proliferating cell nuclear antigen (PCNA) relative to controls. The expression of PCNA was most marked in the central area of the filamental epithelium, from where the chloride cells are thought to originate and migrate. In control fish, chloride cells were associated exclusively with the filamental epithelium. In both controls and copper-exposed fish, two chloride cell populations were seen after Na(+)/K(+)-ATPase immunostaining. These probably represent subpopulations of newly emerged chloride cells: (1) strongly stained cells (mature chloride cells) in the filamental and lamellar epithelium and (2) weakly stained cells, identified by electron microscopy as apoptotic and necrotic chloride cells, mainly in the filamental epithelium. Absolute numbers of mature chloride cells fell, while necrotic and apoptotic chloride cell numbers increased, in copper-exposed fish. A strong correlation could be established for gill Na(+)/K(+)-ATPase specific activity and the number of strongly stained chloride cells in controls and copper-exposed fish and for Na(+)/K(+)-ATPase specific activity and total numbers of immunoreactive cells in copper-exposed fish owing to an increased incidence of weakly staining cells.

Na(+) and Ca(2+) pumps in the gills, epipodites and branchiostegites of the european lobster Homarus gammarus: effects of dilute sea water.

Crude homogenates and plasma-membrane-enriched fractions were prepared from the epithelium of the gills, epipodites and branchiostegites of intermoult European lobsters Homarus gammarus, and Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Na(+)/Ca(2+) exchange activities were quantified in these tissues. Lobsters were kept in sea water (salinity 35 ) or were adapted to dilute sea water (22.1 ). The lobster hyperregulates haemolymph osmolarity and Ca(2+) levels in both media. Homogenates of the podobranchs, arthrobranchs and pleurobranchs had comparable Na(+)/K(+)-ATPase specific activities, and mean activities increased significantly for all three types of gills when the animals were kept in dilute sea water. In the epipodites and branchiostegites, Na(+)/K(+)-ATPase specific activities exceeded those in the gills, and exposure to dilute sea water greatly enhanced these activities. In sea water, 80 % of the total Na(+)/K(+)-ATPase activity is associated with the gills and epipodites (each tissue containing 40 %) and 20 % with the branchiostegites; in dilute sea water, the gills contained approximately 25 %, the epipodites 40 % and the branchiostegites approximately 35 % of the total activity, indicating the relative importance of the epipodites and branchiostegites for ionic hyperregulation in dilute media. In plasma membrane vesicles isolated from the gills, epipodites and branchiostegites, Ca(2+) transport driven by ATP and by a Na(+ )gradient was demonstrated. Exposure to dilute sea water enhanced Na(+)/Ca(2+ )exchange and Ca(2+)-ATPase activities in the epipodites and branchiostegites; in the gills, however, Ca(2+) transport activities decreased. The role of these tissues and enzymes in Na(+) and Ca(2+) handling by the lobster is discussed.

Hera from Thermus thermophilus: the first thermostable DEAD-box helicase with an RNase P protein motif

DEAD-box proteins have been implicated in a wide array of cellular processes ranging from initiation of protein synthesis and ribosome biogenesis to mRNA splicing. Here, we report the isolation, biochemical characterization and crystallization of the first thermophilic DEAD box protein, Hera (heat-resistant RNA-dependent ATPase) from Thermus thermophilus HB8. The molecular mass of the deduced Hera protein sequence (510 amino acid residues) is 55.95 kDa. Hera possesses all of the conserved motifs found among the, DEAD-box RNA helicases. In addition, it also has a motif characteristic of the protein component of ribonuclease P at its C-terminal region (residues 372–386). Hera appears to be non-specific with respect to the RNA species that triggers ATPase activity. Nevertheless, at high temperature, ATPase activity is at a maximum when bacterial 16 S rRNA or 23 S rRNA are used as the substrates. Moreover, a deletion of the RNase P protein motif significantly reduces the ability of Hera to hydrolyze ATP in the presence of RNase P RNA. Hera has a specific ATPase activity of 480 units/μg and therefore, displays the highest ATPase specific activity reported for a protein of the RNA helicase family. We determined that Hera shows helix-destabilizing activity, and that the RNA-unwinding or helix-destabilizing activity of Hera is coupled to ATP hydrolysis. Since Hera is a stable thermophilic protein and we have obtained crystals of it diffracting beyond 2.6 Å, the possibilities for structure determination of a full-length RNA-helicase are open.

Large Scale Purification of Detergent-soluble P-glycoprotein fromPichia pastoris Cells and Characterization of Nucleotide Binding Properties of Wild-type, Walker A, and Walker B Mutant Proteins

P-glycoprotein (Pgp; mouse MDR3) was expressed in Pichia pastoris, grown in fermentor culture, and purified. The final pure product is of high specific ATPase activity and is soluble at low detergent concentration. 120 g of cells yielded 6 mg of pure Pgp; >4 kg of cells were obtained from a single fermentor run. Properties of the pure protein were similar to those of previous preparations, except there was significant ATPase activity in absence of added lipid. Mutant mouse MDR3 P-glycoproteins were purified by the same procedure after growth of cells in flask culture, with similar yields and purity. This procedure should open up new avenues of structural, biophysical, and biochemical studies of Pgp. Equilibrium nucleotide-binding parameters of wild-type mouse MDR3 Pgp were studied using 2'-(3')-O-(2,4,6-trinitrophenyl)adenosine tri- and diphosphate. Both analogs were found to bind with K(d) in the low micromolar range, to a single class of site, with no evidence of cooperativity. ATP displacement of the analogs was seen. Similar binding was seen with K429R/K1072R and D551N/D1196N mutant mouse MDR3 Pgp, showing that these Walker A and B mutations had no significant effect on affinity or stoichiometry of nucleotide binding. These residues, known to be critical for catalysis, are concluded to be involved primarily in stabilization of the catalytic transition state in Pgp.

Characterization of outer arm dynein in sea anemone, Anthopleura midori.

Outer arm dynein was purified from sperm flagella of a sea anemone, Anthopleura midori, and its biochemical and biophysical properties were characterized. The dynein, obtained at a 20S ATPase peak by sucrose density gradient centrifugation, consisted of two heavy chains, three intermediate chains, and seven light chains. The specific ATPase activity of dynein was 1.3 micromol Pi/mg/min. Four polypeptides (296, 296, 225, and 206 kDa) were formed by UV cleavage at 365 nm of dynein in the presence of vanadate and ATP. In addition, negatively stained images of dynein molecules and the hook-shaped image of the outer arm of the flagella indicated that sea anemone outer arm dynein is two-headed. In contrast to protist dyneins, which are three-headed, outer arm dyneins of flagella and cilia in multicellular animals are two-headed molecules corresponding to the two heavy chains. Phylogenetic considerations were made concerning the diversity of outer arm dyneins.

Acid Tolerance ofLactobacillus plantarumfromKimchi

Acid tolerance ofLactobacillus plantarumKFRI 815, isolated fromkimchi, was investigated in terms of Mg and K release, viable cell count, proton permeability, glycolysis, and membrane ATPase activity. The release of Mg from whole cells occurred significantly at pH values below about 3 and rapidly increased at a pH value of about 2. The injured cells significantly increased with time by losing their salt tolerance at a pH of below 4. The proton permeability was minimal at a pH value of about 4, at which the average half time for pH equilibration across the cell membrane was about 70 min. The pH profile for glycolysis of intact cells showed thatL. plantarumcompletely consumed glucose at the pH range of 4.0 to 6.0 and that glucose degradation was reduced by over 50% at pH 2.0. The maximal specific activity of membrane ATPase was about 3.6 units per mg of protein at a pH value close to 5.0. Overall, acid tolerance ofL. plantarumwas found to be closely related to the gross membrane damage and proton permeability of whole cells as well as the specific activity of membrane ATPase.

Osmoregulation and branchial Na +,K +-ATPase in the lobster Homarus gammarus acclimated to dilute seawater

Osmoregulatory ability and Na +,K +-ATPase activity in gills and epipodites were examined following transfer of the lobster Homarus gammarus from sea water (SW; 1292 mOsmol l −1; 38 ppt salinity) to dilute seawater (DSW; 680±5 mOsmol l −1; 20 ppt salinity). The lobster H. gammarus behaves as an osmoconformer in sea water and a poor hyperosmoregulator when equilibrated in dilute seawater, where haemolymph was maintained 154 mOsmol l −1 above that of the medium. Lobsters could withstand direct transfer from SW to DSW and, after about 12 h, the initial drop in blood osmoconcentration and principal inorganic osmolytes ceased and a gradual increase followed during the next 15 days. Chloride and sodium were the main osmotic effectors responsible for the slow gradual adjustment of the blood hyperosmoticity. In DSW-acclimated lobsters, the Na +,K +-ATPase specific activities of gills (arthrobranchia and podobranchia) homogenates and partially purified membrane vesicle fractions were, respectively, 2.0 and 3.5 to 3.9-fold higher that those of the SW-acclimated animals. Enzyme specific activities of homogenate and membrane vesicle fractions isolated from DSW pleurobranchia were 1.4-fold higher than those of the SW-acclimated animals. In trichobranchiate gills and epipodites, saponin treatment increased the Na +,K +-ATPase activity over the native enzyme activity of the respective tissues. The plasma membrane vesicles from trichobranchiate gills and epipodites were better purified when isolated from DSW- than from SW-acclimated lobsters. Homogenates and membrane vesicles (enrichment factors ranging from seven to eight) from epipodites allowed a several-fold increase in enzyme specific activity over trichobranchiate gills. When lobsters were acclimated to DSW, the Na +,K +-ATPase specific activity measured in saponin-treated homogenates of epipodites was positively correlated with the sodium concentration gradient between haemolymph and DSW. The results suggest that epipodites Na +,K +-ATPase activation in DSW should be correlated with the blood sodium gradient, i.e. osmoregulatory ability. Short-circuit current of the hemiepipodite isolated from lobsters acclimated to DSW and mounted in a micro-Ussing chamber was −232 μA cm −2 (negative charge flow driven from apical to basolateral side of preparation) and conductance was 65 mS cm −2, a value characteristic of a leaky epithelium. Half-maximal inhibition of current by the specific Na +,K +-ATPase inhibitor ouabain occurs at 0.78 mM. At 1 mM ouabain, more then 70% of the inhibited current is Na +,K +-ATPase-related ion transport in epipodites. These results provide, for the first time, physiological evidence that, besides trichobranchiate gills, epipodites have an osmoregulatory role in the lobster Homarus gammarus.

Isolation of Supernumerary Yeast ATP Synthase Subunits e and i: CHARACTERIZATION OF SUBUNIT i AND DISRUPTION OF ITS STRUCTURAL GENE ATP18

Two subunits of the yeast ATP synthase have been isolated. Subunit e was found loosely associated to the complex. Triton X-100 at a 1% concentration removed this subunit from the ATP synthase. The N-terminal sequencing of subunit i has been performed. The data are in agreement with the sequence of the predicted product of a DNA fragment of Saccharomyces cerevisiae chromosome XIII. The ATP18 gene encodes subunit i, which is 59 amino acids long and corresponds to a calculated mass of 6687 Da. Its pI is 9.73. It is an amphiphilic protein having a hydrophobic N-terminal part and a hydrophilic C-terminal part. It is not apparently related to any subunit described in other ATP synthases. The null mutant showed low growth on nonfermentable medium. Mutant mitochondria display a low ADP/O ratio and a decrease with time in proton pumping after ATP addition. Subunit i is associated with the complex; it is not a structural component of the enzyme but rather is involved in the oxidative phosphorylations. Similar amounts of ATP synthase were measured for wild-type and null mutant mitochondria. Because 2-fold less specific ATPase activity was measured for the null mutant than for the wild-type mitochondria, we make the hypothesis that the observed decrease in the turnover of the mutant enzyme could be linked to a proton translocation defect through F0.

Changes in physiological indices of smolting during seaward migration of wild coho salmon, Oncorhynchus kisutch

Wild juvenile coho salmon ( Oncorhynchus kisutch) were captured in traps or by seining during the course of parr–smolt transformation and migration from a small coastal stream in Oregon. Physiological measurements indicated that gill (Na+K)-ATPase specific activity was significantly greater in migrants than in non-migrants, but there was no significant difference in kidney (Na+K)-ATPase specific activity in the two populations. Glycogen concentrations and triglyceride concentrations were significantly lower in migrant populations than in non-migrants. Guanine concentrations tended to be lower in non-migrant fish than in migrant fish after May. There were no significant differences in plasma thyroxine or tri-iodothyronine concentrations between migrant and non-migrant fish. The decrease in energy storage compounds supports the hypothesis that migration is caused by an ionic imbalance during smolting, but the lack of changes in kidney (Na+K)-ATPase specific activity during smolting of wild fish does not support this hypothesis or the results from studies with laboratory reared coho salmon.

Effects of water-borne copper on branchial chloride cells and Na +/K +-ATPase activities in Mozambique tilapia ( Oreochromis mossambicus)

Freshwater tilapia ( Oreochromis mossambicus) were exposed for different periods up to 28 days to 3.2 μM of water-borne Cu. Electron microscopical analysis of the gills demonstrated significant changes in the structure and number of chloride cells (CCs) from Cu-exposed fish when compared to controls. These cells, which are the main location of the Na +/K +-ATPase of the gills and which play a crucial role in transepithlial Na + transport, showed a time-related increase of degeneration by apoptosis and necrosis in the Cu-exposed fish. After 28 days of Cu exposure, apoptotic CCs had doubled in number while necrotic CCs had even increased by a factor of ten. The activity of the gill Na +/K +-ATPase and the plasma Na + concentration decreased in time and in parallel. An inverse relationship between the Na +/K +-ATPase specific activity and the branchial Cu content further supports the notion that this enzyme is very sensitive to Cu 2+ inhibition. In contrast to controls, no significant correlation was found in the Cu-exposed fish between the opercular CC number and the gill Na +/K +-ATPase total activities, despite the large increase in number of these cells. This study provides further evidence that not only the number but also the quality of the CCs, may determine to a large extent the branchial capacity of a freshwater fish to absorb Na + from the surrounding water.

Orientation of NAHD-linked ferric chelate (turbo) reductase in plasma membranes from roots ofPlantago lanceolata

Plasma membrane vesicles isolated from roots ofPlantago lanceolata L. revealed approximately 70% right-side-out orientation based on structure-linked latency with H+-ATPase as a marker. Incubation with 0.05% Brij 58 caused the formation of sealed insideout vesicles, evidenced by assaying ATP-dependent proton pumping activity with the optical pH probe acridine orange. NADH-linked FeEDTA reductase activity was stimulated by including either Triton X-100 or Brij 58 in the assay medium. The activity of inverted (Brijtreated) vesicles was not further increased by the addition of Triton, suggesting that maximum activity was obtained in inside-out vesicles. Iron deficiency resulted in a ca. 2-fold increase in the specific activity of both ATPase and Fe(III) chelate reductase but did not cause significant alterations with respect to the effect of detergents. It is concluded that in vitro both donor and acceptor sites of NADH-FeEDTA reductase are located on the cytosolic face of the membrane and trans-oriented flow of electrons is not detectable in plasma membrane vesicles. Unlike Fe chelate reduction in vivo, the plasma membrane-bound reductase activity was insensitive towards application of the translation inhibitor cycloheximide prior to isolation of the membranes, implying the involvement of a regulatory enzyme in the electron transport in vivo.