Uninduced Wild Type Research Articles

Critical Role of SHP2 in the Initiation and Maintenance of Myeloproliferative Neoplasms Evoked By JAK2V617F

The JAK2V617F mutation has been found in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Expression of JAK2V617F results in constitutive activation of the JAK2 tyrosine kinase and its downstream signaling pathways. JAK2 inhibitor therapy can reduce splenomegaly and constitutional symptoms but is not sufficient to cure or produce complete remission in patients with MPNs. Identification of critical signaling pathways/molecules required for the initiation and maintenance of MPNs will facilitate the development of more effective targeted therapies.To determine the role of JAK2V617F mutation in MPNs, we previously generated a conditional Jak2V617F knock-in mouse. We observed that expression of heterozygous Jak2V617F in mice hematopoietic compartments is sufficient to induce a PV-like MPN, whereas homozygous Jak2V617F expression accelerates the development of myelofibrosis. We have found that SHP2, a protein tyrosine phosphatase that positively regulates hematopoietic signaling, is constitutively phosphorylated in mouse and human hematopoietic cells expressing JAK2V617F. However, the contribution of SHP2 in the pathogenesis of MPNs induced by JAK2V617F remains elusive. Here, we sought to determine the role of SHP2 in JAK2V617F-evoked MPNs using conditional SHP2 knockout (SHP2 floxed) and Jak2V617F knock-in mice and MxCre line.Whereas expression of heterozygous Jak2V617F induced a PV-like MPN characterized by increase in red blood cells (RBC), white blood cells (WBC), neutrophils and platelets in the peripheral blood and splenomegaly, deletion of SHP2 normalized the blood parameters and spleen size in Jak2V617F knock-in mice. Flow cytometric analysis showed that deletion of SHP2 inhibited the expansion of erythroid, megakaryocytic, and granulocytic precursors in the bone marrow (BM) and spleens of Jak2V617F mice. Deletion of SHP2 also inhibited the expansion of hematopoietic stem cells and megakaryocyte-erythroid progenitors in the BM and spleens of Jak2V617F knock-in mice. Furthermore, deletion of SHP2 markedly inhibited the erythropoietin (Epo)-independent CFU-E colonies in the BM and spleens of Jak2V617F mice.In order to determine whether the effects of SHP2 deletion in Jak2V617F mice were cell autonomous, BM cells from uninduced control, MxCre;V617F/+ and MxCre;V617F/+;SHP2fl/fl mice were transplanted into lethally irradiated syngeneic recipient mice. Four weeks after transplantation, mice were injected with pI-pC to induce deletion of SHP2 and expression of Jak2V617F simultaneously. Transplanted animals receiving MxCre;V617F/+ BM developed a PV-like disease within 6 weeks after pI-pC induction. However, recipients of MxCre;V617F/+;SHP2fl/fl BM failed to induce the PV disease due to deletion of SHP2. Together, these data suggest that SHP2 is required for the initiation of PV-like MPN mediated by Jak2V617F.To determine if SHP2 is required for the maintenance of MPN evoked by Jak2V617F, uninduced wild type or MxCre;SHP2fl/fl mice BM cells were transduced with retroviruses expressing Jak2V617F and transplanted into lethally irradiated syngeneic recipient animals. Five weeks after transplantation, we assessed the peripheral blood counts to confirm the development of MPN disease. Transplanted animals receiving Jak2V617F-transduced wild type or MxCre;SHP2fl/fl mice BM were then divided into two groups- one group was injected with pI-pC (to induce deletion of SHP2 after establishment of the MPN disease); another group (control group) was injected with saline. We observed that deletion of SHP2 by pI-pC induction significantly inhibited the RBC, hematocrit, WBC and platelet counts in the peripheral blood and reduced the spleen size in transplanted animals expressing Jak2V617F. Moreover, deletion of SHP2 markedly inhibited the Epo-independent erythroid colonies in the BM and spleens of transplanted animals expressing Jak2V617F. Thus, SHP2 is not only required for the initiation of MPN but also required for the maintenance of MPN mediated by Jak2V617F. We have also observed that deletion of SHP2 blocks the development of myelofibrosis in Jak2V617F knock-in mice. In conclusion, our results suggest that SHP2 plays a critical role in the initiation and maintenance of MPNs evoked by Jak2V617F. DisclosuresNo relevant conflicts of interest to declare.

Altered metabolic flux due to deletion of odhA causes L-glutamate overproduction in Corynebacterium glutamicum.

L-glutamate overproduction in Corynebacterium glutamicum, a biotin auxotroph, is induced by biotin limitation or by treatment with certain fatty acid ester surfactants or with penicillin. We have analyzed the relationship between the inductions, 2-oxoglutarate dehydrogenase complex (ODHC) activity, and L-glutamate production. Here we show that a strain deleted for odhA and completely lacking ODHC activity produces L-glutamate as efficiently as the induced wild type (27.8 mmol/g [dry weight] of cells for the ohdA deletion strain compared with only 1.0 mmol/g [dry weight] of cells for the uninduced wild type). This level of production is achieved without any induction or alteration in the fatty acid composition of the cells, showing that L-glutamate overproduction can be caused by the change in metabolic flux alone. Interestingly, the L-glutamate productivity of the odhA-deleted strain is increased about 10% by each of the L-glutamate-producing inductions, showing that the change in metabolic flux resulting from the odhA deletion and the inductions have additive effects on L-glutamate overproduction. Tween 40 was indicated to induce drastic metabolic change leading to L-glutamate overproduction in the odhA-deleted strain. Furthermore, optimizing the metabolic flux from 2-oxoglutarate to L-glutamate by tuning glutamate dehydrogenase activity increased the l-glutamate production of the odhA-deleted strain.

Insertional mutation of marA vitiates inducible multiple antimicrobial resistance in Salmonella enterica subsp. enterica serovar Choleraesuis

marA has been shown to mediate a multiple antimicrobial resistance (MAR) phenotype following induction in some members of the Enterobacteriaceae. When Salmonella Choleraesuis was exposed to inducing agents they displayed higher minimal inhibitory concentrations (MIC) to multiple antimicrobial agents and an increase in marA expression as determined by northern hybridization analysis. The objective of the present study was to determine if mutation of marA vitiated multiple antimicrobial resistance inducibility in S. Choleraesuis. A loss-of-function mutation of marA in a single S. Choleraesuis isolate was created by insertion of the dihydrofolate reductase (DHFR) gene cassette within marA using double homologous recombination. This mutation was complemented with an expression plasmid possessing marA under the control of an IPTG-inducible promoter. Mutation and complementation of marA was verified using polymerase chain reaction, Northern hybridization, and Western blotting assays. Minimum inhibitory concentrations (MICs) of tetracycline, chloramphenicol, nalidixic acid, and rifampin were determined against induced and uninduced wildtype, marA-disrupted and marA-complemented strains using a microbroth dilution assay. Minimum inhibitory concentrations against induced wildtype and marA-complemented strains increased four- to eight-fold for all antimicrobials tested when compared to the uninduced strains while the MICs of the induced marA-disrupted mutant remained the same. However, this increase was abrogated when the cells were grown in the presence of the efflux pump inhibitor compound EPI phe-arg-naphthylamide. The results indicate that a functional marA is solely required for an inducible multiple antimicrobial resistance phenotype in S. Choleraesuis.

Role of Chitin-Binding Proteins in the Specific Attachment of the Marine Bacterium Vibrio harveyi to Chitin

We examined the mechanism of attachment of the marine bacterium Vibrio harveyi to chitin. Wheat germ agglutinin and chitinase bind to chitin and competitively inhibited the attachment of V. harveyi to chitin, but not to cellulose. Bovine serum albumin and cellulase do not bind to chitin and had no effect on bacterial attachment to chitin. These data suggest that this bacterium recognizes specific attachment sites on the chitin particle. The level of attachment of a chitinase-overproducing mutant of V. harveyi to chitin was about twice as much as that of the uninduced wild type. Detergent-extracted cell membranes inhibited attachment and contained a 53-kDa peptide that was overproduced by the chitinase-overproducing mutant. Three peptides (40, 53, and 150 kDa) were recovered from chitin which had been exposed to membrane extracts. Polyclonal antibodies raised against extracellular chitinase cross-reacted with the 53- and 150-kDa chitin-binding peptides and inhibited attachment, probably by sterically hindering interactions between the chitin-binding peptides and chitin. The 53- and 150-kDa chitin-binding peptides did not have chitinase activity. These results suggest that chitin-binding peptides, especially the 53-kDa chitin-binding peptide and chitinase and perhaps the 150-kDa peptide, mediate the specific attachment of V. harveyi to chitin.

The molecular cloning, sequence and expression of the hdcB gene from Lactobacillus 30A

We previously cloned the structural gene hdcA, which encodes the enzyme histidine decarboxylase (HDC; EC 4.1.1.22), from Lactobacillus 30a and found what appeared to be the start of a second gene 59 nucleotide (nt) downstream from the hdcA stop codon [Vanderslice et al., J. Biol. Chem. 32 (1986) 15186–15191]. Here we report the complete nt sequence of this second gene, which we have named hdcB, and show that it encodes a 20-kDa protein, HDCB, which was purified from Escherichia coli. The hdcA and hdcB genes together comprise an operon, the transcription from which is shown to be increased threefold by the presence of histidine in the growth medium. Western blots were used to quantitate the rise in concentrations of both gene products during histidine induction of the hdc operon. This increase was found to be proportional to the observed threefold increase in the concentration of the respective mRNAs. Transcription of the hdc operon in the mutant-3 strain of Lactobacillus 30a [Recsei and Snell, Biochemistry 12 (1973) 365-371] was shown to be constitutively 15-fold greater than in uninduced wild type cells and was unaffected by histidine. The transcription start point was defined as a guanine 73 nt 5′ to the start codon of the hdcA gene. Of the transcripts initiated at this promoter, 15% include both hdcA and hdcB sequences, the remainder terminate in the intergenic region and thus encode only hdcA.

Reduced Cd 2+ accumulation and elevated metallothionein levels in a Cd 2+ and Zn 2+-resistant clonal cho-K1 cell line

A clonal cell line, R40F, was selected from a heterogenous population of Cd 2+ and Zn 2+-resistant CHO-K1 cells. These R40F cells demonstrated resistance to 120- and 4-fold higher concentrations of Cd 2+ and Zn 2+, respectively, than did wild type CHO-K1 cells. When cultured in the presence of low concentrations of Cd 2+ (0.5–1.0 μM), the accumulation of intracellular Cd 2+ in R40F cells appears to be significantly less than in wild type cells. Since R40F cells maintained in medium containing high concentrations of Cd 2+ (200 μM) retain levels of Cd 2+ equivalent to the intracellular concentration observed in wild type cells exhibiting cytotoxicity, it is assumed that reduced Cd 2+ transport alone is unlikely to account for the resistance to Cd 2+ toxicity. Exposure of R40F cells to non-toxic (2 μM or 100 μM) or toxic (200 μM) Zn 2+ levels resulted in an accumulation of Zn 2+ equal to, or greater than, that observed in the wild type cell. When compared to the basal level in uninduced wild type cells, metallothionein levels were elevated 14- and 23-fold, respectively, in R40F cells cultured in the presence of 0.5 μM Cd 2+ and 100 μM Zn 2+. These results are consistent with the hypothesis that R40F cells express Cd 2+ and Zn 2+ resistance as a consequence of a reduction in unbound intracellular Cd 2+ levels and an elevation of metallothionein synthesis.

Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid.

Mutants of Kluyveromyces lactis with elevated uninduced levels of beta-galactosidase (EC 32.1.2.3) activity, constitutive mutants (lac10c), were isolated and characterized to determine the basis for their constitutiveness. These lesions are not operator-type regulatory mutants because they are not closely linked to the beta-galactosidase structural gene. In a constitutive strain having a 7-fold increase in beta-galactosidase activity, the concentration of beta-galactosidase messenger ribonucleic acid (mRNA) was 8- to 10-fold higher than uninduced wild type. The half-life of beta-galactosidase mRNA was the same in the mutant strain (t1/2 = 4.5 +/- 0.2 min) as in uninduced wild-type cells (t1/2 = 4.8 +/- 0.1 min), indicating that the elevated mRNA level in the mutant was not due to a decreased rate of mRNA degradation. Consequently, we hypothesize that the LAC10 product regulates transcription of the beta-galactosidase gene; it probably affects the rate of transcription initiation. Parallel increases in enzyme protein, in constitutive levels of beta-galactosidase activity, and in mRNA further support this position, making translational or posttranslational control by LAC10 unlikely. Several types of data suggest that the LAC10 product functions as a negative regulatory element to prevent transcription. Other data demonstrate that lac10c mutations have pleiotrophic effects, there being constitutive levels not only of beta-galactosidase activity, but also the other lactose-inducible activities of galactokinase (EC 2.7.5.1), galactose-1-phosphate uridyl transferase (EC 2.7.7.10), and lactose transport. It would appear that LAC10 regulates lactose-inducible proteins.

Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid.

Mutants of Kluyveromyces lactis with elevated uninduced levels of beta-galactosidase (EC 32.1.2.3) activity, constitutive mutants (lac10c), were isolated and characterized to determine the basis for their constitutiveness. These lesions are not operator-type regulatory mutants because they are not closely linked to the beta-galactosidase structural gene. In a constitutive strain having a 7-fold increase in beta-galactosidase activity, the concentration of beta-galactosidase messenger ribonucleic acid (mRNA) was 8- to 10-fold higher than uninduced wild type. The half-life of beta-galactosidase mRNA was the same in the mutant strain (t1/2 = 4.5 +/- 0.2 min) as in uninduced wild-type cells (t1/2 = 4.8 +/- 0.1 min), indicating that the elevated mRNA level in the mutant was not due to a decreased rate of mRNA degradation. Consequently, we hypothesize that the LAC10 product regulates transcription of the beta-galactosidase gene; it probably affects the rate of transcription initiation. Parallel increases in enzyme protein, in constitutive levels of beta-galactosidase activity, and in mRNA further support this position, making translational or posttranslational control by LAC10 unlikely. Several types of data suggest that the LAC10 product functions as a negative regulatory element to prevent transcription. Other data demonstrate that lac10c mutations have pleiotrophic effects, there being constitutive levels not only of beta-galactosidase activity, but also the other lactose-inducible activities of galactokinase (EC 2.7.5.1), galactose-1-phosphate uridyl transferase (EC 2.7.7.10), and lactose transport. It would appear that LAC10 regulates lactose-inducible proteins.

Effect of Tungsten and Vanadium on the in Vitro Assembly of Assimilatory Nitrate Reductase Utilizing Neurospora Mutant nit-1

Abstract Tungsten or vanadium in a nutrient medium containing nitrate, but not ammonia, as the sole nitrogen source inhibited the formation of assimilatory NADPH-nitrate reductase (but not nitrate-inducible NADPH-cytochrome c reductase) and growth of wild type Neurospora crassa. Inhibition was prevented by high levels of molybdate in the nutrient medium but was not reversed by adding molybdate to subsequently prepared cell-free enzyme preparations. Neither tungsten nor vanadium was inhibitory when added to partially purified wild type Neurospora NADPH-nitrate reductase. Cell-free preparations of uninduced wild type grown in increasing concentrations of tungsten or vanadium showed progressive inhibition of ability to assemble nitrate reductase in vitro (with extracts of nitrate-induced Neurospora mutant nit-1) which was partially or entirely restored by subsequent addition of molybdate to the extracts of the tunsgten- or vanadium-grown uninduced wild type. Tungsten or vanadium added to acid-treated bovine milk xanthine oxidase or to extracts of uninduced wild type also inhibited in vitro enzyme assembly, with added molybdate partially preventing inhibition. The tungsten or vanadium analogue of nitrate reductase formed in vivo or by in vitro assembly is considerably more labile than its molybdenum counterpart as indicated by the complete loss of 185W or 48V from the enzyme as a result of trichloroacetic acid precipitation or heat treatment. Sucrose density gradient profiles of the Neurospora NADPH-nitrate reductase formed in vitro with 185W- or 48V-grown uninduced wild type or induced nit-1 showed, as in the case of 99Mo, that uninduced wild type and not nit-1 can be the source of tungsten or vanadium in the in vitro assembly of the enzyme. Exogenous 185W added to cell-free extracts of uninduced wild type or acid treated bovine milk xanthine oxidase prior to in vitro assembly of nitrate reductase using nit-1 failed to appear in the subsequently formed enzyme, whereas 48V experienced a small but consistent incorporation only with acid-treated xanthine oxidase. There was no in vitro exchange or incorporation of 185W or 48V into the already formed wild type enzyme. The data implicate tungsten and vanadium as competitive inhibitors of molybdenum in nitrate reductase structure and function.

Involvement of Molybdenum and Iron in the in Vitro Assembly of Assimilatory Nitrate Reductase Utilizing Neurospora Mutant nit-1

Abstract Addition of high levels of sodium molybdate (10-2 m, final concentration) to acid-treated purified bovine milk xanthine oxidase prior to incubation with an extract of the nitrate-induced nitrate reductase Neurospora crassa mutant nit-1 resulted in a significant enhancement of the subsequent in vitro assembly of wild type N. crassa NADPH-nitrate reductase (EC 1.6.6.2). Added molybdate had no effect on the wild type enzyme itself. The enhancement is highly specific for molybdenum, is independent of added phosphate, and also occurs with cell-free preparations of uninduced wild type and with other known acid-treated molybdenum enzymes including chicken liver xanthine dehydrogenase, rabbit liver aldehyde oxidase, bovine sulfite oxidase, and the nitrogenases of Azotobacter and Clostridium. Preincubation at 23° of acid-treated xanthine oxidase preparations resulted in a considerable loss of ability to form nitrate reductase and in a more striking enhancement by subsequently added molybdate. Prior addition of molybdate during the above 23° preincubation prevented in part the loss of the ability of the enzyme to form nitrate reductase, suggesting that the enhancement by molybdate is a restoration effect rather than a stimulatory one. Inclusion of molybdate in the buffer used for homogenization of the uninduced wild type also gave a significant increase in the subsequent in vitro assembly of the enzyme. Various forms and derivatives of molybdenum enhanced to varying degrees the ability of acid-treated xanthine oxidase to form nitrate reductase. Aqueous solutions of several complexes of Mo(V) with cysteine or glutathione under anaerobic conditions failed to substitute for the presumed molybdenum cofactor in the in vitro assembly of nitrate reductase. Sucrose density gradient profiles of NADPH-nitrate reductase formed in vitro by incubation of induced nit-1 extract with a 99Mo-labeled fraction of uninduced wild type (grown on 99MoO4=-containing medium) coincided with that of radioactivity. The addition of 99MoO4= to a cell-free preparation of unlabeled uninduced wild type or to acid-treated xanthine oxidase prior to its incubation with an extract of induced nit-1 gave similar results. Reciprocal experiments using an unlabeled fraction of uninduced wild type and an extract of nit-1 grown and induced in the presence of 99MoO4= showed no incorporation of radioactivity in the in vitro assembled enzyme. Nor was there in vitro exchange or incorporation of 99Mo into the already formed wild type enzyme. Comparable experiments with 55Fe showed that nit-1 and not uninduced wild type or xanthine oxidase is the source of iron in the in vitro assembly of Neurospora NADPH-nitrate reductase.

Evidence for a molybdenum cofactor common to all molybdenum enzymes based on the in vitro assembly of assimilatory NADPH-nitrate reductase using the Neurospora mutant nit-1

The in vitro assembly of an active Neurospora-like assimilatory NADPH—nitrate reductase (E.C. 1.6.6.2) has been effected by incubation of cell-free extracts of the Neurospora crassa nitrate reductase mutant nit-1 with (a) cell-free preparation of certain non-allelic mutants or uninduced wild type or (b) acid-treated molybdenum enzyme from diverse phylogenetic sources (20 different non-molybdenum enzymes were found to be inactive under the same experimental conditions). There is also a specific stimulatory effect of added molybdenum compounds on the in vitro assembly of NADPH-nitrate reductase. The incorporation of 99Mo, or its in vivo competitive inhibitor 185W, occurred in the in vitro assembly of nitrate reductase from a labelled, cell-free preparation of uninduced wild type (obtained by growth on a 99MoO 4 2− or 185WO 4 2−-containing medium) when incubated with an extract of unlabelled induced nit-1. Reciprocal experiments using labelled nit-1 and unlabelled, uninduced wild type showed no incorporation of radioactivity in the in vitro assembly of the enzyme. The results provide direct evidence for the view that molybdenum, presumably as a component of a larger, and as yet, unidentified cofactor, is furnished exclusively by uninduced wild type or acid-treated molybdenum enzymes in the in-vitro assembly system. It is postulated that the molybdenum cofactor of molecular weight approximately 1000 serves as both a link that binds the enzyme sub-units of nit-1 to yield the active enzyme and as an electron carrier.

Invitro formation of assimilatory reduced nicotinamide adenine dinucleotide phosphate: nitrate reductase from a Neurospora mutant and a component of molybdenum-enzymes.

An active Neurospora-like assimilatory NADPH-nitrate reductase (EC 1.6.6.2), which can be formed in vitro by incubation of extracts of nitrate-induced Neurospora crassa mutant nit-1 with extracts of (a) certain other nonallelic nitrate reductase mutants, (b) uninduced wild type, or (c) xanthine oxidizing and liver aldehyde-oxidase systems was also formed by combination of the nit-1 extract with other acid-treated enzymes known to contain molybdenum. These molybdenum enzymes included (a) nitrogenase, or its molybdenum-iron protein, from Clostridium, Azotobacter, and soybeannodule bacteroids, (b) bovine liver sulfite oxidase, (c) respiratory formate-nitrate reductase from Escherichia coli, (d) NADH-nitrate reductase from foxtail grass (Setaria faberii), and (e) FADH(2)- and reduced methyl viologennitrate reductase preparations from certain Neurospora mutants. Several molybdenum-amino-acid complexes, as possible catalytic models of nitrogenase, were inactive (as were some previously tested 20 nonmolybdenum enzymes) in place of the acid-treated molybdenum-containing enzymes. The results imply the existence of a molybdenum-containing component shared by the known molybdenum-enzymes.

In vitro assembly of Neurospora assimilatory nitrate reductase from protein subunits of a Neurospora mutant and the xanthine oxidizing or aldehyde oxidase systems of higher animals.

In vitro assembly or complementation of a hybrid assimilatory nitrate reductase was attained by mixing a preparation of nitrate-induced N. crassa mutant nit-1 specifically with acid-treated (pH 2.5) bovine milk or intestinal xanthine oxidase, rabbit liver aldehyde oxidase, or chicken liver xanthine dehydrogenase. The complementation reaction specifically required induced nit-1, the only nitrate reductase mutant of Neurospora that lacked xanthine dehydrogenase and was unable to use hypoxathine or nitrate as a sole nitrogen source. The complementing activities of the above acid-treated enzymes correspond to their xanthine or aldehyde oxidizing activity profiles on sucrose density gradients. The resulting soluble, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductases are the same as the Neurospora wild type enzyme in sucrose density gradient profile, molecular weight, substrate affinities, and sensitivity to inhibitors and temperature. By analogy to a similar in vitro complementation of nitrate reductase in mixtures of induced nit-1 and individual nonalleic Neurospora mutants, or uninduced wild type, the complemented nitrate apparently consists of an inducible protein subunit (possessing inducible NADPH-cytochrome c reductase) furnished by nit-1 and a subunit from the acid-treated xanthine or aldehyde oxidizing system which can substitute for the constitutive component furnished by the other mutants or uninduced wild type. The data suggest that Neurospora nitrate reductase and the xanthine oxidizing system and aldehyde oxidase of animals, all of which are molybdenum-containing enzymes catalyzing the reduction of nitrate to nitrite, share a highly similar protein subunit.

Formation of assimilatory nitrate reductase by in vitro inter-cistronic complementation in Neurospora crassa.

In vitro complementation of the soluble assimilatory nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-nitrate reductase was attained by mixing cell-free preparations of certain Neurospora nitrate reductase mutants: induced nit-1 (uniquely possessing inducible NADPH-cytochrome c reductase) with (a) uninduced or induced nit-2 or nit-3, or (b) uninduced wild type. The complementing activity of induced nit-1 is soluble while that of nit-2, nit-3, and wild type is particulate but not of mitochondrial origin. All fractions are inactivated by heat or trypsin. The NADPH-nitrate reductase enzymes formed in the above three complementing mixtures are similar to the wild-type enzyme in sucrose density gradient profiles, molecular weight, substrate affinity, sensitivity to inhibitors and temperature, but show different ratios of associated enzyme activities. The data suggest that nitrate reductase consists of at least two protein subunits: a nitrate-inductible subunit as reflected by inductible NADPH-cytochrome c reductase, and a constitutive protein which is activated (as indicated by the appearance of flavine adenine dinucleotide, reduced form (FADH(2))- and reduced methyl viologen-nitrate reductase activities) when it combines with the inductible subunit.