A protein kinase activated by darkness phosphorylates nitrate reductase in Komatsuna (Brassica campestris) leaves

Although it has been shown that leaf nitrate reductase (NR: EC 1.6.6.1) is phosphorylated by subjecting plants to darkness, there is no evidence for the existence of dark-activated or dark-induced NR kinase. This study was undertaken to investigate the occurrence of a protein kinase phosphorylating NR in response to dark treatments. Immediately after transferring Komatsuna (Brassica campestris L.) plants to darkness, we observed rapid increases in the phosphorylating activity of the synthetic peptide, which is designed for the amino acid sequence surrounding the regulatory serine residue of the hinge 1 region of Komatsuna NR, in crude extracts from leaves. The activity reached a maximum after 10 min of darkness. Inactivation states of NR estimated from relative activities with or without Mg2+ were correlated to activities of the putative dark-activated protein kinase. Using the synthetic peptide as a substrate, we purified a protein kinase from dark-treated leaves by means of successive chromatographies on Q-Sepharose, Blue Sepharose, FPLC Q-Sepharose, and ATP-gamma-Sepharose columns. The purified kinase had an apparent molecular mass of 150 kDa with a catalytic subunit of 55 kDa, and it was Ca2+-independent. The purified kinase phosphorylated a recombinant cytochrome c reductase protein, a partial protein of NR, and holo NR, and inactivated NR in the presence of both 14-3-3 protein and Mg2+. The kinase also phosphorylated synthetic peptide substrates designed for sucrose phosphate synthase and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase. Among inhibitors tested, only K252a, a potent and specific serine/threonine kinase inhibitor, completely inhibited the activity of the dark-activated kinase. The activity of the purified kinase was also specifically inhibited by K252a. Taken together with these findings, results obtained suggest that the putative dark-activated protein kinase may be the purified kinase itself, and may be responsible for in vivo phosphorylation of NR and its inactivation during darkness.     

The protein kinase, protein phosphatase and inhibitor protein of nitrate reductase are ubiquitous in higher plants and independent of nitrate reductase expression and turnover

Nitrate reductase activity and NR protein levels in various leaf tissues were drastically decreased (<3.5% of normal activity) either by keeping detached leaves in continuous darkness for up to 6 d (spinach), or by growing plants (pea, squash) hydroponically on ammonium as the sole N-source, or by germinating and growing etiolated seedlings in complete darkness (squash). The presence of nitrate reductase protein kinase (NRPK), nitrate reductase protein phosphatase (NRPP) and inhibitor protein (IP) was examined by measuring the ability of NR-free desalted extracts to inactivate (ATP-dependent) and reactivate (5-AMP/EDTA-dependent) added purified spinach NR in vitro. Extracts from low-NR plants (ammonium-grown pea and squash) were also prepared from leaves harvested at the end of a normal light or dark phase, or after treating leaves with anaerobiosis, uncouplers or mannose, conditions which usually activate NR in nitrategrown normal plants. Without exception, extracts from NR-deficient plant tissues were able to inactivate and reactivate purified spinach NR with normal velocity, irrespective of pretreatment or time of harvest. Considerable NRPK, NRPP and IP activities were also found in extracts from almost NR-free ripe fruits (cucumber and tomato). Activities were totally absent, however, in extracts from isolated spinach chloroplasts. The NRPK and IP fractions were partially purified with normal yields from NR-deficient squash or spinach leaves, following the purification protocol worked out for nitrate-grown spinach. The Ca²⁺/Mg²⁺-dependent kinase fraction from NR-deficient squash or spinach phosphorylated added purified spinach NR with -[³²P]ATP and inactivated the enzyme after addition of IP. It is suggested (i) that the auxiliary proteins (NRPK, IP, NRPP) which modulate NR are rather species- or organ-unspecific, (ii) that they do not turn over as rapidly as does NR, (iii) that they are probably expressed independently of NR, and (iiii) that they are not covalently modulated, but under control of metabolic and/or physical signals which are removed by desalting.Abbreviations IP inhibitor protein - NR NADH-nitrate reductase - NRA nitrate reductase activity - NRPK nitrate reductase protein kinase - NRPP nitrate reductase protein phosphatase - PK protein kinase This work was supported by the Deutsche Forschungsgemeinschaft (SFB 251).     

Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase

Spinach (Spinacea oleracea L.) nitrate reductase (NR) is inactivated by phosphorylation on serine-543, followed by binding of the phosphorylated enzyme to 14-3-3 proteins. We purified one of several chromatographically distinct NR_serine-543 kinases from spinach leaf extracts, and established by Edman sequencing of 80 amino acid residues that it is a calcium-dependent (calmodulin-domain) protein kinase (CDPK), with peptide sequences very similar to Arabidopsis CDPK6 (accession no. U20623; also known as CPK3). The spinach CDPK was recognized by antibodies raised against Arabidopsis CDPK. Nitrate reductase was phosphorylated at serine-543 by bacterially expressed His-tagged CDPK6, and the phosphorylated NR was inhibited by 14-3-3 proteins. However, the bacterially expressed CDPK6 had a specific activity approx. 200-fold lower than that of the purified spinach enzyme. The physiological control of NR by CDPK is discussed, and the regulatory properties of the purified CDPK are considered with reference to current models for reversible intramolecular binding of the calmodulin-like domain to the autoinhibitory junction of CDPKs. Received: 12 February 1998 / Accepted: 28 May 1998     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

Regulation of spinach-leaf nitrate reductase by reversible phosphorylation.

Okadaic acid and microcystin (but not the inactive methyl esters of these toxins) prevented the rapid light-induced activation of nitrate reductase (NR) in intact spinach leaves. In vitro, nitrate reductase was inactivated by a protein kinase and activated by PP2A. The role of reversible protein phosphorylation in regulation of light-coupled cytoplasmic metabolism is discussed.     

Nitrate reductase activity in chicory roots following excision

In young chicory plantlets (Cichorium intybus L. Witloof cv.Flash), nitrate assimilation takes place mainly in the roots.Nitrate reductase activity (NRA) was measured in roots deprivedof shoot control by excision and transferred into a sucrose-containingmedium. Such a treatment resulted in a drop of about 60% ofNRA within 3 h. The level of NR protein decreased after 12 hand the level of NR-mRNA after several days. This adaptationof nitrate assimilation to excision was affected by a phosphorylation-dephosphorylationmechanism as shown by increased sensitivity to magnesium ofin vitro NRA. Okadaic acid, a serinethreonine protein phosphatasesinhibitor, enhanced the decrease of NRA. Conversely, staurosporine,a serine-threonine protein kinases inhibitor, antagonized theinhibition of NRA. This suggests that excision caused a rapidinactivation of NRA in roots of chicory by modifying the phosphorylationbalance towards a phosphorylated NR form which could enter aninactive complex. Key words: Chicory, nitrate reductase, staurosporine     

Regulation of Nitrate Reductase in Neurospora crassa: Stability In Vivo

Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-nitrate reductase and its related enzyme activities, NADPH-cytochrome c reductase and reduced benzyl viologen-nitrate reductase, are all induced following the transfer of ammonia-grown wild-type Neurospora mycelia to nitrate medium. After nitrate reductase is induced to the maximal level, the addition of an ammonium salt to, or the removal of nitrate from, the cultures results in a rapid inactivation of nitrate reductase and its two partial component activities. This rapid inactivation is slowed down by the protein synthesis inhibitor, cycloheximide. Experiments on the mixing of extracts in vitro rule out the presence of an inhibitor of nitrate reductase in free form in extracts containing inactivated nitrate reductase. Ammonia does not inhibit the uptake of nitrate by the mycelia. Inactivation of nitrate reductase in vivo by ammonia depends on the concentration of the ammonium salt and is not reversed by increasing the nitrate concentration of the medium. The nitrate-inducible NADPH-cytochrome c reductase activity and reduced benzyl viologen-nitrate reductase activity respectively of the nitrate-nonutilizing mutants nit-1 and nit-3 are not inactivated in vivo by the addition of an ammonium salt or the withdrawal of nitrate. This finding suggests that the integrity of the nitrate reductase complex is required for the in vivo inactivation of nitrate reductase and its associated activities.     

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in constitutive activation of the enzyme in vivo and nitrite accumulation

In wild-type Nicotiana plumbaginifolia and other higher plants, nitrate reductase (NR) is rapidly inactivated/activated in response to dark/light transitions. Inactivation of NR is believed to be caused by phosphorylation at a special conserved regulatory Ser residue, Ser 521, and interactions with divalent cations and inhibitory 14-3-3 proteins. A transgenic N. plumbaginifolia line (S(521)) was constructed where the Ser 521 had been changed by site-directed mutagenesis into Asp. This mutation resulted in complete abolishment of inactivation in response to light/dark transitions or other treatments known to inactivate NR. During prolonged darkness, NR in wild-type plants is in the inactivated form, whereas NR in the S(521) line is always in the active form. Differences in degradation rate between NR from S(521) and lines with non-mutated NR were not found. Kinetic constants like Km values for NADH and NO3(-) were not changed, but a slightly different pH profile was observed for mutated NR as opposed to non-mutated NR. Under optimal growth conditions, the phenotype of the S(521) plants was not different from the wild type (WT). However, when plants were irrigated with high nitrate concentration, 150 mM, the transgenic plants accumulated nitrite in darkness, and young leaves showed chlorosis.     

Action of Corn and Rice-inactivating Proteins on a Purified Nitrate Reductase from Chlorella vulgaris

When nitrate reductase (NR) purified from Chlorella was incubated with NR-inactivating proteins purified from corn roots and rice cell suspension cultures or with trypsin there was a loss in NADH-NR and NADH cytochrome c reductase (NADH-CR) activities with time whereas the reduced methylviologen NR (MV-NR) remained active. When NADH-NR and NADH-CR activities were inactivated completely by the incubation with corn protein, the major protein band obtained by polyacrylamide gel electrophoresis shifted from an R_F value of 0.12 to an R_F of 0.25 and reduced MV-NR activity moved to the new position on the gel. When NADH-NR and NADH-CR activities were partially inactivated by the corn protein, NADH-NR activity was detected in an intermediate position (R_F value of 0.18). Incubation with trypsin also caused a change in the NR protein migration pattern (R_F value of 0.20). This protein band also had reduced MV-NR activity. Thus, the corn inactivator degrades NR in a fashion similar to but not identical with trypsin. The incubation of NR with rice inactivating protein resulted in a loss of NADH-NR but had no effect on the migration of NR protein or on the reduced MV-NR activity or mobility suggesting that the rice protein binds to Chlorella NR.     

Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers

Assimilatory nitrate reductase (NR) of higher plants is a most interesting enzyme, both from its central function in plant primary metabolism and from the complex regulation of its expression and control of catalytic activity and degradation. Here, present knowledge about the mechanism of post-translational regulation of NR is summarized and the properties of the regulatory enzymes involved (protein kinases, protein phosphatases and 14-3-3-binding proteins) are described. It is shown that light and oxygen availability are the major external triggers for the rapid and reversible modulation of NR activity, and that sugars and/or sugar phosphates are the internal signals which regulate the protein kinase(s) and phosphatase. It is also demonstrated that stress factors like nitrate deficiency and salinity have remarkably little direct influence on the NR activation state. Further, changes in NR activity measured in vitro are not always associated with changes in nitrate reduction rates in vivo, suggesting that NR can be under strong substrate limitation. The degradation and half-life of the NR protein also appear to be affected by NR phosphorylation and 14-3-3 binding, as NR activation always correlates positively with its stability. However, it is not known whether the molecular form of NR in vivo affects its susceptibility to proteolytic degradation, or whether factors that affect the NR activation state also independently affect the activity or induction of the NR protease(s). A second and potentially important function of NR, the production of nitric oxide (NO) from nitrite is briefly described, but it remains to be determined whether NR produces NO for pathogen/stress signalling in vivo.     

Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum

BACKGROUND: The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented. METHODS: Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source. RESULTS: NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein. CONCLUSIONS: The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities.     

Nitrate assimilation in the forage legume Lotus japonicus L.

Nitrate assimilation in the model legume, Lotus japonicus, has been investigated using a variety of approaches. A gene encoding a nitrate-inducible nitrate reductase (NR) has been cloned and appears to be the only NR gene present in the genome. Most of the nitrate reductase activity (NRA) is found in the roots and the plant assimilates the bulk of its nitrogen in that tissue. We calculate that the observed rates of nitrate reduction are compatible with the growth requirement for reduced nitrogen. The NR mRNA, NRA and the nitrate content do not show a strong diurnal rhythm in the roots and assimilation continues during the dark period although export of assimilated N to the shoot is lower during this time. In shoots, the previous low NR activity may be further inactivated during the dark either by a phosphorylation mechanism or due to reduced nitrate flux coincident with a decreased delivery through the transpiration stream. From nitrate-sufficient conditions, the removal of nitrate from the external medium causes a rapid drop in hydraulic conductivity and a decline in nitrate and reduced-N export. Root nitrate content, NR and nitrate transporter (NRT2) mRNA decline over a period of 2 days to barely detectable levels. On resupply, a coordinated increase of NR and NRT2 mRNA, and NRA is seen within hours.     

Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase.

Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels. To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed. Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis. Active NR protein was produced in a Pichia pastoris expression system. Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA. Mutant forms of NR were constructed, produced in P. pastoris, and tested in the in vitro inactivation assay. Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach. A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect. The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated. These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for post-translational regulation in vitro.     

Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation.

Spinach (Spinacia oleracea L.) leaf nitrate reductase (NADH:NR;NADH:nitrate oxidoreductase, EC 1.6.6.1) activity was found to rapidly change during light/dark transitions. The most rapid and dramatic changes were found in a form of NR which was sensitive to inhibition by millimolar concentrations of magnesium. This form of NR predominated in leaves in the dark, but was almost completely absent from leaves incubated in the light for only 30 min. When the leaves were returned to darkness, the NR rapidly became sensitive to Mg2+ inhibition. Modulation of the overall reaction involving NADH as electron donor was also found when reduced methyl viologen was the donor (MV:NR), indicating that electron transfer had been blocked, at least in part, at or near the terminal molybdenum cofactor site. Changes in activity appear to be the result of a covalent modification that affects sensitivity of NR to inhibition by magnesium, and our results suggest that protein phosphorylation may be involved. NR was phosphorylated in vivo after feeding excised leaves [32P]Pi. The NR subunit was labeled exclusively on seryl residues in both light and dark. Tryptic peptide mapping indicated three major 32P-labeled phosphopeptide (Pp) fragments. Labeling of two of the P-peptides (designated Pp1 and 3) was generally correlated with NR activity assayed in the presence of Mg2+. In vivo, partial dephosphorylation of these sites (and activation of NR assayed with Mg2+) occurred in response to light or feeding mannose in darkness. The light effect was blocked completely by feeding okadaic acid via the transpiration stream, indicating the involvement of type 1 and/or type 2A protein phosphatases in vivo. While more detailed analysis is required to establish a causal link between the phosphorylation status of NR and sensitivity to Mg2+ inhibition, the current results are highly suggestive of one. Thus, in addition to the molecular genetic mechanisms regulating this key enzyme of nitrate assimilation, NR activity may be controlled in leaves by phosphorylation/dephosphorylation of the enzyme protein resulting from metabolic changes taking place during light/dark transitions.     

Glucose-signalled inhibition of cysteine-proteases involved in nitrate reductase degradation in oat leaf segments

Regulation of nitrate reductase (NR; EC 1.6.6.1) breakdown, measured as loss of maximal activity (MNRA), was studied in leaf segments of 7-day-old oat plants in the light for up to 4 h. In segments floating on 1 mM tungstate, NR lost more than 40% of its initial maximal activity. Cycloheximide, high (300 mM) glucose (Glc) and inhibitors of cysteine proteases stabilized NR in situ , suggesting that MNRA decrease was due to the hydrolysis of NR by a short-lived, glucose-modulated cysteine protease. Loss of MNRA was accelerated by cantharidin (CTHR) and inhibited by staurosporine, suggesting that NR breakdown required continuous phosphorylation. High glucose inhibited any further MNRA decrease when supplied after a 30-min pretreatment with CTHR, suggesting that a phosphorylated protein was its target. Isoosmolar polyethylene glycol also stabilized NR but not in the presence of CTHR. Low (30 mM) Glc stabilized NR only in the presence of Ca²⁺, and CTHR inhibited its effect. EGTA and LaCl₃ completely arrested the effects of both high- and low- Glc. Like low D-Glc, low L-Glc (glucose analog not transported) inhibited NR breakdown in the presence of Ca²⁺, but at high concentration only 2-deoxyglucose, that is phosphorylated but not further metabolized, and glucose-6P were effective in the presence of CTHR, suggesting that receptors for high- and low- Glc were located in different cell compartments. It is proposed that high- and low- Glc trigger different signalling pathways, with calcium as a common upstream secondary messenger and protein kinases and protein phosphatases being downstream components in the cascade of reactions that modulates NR proteolysis.     

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase.

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.     

Activation and stabilization of nitrate reductase by NADH in wheat and maize

Preincubation of nitrate reductase (NR) extracted from wheat shoot tips with NADH in vitro, activated and stabilized activity at both O° and 25°. However, preincubation with potassium ferricyanide inactivated the NR in vitro. NADH also stabilized the NR activity in extracts from maize shoot tips. It was observed that NR from both wheat and maize was active at low temperatures.     

Isolation and characterization of nitrate reductase from the halophilic sulfur-oxidizing bacterium <Emphasis Type="Italic">Thioalkalivibrio nitratireducens</Emphasis>

A novel nitrate reductase (NR) was isolated from cell extract of the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens strain ALEN 2 and characterized. This enzyme is a classical nitrate reductase containing molybdopterin cofactor in the active site and at least one iron-sulfur cluster per subunit. Mass spectrometric analysis showed high homology of NR with the catalytic subunit NarG of the membrane nitrate reductase from the moderately halophilic bacterium Halomonas halodenitrificans. In solution, NR exists as a monomer with a molecular weight of 130–140 kDa and as a homotetramer of about 600 kDa. The specific nitrate reductase activity of NR is 12 μmol/min per mg protein, the maximal values being observed within the neutral range of pH. Like other membrane nitrate reductases, NR reduces chlorate and is inhibited by azide and cyanide. It exhibits a higher thermal stability than most mesophilic enzymes.     

Different forms of molybdenum cofactor inVicia faba seeds: The presence of molybdenum cofactor carrier protein and its purification

There were significant differences in the contents of molybdenum cofactor (Mo-co), both in a low-molecular-mass form (free Mo-co) and in a protein-bound form, in seeds of sevenVicia faba genotypes. Low-molecular-mass Mo-co species present in the extracts were detected by their ability to reactivate, through a dialysis membrane, aponitrate reductase from theNeurospora crassa nit-1 mutant. In extracts of all genotypes tested, the amount of Mo-co capable of directly reactivating nitrate reductase of theN. crassa nit-1 mutant was always much higher than that of low-molecular-mass Moco. These data cannot be explained by considering, as traditionally, that Mo-co detected directly, i.e. without any previous treatment for its release from Mo-coproteins, corresponds to free low-molecular mass Mo-co. A protein which bound Mo-co was purified to electrophoretic homogeneity. This protein consisted of a single 70-kDa polypeptide chain and carried a Mo-co that could be efficiently released when in contact with aponitrate reductase.Abbreviations CP carrier protein - Mo-co molybdenum cofactor - NR nitrate reductase - XO xanthine oxidase