Activation in vitro of respiratory nitrate reductase of Escherichia coli K12 grown in the presence of tungstate. Involvement of molybdenum cofactor

The chlorate-resistant (chlR) mutants are pleiotropically defective in molybdoenzyme activity. The inactive derivative of the molybdoenzyme, respiratory nitrate reductase, present in the cell-free extract of a chlB mutant, can be activated by the addition of protein FA, the probable active product of the chlB locus. Protein FA addition, however, cannot bring about the activation if 10 mM sodium tungstate is included in the culture medium for the chlB strain. The inclusion of a heat-treated preparation of a wild-type or chlB strain prepared after growth in the absence of tungstate, restores the protein-FA-dependent activation of nitrate reductase. All attempts to activate nitrate reductase in extracts prepared from tungstate-grown wild-type Escherichia coli strains failed. It appears that during growth with tungstate, the possession of the active chlB gene product leads to the synthesis of a nitrate reductase derivative which is distinct from that present in the tungstate-grown chlB mutant. Heat-treated preparations from chlA and chlE mutants which do not possess molybdenum cofactor activity fail to restore the activation. Fractionation by gel filtration of the heat-treated preparation from a wild-type strain produced two active peaks in the eluate of approximate Mr 12000 and less than or equal to 1500. The active material in the heat-treated extract was resistant to exposure to proteinases, but after such treatment the active component, previously of approximate Mr 12000, eluted from the gel filtration column with the material of Mr less than or equal to 1500. The active material is therefore of low molecular mass and can exist either in a protein-bound form or in an apparently free state. Molybdenum cofactor activity, assayed by the complementation of the apoprotein of NADPH:nitrate oxidoreductase in an extract of the nit-1 mutant of Neurospora crassa, gave a profile following gel filtration similar to that of the ability to restore respiratory nitrate reductase activity to the tungstate-grown chlB mutant soluble fraction. This was the case even after proteinase treatment of the heat-stable fraction. Analysis of the chlC (narC) mutant, defective in the structural gene for nitrate reductase, revealed that heat treatment is not necessary for the expression of the active component. Furthermore both the active component and molybdenum cofactor activity are present in corresponding bound and free fractions in the non-heat-treated soluble subcellular fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12

We report some properties of Protein PA which has been isolated from the soluble fraction of a chlB mutant after anaerobic growth in the presence of KNO3. This protein has been identified by its capacity to reactivate nitrate reductase present in the soluble fraction of a chlA mutant by the complementation process. The presence of active Protein PA in the chlB mutant is independent of the presence of oxygen or of nitrate during growth. In contrast, the addition of sodium tungstate to the growth medium leads to the formation of inactive Protein PA which is not able to activate nitrate reductase in the chlA-soluble extract by complementation. Inactive Protein PA has been quantitated immunologically. The partial purification of Protein PA has been achieved from various chlorate-resistant mutants (chlA-chlG). The establishment of particular complementation systems comprising the soluble extracts of chlA or chlB mutants and partially purified Protein PA from soluble fractions of different chlorate-resistant mutants, has allowed the quantitative estimation of this protein. The analysis by 'rocket immunoelectrophoresis' using an antiserum specific for Protein PA has shown that inactive Protein PA is present in approximately equivalent amounts in the chlA, chlE, chlG and chlD mutants.     

Molybdoenzyme biosynthesis in Escherichia coli: in vitro activation of purified nitrate reductase from a chlB mutant.

All molybdoenzyme activities are absent in chlB mutants because of their inability to synthesize molybdopterin guanine dinucleotide, which together with molybdate constitutes the molybdenum cofactor in Escherichia coli. The chlB mutants are able to synthesize molybdopterin. We have previously shown that the inactive nitrate reductase present in a chlB mutant can be activated in a process requiring protein FA and a heat-stable low-molecular-weight substance. We show here that purified nitrate reductase from the soluble fraction of a chlB mutant can be partially activated in a process that requires protein FA, GTP, and an additional protein termed factor X. It appears that the molybdopterin present in the nitrate reductase of a chlB mutant is converted to molybdopterin guanine dinucleotide during activation. The activation is absolutely dependent upon both protein FA and factor X. Factor X activity is present in chlA, chlB, chlE, and chlG mutants.     

Analysis of purity of Clostridium difficile toxin A derived by affinity chromatography on immobilized bovine thyroglobulin

Abstract The chlorate resistance mutants are pleitropically defective in the activity of all molybdoenzymes in Escherichia coli . Protein FA addition to the soluble fraction of a chlB mutant, brings about the activation of the molybdoenzyme, respiratory nitrate reductase, an inactive precursor of which is present in the chlB fraction. The rate of the activation process but not its extent is dependent upon the quantity of protein FA present. Protein FA activity is constitutively expressed and was present in normal amounts in chlA, D, E, F and G mutants but was absent from all chlB strains examined. This is consistent with protein FA being the active product of the chlB locus. Sodium tungstate (10 mM) in the growth medium has no effect on protein FA activity. Protein FA does not function as a source of molybdenum cofactor activity in the activation process.     

Identification of the molybdenum cofactor in chlorate-resistant mutants of Escherichia coli.

Experiments were performed to determine whether defects in molybdenum cofactor metabolism were responsible for the pleiotropic loss of the molybdoenzymes nitrate reductase and formate dehydrogenase in chl mutants of Escherichia coli. In wild-type E. coli, molybdenum cofactor activity was present in both the soluble and membrane-associated fractions when the cells were grown either aerobically or anaerobically, with and without nitrate. Molybdenum cofactor in the soluble fraction decreased when the membrane-bound nitrate reductase and formate dehydrogenase were induced. In the chl mutants, molybdenum cofactor activity was found in the soluble fraction of chlA, chlB, chlC, chlD, chlE, and chlG, but only chlB, chlC, chlD, and chlG expressed cofactor activity in the membrane fraction. The defect in the chlA mutants which prevented incorporation of the soluble cofactor into the membrane also caused the soluble cofactor to be defective in its ability to bind molybdenum. This cofactor was not active in the absence of molybdate, and it required at least threefold more molybdate than did the wild type in the Neurospora crassa nit-1 complementation assay. However, the cofactor from the chlA strain mediated the dimerization of the nit-1 subunits in the presence and absence of molybdate to yield the 7.9S dimer. Growth of chlA mutants in medium with increased molybdate did not repair the defect in the chlA cofactor nor restore the molybdoenzyme activities. Thus, molybdenum cofactor was synthesized in all the chl mutants, but additional processing steps may be missing in chlA and chlE mutants for proper insertion of cofactor in the membrane.     

Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12

We report some properties of Protein PA which has been isolated from the soluble fraction of a chlB mutant after anaerobic growth in the presence of KNO₃. This protein has been identified by its capacity to reactivate nitrate reductase present in the soluble fraction of a chlA mutant by the complementation process. The presence of active Protein PA in the chlB mutant is independent of the presence of oxygen or of nitrate during growth. In contrast, the addition of sodium tungstate to the growth medium leads to the formation of inactive Protein PA which is not able to activate nitrate reductase in the chlA-soluble extract by complementation. Inactive Protein PA has been quantitated immunologically. The partial purification of Protein PA has been achieved from various chlorate-resistant mutants (chlA?chlG). The establishment of particular complementation systems comprising the soluble extracts of chlA or chlB mutants and partially purified Protein PA from soluble fractions of different chlorate-resistant mutants, has allowed the quantitative estimation of this protein. The analysis by ‘rocket immunoelectrophoresis’ using an antiserum specific for Protein PA has shown that inactive Protein PA is present in approximately equivalent amounts in the chlA, chlE, chlG and chlD mutants     

Involvement of a low-molecular-weight substance in in vitro activation of the molybdoenzyme respiratory nitrate reductase from a chlB mutant of Escherichia coli.

The soluble subcellular fraction of a chlB mutant contains an inactive precursor form of the molybdoenzyme nitrate reductase, which can be activated by the addition to the soluble fraction of protein FA, which is thought to be the active product of the chlB locus. Dialysis or desalting of the chlB soluble fraction leads to the loss of nitrate reductase activation, indicating that some low-molecular-weight material is required for the activation. The protein FA-dependent activation of nitrate reductase can be restored to the desalted chlB soluble fraction by the addition of a clarified extract obtained after heating the chlB soluble fraction at 100 degrees C for 8 min. The heat-stable substance present in this preparation has a molecular weight of approximately 1,000. This substance is distinct from the active molybdenum cofactor since its activity is unimpaired in heat-treated extracts prepared from the organism grown in the presence of tungstate, which leads to loss of cofactor activity. Mutations at the chlA or chlE locus, which are required for molybdenum cofactor biosynthesis, similarly do not affect the activity of the heat-treated extract in the in vitro activation process. Moreover, the active material can be separated from the molybdenum cofactor activity by gel filtration. None of the other known pleiotropic chlorate resistance loci (chlD, chlG) are required for the expression of its activity. Magnesium ATP appears to have a role in the formation of the active substance. We conclude that a low-molecular-weight substance, distinct from the active molybdenum cofactor, is required to bestow activity on the molybdoenzyme nitrate reductase during its biosynthesis.     

Molybdenum cofactor requirement for in vitro activation of apo-molybdoenzymes of Escherichia coli

The apo-nitrate reductase precursor in an Escherichia coli chlB mutant preparation obtained following growth in the presence of tungstate is activated by incubation with protein FA and a heat-treated preparation from an E. coli crude extract. We show that the requirement for heat-treated E. coli crude extract can be fulfilled by material obtained from either of two heat-denatured purified E. coli molybdoenzymes, namely nitrate reductase or trimethylamine N-oxide reductase. Apo-trimethylamine N-oxide reductase precursor in the tungstate-grown chlB preparation can be activated in a similar manner with material from either heat-denatured molybdoenzyme. The active component in the denatured molybdoenzyme preparations is shown to be the molybdenum cofactor by Neurospora crassa nit1 molybdenum cofactor assay, size estimation and fluorimetric analysis. The direct demonstration of the requirement for molybdenum cofactor in the E. coli tungstate-grown chlB complementation system is an important step towards the molecular definition of the activation process and an understanding of the mechanism of cofactor acquisition during molybdoenzyme biosynthesis.     

Genetic and biochemical evidence for the involvement of a molybdenum-dependent enzyme in one of the selenite reduction pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106

Selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans was observed under photosynthetic conditions, following a 100-h lag period. This adaptation period was suppressed if the medium was inoculated with a culture previously grown in the presence of selenite, suggesting that selenite reduction involves an inducible enzymatic pathway. A transposon library was screened to isolate mutants affected in selenite reduction. Of the eight mutants isolated, two were affected in molybdenum cofactor synthesis. These moaA and mogA mutants showed an increased duration of the lag phase and a decreased rate of selenite reduction. When grown in the presence of tungstate, a well-known molybdenum-dependent enzyme (molybdoenzyme) inhibitor, the wild-type strain displayed the same phenotype. The addition of tungstate in the medium or the inactivation of the molybdocofactor synthesis induced a decrease of 40% in the rate of selenite reduction. These results suggest that several pathways are involved and that one of them involves a molybdoenzyme. Although addition of nitrate or dimethyl sulfoxide (DMSO) to the medium increased the selenite reduction activity of the culture, neither the periplasmic nitrate reductase NAP nor the DMSO reductase is the implicated molybdoenzyme, since the napA and dmsA mutants, with expression of nitrate reductase and DMSO reductase, respectively, eliminated, were not affected by selenite reduction. A role for the biotine sulfoxide reductase, another characterized molybdoenzyme, is unlikely, since its overexpression in a defective strain did not restore the selenite reduction activity.     

A common pathway for the activation of several molybdoenzymes in Escherichia coli K12

Three molybdoenzymes, nitrate reductase, formate benzyl-viologen oxidoreductase and trimethylamine-N-oxide reductase which form part of different systems, have been studied in a parental strain of Escherichia coli K12. When the organism is grown in the presence of 10 mM tungstate, these three enzymes are present in an inactive form which may be activated in vivo by the addition of 1 mM sodium molybdate. The mixing of soluble fractions from chlA and chlB mutants grown under the appropriate conditions leads to the activation of nitrate reductase, formate benzyl-viologen oxidoreductase and trimethylamine-N-oxide reductase. The activation of each enzyme is maximal when the mutants are grown under conditions that lead to the induction of that enzyme in the wild-type strain. The employment of purified proteins, the association factor FA and the Protein PA, which are presumed to be the products of the chlA and chlB genes, has shown that these proteins are responsible for the activation of the three enzymes during the complementation process.     

Nitrate reductase-deficient mutants in barley : immunoelectrophoretic characterization

Nitrate reductase-deficient barley (Hordeum vulgare L.) mutants were assayed for the presence of a functional molybdenum cofactor determined from the activity of the molybdoenzyme, xanthine dehydrogenase, and for nitrate reductase-associated activities. Rocket immunoelectrophoresis was used to detect nitrate reductase cross-reacting material in the mutants. The cross-reacting material levels of the mutants ranged from 8 to 136% of the wild type and were correlated with their nitrate reductase-associated activities, except for nar 1c, which lacked all associated nitrate reductase activities but had 38% of the wild-type cross-reacting material. The cross-reacting material of two nar 1 mutants, as well as nar 2a, Xno 18, Xno 19, and Xno 29, exhibited rocket immunoprecipitates that were similar to the wild-type enzyme indicating structural homology between the mutant and wild-type nitrate reductase proteins. The cross-reacting materials of the seven remaining nar 1 alleles formed rockets only in the presence of purified wild-type nitrate reductase, suggesting structural modifications of the mutant cross-reacting materials. All nar 1 alleles and Xno 29 had xanthine dehydrogenase activity indicating the presence of functional molybdenum cofactors. These results suggest that nar 1 is the structural gene for nitrate reductase. Mutants nar 2a, Xno 18, and Xno 19 lacked xanthine dehydrogenase activity and are considered to be molybdenum cofactor deficient mutants. Cross-reacting material was not detected in uninduced wild-type or mutant extracts, suggesting that nitrate reductase is synthesized de novo in response to nitrate.     

Genetic and Biochemical Evidence for the Involvement of a Molybdenum-Dependent Enzyme in One of the Selenite Reduction Pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106

Selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans was observed under photosynthetic conditions, following a 100-h lag period. This adaptation period was suppressed if the medium was inoculated with a culture previously grown in the presence of selenite, suggesting that selenite reduction involves an inducible enzymatic pathway. A transposon library was screened to isolate mutants affected in selenite reduction. Of the eight mutants isolated, two were affected in molybdenum cofactor synthesis. These moaA and mogA mutants showed an increased duration of the lag phase and a decreased rate of selenite reduction. When grown in the presence of tungstate, a well-known molybdenum-dependent enzyme (molybdoenzyme) inhibitor, the wild-type strain displayed the same phenotype. The addition of tungstate in the medium or the inactivation of the molybdocofactor synthesis induced a decrease of 40% in the rate of selenite reduction. These results suggest that several pathways are involved and that one of them involves a molybdoenzyme. Although addition of nitrate or dimethyl sulfoxide (DMSO) to the medium increased the selenite reduction activity of the culture, neither the periplasmic nitrate reductase NAP nor the DMSO reductase is the implicated molybdoenzyme, since the napA and dmsA mutants, with expression of nitrate reductase and DMSO reductase, respectively, eliminated, were not affected by selenite reduction. A role for the biotine sulfoxide reductase, another characterized molybdoenzyme, is unlikely, since its overexpression in a defective strain did not restore the selenite reduction activity.     

Characterisation of the mob locus of Rhodobacter sphaeroides WS8: mobA is the only gene required for molybdopterin guanine dinucleotide synthesis

The mob genes of several bacteria have been implicated in the conversion of molybdopterin to molybdopterin guanine dinucleotide. The mob locus of Rhodobacter sphaeroides WS8 comprises three genes, mobABC. Chromosomal in-frame deletions in each of the mob genes have been constructed. The mobA mutant strain has inactive DMSO reductase and periplasmic nitrate reductase activities (both molybdopterin guanine dinucleotide-requiring enzymes), but the activity of xanthine dehydrogenase, a molybdopterin enzyme, is unaffected. The inability of a mobA mutant to synthesise molybdopterin guanine dinucleotide is confirmed by analysis of cell extracts of the mobA strain for molybdenum cofactor forms following iodine oxidation. Mutations in mobB and mobC are not impaired for molybdoenzyme activities and accumulate wild-type levels of molybdopterin and molybdopterin guanine dinucleotide, indicating they are not compromised in molybdenum cofactor synthesis. In the mobA mutant strain, the inactive DMSO reductase is found in the periplasm, suggesting that molybdenum cofactor insertion is not necessarily a pre-requisite for export.     

Molybdenum cofactor: a compound in the in vitro activation of both nitrate reductase and trimethylamine-N-oxide reductase activities in Escherichia coli K12

Nitrate reductase (nitrite: (acceptor) oxidoreductase, EC 1.7.99.4) and trimethylamine N-oxide reductase (NADH : trimethylamine-N-oxide oxidoreductase, EC 1.6.6.9) activities were reconstituted by incubation of the association factor FA (the putative product of the chlB gene) with the soluble extract of the chlB mutant grown anaerobically in the presence of trimethylamine N-oxide. When soluble extracts of the chlB mutant grown on 10 mM sodium tungstate, a molybdenum competitor, were used in complementation systems, no enzymatic reactivation was observed. Heated extracts of the parental strain 541 were shown to contain a thermoresistant molybdenum cofactor by their ability to reactivate NADPH-nitrate reductase activity in the nit1 mutant of Neurospora crassa. By complementation of parental strain heated extract with association factor FA and soluble extract of the chlB mutant grown in the presence of sodium tungstate, we were able to show for the first time that the molybdenum cofactor is an activator common to the in vitro reconstitution of both nitrate reductase and trimethylamine-N-oxide reductase activities.     

Presence of the molybdenum-cofactor in nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

Summary Nicotiana tabacum mutant cell cultures lacking nitrate reductase activity were assayed for the presence of the molybdenum-cofactor using its ability to restore NADPH-nitrate reductase activity in extracts of Neurospora crassa nit-1 mycelia. The molybdenum-cofactor of the tobacco wild-type line was shown to complement efficiently the N. crassa nit-1 mutant in vitro. The molybdenum-cofactor seems to exist in a bound form, as acid-treatment was required for release of cofactor activity. Molybdate (5–10 mM), ascorbic acid, and anaerobic conditions greatly increased the activity of the cofactor, demonstrating its high lability and sensitivity to oxygen. Similar results were obtained with two tobacco nia mutants, which are defective in the apoprotein of nitrate reductase. The four cnx mutants studied were shown to contain exclusively an inactive form of the molybdenum-cofactor. This inactive cofactor could be reactivated in vitro and in vivo by unphysiologically high concentrations of molybdate (1–10 mM), thereby converting the cnx cells into highly active cofactor sources in vitro, and restoring nitrate reductase and xanthine dehydrogenase in vivo to partial acitivity. Thus the defect of the cnx mutants resides in a lack of molybdenum as a catalytically active ligand metal for the cofactor, while the structural moiety of the cofactor seems not to be impaired by the mutation. The subunit assembly of the nitrate reductase was found to be independent of the molybdenum content of the cofactor.     

Biochemical genetics of further chlorate resistant, molybdenum cofactor defective, conditional-lethal mutants of barley

Summary Three plants, R9201 and R11301 (from cv. Maris Mink) and R12202 (from cv. Golden Promise), were selected by screening M₂ populations of barley (Hordeum vulgare L.) seedlings (mutagenised with azide in the M₁) for resistance to 10 mM potassium chlorate. Selections R9201 and R11301 were crossed with the wild-type cv. Maris Mink and analysis of the F₂ progeny showed that one quarter lacked shoot nitrate reductase activity. These F₂ plants also withered and died in the continuous presence of nitrate as sole nitrogen source. Loss of nitrate reductase activity and withering and death were due in each case to a recessive mutation in a single nuclear gene. All F1 progeny derived from selfing selection R12202 lacked shoot nitrate reductase activity and also withered and subsequently died when maintained in the continuous presence of nitrate as sole nitrogen source. All homozygous mutant plants lacked not only shoot nitrate reductase activity but also shoot xanthine dehydrogenase activity. The plants took up nitrate, and possessed wild-type or higher levels of shoot nitrite reductase activity and NADH-cytochrome c reductase activity when treated with nitrate for 18 h. We conclude that loss of shoot nitrate reductase activity, xanthine dehydrogenase activity and withering and death, in the three mutants R9201, R11301 and R12202 is due to a mutation affecting the formation of a functional molybdenum cofactor. The mutants possessed wild-type levels of molybdenum and growth in the presence of unphysiologically high levels of molybdate did not restore shoot nitrate reductase or xanthine dehydrogenase activity. The shoot molybdenum cofactor of R9201 and of R12202 is unable to reconstitute NADPH nitrate reductase activity from extracts of the Neurospora crassa nit-1 mutant and dimerise the nitrate reductase subunits present in the respective barley mutant. The shoot molybdenum cofactor of R11301 is able to effect dimerisation of the R11301 nitrate reductase subunits and can reconstitute NADPH-nitrate reductase activity up to 40% of the wild-type molybdenum cofactor levels. The molybdenum cofactor of the roots of R9201 and R11301 is also defective. Genetic analysis demonstrated that R9201, but not R11301, is allelic to R9401 and Az34 (nar-2a), two mutants previously shown to be defective in synthesis of molybdenum cofactor. The mutations in R9401 and R9201 gave partial complementation of the nar-2a gene such that heterozygotes had higher levels of extractable nitrate reductase activity than the homozygous mutants.We conclude that: (a) the nar-2 gene locus encodes a step in molybdopterin biosynthesis; (b) the mutant R11301 represents a further locus involved in the synthesis of a functional molybdenum cofactor; (c) mutant Rl2202 is also defective in molybdopterin biosynthesis; and (d) the nar-2 gene locus and the gene locus defined by R11301 govern molybdenum cofactor biosynthesis in both shoot and root.     

Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase

An assay method is described for measurement of absolute concentrations of the molybdenum cofactor, based on complementation of the defective nitrate reductase ('apo nitrate reductase') in extracts of the nit-1 mutant of Neurospora crassa. A number of alternative methods are described for preparing, anaerobically, molybdenum-cofactor-containing solutions from sulphite oxidase, xanthine oxidase and desulpho xanthine oxidase. For assay, these were mixed with an excess of extract of the nit-1 mutant, incubated for 24 h at 3.5 degrees C then assayed for NADPH:nitrate reductase activity. In all cases, the specific activity of the molybdenum cofactor, expressed as mumol of NO2-formed/min per ng-atom of Mo added from the denatured molybdoenzyme , was 25 +/- 4, a value that agrees with the known catalytic activity of the nitrate reductase of wild-type Neurospora crassa. This indicates that, under our conditions, there was quantitative transfer of the molybdenum cofactor from denatured molybdoenzyme to yield fully active nitrate reductase. Comparable cofactor assay methods of previous workers, apparently indicating transfer efficiencies of at best a few per cent, have never excluded satisfactorily the possibility that cofactor activity arose, not from stoichiometric constituents of the molybdoenzymes , but from contaminants. The following factors were investigated separately in developing the assay:the efficiency of extraction of the cofactor from the original enzyme, the efficiency of the complementation reaction between cofactor and apo nitrate reductase, and the assay of the resultant nitrate reductase, which must be carried out under non-inhibitory conditions. Though the cofactor is unstable in air (t1/2 about 15 min at 3.5 degrees C), it is stable when kept anaerobic in the presence of sodium dithionite, in aqueous solution or in dimethyl sulphoxide (activity lost at the rate of about 3%/24 h at 20-25 degrees C). Studies of stabilities, and investigations of the effect of added molybdate on the assay, permit conclusions to be drawn about the ligation of molybdenum to the cofactor and about steps in incorporation of the cofactor into the apoenzyme. Though the development of nitrate reductase activity is slow at 3.5 degrees C (t1/2 1.5-3 h) the complementation reaction may be carried out in high yield, aerobically. This is ascribed to rapid formation of an air-stable but catalytically inactive complex of the cofactor, as a precursor of the active nitrate reductase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification and purification of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K12

The isolation and purification of a protein which is the presumed product of the chlA gene has been achieved. This protein, which we have named Protein PA, has been isolated from the soluble fraction of a chlB mutant. The protein was identified by its ability to activate nitrate reductase (EC.1.7.99.4) when mixed with a soluble fraction derived from a chlA mutant. The protein has a molecular weight of about 72 000 and is composed of a single polypeptide chain. Antiserum specific for Protein PA has been produced. Removal of Protein PA from the soluble fraction of chlB mutant by immunoprecipitation with this antiserum leads to the loss of the ability of the preparation to activate nitrate reductase when mixed with a soluble fraction from a chlA mutant. Protein PA, therefore, performs an essential but as yet undefined role in the activation process. Employing this antiserum Protein PA could be quantified by rocket immunoelectrophoretic analysis. The activity of the isolated Protein PA is low, since comparatively large amounts of Protein PA are required to activate the nitrate reductase present in the soluble fraction of the chlA mutant. The mixing of Protein PA with the chlA mutant soluble fraction leads to activation of nitrate reductase in both a soluble and a membranous form, as is the case when the complete soluble fraction of the chlB mutant is used in place of Protein PA. After activation, however, only a small proportion (15%) of the Protein PA is associated with the newly formed membranous material.     

Biochemical characterization of a singular mutant of nitrate reductase from Chlamydomonas reinhardii. New evidence for a heteropolymeric enzyme structure

A singular mutant strain from Chlamydomohas reinhardii defective in nitrate reductase has been characterized. Mutant 301 possesses an ammonia-repressible NAD(P)H-cytochrome c reductase with the same charge and size properties as the low molecular weight ammonia-repressible diaphorase present in the wild-type strain 6145c and is also able to reconstitute NAD(P)H-nitrate reductase activity by in vitro complementation with reduced benzyl viologen-nitrate reductase from mutant 305. Furthermore, a heat-labile costitutive molybdenum cofactor which is fuctionally active is also present in mutant 301. Mutant 301 has the two requirements exhibited by the active nitrate reductase complex from fungi, namely, NAD(P)H-cytochrome c reductase activity and molybdenum cofactor, but lacks NAD(P)H-nitrate reductase activity. This fact together with biochemical data presented from other C. reinhardii mutants strongly suggest a heteropolymeric model for the nitrate reductase complex of the alga.     

Reconstitution of Nitrate Reductase Activity and Formation of Membrane Particles from Cytoplasmic Extracts of Chlorate-Resistant Mutants of Escherichia coli

The reconstitution of nitrate reductase activity in mixtures of cytoplasmic fractions from the chlorate-resistant mutants chlA, B, C, and E which are lacking this activity was investigated, and the membrane-like particulate material which formed during this reconstitution was analyzed by polyacrylamide gel electrophoresis. When chlA and chlB extracts are incubated together, the cytoplasmic membrane proteins present in the particles which are formed are contributed by both mutants, and the proteins are essentially the same as the proteins in the cytoplasmic membrane fractions of the two mutants. Identical amounts of protein become particulate when cytoplasmic extracts of any of the mutant strains or wild-type strains are incubated at 32 C either singly or in mixtures, and the formation of particulate material does not appear to be a consequence of nitrate reductase reconstitution. Experiments with wild-type strains indicate that the membrane proteins in the cytoplasmic extract are derived from the cytoplasmic membrane during cell breakage. Reconstitution experiments involving various combinations of preincubated and unincubated extracts of the mutants have allowed a preliminary identification of three types of components which are necessary for the formation of active nitrate reductase: (i) a soluble factor present only in extracts from induced chlB; (ii) a different soluble factor which is missing in chlB but is present in extracts from wild-type, chlA, chlC, and chlE; and (iii) a complex including the nitrate reductase protein which is inactivated by preincubation of the mutant extracts.