Immunological studies with nitrogenase from Azotobacter and bacterial nitrate reductases

ANALYTICAL

95, 24-31 (1979)

BIOCHEMISTRY

Immunological D.J.D.

Studies with Nitrogenase from Azotobacter Bacterial Nitrate Reductasesl NICHOLAS,JUDY V. FERRANTE,ANDG.

Department of Agricultural

and

R. CLARKE

Biochemistry, Waite Agricultural Research Institute, The University of Adelaide, Glen Osmond, 5064 South Australia Received October 1978

Antibodies raised to purified Component I of nitrogenase (MO-Fe-S) protein from Azotobatter vinelandii cross-reacted not only with this protein but also with nitrate reductases from a number of bacteria. Antibodies raised for a purified nitrate reductase fromEscherichia coli also formed precipitin bands with this Component I of nitrogenase. Antibodies to Component I, however, did not react with nitrate reductases from either a blue-green alga Anabaena cylindrica or with higher plants, or with aldehyde dehydrogenase and xanthine oxidase from animal sources.

About 25 years ago, nitrate reductase living Rhizobium japonicum to antisera prefrom Neurospora crassa was first identified pared against Component I of nitrogenase as a NADPH-linked flavoprotein containing from bacteroids of soya bean nodules. molybdenum and a mechanism of action for In this paper, techniques are described it was proposed (1,2). Subsequent work with for raising antibodies to purified Component bacteria, fungi, and green plants has amply I of nitrogenase from Azotobacter and to confirmed these basic findings (3-5). Later, purified nitrate reductase from E. coli. It is Nason and his collaborators reported that shown that each antibody type crosscytochrome b557 was also involved in elec- reacted not only with its corresponding tron transfer to nitrate in Neurospora (6,7) antigen protein, but also with the other and McGregor (8) identified cytochrome b, protein as well. as an electron carrier for nitrate reduction in Escherichia coli. MATERIALS AND METHODS Nitrogenase is known to be composed of Materials. Xanthine oxidase (EC 1.2.3.2) two proteins, namely Component I containfrom butter milk and aldehyde dehydrogening MO-Fe and S2 and Component II conase (EC 1.2.1.3) from bakers’ yeast were taining Fe and S (9). Antibodies raised in purchased from Sigma Chemical Company mice and rabbits to Component I of nitro- and Sepharose 6B from Pharmacia Chemical genase from Azotobacter have been sucCompany. All other reagents used were of cessfully used to follow the fate of this proanalar grade. tein during its repression with ammonium Culturing the bacteria. Escherichia coli ions (10). Immunodiffusion tests were also B, P903 and E. coli K-12 P801 were grown used by Bishop et al. (11) to demonstrate as described by Lund and DeMoss (12). cross-reacting material in extracts of freeAzotobacter vine/an&i strain OP was grown in modified Burk’s medium without com1 This paper is dedicated to the memory of Dr. Alvin bined nitrogen as described by Nicholas and Nason. Deering (10). It was also grown in a modified * Abbreviations used: S, Supematant fraction; IgG, immunoglobulin; SDS, sodium dodecyl sulfate. Burk’s medium supplemented with either 8 0003-2697/79/070024-08$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

24

Azo~obacter

NITROGENASE

AND

BACTERIAL

mM KN03 or 300 mM ammonium chloride. Thiobacillus denitri$cans (9547, NCIB strain AB5) was grown and harvested as described by Adams et al. (13). The blue-green alga, Anabuenu cyfindricu (Lemmerman strain 1403/2A), was cultured in the medium containing 100 mM KN03 as described by Brownell and Nicholas (14). Neurosporu uussu (wild type) strain 5297a was grown as described by Nicholas and Nason (4). Agrobucterium tumefuciens (strain 229) was grown under aerobic conditions with vigorous shaking in loo-ml batch cultures of the following medium at pH 7.0 containing per liter: 1.3 g glutamic acid, 0.5 g trisodium citrate, 1.0 g K2HPOI, 0.5 g KH,PO,, 9.5 mg FeSO,, 12.0 mg MnCl,, 3.0 mg H3B03, 3.0 mg ZnSO,, 0.3 mg Na,MoO,, 0.3 mg CuS04, 0.3 mg CoCl,, 10 ml of 20% (w/v) glucose, and 10 ml of 2% (w/v) MgSO,. Biotin and thiamine were filter sterilized through a 0.22 pm millipore unit and added at 0.2 mg and 5.0 mg/liter, respectively. Cells harvested at 2°C during the log phase of growth were washed with 0.025 M Tris-HCl, pH 7.5. Preparation of cell-free extracts. Crude extracts of the various microorganisms were prepared by passage through an Aminco French Pressure Cell at 2°C at a pressure of 200 MPa (10). The homogenates were centrifuged at 3000g for 5 min and the supernatant fractions (S,) were used for the immunodiffusion tests. In the case of A. vinelundii grown with ammonia or nitrate, the S,,, fraction was used (10). Cell-free extracts of N. crussu were prepared by the method of Nicholas and Nason (4). TABLE PURIFICATION

Fraction

Volume

Total protein (mg)

1 2 3

17.0 15.5 13.0

911 302 72

OF NITRATE

Total enzyme (units) 41,430 31,060 23.990

NITRATE

REDUCTASES

2.5

Production undpuri$cution ofantibodies. The IgG fraction of the goat antiserum raised to purified Component I was prepared as described previously (15). A rabbit antiserum to heat-released nitrate reductase from E. co/i B (Fraction 3, Table 1) was raised by the following procedure. Three subcutaneous injections of 1.5 mg protein emulsified in Freund’s complete adjuvant were administered. This was followed by two intravenous injections of 1.5 and 3.0 mg antigen, respectively. All injections were conducted at fortnightly intervals. The animal was bled 10 days after the final injection and the IgG fraction of the whole antiserum prepared by a neutral salt fractionation technique (10). Assays for enzyme activities. The C,H, reduction assay was used to monitor nitrogenase activity (10). Nitrate reductase activity was assayed at 30°C. The following components of the reaction mixture (pmol) were contained in a final volume of 2 ml in 1.1 x lo-cm test tubes: phosphate 320, sodium nitrate 10, benzyl viologen 0.4. An appropriate aliquot of the enzyme was added and the liquid surface was layered with liquid paraffin. Then 100 ~1 of freshly prepared Na,S,O, (1.56 pmol) in 2% (w/v) sodium bicarbonate was added below the paraffin layer via a gas-tight microsyringe. After a lo-min incubation period, l-ml aliquots were dispensed into test tubes in air and 1 ml of 1% (w/v) sulfanilamide in N HCl and 1 ml of 0.01% (w/v) a-naphthylethylenediamine-HCl were added. After 10 min, the absorbance at 540 nm was determined in lcm cuvettes in a Shimadzu spectrophotom1 REDUCTASE

FROM E. cd

Specific activity (nmol NOz. produced/min/ mg protein) 45.5 111 332

B Percentage recovery enzyme activity 100 15 58

Purification I 2.4 7.3

26

NICHOLAS,

FERRANTE,

eter. The specific activity of nitrate reductase is expressed in nanomoles nitrite produced per minute per milligram protein. Nitrate reductase activity in polyacrylamide gels was detected as a pink band by the staining method of Lund and DeMoss (12). Protein was determined by the microbiuret method of Itzhaki and Gill (16) using bovine serum albumin as a standard. Zrnmunodiffusion techniques. A system of diffusion in agar was employed as described previously (10). Polyacrylamide gel electrophoresis. Discontinuous electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate (SDS) was carried out using the system described by Williams and Reisfeld (17), except that stacking and separating gels and electrode buffers contained 0.1% (w/v) SDS. Samples were reduced and dissociated prior to discontinuous electrophoresis by treatment with 4 mg SDSlmg protein and 1% (v/v) 2-mercaptoethanol in a boiling water bath for 5-7 min. Bromophenol blue 0.05% (w/v) was used as the tracking dye, and the gels were stained with Coomassie brilliant blue R250 as described previously (10). A nondissociating polyacrylamide gel system was used to locate the nitrate reductase enzyme. The procedure described was modified by omitting the stacking gels and SDS. Enzyme fractions containing 10% (v/v) glycerol were loaded directly onto the separating gels. After electrophoresis, the gels were either stained for protein with Coomassie brilliant blue R250 or assayed for nitrate reductase activity as described by Lund and DeMoss (12).

AND CLARKE

Rm 0.24

a

b

Rm 0.25

c

FIG. 1. Detection of nitrate reductase in polyacrylamide gels. (a) Protein pattern of a heat-released nitrate reductase (250 pg Fraction 3, Table 1) run in a nondissociating polyacrylamide gel electrophoresis system and stained with Coomassie brilliant blue R250. (b) Nitrate reductase activity of the heat-released enzyme (500 pg Fraction 3, Table 1) run in a nondissociating polyacrylamide gel electrophoresis system. (c) Protein pattern of a heat-released nitrate reductase (100 pg, Fraction 3, Table 1) run in an SDS-polyacrylamide disc gel electrophoresis system. Nitrate reductase activity indicated by arrows. Experimental details are given under Materials and Methods.

7 g to 15 ml of 0.025 M Tris-HCI, pH 7.5, containing 5 mM MgS04, 5 fig/ml ribonuclease, and 50 &ml deoxyribonuclease were ruptured in an Aminco French Pressure Cell at 200 MPa. The crude extract was centrifuged at 20,OOOg for 30 min in a Sorvall RC2B centrifuge (SS-34 Rotor) and the supematant (Fraction 1) retained. Fraction 1 was centrifuged at 144,OOOg for 120 min in a Beckman Type 65 rotor in a Spinco ultracentrifuge and the pellet washed repH 7.5. peatedly with 0.025 M Tris-HCl, The pellet (Fraction 2) was then resuspended pH 8.3, and placed in a in 0.1 M Tris-HCl, waterbath at 60°C for 20 min. The heattreated extract was then cooled in ice and RESULTS again centrifuged at 120,OOOg for 90 min. The supematant contained the heat-released Purijicalion of Nitrate Reductase enzyme (Fraction 3), which was approxifrom E. coli B mately 80% of the nitrate reductase activity The purification procedures in Table 1 of Fraction 2. This enzyme (Fraction 3) was were carried out at 2°C unless otherwise resolved into two major proteins by nondisstated. The method follows closely on that sociating polyacrylamide gel electrophoresis (Fig. la) as described under Materials and described by Lund and DeMoss (12). Cells of E. coli B suspended in the ratio of Methods. In duplicate gels it was established

Azorobactcr

NITROGENASE

AND BACTERIAL

NITRATE

REDUCTASES

27

0.21D-

O.l! 5

-

04

O-

0.0

59

; $ .f s B

: : I

I

50

60

‘\

b

-4

70 Fraction

.--

80

--¤*

90

I 100

110

Number

FIG. 2. Purification of a heat-released nitrate reductase (Fraction 3, Table 1) on a Sepharose 6B gel column equilibrated with 0.1 M NaCl in 0.05 M Tris-HCI, pH 7.5. Nitrate reductase activity (Nmol NO,- produced/ml) and protein (mg/ml). Protein patterns of eluted fractions run in a nondissociating polyacrylamide gel electrophoresis system and stained with Coomassie brilliant blue R250. Amounts loaded were 150, 150, and 145 pg for (a), (b), and (c), respectively. Nitrate reductase activity indicated by arrows. Experimental details are given under Materials and Methods.

that nitrate reductase activity was associated phoresis showed that the protein composiwith the protein band at R, = 0.24-0.25 tion changed across the peak (Fig. 2). Nitrate (Fig. lb). When Fraction 3 was dissociated reductase activity was associated with both and reduced with SDS and 2-mercaptoethamajor protein bands in fraction (70) (Fig. 2b) no1 and run on SDS-polyacrylamide gel disc which had the highest specific activity for the electrophoresis, although the protein band enzyme. Initial and late fractions of this at R, = 0.24-0.25 remained it was less in- peak (65) (Fig. 2a) and (75) (Fig. 2C) had tense, while other bands appeared at R, lower specific activities and each contained = 0.42, 0.47, and 0.48 (Fig. lc). The most one of the two major proteins present in (b). prominent band in the solubilized enzyme An interesting result of the staining procewas a fast-moving component at R, = 0.98 dure for nitrate reductase activity in gels for (Fig. lc). Fraction 3 was separated on a late fractions (97- 102) from the second peak Sepharose 6B gel column (2.5 x 100 cm) of the elution profile was the formation of a and 4-ml fractions were collected (Fig. 2). dense white band at a position immediately Nitrate reductase activity was located en- prior to the bromophenol blue marker (R, tirely in the first peak of the elution profile. = 0.98). These fractions contained FMN An analysis of the proteins in this area by identified by high voltage paper electronondissociating polyacrylamide gel electrophoresis and paper chromatography (18).

28

NICHOLAS,

Immunological Reductase

Reactions

FERRANTE,

of Nitrate

An antiserum to the heat-released nitrate reductase (Fraction 3, Table 1) was raised in a rabbit and the IgG fraction was prepared as described under Materials and Methods. The reaction of this immunoglobulin with its immunogen consisted of a heavy precipitin line and a less discrete band (Fig. 3). In immunodiffusion reactions where cell-free extracts of A. vinelandii grown with nitrate and heat-released nitrate reductase from 25. co/i B (Fraction 3, Table 1) were placed in adjacent wells (H and G, respectively) and reacted against the antibodies to nitrate reductase, a spurring reaction of partial identity was observed as shown in Fig. 3. The A. vinelandii extract did not contain ComTABLE

AND CLARKE

ponent I because, when supplemented with purified Component II [Fraction 5, Table 1, Ref. (lo)] it did not reduce C,H, to CzH4, neither was it detected by gel electrophoresis. It is of interest that purified Component I from A. vinelandii [Fraction 5, Table 1, Ref. (lo)] yielded a single precipitin line crossreaction with antibody to nitrate reductase (Fig. 4). Immunological Reactions Of Nitrogenase

of Component

Z

An antiserum to purified Component I of A. vinetandii again gave a characteristic double precipitin line after immunodiffusion against the immunogen as shown in Fig. 5 and described previously (10). Reactions of partial identity between antibody to Com2

RESULTS ok IMMUNODIFFLJSION TESTS WITH ANTIBODY TO PURIFIED COMPONENT I FROM A. vinelandii AND ANTIBODYTO PURIFIED NITRATE REDUCTASE FROM E.coli B WITH ANTIGENS FROM VARIOUS SOURCES Antibody to Component I from Antigen Purified from Purified from

A. vinelandii

Antibody to nitrate reductase from E. coli B

Component I

BVH-linked nitrate reductase activity

A. vinelandii

+

+

-

nitrate reductase E. coli B

+

+

+

+

+

+

+

+

+

+

+

+

Crude extracts of Agrobacterium tumefaciens E, co/i K-12 Thiobacillus denitrificans Anabaena cylindrica

(grown with nitrate) Neurospora A. vinelandii

crassa

(grown with ammonia) A. vinelandii (grown with nitrate) Aldehyde dehydrogenase (from yeast) Xanthine oxidase (from buttermilk)

-

-

+

-

-

+

-

-

-

+

+

+

-

-

-

-

Azotobacrer

NITROGENASE

AND BACTERIAL

NITRATE

REDUCTASES

29

nor nitrogenase was detected in extracts of these cells. Purified aldehyde dehydrogenase from yeast and xanthine oxidase from buttermilk which contain molybdenum and nonheme iron did not react immunologically with the antiserum for either Component I of nitrogenase or nitrate reductase nor did they contain a reduced benzyl viologen-linked nitrate reductase activity. DISCUSSION

FIG. 3. Ouchterlony immunodiffusion assays in agar gel. Comparison of precipitin reaction of (a) 60 pg heatreleased nitrate reductase (Fraction 3, Table 1) in wells A, B, C, and G and (b) 380 pg of a cell-free extract of A. vinelandii grown with nitrate (S,,,) in wells D, E, F, H, I, and J. Five hundred micrograms of the IgG fraction of antiserum to heat-released nitrate reductase was contained in the three center wells. Experimental details are given under Materials and Methods.

ponent I of nitrogenase and cell-free extracts of bacteria containing nitrate reductase, namely, E. coli K-12, Thiobacillus denitrifcans, and Agrobacterium tumefaciens were also observed (Figs. 5 and 6). Extracts of these bacteria did not have any nitrogenase activity as determined by the C,H, reduction method and by gel electrophoresis. Extracts of the alga Anabaena cylindrica and of the fungus Neurospora crassa, respectively, which contained nitrate reductase activity, did not react immunologically either with antibodies to Component I from A. vinelandii or those of nitrate reductase from E. was not detected in cofi B. Nitrogenase Anabaena grown with nitrate because heterocysts were absent and extracts of the alga did not reduce C,H, to C,H,. Cell-free extracts of A. vinelandii grown with ammonium did not react immunologically with antiserum to either Component I or nitrate reductase. Neither nitrate reductase activity

The data in this paper show that Component I of nitrogenase from Azotobacter shares common antigenic sites with a number of bacterial nitrate reductases. It is of interest that this relation does not hold for nitrate reductases from either a blue-green alga Anabaena cylindrica or higher plants. Moreover, antibodies for Component I did not cross-react with the following molybdoenzymes from animal sources namely aldehyde dehydrogenase and xanthine oxidase. Thus, it is only in bacteria that this antigenic similarity between two molybdenum-containing proteins has been observed. This indicates that during evolution both Com-

FIG. 4. Ouchterlony immunodiffusion assays in agar gel. Top row: purified Component I[ 100 fig Fraction 5, Table 1, Ref. (IO)]. Bottom row: heat-released nitrate reductase (60 pg Fraction 3, Table 1). Middle row: 500 pg IgG fraction of antiserum to heat-released nitrate reductase.

30

NICHOLAS,

FERRANTE,

ponent I of nitrogenase and bacterial nitrate reductases could well have been derived from a common ancestor protein. The concept that there is a common cofactor for a range of molybdoenzymes has been supported by genetic (19-21) and biochemical evidence (22-24). Thus, Ketchum et al. (22) and Nason et al. (25) demonstrated that an in vitro activation of nitrate reductase in extracts of a mutant strain (nit-l) of N. crussa could be achieved by adding various acid-treated molybdoenzymes. They suggested that a molybdenum moiety associated with a small organic molecule or polypeptide was the common factor for these enzymes. Subsequently Shah and Brill(26) isolated an Fe-MO cofactor from Component I of nitrogenase of Azotobucter and Clostridium pusteuriunum , respectively, which reactivated inactive Component I from a mutant strain ofAzotobucter. Pienkos et al. (27) isolated a molybdenum cofactor from xanthine oxidase which also activated nitrate reductase in ex-

FIG. 5. Ouchterlony immunodiffusion assays in agar gel. Comparison of purified Component I [50 pg Fraction 5, Table 1, Ref. (IO)] (E F); 450 pg cell-free extract (S3) of E. coli K-12 (C, D) and 400 pg cell-free extract (S3) of T. denitriJcans (A.B) with 50 pg of IgG fraction of antiserum to purified Component I from A. vinelandii [Fraction 5, Table 1, Ref. (lo)].

AND CLARKE

FIG. 6. Ouchterlony immunodiffusion assays in agar gel. The precipitin reaction of 200 pg cell-free extract (S,) of Agrobacterium tumefaciens (wells A and B) with 50 pg of IgG fraction of antiserum to purified Component I from A. vinelandii [Fraction 5, Table 1, Ref. (lo)]. Wells C and D, no antigen.

tracts ofN. crussu mutant strain (nit-Z) but it had no effect on an inactive nitrogenase in extracts of Azotobucter mutant strain UW45. Bishop et al. (11) have shown that antiserum prepared against the MO-Fe protein component of nitrogenase from soybean nodule bacteroids cross-reacted with extracts of free-living Rhizobium juponicum grown under various cultural conditions. The most intense precipitin bands resulted from cross-reaction of the antiserum with extracts of cells grown anaerobically with nitrate or anaerobically with nitrate and ammonia. Our observations that antiserum to Component I of nitrogenase cross-reacts with bacterial nitrate reductases and vice versa are relevant to the interpretation of the data of Bishop et al. because Cheniae and Evans (28) have shown that bacteroids from soybean contain nitrate reductase, as do cells of Rhizobium juponicum grown with nitrate (29). Thus, it would be of interest to establish whether antibodies raised to a bacterial nitrate reductase would also crossreact with extracts of bacteroids from soybean. ACKNOWLEDGMENTS This work was supported by a generous grant from the Australian Research Grants Committee. The assist-

A;otobacrer

NITROGENASE

AND BACTERIAL

ante of Mr. David Hein and Mr. Jan Rohozinski gratefully acknowledged.

is

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69, 580-583.

4. Nicholas, D. J. D., and Nason, A. (1954) J. Biol. Chem. 211, 183-197. 5. Nicholas, D. J. D., and Nason. A. (1955) Plunt Physiol. 30, 135-138. 6. Garrett, R. H.. and Nason, A. (1967) Proc. Nut. Acud. Sci. USA 58, 1603-1610. 7. Garrett, R. H., and Nason, A. (1969)J. Biol. Chem. 244, 2870-2882. 8. MacGregor, C. H. (1975) J. Bocterid. 121, 11 I I1116. 9. Burris. R. H. (1971) in The Chemistry and Biochemistry of Nitrogen Fixation (Postgate, J. R., ed.), chap. 4, Plenum, London. 10. Nicholas, D. J. D.. and Deering, J. V. (1976) Aust. J. Bid. SC;. 29, 147-161. I I. Bishop, P. E., Evans, H. J., Daniel, R. M., and Hampton. R. 0. (1975) Biochim. Biophys. Acra 381, 248-256. 12. Lund, K., and DeMoss. J. A. (1976)3. Bid/. Chem. 251, 2207-2216. 13. Adams. C. A.. Warnes. G. M., and Nicholas, D. J. D. (1971) Biochim. Biophys. Actu 235, 398-406. 14. Brownell, P. F.. and Nicholas, D. J. D. (1967) Planf Physiol. 42, 915-921.

NITRATE

REDUCTASES

31

15. Ferrante. J. V.. and Nicholas, D. J. D. (1976)FEB.S Lrtt. 66, 187-190. 16. Itzhaki, R. F., and Gill, D. M. (1964) Anal. Eiothem. 9, 401-410. 17. Williams. D. E., and Reisfeld, R. A. (1964) Ann. N. Y. Acad. Sci. 121, 373-381. 18. Sawhney. V.. and Nicholas, D. J. D. ( 1977)5. (;rn. Microbial. 100, 49-58. 19. Pateman. J. A., Cove. J., Rever, B. M.. and Roberts, D. B. (1964) Nature (London) 201, 58-60. 20. Kondorosi, A., Barabas, 1.. Svab. A.. Orosz, L.. Sik. T., and Hotchkiss, R. D. (1973) Nature NW Biol. 246, 153-154. 21. Scazzocchio,G.,Holl,F.G..andFoguelman.A.I. (1973) Eur. 1. Biochem. 36, 428-445. 22. Ketchum, P. A., Cambier. H. Y.. Frazier, W. A., 111, Madansky, C. H., and Nason. A. (1970) Proc. NM. Acud. Sci. USA 66, 1016-1023. 23. Nason. A., Lee, K. Y.,Pan, S. S.. Ketchum, P. A.. Lamberti, A., and De Vries, J. (1971) Proc. Nur. Acud.

Sri.

USA

68, 3242-3246.

24. Cheniae. G. M.. and Evans, H. J. (1960) Plant Physiol.

35, 454-462.

25. Nason. A., Antoine, A. D., Ketchum. P. A.. Frazier, W. A., III, and Lee, D. K. (1970) Proc-. Nut. Acud. Sci. USA 65, 137-144. 26. Shah, V. K., and Brill, W. J. (1977) Proc. Nut. Acud.

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14, 3249-3253.

27. Pienkos, P. T.. Shah, V. K., and Brill, W. J. (1977) Proc. Nut. Acad. Sci. USA 74, 5468-5471. 28. Cheniae. G. M., and Evans, H. J. (1957) B&him. Bhphys.

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29. Nicholas, D. J. D., Maruyama. Y., and Fisher. D. J. (1%2) Biorhim. Biophys. Acta 56,623-626.