The identification of nitrate reductase fromEscherichia coli as the antigen for a monoclonal antibody of unknown specificity

The immunoaffinity chromatography of total membrane proteins fromEscherichia coli helped determine the specificity of the monoclonal antibody 3A6 that was obtained upon immunization of mice with nicotinamide nucleotide transhydrogenase preparations and reacted with an unknownE. coli antigen. Proteins with apparent molecular masses of 150, 45, and 20 kDa were isolated and identified byN-terminal sequencing as the subunits of nitrate reductase. This conclusion was confirmed by immunoblotting with the 3A6 antibody of the proteins from theE. coli cells grown upon induction of nitrate reductase. It was shown that the 3A6 antibody specifically recognizes the α subunit of nitrate reductase, and the formation of the enzyme-antibody complex does not result in a loss of the enzyme catalytic activity.     

Characterisation of a monoclonal antibody against the fimbrial F8 antigen of Escherichia coli

Abstract A monoclonal IgG1 antibody against F8 fimbriae was obtained with the hybridoma technique using spleen cells from C3H/f mice immunised with a fimbrial preparation of Escherichia coli 2980 (O18ac:K5:H⁻:F1C, F8) and Sp 2/0 Ag8 myeloma cells. The hybrid cells were cloned twice by limiting dilution and grown in tissue culture. The monoclonal antibody was purified from culture supernatants on Protein A Sepharose. It reacted with F8 fimbriae in colony blot, enzyme-linked immunosorbent assay (ELISA) and immunoblot after electrotransfer from sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of fimbrial preparations. The antibody bound to and agglutinated F8-fimbriated bacteria.     

Solubilization of Escherichia coli nitrate reductase by a membrane-bound protease.

Nitrate reductase extracted from the membrane of Escherichia coli by alkaline heat treatment was purified to homogeneity and used to prepare specific antibody. Nitrate reductase, precipitated by this antibody from Triton extracts of the membrane, contained a third subunit not present in the purified enzyme used to prepare the antibody. Nitrate reductase precipitated by antibody from alkaline heat extracts was composed of peptide fragments of various sizes. These fragments were produced by a membrane-bound protease which was activated by alkaline pH and heat. It is the action of this protease that releases the enzyme from the membrane, as shown by the observations that protease inhibitors decreased the amount of solubilization of the enzyme, and the enzyme remaining in the membrane after heating showed much less proteolytic cleavage than that which was released.     

The iron-sulfur cluster composition of Escherichia coli nitrate reductase

Nitrate reductase from Escherichia coli has been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance (EPR) spectroscopies, as well as by Fe-S core extrusion, to determine the Fe-S cluster composition. The results indicate approximately one 3Fe and three or four [4Fe-4S]2+,1+ centers/molecule of isolated enzyme. The magnetic circular dichroism spectra and magnetization characteristics show the oxidized and reduced 3Fe and [4Fe-4S] centers to be electronically analogous to those in bacterial ferredoxins. The form and spin quantitation of the EPR spectra from [4Fe-4S]1+ centers in the reduced enzyme were found to vary with the conditions of reduction. For the fully reduced enzyme, the EPR spectrum accounted for between 2.9 and 3.5 spins/molecule, and comparison with partially reduced spectra indicates weak intercluster magnetic interactions between reduced paramagnetic centers. In common with other Fe-S proteins, the 3Fe center was not extruded intact under standard conditions. The results suggest that nitrate reductase is the first example of a metalloenzyme where enzymatic activity is associated with a form that contains an oxidized 3Fe center. However, experiments to determine whether or not the 3Fe center is present in vivo were inconclusive.     

Biochemical and genetic studies with nitrate reductase C-gene mutants of Escherichia coli

Summary Twenty-eight narC (chlC) mutants of Escherichia coli were isolated and characterised by their resistance to chlorate, inability to use nitrate as terminal electron acceptor and positive gas reaction. The extent of gas production by the majority of mutants was almost normal but quantitative differences ranging from 40 to 100% of wild-type activity were found. Biochemical studies showed that all the mutants lacked nitrate reductase, decreasing gas production was correlated with a simultaneous decrease in formate dehydrogenase activity and the lowest gas production was due to deficiencies in formate dehydrogenase and hydrogenase. The position of narC relative to other loci was determined as: purB ... hemA ... narC ... supIII,C ... galU ... att80 ... tonB ... trp ... cysB by transduction analysis, and the mutant sites of 6 strains representing the complete range of gas reactions were clustered at this position. It is suggested that narC is the structural gene for nitrate reductase and the variations in phenotype may be due to polarity effects on neighbouring genes specifying components of the formate hydrogenlyase system. Transduction of narC by 80 could not be detected but an effect of galU ^– on phage P1kc susceptibility was demonstrated.     

Synthesis and degradation of nitrate reductase in Escherichia coli.

The biosynthesis, insertion, and in vivo stability of nitrate reductase were examined by following the amount of labeled enzyme present in both membranes and cytoplasm at varying times after a short pulse of radioactive sulfate. Nitrate reductase levels were measured by autoradiography of immunoprecipitated material after fractionation on sodium dodecyl sulfate-polyacrylamide gels. These experiments demonstrated that subunits A and B were synthesized in the cytoplasm and subsequently inserted into membranes. The insertion of these subunits was dependent upon the synthesis of another protein, and the rate of synthesis of this protein determined the rate of insertion of subunits A and B. The nitrate reductase produced by the chlA mutant was inserted into membranes in the normal fashion, whereas the nitrate reductase produced by the chlC and chlE mutants was poorly incorporated. The nitrate reductase in the wild type was completely stable in vivo under inducing or noninducing conditions, whereas in the chlC and chlE mutants nitrate reductase was degraded extensively in both the cytoplasm and membranes, even under inducing conditions. Under similar conditions, nitrate reductase was stable in the chlA mutant.     

Nitrous oxide production by Escherichia coli is correlated with nitrate reductase activity

Nitrous oxide production by Escherichia coli seems to result from the reduction of NO2- by NO3- reductase. This hypothesis is consistent with previous observations and with the observation that molybdenum was required for both NO3- reduction and N2O production. Several E. coli NO3- reductase mutants were assayed for both N2O-producing and NO3--reducing activity. The hypothesized role of NO3- reductase is supported by the correlation of these two activities. Nitrate reduction to NH4+ enhanced growth, but NO2- reduction to N2O apparently did not. Therefore, this process differs significantly from respiratory denitrification.     

Identification of antigen in rat spermatogenic cells interacting with an anti-human sperm monoclonal antibody

A monoclonal antibody (MAb) raised against human sperm protein, designated YWK-II, was used to determine the distribution of antigens in rat spermatozoa and rat testicular germ cells. By an indirect immunofluorescent method, the antibody localized over the rat spermatozoal head, except for the postacrosomal region. In paraffin sections of adult and immature rat testis, germ cells, at every developmental stage, and Sertoli cells stained, while interstitial cells and peritubular myoid cells remained unstained. When cocultures of Sertoli and germ cells were tested, only the germ cells stained intensely. Sertoli cells and peritubular myoid cells in cultures did not stain. In the epididymal sections, strong staining occurred with spermatozoa in the lumen and epididymal epithelial cells, with moderate staining in the myoid layers of epididymis. To determine the sperm antigen interacting with the YWK-II antibody, rat spermatozoa proteins were prepared and analyzed by an immunoblot technique. The monoclonal antibody interacted with a single protein, with an estimated molecular weight of 115,000, present in the cauda epididymal spermatozoa. Among the proteins of the caput epididymal spermatozoa, however, the antibody interacted with a major and a minor band with molecular weights of 115,000 and 88,000, respectively. On the other hand, with proteins prepared from the membrane fraction of adult and immature rat testis, the antibody reacted with two bands with estimated molecular weights of 88,000 and 115,000. In the lysate prepared from germ cells dissociated from Sertoli-germ cell cocultures, the antibody recognized only the 88,000 protein. The present results show that the YWK-II MAb interacts with two proteins with different molecular weights. The amount of the interacting proteins in spermatozoa varied with their location within the epididymis.     

Enzymic properties of a mutant of Escherichia coli K 12 lacking nitrate reductase

Summary A pleiotropic mutant of Escherichia coli K ₁₂ lacking reduced NAD: nitrate oxidoreductase, soluble formate dehydrogenase and membrane-bound formate:ferricytochrome b₁ oxidoreductase is described. Levels of several other enzymes and cytochromes have been measured and found to differ little from those normally present in the wild type with the exceptions of cytochrome c₅₂₂, reduced NAD:cytochrome c oxidoreductase and reduced NAD:nitrite oxidoreductase which are very high. Although the affected gene maps in a different position from that reported for chl A by other workers it seems likely that the two loci are identical.     

Escherichia coli mutants with altered control of alcohol dehydrogenase and nitrate reductase.

Mutants of Escherichia coli which overproduce alcohol dehydrogenase were obtained by selection for the ability to use ethanol as an acetate source in a strain auxotrophic for acetate. A mutant having a 20-fold overproduction of alcohol dehydrogenase was able to use ethanol only to fulfill its acetate requirement, whereas two mutants with a 60-fold overproduction were able to use ethanol as a sole carbon source. The latter two mutants produced only 25% of the wild-type level of nitrate reductase, when grown under anaerobic conditions. Alcohol dehydrogenase production was largely unaffected by catabolite repression but was repressed by nitrate under both aerobic and anaerobic conditions. The genetic locus responsible for alcohol dehydrogenase overproduction was located at min 27 on the E. coli genetic map; the gene order, as determined by transduction, was trp tonB adh chlC hemA. The possible relationship of alcohol dehydrogenase to anaerobic redox systems such as formate-nitrate reductase is discussed.     

Biosynthesis of membrane-bound nitrate reductase in Escherichia coli: evidence for a soluble precursor.

Membrane-bound nitrate reductase of Escherichia coli consists of three subunits designated as A, B, and C, with subunit C being the apoprotein of cytochrome b, A hemA mutant that cannot synthesize delta-aminolevulinic acid (ALA) produces a normal, stable, membrane-bound enzyme when grown with ALA. When grown without ALA, this mutant makes a reduced amount of membrane-bound enzyme that is unstable and contains no C subunit. Under the same growth conditions, this mutant accumulates a large amount of a soluble form of the enzyme in the cytoplasm. Accumulation of this cytoplasmic form begins immediately upon induction of the enzyme with nitrate. The cytoplasmic form is very similar to the soluble form of the enzyme obtained by alkaline heat extraction. It is a high-molecular-weight complex with a Strokes radius of 8.0 nm and consists of intact A and B subunits. When ALA is added to a culture growing without ALA, the cytoplasmic form of the enzyme is incorporated into the membrane in a stable form, coincident with the formation of functional cytochrome b. Reconstitution experiments indicate that subunit C is present in cultures grown without ALA but is reduced in amount or unstable. These results indicate that membrane-bound nitrate reductase is synthesized via a soluble precursor containing subunits A and B, which then binds to the membrane upon interaction with the third subunit, cytochrome b.     

Molybdenum effector of fumarate reductase repression and nitrate reductase induction in Escherichia coli.

In Escherichia coli the presence of nitrate prevents the utilization of fumarate as an anaerobic electron acceptor. The induction of the narC operon encoding the nitrate reductase is coupled to the repression of the frd operon encoding the fumarate reductase. This coupling is mediated by nitrate as an effector and the narL product as the regulatory protein (S. Iuchi and E. C. C. Lin, Proc. Natl. Acad. Sci. USA 84:3901-3905, 1987). The protein-ligand complex appears to control narC positively but frd negatively. In the present study we found that a molybdenum coeffector acted synergistically with nitrate in the regulation of frd and narC. In chlD mutants believed to be impaired in molybdate transport (or processing), full repression of phi(frd-lac) and full induction of phi(narC-lac) by nitrate did not occur unless the growth medium was directly supplemented with molybdate (1 microM). This requirement was not clearly manifested in wild-type cells, apparently because it was met by the trace quantities of molybdate present as a contaminant in the mineral medium. In chlB mutants, which are known to accumulate the Mo cofactor because of its failure to be inserted as a prosthetic group into proteins such as nitrate reductase, nitrate repression of frd and induction of narC were also intensified by molybdate supplementation. In this case a deficiency of the molybdenum coeffector might have resulted from enhanced feedback inhibition of molybdate transport (or processing) by the elevated level of the unutilized Mo cofactor. In addition, mutations in chlE, which are known to block the synthesis of the organic moiety of the Mo cofactor, lowered the threshold concentration of nitrate (< 1 micromole) necessary for frd repression and narC induction. These changes could be explained simply by the higher intracellular nitrate attainable in cells lacking the ability to destroy the effector.     

Autoregulation of the nar operon encoding nitrate reductase in Escherichia coli

Summary Nitrate reductase is demonstrated to exert an autogenous control on its own synthesis. This effect requires the participation of the molybdenum cofactor. Use of strains in which the control region of the nar operon is mutated reveals two loci in this region: one, affected in strain LCB94, is common to both autoregulation and induction by nitrate while the other, mutated in strain LCB188, is specific for the induction by nitrate. It is proposed that the autogenous control prevents the unnecessary accumulation of the nitrate reductase subunits in the cytoplasm.     

Kinetic analysis of respiratory nitrate reductase from Escherichia coli K12

Purified respiratory nitrate reductase from Escherichia coli is able to use either reduced viologen dyes or quinols as the electron donor and nitrate, chlorate, or bromate as the electron acceptor. When reduced viologen dyes act as the electron donor, the enzyme follows a compulsory-order, "Theorell-Chance" mechanism, in which it is an enzyme-nitrate complex that is reduced rather than the free enzyme. In contrast, if quinols are used as the electron donor, then the enzyme operates by a two-site, enzyme-substitution mechanism. Partial proteolysis of the cytochrome b containing holoenzyme by trypsin results in loss of cytochrome b and in cleavage of one of the enzyme's subunits. The cytochrome-free derivative exhibits a viologen dye dependent activity that is indistinguishable from that of the holoenzyme, but it is incapable of catalyzing the quinol-dependent reaction. The quinol-dependent, but not the viologen dye dependent, activity is inhibited irreversibly by exposure to diethyl pyrocarbonate and reversibly by treatment with 2-n-heptyl-4-hydroxyquinoline N-oxide. We conclude that the holoenzyme has two independent and spatially distinct active sites, one for quinol oxidation and the other for nitrate reduction.     

Proton translocation and the respiratory nitrate reductase of Escherichia coli.

Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.     

Nitrous oxide production by Escherichia coli is correlated with nitrate reductase activity.

Nitrous oxide production by Escherichia coli seems to result from the reduction of NO2- by NO3- reductase. This hypothesis is consistent with previous observations and with the observation that molybdenum was required for both NO3- reduction and N2O production. Several E. coli NO3- reductase mutants were assayed for both N2O-producing and NO3--reducing activity. The hypothesized role of NO3- reductase is supported by the correlation of these two activities. Nitrate reduction to NH4+ enhanced growth, but NO2- reduction to N2O apparently did not. Therefore, this process differs significantly from respiratory denitrification.