Immuno chemical characterization of nitrate reductase from spinach leaves and roots

Vol. 113, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

June 29, 1983

Pages

733-737

IMMUNO CHEMICAL CHARACTERIZATION OF NITRATE REDUCTASE FROM SPINACH LEAVES AND ROCTS Sylvie

Ferrario,

Laboratoire no 799,Universite

Received

February

Bertrand

Hire1

and Pierre

Gadal

de Physiologie Vegetale Metabolique, de Paris-Sud, Centre d'Orsay - Bat. ORSAY Cedex, France

ERA CNRS 430 - 91405

29, 1983

SUMMARY: Nitrate reductase from spinach (Spinacia oleracea L.) leaves was purified to homogeneity and specific antibodies against the protein Using immunodiffusion and were raised by the immunization of rabbits. immunoprecipitation techniques, the enzymes from leaves and roots were compared. It was demonstrated that the two nitrate reductases were different proteins on the basis of their antigenic behaviour.

In higher

plants,

in the roots the nitrate 1.6.6.1)

nitrate

or transported is

first

reduced

and complete

(NiR) glutamine

by glutamine

acids

synthetase

aspects

of nitrate

including

work

(4,9,10,11,12)

(3).

Different

methods

enzyme

(4,13,14,15).

in

squash

cotyledons

reductase method

have also Very

enzyme was purified the protein

were

for

regulation

been

plant

leaf

used to compare

higher for

glutaamino

on different properties

plants

and algae.

the purification

from

leaf

NR

nitrate

work

reports

reductase.

antibodies

of

against

to compare

The present nitrate

and specific the enzymes

formed

antibodies

and used (16).

into

of various

of studies

specific

leaves

reductase

have been described,

reported

of spinach

to homogeneity

synthesis

and regulatory

in various

recently

(NR) (E.C.

incorporated and the

the

either

and leaves

by nitrite

then

number

have been obtained

the purification

is

roots

reductase

achieved

(4,5,6,7,8)

reductase

from different for

and its

structural

of nitrate

the

donor

An increasing

reduction

on the

by,nitrate ammonia

can be reduced

In both

(E.C.6.3.1.2)(2)

as an amino

and nucleotides

(1).

to NH4+ is

The resulting

can be utilized

by the roots

leaves

to nitrite

reduction

(E.C.l.7.7.1).

mine

absorbed to the

a

Th,e raised

and root

against tissues.

WXTERIALS AND METHODS Plant Material. Fresh spinach leaves (Spinacia oleracea L. C.V. VirofZay) for the enzyme purification and immunological investigations were purchased at a local market. Enzyme purification. leaves (2 Kg) were

All ground

operations were carried out in 6 1 of 100 mM K-phosphate

at 4OC. buffer,

Spinach pH 7.5

0006-291X/83 .733

$1.50

Copyright 0 1983 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Vol. 113, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

containing the following protective agents: 10 mM L-cysteine, 1 mM EDTA, 1 pM Na2MoO and 1 p FAD in a Waring Blender for 3 x 1 min at maximum speed. The'homogenate was filtered through one layer of nylon net (mesh size, 150 pm) and centrifuged for 1 h at 30,000 g using a Beckman J 21 C centrifuge equipped with a JA 14 rotor. The supernatant was then ammonium sulphate precipitated to between 20 and 40% of the saturation. The pellet of proteins was resuspended in a minimal volume of 10 mM Kphosphate buffer, pH 6.8 containing 10 mM L-cysteine, 1 mM EDTA, 1 )iM Na2Mo04 and 1 PM FAD. Hydroxyapatite, in powder form (Bio-rad Laboratories, Richmond, California), was then added to the protein solution and stirred until all the enzyme activity was fixed onto the gel. The supernatant was discarded after centrifugation for 15 min at 10,000 g and the pellet was resuspended with 200 ml of 100 mM K-phosphate buffer pH 6.8 containing the protective agents. After another centrifugation for 15 min at 10,000 g the gel pellet was discarded and 200 mM @JH4)2S0 was This solution was then fixed onto a PR enyl added to the supernatant. Sepharose column (15 x 1.5 cm, Pharmacia, Uppsala) previously equiliThe column was washed with rebrated in the resuspending buffer. The proteins suspending buffer until all the unbound protein was removed. were then eluted with a linear gradient of decreasing (NH ) SO to 0 mM) and increasing ethyleneglycol (0 to 60%, v/v), biti? o+ ::::h were dissolved in 100 ml 10 mM K-phosphate buffer at pH 7.5 containing the protective agents. 2 ml fractions were collected. Fractions containing peak enzyme activity were pooled and then applied onto the top of a 6 % cylindrical gel (6 x 2.5 cm) for preparative polyacrylamide of 2 mm gel electrophoresis. After the run, the gel was cut into slices in width with a razor blade. Each gel slice was incubated in the assay medium and nitrite formation was determined in the medium. The slice containing maximum activity was selected and the protein was extracted from the gel by electrophoresis as previously described (17). Immunization procedure, specificity

Procedure and Specificity of the Serum. The immunization the control of purity of the enzyme preparation and the of the antibodies were performed as described previously

(17).

Enzyme assay. NADH-dependent NR activity was assayed as described by Hageman and Flesher. (18). Methyl viologen (MV) dependent NR activity was assayed according to Paneque et al. (19). RESULTS AND DISCUSSION Purification

of NR from

obtained

(specific

activity

to immunize

the rabbits.

polyacrylamide

gel

Specificity checked double

with

extracts test

in which

increasing

quantities

precipitated result

entirely

shows clearly

Purity

leaves

in Fig.

of spinach constant

leaf

that

using Only

and were

used

was checked

by

preparation

was

1A.

the Ouchterlony

one sharp precipitin on the agar plate..

Fig.

NR in an immunoprecipitation

amounts either

1B.

of antibodies of NR activity

of antiserum. with

in Fig.

of the antiserum

of spinach

as shown

enzyme were

per mg of protein)

of the preparation as shown

in the presence

the behaviour

experiment

Two mg of purified

of 16 nKat The purity

antibodies.

crude

band was detected 2 shows

Leaves.

electrophoresis

of the

diffusion

Spinach

Enzyme activity

were

incubated

(0.05

NADH or MV as the electron

the antibodies 734

were

raised

against

nKat)

with was

donor. an intact

The

BIOCHEMICAL

Vol. 113, No. 3, 1983

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A

Fig.

l.A:

Control cylindrical B:

of

Moreover

the

that

about

immune are

with

50%

of

complex

Fig.

of

the

not

1C that

spinach

closely

leaves

on a 6%

of

the

antibodies

leaf

root

catalytic

and

root

NR.

recognize

employed NR from the

735

by

in

the site

to

of

compare

NR with

It

fact the

antigenic the

Immunodiffusion

spinach. root

the

recovered that

the

were and

was

to

(20,21).

confirmed

demonstrates

test. the

NR from spinach roots = antiserum; (R) = root

components

was

activity

also

situated

leaf

molybdenum

enzyme

procedures of

and

of (AS)

antibodies

This

2).

comparison

behaviour

FAD the

initial

(Fig.

immunoprecipitation logical

both

specificity

Immunological

NR from gel.

Immune-chemical characterization and leaves by immuncdiffusion: NR and (L) = leaf RR.

enzyme

probably

the purity of polyacrylamide

Control of the purity of the antiserum prepared against NR from spinach leaves by Ouchterlony double diffusion The control well contained 8 pl of antiserum (AS) and peripheral wells contained 1.2 nKat of NR activity.

C:

molecule

of

sites protein.

and the

immuno-

can

be

a faint

seen

in

band

of

BIOCHEMICAL

Vol. 113, No. 3, 1983

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ANTISERUM Fig.

2

Inununoprecipitation leaves. M

curves

M

precipitate. between

It the

can also

two proteins

of NH from spinach

root

NH (NASH-dependent

root

N-R ( M-J-

U

II

H

leaf NH activity complex

recovered

from the immune

M

Control

be observed with

inhibited and root during

with

l/30

MH from

structural

with

non-immune

that

a spur

a partial

occurring

steps

procedure

of nitrogen

variously

reported.

glutamate

synthase

assimilation

For example, (25,

26) Possess

results

a specific

736

completely leaf

However

of the root

enzyme

excluded.

plants

synthetase root

the

(22).

enzymes

in higher

20%

serum

show that

be completely

glutamine

enzyme, about

is

instability

and root

activity,

leaf

l/65

proteins

relative

leaf

with

enzyme activity

different

cannot

of different

exists two precipitin

of antiserum,

These

are probably due to the

the

of the

inhibited

leaf

serum dilution.

spinach

identity

NH (NADH or W-dependent)

was only the

serum.

between

concentrations

In contrast

extraction

The occurrence primary

increasing

modifications the

the

enzyme activity 2).

)

NH ( MV-

of root

(Fig.

)

leaf

immunoprecipitation

initial

)

-

amounts

of the

II

'1

for

dilution

"

NH (NADH-

When constant with

activity)

leaf

to those incubated

and

H

similar were

roots

II

bands.

used

DILUTION

involved

in the

has already (23,

been

24)and

enzyme with

different

Vol. 113, No. 3, 1983 a spatial organs These

distribution of several

kinetic

BIOCHEMICAL of various higher

and immunological results

metabolic

and those

pathways,

AND BIOPHYSICAL

plant

isoenzymes

may occur

in the different

species.

properties

when compared

of the present

especially

RESEARCH COMMUNICATIONS

those

study involved

confirm

to the that

in nitrogen

leaf

enzyme.

in a number

of

assimilation,

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Beevers, L. and Hageman, R.H. (1969) Ann. Rev. Plant Physiol., 20, 495 - 522. Miflin, B.J. and Lea, P.J. (1976) Phytochem. 15, 873 - 885. Stadtman, E.R. (1973) In: The enzymes of glutazne metabolism. Prusiner and Stadtman Eds., Acad. Press New York, London, 1 - 6. Hewitt, E.J. (1975) Ann. Rev. Plant Physiol., 26, 73 - 100. A. and Varner, R.L. (1980) Plant Sci. Lett., Kuo, J., Kleinhoffs, II, 371 - 381. Campbell, W.H. (1982) Plant Physiol., 69, Suppl. No. 643. Solomonson, L.P. (1979) In: Nitrogen assimilation of plants. Hewitt and Cutting Eds., Acad. Press New York, London, San Francisco, 199 - 205. Notton, B.A. and Hewitt, E.J. (1979) In: Nitrogen assimilation of plants. Hewitt and Cutting Eds., Acad. Press New York, London, San Francisco, 227 - 244. Srivastava, H.S. (1980) Phytochem., 19, 725 - 753. Sielke, H.R. and Filner, P. (1971) J. Biol. Chem. 246, 1772 - 1773. Funkhouser, E-A., Shen, T.C. and Ackermann, R. (1980) Plant Physiol., 65, 944 - 948. Naik, M.S., Abrol, Y-P., Nair, T.V.R. and Ramarao, C.S. (1982) Phytochem., 21, 495 - 504. Solomonson, L.P. (1975) Plant Physiol., 56, 853 - 855. Campbell, W.H. (1979) In: Nitrogen assimzation of plants. Hewitt and Cutting Eds., Acad. Press New York, London, San Francisco, 321 - 330. Mendel, R.R. and Muller, A.J. (1978) Molec. Gen. Genet., -161, 77 - 80. Smarelli, J. and Campbell, W.H. (1982) Plant Physiol., 68, 1226 1230. Hirel, B., Perrot-Rechenmann, C., Suzuki, A., Vidal, J. and Gadal, P. (1982) Plant Physiol., 69, 983 - 987. Hageman, R.H. and Flesher, D. (1960) Plant Physiol., 2, 700 - 708. Panegue, A., Del Campo, F.F., Ramirez, J.M. and Losada, M. (1965) Biochem. Biophys. Acta, 109, 79 - 85. Hewitt, E.J., Hucklesby, D.P. and Notton, B.A. (1976) In: Plant Biochemistry. Bonner and Varner Eds. Acad. Press New York, 633 - 672. Guerrero, M.G., Vega, J.M. and Losada, M. (1981) Ann. Rev. Plant Physiol., 32, 169 - 204. Oaks, A. (1979) In: Nitrogen assimilation of plants. Hewitt and Cutting Eds., Acad. Press, New York, London, San Francisco, 217 - 226. Mann, A.F., Fentem, P.A. and Stewart, G.R. (1979) FEBS Lett., 110, 265 - 267. Hirel, B. and Gadal, P. (1980) Plant Physiol., 66, 619 - 623. J. and Gadal, P. (1982) Plant Physiol., 2, Suzuki, A., Vidal, 027 - 832. 20, 597 - 600. Cullimore, J.V. and Sims, A.P. (1981) Phytochemistry,

737