Planta

Planta 144, 137- 141 (1979)

9 by Springer-Verlag 1979

Pyridine Nucleotide Specificity and Other Properties of Purified Nitrate Reductase from Chlorella variegata C.R. Hipkin, B.A. A1-Bassam, and P.J. Syrett Department of Botany and Microbiology, University College of Swansea, Singleton Park, Swansea SA2 8PP, U.K.

Abstract. Nitrate reductase (NR) (EC 1.6.6.2.) from Chlorella variegata 211/10d has been purified by blue sepharose affinity chromatography. The enzyme can utilise N A D H or N A D P H for nitrate reduction with apparent Km values of 11.5 laM and 14.5 gM, respectively. Apparent Km values for nitrate are 0.13 mM ( N A D H - N R ) and 0.14 m M ( N A D P H - N R ) . The diaphorase activity of the enzyme is inhibited strongly by parachtoromercuribenzoic acid; N A D H or N A D P H protects the enzyme against this inhibition. N R proper activity of the enzyme is partially inactive after extraction and may be activated after the addition of ferricyanide. The addition of N A D ( P ) H and cyanide causes a reversible inactivation of the N R proper activity although preincubation with either N A D H or N A D H and A D P has no significant effect. Key words: ChlorelIa - Nitrate reductase - Pyridine nucleotide specificity.

as is apparently so in many other species of algae, particularly non-chlorophytes (Hipkin et al., 1978), and most higher plants (Beevers et al., 1964). In other unicellular algae such as Dunaliella (Le Claire and Grant, 1972; Heimer, 1976), Ankistrodesmus (Zumft et al., 1972; Ahmed and Spiller, 1976; Hipkin and Syrett, 1977), Cyanidium (Rigano, 1971) and ChIamydomonas (Sosa and Cardenas, 1977; Hipkin et al., 1978), either N A D H or N A D P H can donate electrons for enzymic nitrate reduction in vitro. We have purified the nitrate reductase from ChlorelIa variegata 211/10d by affinity chromatography and show here that the enzyme can utilise either N A D H or N A D P H as electron donor. Other properties of the enzyme such as its activation, inactivation and reactivation are also described and discussed.

Materials and Methods Introduction In algae, the molybdoflavoprotein, nitrate reductase, catalyses the transfer of electrons from a reduced pyridine nucleotide to nitrate in a sequential redox reaction involving a FAD-dependent N A D ( P ) H - d i a p h o rase and a molybdenum containing nitrate reductaseproper (Losada, 1973). In algae, particularly freshwater chlorophytes, certain properties of the enzyme have been studied thoroughly. In most species of Chlorella studied so far, nitrate reductase has been shown to be a NADH-specific enzyme (Shafer et al., 1961; Syrett and Morris, 1963; Zumft et al., 1969) Abbreviations. NR~Nitrate reductase; FAD=Flavin-adenine dinucleotide; FMN=Riboflavin 5'-phosphate; p-CMB=para-Chloromercuribenzoic; BV= Benzyl viologen

Growth of Organism Chlorella variegata (Cambridge Culture Collection strain number 211/10d) was grown under conditions described elsewhere for C. fusca vat. vacuolata (Syrett, 1973).

Extraction and Purification of Nitrate Reductase

Crude extracts of C. variegata were prepared in 0.05 M phosphate buffer, pH 7.5, containing 5 ~tM FAD (unless stated otherwise). Cells were broken after passage through a French pressure cell at 4~ C and 100 MPa and extracts were clarified by centrifugation at 36,000g at 3~ C. Subsequent purification steps involving protamine sulphate, ammonium sulphate and blue dextran affinity chromatography were as described by Solomonson (1975) except that the affinity gel was commercial Blue Sepharose CL-6B (Pharmacia Fine Chemicals) and extracts were allowed to equilibrate with the gel in the column for 16 h prior to elution procedures.

0032-0935/79/0144/0137/$01.00

C.R. Hipkin et al. : Nitrate Reductase from Chlorella

138

Table 1. Purification of nitrate reductase from Chlorella variegata Fraction

Crude extract Protamine sulphate Ammonium sulphate Blue sepharose (NADH-elution)

NADH-nitrate reductase

NADPH-nitrate reductase

Specific activity

Purification

Recovery %

Specific activity

Purification

Recovery %

3.2 6.4 18.1 718

1 2 5.7 224

100 78 53 28

4.2 8.7 34.1 766

1 2.1 8.1 182

100 83 77 21

Cell free extracts from nitrate-grown cells of Chlorella variegata were prepared as described in Materials and Methods and purification procedures were followed, as described by Solomonson (1975). Specific activity is nmoles NO'2 formed rain i m g protein- t

Enzyme Assays NAD(P)H-nitrate-reductase activity was assayed in a final volume of 1.3 ml containing 50 gmol of phosphate buffer, pH 7.5, 5 nmol FAD, 10 gmol KNOa and 0.8 gmol NAD(P)H. Reactions were stopped after 10 min at 30~ by the addition of 200 ~tmol zinc acetate and 1.0 ml 95% ethanol. After clarification by centrifugation nitrite was estimated by the Griess-Ilosvay method as described elsewhere (Hipkin and Syrett, 1977). FMN-nitrate reductase and FAD-nitrate reductase were assayed in a final volume of 1.55 ml as described above except that NAD(P)H was replaced by 17 ~tmol FMN or 17 gmol FAD chemically reduced after the addition of 2 mg of sodium dithionite and 0.25 mg sodium bicarbonate. Benzyl viologen-nitrate reductase was assayed in a final volume of 1.6 ml as described above except that 0.5 gmoles benzyl viologen chemically reduced by the addition of 2 mg dithionite and 2 mg sodium bicarbonate was used as electron donor in the presence of 50 gmol KNO3. Cytochrome " c " rednctase activity was measured in a final volume of 2.8 ml containing 90 gmol phosphate buffer pH 7.5, 0.8 ml 1% cytochrome " c " . Reactions were initiated by the addition of 0.4pmol NADH and the reduction of cytochrome "c" was followed at 550nm in a Pye Unicam S.P. 2000 spectrophotometer.

211/10d we discovered that in cell-free extracts from both strains nitrate was enzymatically reduced to nitrite when either N A D H or N A D P H was added as electron donor. Since pyridine nucleotide bispecificity is an unusual feature of nitrate reductases from Chlorella spp. we decided to purify the nitrate reductase from strain 211/10d by blue sepharose affinity

500

400

~300 .~ "5 ID

Protein Estimation Protein was measured in extracts by the method of Lowry et al. (1951).

U

B

A NADPH

20o

-I Activation, Inactivation and Reactivation of Nitrate Reductase Enzyme preparations were activated after 10 rain incubation with 0.6 mM potassium ferricyanide at 20 ~ C, inactivated by incubation with 0.3 mM NAD(P)H and 3 gM potassium cyanide at 20~ and reactivated by the preincubation of inactivated enzyme with 0.6 mM potassium ferricyanide at 20 ~ C.

-i

100

,

z,

Results

Purification and Pyridine Nucleotide Specificity In some preliminary experiments on the properties of nitrate reductase in Chlorella variegata 211/10a and

8 12 14 16 Fraction number

20

Fig. I. Elution profile of nitrate reductase from Blue Sepharose CL-6B. The elution procedure used involving low ionic buffer (A) and high ionic buffer (B) and reduced pyridine nucleotide buffer, was that described by Solomonson (1975) except that NADPH was used as the reduced pyridine nucleotide. Specific activity is nmol NO2 formed rain- 1 mg protein- ~

139

C.R, Hipkin et al. : Nitrate Reductase from Chlorella Table 2. Activities of nitrate reductase (NR) from Chlorella variegata Assay

Specific activity

NADH-NR NADPH-NR BV-NR FMN-NR FAD-NR Cytochrome 'c' reductase

206 249 144 14l 177 262

Table 4. In vitro activation, inactivation and reactivation of nitrate reductase (NR) in Chlorella variegata Additions

NADHNR

Nitrate reductase purified after NADH-elution of the blue sepharose column was assayed for the stated activities as described in Materials and Methods. Specific activity is nmol NO'2 formed min- * mg protein-

Table 3. Apparent Km values of substrates for NAD(P)H-nitrate

reductase (NR) from Chlorella variegata Substrate

Km app. gM NADH-NR

NADH NADPH NO'a

% Activity

NADPH-NR

None 100 Ferricyanide 168 KCN + NADH 4 KCN + NADPH 4 KCN + NADH + Nitrate 100 KCN + NADPH + Nitrate 92 KCN + NADH + Ferricyanide 72 KCN+NADPH+ Ferricyanide 88

NADPHNR

BVNR

100 189 4 4 114 102 82 93

100 129 19 22 97 94 69 69

Partially purified preparations of nitrate reductase ((NH,)2SO4fractions) were activated, inactivated and reactivated as described in Materials and Methods. KNO3 was added to a concentration of 10 raM. 100% activities were as follows: NADH-NR= t5 nmol NOz formed min i mg protein- 1; NADPH-NR = 19.8nmol NO2 formed rain -~ mg protein; BV-NR=24.3nmol NO2 formed rain- ~ mg protein- 1

11.5 14.5 140

130

Nitrate reductase was purified 200-fold after blue sepharose affinity chromatography. Assay conditions were as described in Materials and Methods and Km values were obtained from Woolf plots

20 16

r~ 8 z g

o"'~" f 6

f 7

P 7.5 pH

I 8

I 9

Fig. 2. Effect of pH on the activity of partially purified nitrate reductase (NR). Partially purified enzyme preparation ((NH4)2SO4-fractions) were incubated at the stated pH's in the presence of 0.05 M phosphate buffer (circles) or tris buffer (triangles) and their NADH (shaded symbols) and NADPH (open symbols) nitrate reductase activities were measured. NR activity is nmol NOe formed min ~ mg protein-J

the p u r i f i c a t i o n procedure N A D P H - n i t r a t e reductase activity exceeded N A D H - n i t r a t e reductase activity. F u r t h e r m o r e , Fig. 1 shows that N A D H nitrate reductase activity could be eluted off the affinity c o l u m n using N A D P H buffer. Similar results were o b t a i n e d in the e l u t i o n of N A D P H - n i t r a t e reductase activity with N A D P H buffer. The activity of the purified enzyme with various electron d o n o r s is s h o w n in T a b l e 2. R e d u c e d F M N , F A D a n d benzyl viologen were less effective as elect r o n d o n o r s t h a n N A D H and N A D P H . S u b s t a n t i a l a m o u n t s of c y t o c h r o m e " c " reductase activity were associated with purified fractions of n i t r a t e reductase and p r e s u m a b l y represented the d i a p h o r a s e activity of the enzyme. A p p a r e n t Km values for N A D H , N A D P H a n d nitrate are s h o w n in T a b l e 3. The enzyme showed a slightly higher affinity for N A D H while the apparent Km values for nitrate, i.e. 130 btM ( N A D H - n i t r a t e reductase) and 140 btM ( N A D P H - n i t r a t e reductase), were n o t significantly different. Both pyridine n u c l e o t i d e - n i t r a t e reductase activities showed p H o p t i m a in a relatively b r o a d range between p H 7.4 a n d p H 7.7 (Fig. 2).

In Vitro Activation and Inactivation c h r o m a t o g r a p h y to study this a n d other properties of the enzyme. Table 1 shows that after affinity chrom a t o g r a p h y with N A D H elution, a n a p p r o x i m a t e 200-fold p u r i f i c a t i o n of N A D ( P ) H - n i t r a t e reductase activity was obtained. Moreover, at each step d u r i n g

U s i n g partially purified enzyme p r e p a r a t i o n s we also studied the in vitro activation, i n a c t i v a t i o n and reactiv a t i o n of the N A D ( P ) H - n i t r a t e reductase activity a n d the benzyl v i o l o g e n - n i t r a t e reductase activity from C.

C.R. Hipkin et al. : Nitrate Reductase from Chlorella

140 Table 5. Effect of preincubation with NADH and ADP on nitrate

reductase (NR) activity in Chlorellavariegata Additions

Specific activity NADH-NR

NADPH-NR

Zerotime None

14.5

18.9

After 100 rain None NADH ADP NADH, ADP NADH, ADP, MgClz

14.25 14.50 12.8 14.4 14.5

18.0 19.2 18.9 18.9 19.4

Partially purified preparations ((NH,)2SO, fractions) of nitrate reductase were incubated with the stated additions for 100 min at 0~ NADH and ADP were added to a concentration of 0.3 mM and MgC12 was added to a concentration of 10 mM. Enzyme activities were assayed as described in Materials and Methods

Table 6. Effect of para-chloro mercuribenzoic acid on nitrate

reductase activity in Chlorella variegata Additions

None p-CMB NADH+p-CMB NADPH +p-CMB

% Activity NADH-NR

NADPH-NR

BV-NR

100 4 72 116

100 28 96 97

t 00 92 104 100

Partially purified preparations of nitrate reductase ((NH4)2SO4fractions) were incubated with stated additions at the following concentrations: p-CMB, 20 gM; NAD(P)H, 0.3 mM; NAD(P)H was always added prior to p-CMB addition

variegata (Table 4). All three enzymic activities from partially purified preparations were partially inactive and could be activated by the addition of ferricyanide. Enzyme activities could be further inactivated by the addition of N A D ( P ) H and cyanide and this effect could be largely reversed by the subsequent addition of ferricyanide. When nitrate was added prior to N A D ( P ) H and cyanide the enzyme was protected from inactivation, in other experiments we have found that chlorate also protects N A D ( P ) H and benzyl viologen-nitrate reductase activities against cyanide-inactivation, but the extent of the protection is much less and results have been very inconsistent, (data not shown). We have also attempted to inactivate N A D ( P ) H nitrate reductase activity in partially purified extracts

from C. variegata using N A D H and A D P (Table 5). Neither N A D H alone nor N A D H and A D P had any effect on enzyme activity. Table 6 shows the effect of para-chloromercuribenzoic acid (p-CMB), and inhibitor of active sulphydryl groups, on partially purified nitrate reductase from C. variegata. Both N A D H and N A D P H - n i t r a t e reductase activities were inhibited by p-CMB although benzyl viologen-nitrate reductase activity was unaffected. Significantly, when either N A D H or N A D P H was added prior to the addition of p-CMB, NAD(P)H-nitrate reductase activity was protected against the inhibition.

Discussion

Since the early reports of pyridine nucleotide specificity of nitrate reductase from algae and higher plants (Shafer et al., 1961 ; Syrett and Morris, 1963; Beevers et al,, 1964) it has c o m m o n l y been assumed that the probable physiological electron donor for nitrate reduction in such plants is N A D H . This assumption, however, may not be valid for some species of green algae (Heimer, 1976; Sosa and Cardenas, 1977; Hipkin and Syfett, 1977). We have shown here that purified nitrate reductase from Chlorella variegata has both N A D H and N A D P H activites. During the purification process N A D P H activity always exceeds N A D H activity and therefore we exclude the possibility of phosphatase interference in our assays. However, we do not exclude the possibility that two separate enzyme components may be involved i.e. a N A D H - n i t r a t e reductase protein and a N A D P H - n i t r a t e reductase protein. For the following reasons, however, we find the possibility of separate enzymes unlikely; (i) N A D ( P ) H nitrate reductase activity may be eluted off the affinity column with either N A D H or N A D P H , (ii) the relative similarities of apparent Km values for the pyridine nucleotides and nitrate, (iii) the similar p H profiles for both activites and (iv) the protection of both activities against p - C M B inactivation by either N A D H or N A D P H . Regarding p-CMB inactivation, it is interesting to note that in Chlorella fusca, an alga with a NADH-specific nitrate reductase activity, N A D H but not N A D P H ist effective in protecting the enzyme (Vega et al., 1972). The enzyme activity from C. fusca, however, resembles the nitrate reductase proper activity from C. variegata (i.e. benzyl viologen-nitrate reductase activity or FNH2-nitrate reductase activity) in that it is little affected by the addition of p-CMB (Moreno et al., 1972). We assume from these experiments that p-CMB exerts its effects on nitrate reduc-

C.R. Hipkin et al. : Nitrate Reductase from Chlorella

tase primarily at the site of NAD(P)H attachment on the diaphorase. Since NADPH-nitrate reductase extracted from cells of C. variegata is partially inactive and may be activated by preincubation with ferricyanide, the behaviour of the enzyme resembles that of the nitrate reductase from C. vulgaris but not that from C. fusca (Vennesland and Solomonson, 1972). Like the NADH-nitrate reductase activities of C. fusca (Vega etal., 1972) C. vulgaris (Solomonson, 1974)and Chlamydomonas reinhardi (Barea et al., 1976) the NAD(P)H-nitrate reductase activity and the benzyl viologen-nitrate reductase activity of C. variegata is inactivated by cyanide in the presence of NADH or NADPH but the enzyme may be largely reactivated after preincubation with ferricyanide or protected by the addition of nitrate prior to the addition of cyanide. We have been unable to inactivate NAD(P)Hnitrate reductase activity in partially purified preparations from C. variegata by long term preincubation with NADH alone or with NADH and ADP. These results therefore differ from those reported with nitrate reductases from C. fusca (Maldonado et al., 1973) and Ankistrodesmus braunii (Diez et al., 1977).

References Ahmed, J., Spiller, H.: Purification and some properties of the nitrate reductase from Ankistrodesmus braunii. Plant Cell Physiol. 17, 1-10 (1976) Barea, J.L., Sosa, F., Cardenas, J. : Cyanide inactivation of Chlamydomonas reinhardi nitrate reductase under reducing conditions. Z. Pfianzenphysiol. 79, 237-245 (1976) Beevers, L., Flesher, D., Hageman, R.H. : Studies on the pyridine nucleotide specificity of nitrate reductase in higher plants and its relationship to sulfhydryl level. Biochem. Biophys. Acta 89, 453-464 (1964) Diez, J., Chaparro, A., Vega, J.M., Relimpio, A.: Studies on the regulation of assimilatory nitrate reductase in Ankistrodesrnus braunii. Planta 137, 231-234 (1977) Heimer, Y.M.: Specificity for nicotinamide adenine dinucleotide and nicotinamide adenine dinuclcotide phosphate of nitrate reductase from the salt-tolerant alga Dunatie[la parva. Plant. Physiol. 58, 57-59 (1976) Hipkin, C.R., Syrett, P.J.: Nitrate reduction by whole cells of Ankistrodesmus braunii and Chlamydomonas reinhardi. New Phytol. 79, 639 648 (1977)

141 Hipkin, C.R., Syrett, P.J., A1-Bassam, B.A. : Nitrate reductase activity in unicellular algae, a comparative study. Brit. Phyc. J. 13 (I978), in press Le Claire, J.A., Grant, B.R.: Nitrate reductase from Dunaliella tertiolecta, purification and properties. Plant Cell Physiol. 13, 899-907 (1972) Losada, M.: Interconversions of nitrate and nitrite reductase of the assimilatory type. In: Metabolic interconversions of enzymes. III Internat. Syrup., p. 257, Fischer, E.H., Krebs, E.G., Neurath, H., Stadtman, E.R. (eds.). Seattle 1973, Berlin, Heidelberg, New York: Springer 1974 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Maldonado, J.M., Herrera, J., Paneque, A., Losada, M.: Reversible inactivation by NADH and ADP of Chlorella fusca nitrate reductase. Biochem. Biophys. Res. Commun. 51, 27-33 (1973) Moreno, C.G., Aparicio, P.J., Palacian, E., Losada, M.: Interconversion of the active and inactive forms of Chlorella nitrate reductase. FEBS Lett. 26, 11-14 (1972) Rigano, C. : Studies on nitrate reductase from Cyanidium cladarium. Arch. Mikrobiol. 76, 265-276 (1971) Shafer, J., Baker, J.E., Thompson, J.F. : A Chlorella mutant lacking nitrate reductase. Am. J. Bot. 48, 896-899 (1961) Solomonson, L.P.: Regulation of nitrate reductase activity by NADH and cyanide. Biochim, Biophys. Aeta 334, 297 308 (l 974) Solomonson, L.P. : Purification of NADH-nitrate reductase by affinity chromatography. Plant. Physiol. 56, 853-855 (1975) Sosa, F.M., Cardenas, J.: NADP as electron donor for nitrate reduction in Chlamydomonas reinhardi. Z. Pflanzenphysiol. 85, 171-175 (1977) Syrett, P.J. : Measurement of nitrate and nitrite reductase activities in whole cells of Chlorella. New Phytol. 72, 37 46 (1973) Syrett, P.J., Morris, I.: The inhibition of nitrate assimilation by ammonium in Chlorella. Biochim. Biophys. Acta. 67, 566-575 (1963) Vega, J.M., Herrera, J., Relimpio, A.M., Aparicio, P.J. : NADHnitrate r6ductase de Chlorella." nouvelle contribution 5. l'6tude de ses propri6t6s. Physiol. V6g. 10, 637-652 (1972) Vennesland, B., Solomonson, L.P.: The nitrate reductase of Chlorella. Species or Strain differences. Plant Physiol. 49, 1029-1031 (1972) Zumft, W.G., Paneque, A., Aparicio, P.J., Losada, M. : Mechanism of nitrate reduction in Chlorella. Biochem. Biophys. Res. Cornmum 36, 980-986 (1969) Zumft, W.G., Spiller, H., Yeboah-Smith, I. : Eisengehalt und Elektronendonator-Spezifisitfit der Nitratreductase yon Ankostrodesmus. Planta 102, 228-236 (1972)

Received 5 June; accepted 28 August 1978