Inactivation Of Nitrate Reductase Research Articles

Nitrate reductase of Chlorella fusca: Partial purification, cytochrome content and presence of HCN after in vivo inactivation

An inactivated nitrate reductase (EC 1.6.6.1) formed in vivo by the green alga Chlorella fusca Shihira and Kraus is shown to be a cyanide complex. The partially purified inactive enzyme releases 0.048 nmol of HCN per unit of enzyme activated. This compares with 0.066 nmol of HCN liberated in similar previous measurements with the inactivated enzyme from Chlorella vulgaris. The nitrate reductase from C. fusca has been purified to a level of 67 μmol nitrate reduced per min per mg enzyme. It contains a cytochrome b557, at a level 1.9-fold higher per unit of active enzyme, than the nitrate reductase from C. vulgaris.

Molybdenum cofactors from molybdoenzymes and in vitro reconstitution of nitrogenase and nitrate reductase.

A molybdenum cofactor (Mo-co) from xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) can be isolated from the enzyme by a technique that has been used to isolate an iron-molybdenum cofactor (FeMo-co) from component I of nitrogenase. N-Methylformamide is used for the extraction of these molybdenum cofactors. Mo-co from xanthine oxidase activates nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2) in an extract from Neurospora crassa mutant strain Nit-1; however, FeMo-co is unable to activate nitrate reductase in strain Nit-1. Mo-co from xanthine oxidase is unable to activate nitrogenase in an extract of Azotobacter vinelandii mutant strain UW45. Inactive component I in this extract can be activated by FeMo-co. These results indicate that nitrate reductase and xanthine oxidase share a common molybdenum cofactor, but this cofactor is different from the molybdenum cofactor in nitrogenase.A. vinelandii synthesizes both Mo-co and FeMo-co. Mo-co is produced when the cells fix N(2) and also when they are repressed for nitrogenase synthesis by growth in a medium containing excess ammonium. However, FeMo-co is not produced when cells are grown in an ammonium-containing medium. Partially purified preparations of component I from A. vinelandii and Klebsiella pneumoniae contain both FeMo-co and Mo-co. The presence of both FeMo-co and Mo-co activities in partially purified preparations of component I explains previous reports of activation of inactive nitrate reductase in strain Nit-1 by acid-treated component I of nitrogenase. The Mo-co can be separated from FeMo-co in these preparations by chromatography on Sephadex G-100 in N-methylformamide. Both FeMo-co and Mo-co are sensitive to oxygen.

Studies on the regulation of assimilatory nitrate reductase in Ankistrodesmus braunii

In the green alga Ankistrodesmus braunii, all the activities associated with the nitrate reductase complex (i.e., NAD(P)H-nitrate reductase, NAD(P)H-cytochrome c reductase and FMNH2-or MVH-nitrate reductase) are nutritionally repressed by ammonia or methylamine. Besides, ammonia or methylamine promote in vivo the reversible inactivation of nitrate reductase, but not of NAD(P)H-cytochrome c reductase. Subsequent removal of the inactivating agent from the medium causes reactivation of the inactive enzyme. Menadione has a striking stimulation on the in vivo reactivation of the inactive enzyme. The nitrate reductase activities, but not the diaphorase activity, can be inactivated in vitro by preincubating a partially purified enzyme preparation with NADH or NADPH. ADP, in the presence of Mg(2+), presents a cooperative effect with NADH in the in vitro inactivation of nitrate reductase. This effect appears to be maximum at a concentration of ADP equimolecular with that of NADH.

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPHdependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by I mM NADP. The inclusion of 1 μM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FADprotected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred.

Nitrate reductase inactivating factor from rice cells in suspension culture

With a crude extract of cultured rice cells, there was no direct relationship between the activity of NADH nitrate reductase measured and the amount of cell extract used, when the amount was large. It appeared that some factor in the cell extract inactivated nitrate reductase. The inactivating factor could be separated from nitrate reductase by (NH4)2SO4 precipitation. The factor seemed to be protein: 1) it was precipitable with (NH4)2SO4 heat labile, and pronase treatment caused loss of activity; 2) cycloheximide reduced the formation of the inactivating factor. The activity of this factor fluctuated during the growth period. The existence of this inactivating factor was further investigated in various other cultured cells.

Nitrate reductase inactivation and reducing power and energy charge in chlorella cells

Ammonia, methylamine and other uncouplers of photosynthetic phosphorylation promote in vivo the reversible conversion of the oxidized active form of Chlorella fusca nitrate reductase into its reduced inactive form. Concomitantly with the in vivo inactivation of the enzyme by the uncoupler, there occurs an increase in the cellular levels of the reduced forms of nicotinamide nucleotides and of the uncharged forms of adenine nucleotides. A general view of the regulation of photosynthesis by reducing power and energy charge is discyssed.

Comparative Studies on the Induction and Inactivation of Nitrate Reductase in Corn Roots and Leaves

A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH(2)-nitrate reductase and NO(3) (-)-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A x 182E). The three activities were induced in parallel in both tissues when NO(3) (-) was supplied. WO(4) (=) suppressed the induction of NADH- and FMNH(2)-nitrate reductase activities in root tips and leaves. The NO(3) (-)-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO(4) (=). In leaves, on the other hand, there was a marked superinduction of the NO(3) (-)-induced NADH-cytochrome c reductase in the presence of WO(4) (=).The half-life values of NADH-nitrate reductase and FMNH(2)-nitrate reductase measured by removing NO(3) (-) and adding WO(4) (=) to the medium, were 4 hours in root tips and 6 hours in excised leaves. Addition of NO(3) (-) in the induction medium together with WO(4) (=) gave partial protection of NADH-nitrate reductase and FMNH(2)-nitrate reductase activities in both root tips and leaves with a t(0.5) of 6 and 8 hours, respectively. NO(3) (-) also reduced the loss of nitrate reductase activity from mature root sections. In the presence of cycloheximide, both NADH-nitrate reductase and NO(3) (-)-induced NADH-cytochrome c reductase activities were lost at similar rates in root tips. NO(3) (-) protected the loss of NO(3) (-)-induced NADH-cytochrome c reductase to the same extent as that of NADH-nitrate reductase.

Nitrate reductase from bacteroids of Rhizobium japonicum: enzyme characteristics and possible interaction with nitrogen fixation

The soluble nitrate reductase of Rhizobium japonicum bacteroids has purified and its properties compared to those of aerobically grown cells. The enzymes from both sources are similar with molecular weights of about 70 000 suggesting no close relationship with the molybdo-protein component of nitrogenase. Nitrite, the product of nitrate reductase, strongly inhibited the nitrogenase activity from bacteroids, at concentrations less than 100 μM. Thus, an interference in the rate of nitrogen fixation is possible as a result of nitrate reductase activity. A study of the distribution of nitrate reductase in bacteroids indicates that a proportion of the total activity is membrane-bound but that this activity is similar to that in the soluble fraction. Purified nitrate reductase required reduced viologen dyes for activity. Neither NADPH or NADH or FAD could substitute as electron donors. Dithionite is a strong inhibitor and inactivated nitrate reductase from all sources examined. This inactivation is prevented by methyl viologen. Purified nitrate reductase from bacteroids and bacteria Rhizobium japonicum is practically unaffected by exposure to oxygen.

Serological estimation of spinach nitrate reductase

An antiserum raised in guinea pigs to a preparation of spinach nitrate reductase inactivated the enzyme. The amount of antiserum required to produce 50% inactivation of a standardized nitrate reductase preparation was increased by addition of enzymically inactive nitrate reductase. A quantitative relationship exists between the original activity of the added enzyme and the required increase in antiserum.

Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora

Evidence is presented which suggests that the NAD(P)H-cytochrome c reductase component of nitrate reductase is the main site of action of the inactivating enzyme. When tested on the nitrate reductase (NADH) from the maize root and scutella, the NADH-cytochrome c reductase was inactivated at a greater rate than was the FADH 2-nitrate reductase component. With the Neurospora nitrate reductase (NADPH) only the NADPH-cytochrome c reductase was inactivated. p-Chloromercuribenzoate at 50 μM, which gave almost complete inhibition of the NADH-cytochrome c reductase fraction of the maize nitrate reductase, had no marked effect on the action of the inactivating enzyme. A reversible inactivation of the maize nitrate reductase has been shown to occur during incubation with NAD(P)H. In contrast to the action of the inactivating enzyme, it is the FADH 2-nitrate reductase alone which is inactivated. No inactivation of the Neurospora nitrate reductase was produced by NAD(P)H alone and also in the presence of FAD. The lack of effect of the inactivating enzyme and NAD(P)H on the FADH 2-nitrate reductase of Neurospora suggests some difference in its structure or conformation from that of the maize enzyme. A low level of cyanide (0.4 μM) markedly enhanced the action of NAD(P)H on the maize enzyme. Cyanide at a higher level (6 μM) did give inactivation of the Neurospora nitrate reductase in the presence of NADPH and FAD. The maize nitrate reductase, when partially inactivated by NADH and cyanide, was not altered as a substrate for the inactivating enzyme. The maize root inactivating enzyme was also shown to inactivate the nitrate reductase (NADH) in the pea leaf. It had no affect on the nitrate reductase from either Pseudomonas denitrificans or Nitrobacter agilis.

Ammonium inactivation of nitrate reductase in Lemna minor L.

The addition of ammonium to nitrate induced plants of Lemna minor L. brings about a rapid loss in extractable nitrate reductase activity. This inactivation is reversible both in vivo and in vitro. Inhibitors of RNA and protein synthesis do not protect nitrate reductase against ammonium inactivation. It is suggested that factors, in addition to ammonium ions, are components of the inactivating system. Inactivation may involve some form of protein-protein interaction. The physiological significance of rapid ammonium inactivation of nitrate reductase is discussed.

Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes

1. 1.|The respiratory nitrate reductase of Klebsiella aerogenes was solubilized from the bacterial membranes by deoxycholate and purified further by means of gel chromatography in the presence of deoxycholate, and anion-exchange chromatography. 2. 2.|Dependent on the isolation procedure two different homogeneous forms of the enzyme, having different subunit compositions, can be obtained. These forms are designated nitrate reductase I and nitrate reductase II. Both enzyme preparations are isolated as tetramers having sedimentation constants ( s 20, w ) of 22.1 S and 21.7 S for nitrate reductase I and II, respectively. The nitrate reductase I tetramer has a molecular weight of about 10 6. 3. 3.|In the presence of deoxycholate both enzyme preparations dissociate reversibly into their respective monomeric forms. The monomeric form of nitrate reductase I has a molecular weight of about 260 000 and a sedimentation constant of 9.8 S. For nitrate reductase II these values are 180 000 and 8.5 S, respectively. 4. 4.|Nitrate reductase I consists of three different subunits, having molecular weights of 117 000; 57 000 and 52 000, which are present in a 1:1:2 molar ratio, respectively. Nitrate reductase II contains only the subunits with a molecular weight of 117 000 and 57 000 in a equimolar ratio. 5. 5.|Treatment at pH 9.5 in the presence of deoxycholate and 0.05 M NaCl or ageing removes the 52 000 M r subunit from nitrate reductase I. This smallest subunit, in contrast to the other subunits, is a basic protein. 6. 6.|The 52 000 M r subunit has no catalytic function in the intramolecular electron transfer from reduced benzylviologen to nitrate. However, it appears to have a structural function since nitrate reductase II, which lacks this subunit, is much more labile than nitrate reductase I. Inactivation of nitrate reductase II can be prevented by the presence of deoxycholate. 7. 7.|The spectrum of the enzyme resembles that of iron-sulfur proteins. No cytochromes or contaminating enzyme activities are present in the purified enzyme. Only reduced benzylviologen was found to be capable of acting as an electron donor. 8. 8.| p- Chlormercuribenzoate enhances the enzymatic activity at concentrations of 0.1 mM and lower. At higher p- chlormercuribenzoate concentrations the enzymatic activity is inhibited non-competitively with either nitrate or benzylviologen as a substrate. The inhibition is not counteracted by cysteine.

The Presence of Bound Cyanide in the Naturally Inactivated Form of Nitrate Reductase of Chlorella vulgaris

Abstract In the presence of NADH and cyanide, NADH-nitrate oxidoreductase (EC 1.6.6.1) from Chlorella vulgaris is converted to an inactive form which is readily reactivated by ferricyanide. Experiments with H14CN indicated that the inactivation process is associated with the firm binding to the protein of 0.066 nmole of cyanide per unit of enzyme inactivated. This cyanide binding was linearly proportional to the amount of inactivation. No firm cyanide binding and no inactivation occurred in the absence of NADH or an equivalent reductant. The bound 14C was released when the enzyme was reactivated by ferricyanide. After the cells had been treated with ammonia for several hours prior to disruption, the crude cell extracts contained the nitrate reductase primarily in the inactive form. Critical examination of the methods of cell disruption suggested that the inactivation of the enzyme had truly occurred in vivo. After 300-fold purification, activation of this in vivo inactivated enzyme resulted in the release of 0.066 nmole of HCN per unit of enzyme activated. We conclude that the inactivation of nitrate reductase in vivo involves the formation of a firmly bound complex of reduced enzyme and cyanide. The reaction between reduced enzyme and HCN may be written: Er + HCN (ka)/⇄/(kd) Er-HCN The measured value for ka was 1.25 x 106 m-1 min-1, for kd, 4.5 x 10-4 min-1, giving a dissociation constant, Kd = 3.6 x 10-10 m.

Inactivation of nitrate reductase by NADH in Nitrobacter agilis

Nitrate reductase from Nitrobacter agilis was inactivated by NADH (but not by NADPH) in the absence of nitrate. The inactivation of the enzyme by over-reduction with NADH was overcome by oxidizing the reduced enzyme with nitrate, ferricyanide, NAD + or NADP +.

Presence of HCN in Chlorella vulgaris and its possible role in controlling the reduction of nitrate

NITRATE reductase of Chlorella vulgaris is present in crude extracts mainly in an inactive form1–3 which may be converted to an active form by the addition of an artificial oxidant such as ferricyanide4. In crude extracts from which low-molecular weight substances have been removed by gel filtration, added NADH (or NADPH) causes a rapid conversion of the active form of nitrate reductase to the inactive form5. Losada and coworkers have also observed an inactivation of the nitrate reductase from Chlorella fusca6 and Chlamydomonas reinhardi7 by added NADH. A low-molecular weight fraction separated from crude extracts of Chlorella vulgaris also causes an inactivation of nitrate reductase when added back to the extract which had been treated with ferricyanide and passed through a Sephadex column to remove excess reagent5. In all cases the enzyme can be reactivated by the addition of the oxidant, ferricyanide4–8. On purification of the enzyme, Solomonson9 found that two substances must be present simultaneously to cause the reversible inactivation: (1) a reducing substance, preferably NADH, and (2) a substance that behaves like HCN. The molar concentration of HCN needed to effect a rapid and reversible inactivation of the enzyme was of about the same magnitude as the enzyme concentration. Thus, activated, suitably purified nitrate reductase with added excess NADH, provides an exceedingly sensitive, though non-specific test for HCN, as shown in Fig. 1.

Purification and properties of a nitrate reductase-inactivating enzyme

The NADH nitrate reductase-inactivating enzyme in the maize root has been purified 460-fold. It was precipitated by 40–60% saturation with (NH 4) 2SO 4 and was soluble at pH 4.0. On a CM-32 cellulose column, equilibrated with 10 mM acetate (ph 5.0), it was eluted after a lag as a single peak with 10 mM acetate containing 50 mM NaCl (pH 5.0). It was then characterized on a Sephadex G-200 column and its molecular weight estimated to be 44 000. The inactivating enzyme, like nitrate reductase, has its optimum activity in the neutral pH range and appears to be located in the cytoplasm of the mature-root cell. It was more stable to heat treatment than nitrate reductase. Inhibition of the inactivating enzyme by phenylmethylsulphonyl fluoride suggested the involvement of a serine residue at its active site. When phenylmethylsulphonyl fluoride was used in the extraction medium for the mature root, the in vitro inactivation of nitrate reductase was prevented. The fraction containing the inactivating enzyme degraded casein by a phenylmethylsulphonyl fluoride-sensitive reaction but it had no peptidase activity.

Reversible inactivation by ammonia of assimilatory nitrate reductase in Cyanidium caldarium.

Reversible inactivation of nitrate reductase by ammonia in intact cells ofCyanidium caldarium is described. In this organism the phenomenon occurs in the light or in the dark, in contrast with green algae where it is reported that it occurs only in the light and in the presence of CO2.

A Nitrate Reductase Inactivating Enzyme from the Maize Root

The nitrate reductase in the mature root extract of 3-day maize (Zea mays) seedlings was relatively labile in vitro. Insoluble polyvinylpyrrolidone used in the extraction medium produced only a slight increase in the stability of the enzyme. Mixing the mature root extract with that of the root tip promoted the inactivation of nitrate reductase in the latter. The inactivating factor in the mature root was separated from nitrate reductase by (NH(4))(2)SO(4) precipitation. Nitrate reductase was found in the 40% (NH(4))(2)SO(4) precipitate, while the inactivating factor was largely precipitated by 40 to 55% (NH(4))(2)SO(4). The latter fraction of the mature root inactivated the nitrate reductase isolated from the root tip, mature root, and scutellum. The inactivating factor, which has a Q(10) 15 to 25 C of 2.2, was heat labile, and hence has been designated as a nitrate reductase inactivating enzyme. The reduced flavin mononucleotide nitrate reductase was also inactivated, while an NADH cytochrome c reductase in nitrate-grown seedlings was inactivated but at a slower rate. The inactivating enzyme had no influence on the activity of nitrite reductase, glutamate dehydrogenase, xanthine oxidase, and isocitrate lyase. The activity of the nitrate reductase inactivating enzyme was not influenced by nitrate and was also found in the mature root of minus nitrate-grown seedlings.

Mechanism of nitrate reductase reversible inactivation by ammonia in Chlamydomonas

Ammonia promotes in vivo the conversion of the active form of nitrate reductase into its inactive form by indirectly causing the reduction of the enzyme. Apparently ammonia acts as an uncoupler of noncyclic photophosphorylation, a process which in turn leads to a rise in the level of reducing power in the cell. The transformation is reversible, and, upon ammonia removal, the enzyme becomes again oxidized and active. Nitrate reductase inactivation by ammonia requires light and does not occur when the noncyclic electron flow is blocked or when the photosynthetically generated reducing power is being simultaneously oxidized. In the dark and in the absence of ammonia, the enzyme appears either active or inactive in response to the degree of aerobicity or anaerobicity of the culture.

Regulation by ammonia of nitrate reductase synthesis and activity in [formula omitted

In the green alga Chlamydomonas reinhardi , all the enzymes of the nitrate-reducing system are nutritionally repressed by ammonia. Besides, ammonia promotes in vivo the reversible inactivation of nitrate reductase in an indirect way by primarily raising the cellular level of reducing power, which in turn determines the reversible reduction of the enzyme. Subsequent removal of ammonia from the medium causes reactivation of the inactive enzyme. Interconversion of the active and inactive forms into one another can also be directly achieved in vitro by reducing and oxidizing nitrate reductase with its own physiological substrates.