Blue light photoreactivation of nitrate reductase from green algae and higher plants

Vol. 70, No. 4,1976

BLUE

BIOCHEMICAL

LIGHT

PHOTOREACTIVATION GREEN

Pedro

J.

Received

AND

Josi

de Universidad

April

OF

ALGAE

Aparicio,

Departamento

AND BIOPHYSICAL

M.

Bioquimica, de

NITRATE

HIGHER

and

Facultad

de

Sevilla,

by

plex

of

bss7

and

high

Sevilla,

donor

(1 ,2).

molecular

can

other

higher

fusca

reactivation

gae

and

(6)

with

be

the

the

in

vivo

of

subsequent enzyme.

more

(7)

of

culture

of growing

inactivation

ammonia

promotes

from in

if

and

cells

reductase

inactivated

effectively

electron

en-

a

rapid

removal

be

from

active

to

the

Nitrate

also

as

one

ammonia

about

enzyme

interconvertible

reinhardi

brings

can

an

com-

cytochrome

NADH

forms:

addition

and

(5).

vitro

reoxidizing

chain, of

in

green

vitro

by

cyanide

inactivated

reactivated by

transport

protein

is

cata-

enzyme

The

requires

Chlamydomonas

the

FAD, (l-4).

algae

is

is

alincu-

simulta-

(g,lO).

acts

inactivation

CSIC,

nitrite an

contains

physiological

nitrate

instantaneously

presumably tron

or

(8),

NAD(P)H

present Since

can

plants

green

The

The of

higher

neously

y

l-6.6.1.),

normaly

two

reductase.

the

bation

in

with

nitrate

Ciencias Spain.

to

carriers

plants

(5).

autotrophically of

Calero

reductase from spinach short periods of time with white or blue light greatly accelerate the suggest that blue light assimilation of nitrate in

nitrate

that

from

exist

inactive

Chlorella

Fernando

in

(EC

electron

reductase

that

of

weight, as

and

Nitrate

the

reduction reductase

molybdenum

algae

zyme

the

NAD(P)H-nitrate

green

FROM

13,1976

eucaryotes,

lyzed

REDUCTASE

PLANTS

Rolddn

SUMMARY. The inactive form of NADH-nitrate and Chlorella fusca is fully reactivated when the enzyme-complex is illuminated but not with red light. Flavin nucleotides photoreactivation process. The results might act as a modulating agent in the green algae and higher plants.

In

RESEARCH COMMUNICATIONS

the

it enzyme

Vennesland’s

Copyright 0 1976 by Academic Press, inc. All rights of reproduction in any form reserved.

by a

has

been is

due group

1071

nitrate

ferricyanide

critical

reductase (lo-12),

component

in

suggested

that

the

primarily

to

over-reduction

is

accumulating

which the

in

evidence

elec-

vivo of that

BIOCHEMICAL

Vol. 70, No. 4, 1976

assigns

ot

a

physiological

(13).

ess

n it

ion

cant

such

rate

process,

temperature several

the

green

algae

diminish light

in green

in or

blue

light or

higher

the

reactivation

the

regulation

algae

and

may

this

a

inactivation

proc-

flavin

role

needed

paper

show in

vitro

in to

that

short

nucle-

the

react

attain

a

Added

i-

signifi-

the

is

time

-secfrom

markedly

physiological

also

and

reductase

nucleotides

assimilatory

plants

light of

nitrate flavin

The

time. of

visible periods

inactivated

plants.

higher

the oxygen,

have are

reactivate in

in pH,

(8,13-16). in

vivo

cyanide

hours

activity

presented

specifically onds-

to phosphate,

and

of

data

as

but

restoration The

in

role

Factors

ides,

vat

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

role reduction

of of

blue nitrate

discussed.

METHODS. Nitrate reductase from spinach was partially purified as previously described (17): a) leaves were blended in a Waring blendor in 100 mM potassium phosphate, pH 7 containing 5 mM KN03, 10 NM FAD, 1 mM EDTA and 0.1 mM dithioerythritol; b) the cell free extract was fractionated with ammonium sulfate between 25 and 45% saturation; c) the resultant preparation was treated with calcium phosphate gel and d) the gel eluate that contains the nitrate reductase was concentrated with 50% ammonium sulphate. Basically a similar procedure was followed to purify the nitrate reductase from Chlorella fusca. 10 ml of the concentrated preparation was applied to a G-25 Sephadex column equilibrated with 100 mM Tris, pH 8.2 containing 10 NM FAD and incubated for 20 min in the dark with 1 .O mM NADH containing 0.3 mM KCN. To eliminate excess of NADH and cyanide the preparation was passed in the dark through a G-25 Sephadex column equilibrated with 100 mM potassium phosphate, pH 6.8. This preparation containing the inactivated nitrate reductase that was stable in the dark for several days was used for the light reactivating experiments. To prepare in vivo inactivated nitrate reductase, Chlorella fusca was cultured autotromy with nitrate as the only source of nitrogen, as previously described (18). At the end of the logarithmic phase of growth 10 &I ammonium chloride was added to the culture medium. After 1.5 hours of incubation the cells were collected by centrifugation and broken in a Buhler vibrator using a buffer solution of 50 mM Tris pH 8.2 containing 1 mM EDTA. The cell-free extract was treated with streptomycin sulfate and centrifuged at 144,000 x p for 1 hour. The resultant supernatant was concentrated under N2 in an Amicon ultrafiltration device. Once the ccl Is were collected al 1 operations were done in the dark at 49C. Light was supplied with a Sylvania 24 V. Lights of different colors were nm for the blue and BT 603 nm for the 4.8 and 270 mW/cm2 for the white, blue The nitrate reductase preparation cm glass cuvette kept at 4sC with taken out for the enzymatic assay. spectrophotometrically by following 340 nm.

tungsten obtained red. The and red

halogen lamp F.C.S. with Balzers filters: light intensity used lights, respectively.

was illuminated iced water. Alfquots Nitrate reductase the nitrate-dependent

1072

in of activity

150 W, BT 447 was 560,

a 3.0 x 1.0 x 1.0 the sample were was estimated NADH oxidation at

Vol.

BIOCHEMICAL

70, No. 4,1976

10

20

AND

BIOPHYSICAL

50 00 TIME OF ILLUYlNATlON

Figure 1. Photoreactivation of light of different colors. 1.0 for activity measurements. When with ferricyanide the enzymatic of NADH oxidized per min.

RESULTS.

Figure

reductase

using

white

light

Blue

‘I ight

ably

due

white

fifty

if of

times

white

if

1 ight. the

the

even that

few

blue

presumthat

of

was

allowed,

did

light

not

After full

a

period

reactivation with

inactivated

the

same

type

reacwas

illuminated

previously

the

intensity

light. light

minutes.

(hours), with

its

red

a

light

subsequently

showed

nitrate With

illumination Red

the

was

cyanide

of

with

enzyme

time compared

though

of

minutes

spinach

in

longer

time

with were used incubated 140 nmoles

wavelengths.

reached.

reductase

of

of

as

was

90

Nitrate

absence

much

sufficient

than of

obtained

a

reductase

higher

reductase of protein samples were reached was

accomplished

intensity

activity

nitrate

nitrate

different

was

light

but, level

120

spinach mg samples similar activity

of

took low

illumination

was

light

COMMUNICATIONS

(ml,,)

photoreactivation

reactivation

its

the

of

the

reactivation to

same

tivate

shows

actinic

full

light,

the

in

1

RESEARCH

with of

NADN

response

to

1 ight. Under or

FMN

did

not

under FAD process,

to

experimental

the

inactivated

affect red

or

the

FMN

conditions spinach

activity

1 ight. (20 reducing

when

By pM)

contrast, greatly

the

used, nitrate

the the

enzyme

required

1073

was

presence

accelerated time

the

seconds

light

of

FAD

dark

or

preparation

kept of

the to

addition

reductase in small

the amounts

of

reactivating (figure

2).

The

Vol.

70, No.

BIOCHEMICAL

4, 1976

AND

BIOPHYSICAL

TINE OF ILLUIIIATION

Effect Figure 2. of spinach nitrate used for activity half the enzymatic aration was made partially deficient

RESEARCH

ImInI

of

flavin nucleotides on the photoreactivation reductase. 2.5 mg samples of protein were measurements. The preparation used had only activity of that used in figure 1. The prepwith a new Sephadex column and was apparently in bound flavin.

I

FAD

10

20 TINE

Figure tivation Conditions

3. of

addition

of of

or

in

blue

1 ight,

when

FAD

of

its

had

was

of

no

00

(ml@

effect

nitrate

not

SO

different reductase

in

on

colors

absence. but

40

2044

colors the

on the presence

photoreacof FAD.

2.

different

spinach

20

OF IUWWIlK)N

light nitrate

figure

nitrate

Lights

tivation FAD

Effect spinach as in

of

ess.

COMMUNICATIONS

the

I ight,

3

present. 1074

effects

either

shows comple

photoreactivation

similar

reductase Figure

red

had

that tel

in both

y

react

procon

the

the

presence

white

light

i vated

the

reacof and enzyme

Vol.

BIOCHEMICAL

70, No. 4, 1976

Table

1.

AND

Photoreactivation reductase

BIOPHYSICAL

of from

the in Chlorella

RESEARCH

vivo

inactivated fusca

Enzymatic

Treatment (nmoles

nitrate

activity

NADH

oxidized

per

min)

40

Dark White

110

light

Ferricyanide

0.5 Other

mg

115

samples experimental

Similar trate

of

and

were from

cyanide

The upon

Chlorella

tivated

of

when by

the

in

minutes.

to

was

DISCUSSION. source

same

It of

damental

is

energy

as

has

been

for

the

In

a

ic

apparatus

of

nitrate I ight

20

degree

of

with

ferr

all

i zed and

of (21).

In

assimilation (22).

FAD.

be

photoreac-

in

can

illuminating FAD

1

full

are

white was

ex-

reactivation the

with

vivo

of

enzyme

light

prepa-

during

obtained

10

when

the

1 ished

that

light organisms

More in

is

recently

metabolism

in

the

basic

for

their

the

importance

the

plant

funof kingdom

.

higher

assimilation

of

i cyanide.

(19). agent

(20)

Table

photosynthetic

processes

with

preparations

in

1.

ni-

inactivated culture

by pM

purified

presence

r eactivation

estab

modulating

real algae

shown

of

in

vital

light

cell

achieved

well

the

measurements. figure

inactivated

reductase the

in

partially

cell-free

As

treated

activity same as

previously

in

nitrate

presence

The

enzyme

fusca

fusca

was

for the

when

illuminated

light.

the

obtained

ammonia

reductase

ration

used were

corresponding

white

nitrate

were

Chlorella

was

addition

cited

protein conditions

results

reductase

NADH

blue

COMMUNICATIONS

plants

most

nitrate

is

1955 in

Apparently

Stoy

of

the

observed

wheat

1075

is

by an

leaves

light

reducing

provided

after also

power the

needed

photosynthet-

increase

in

the

illumination an

essential

rate with

factor

Vol.

70, No. 4, 1976

for

the

induction

other

investigators

light

on

treated

exciting be

the

actinic electron of and

the

the

1 ight

this

the

the

in and

light

white

nitrate

reductase

for

of

the

convert one

enzyme.

process,

bound

directcan

effect

of

component(s)

The

absorb flavins

the

by

reductase

some

exogenous

two

blue

known

light have

FAD

comare

a

may

clear act

as

process.

to

determine

eventually

induced

to

active

the

photooxidize

this

progress

able

to

changes

the

action

elucidate in

the

its

spectrum

mechanism

prosthetic

by

groups

of

enzyme. The

fact

nitrate

that

reductase

light

is

trate

reductase

enhances thet

pigment are

Recently of

ferricyanide,

which

Since

photoreactivation

receptor

(23).

effects

nitrate

to

reductase

phenomenon

studying

chain

b557.

is the

inactive by

transport

Experiments of

Since

NADH-nitrate

on

1 ight into

presumably

cytochrome

effect

blue

reductase

is

COMMUNICATIONS

(14).

reactivated

light

plants

vitro

vulgaris

that

protein.

RESEARCH

in in

Chlorella

nitrate

chemically

ponents FAD

of the

the

some

show

form

also

reductase

monoxide

experiments

BIOPHYSICAL

nitrate

of

carbon

AND

mentioned activity

with Our

of

of

the

inactive ly

BIOCHEMICAL

ic

one

the organisms

of

white

light

from

Chlorella

the

supports

physiological

activity. formation

reactivates

agents It

of

is

worth

protein

and

the

in

the

idea

which noting nucleic

can that acids

vivo

inactivated that

blue

modulate blue

nilight

in

photosyn-

(24).

ACKNOWLEDGMENTS. The authors wish to thank Prof. M. Losada for his great interest and encouragement. The helpful collaboration of Dr. B.D. McSwain in the early stages of this work was greatly appreciated. This investigation was aided in part by grants to M. Losada from the National Science Foundation GF 44115 and from the Philips Research Laboratories. We thank Miss M.J. Pe’rez de LeBn for her skilled technical assistance.

REFERENCES 1. 2. 3. 4. 5.

Beevers, L., and Hageman, R.H. (1969) Ann. Rev. Plant Physiol., 20, 495-522. Hewitt, E.J. (1975) Ann. Rev. Plant Physiol., 26, 73-100. Losada, M. (1976) J. Mol. Catal., in press. Solomonson, L.P., Lorimer, G.H., Gal 1, R.L., Borchers, R., and Bailey, J.L. (1975) J. Biol. Chem., 250, 4120-4127. Losada, M. (1974) Metabolic Interconversion of Enzymes, pp. 257, Fischer, E.H., Krebs, F. G., Neurath, H., and Stadtman, E.R. ed., Springer-Verlag, Berlin.

1076

Vol.

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12.

13. 14.

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AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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De ia-Rosa, F.F., Doctoral Thesis, Universidad de Sevilla (1975). Zumft, W.G., Paneque, A., Aparicio, P.J., and Losada, M. (1969) Biochem. Biophys. Res. Commun., 36, 980-986. Arnon, D. I. (1971) Proc. Nat. Acad. Sci. USA, 68, 2883-2892. (1972) Ann. Rev. Plant Physiol., 23, 133-156. Zuker, M. Losada, M. (1976) Reflections on Biochemistry, in press, Kornberg, A., Horecker, B.L., and Or-d, J. ed., Pergamon Press, Oxford. stay, v. (1955) Physiol. Plant., 8, 963-986. Beevers, L., and Hageman, R.H. (1972) Photophysiology, 7, 85-113., Giese, A.C. ed., Academic Press, New York, London. Voskresenskaya, N.P. (1972) Ann. Rev. Plant Physiol., 23, 219-234.

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