Invitro formation of assimilatory nitrate reductase: Presence of the constitutive component in bacteria

BIOCHEMICAL

Vol. 52, No. 4, 1973

--In Vitro

Formation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Assimilatory

of the Constitutive

Nitrate Component

Reductase:

Presence

in Bacteria.

Paul A. Ketchum and Ruth S. Swarin Department of Biological Sciences Oakland University Rochester, Michigan 48063

Received

April

23,

1973 ABSTRACT

Cell-free extracts of a selected group of bacteria which are capable of metabolyzing dinitrogen and/or nitrate contain a soluble form of the constitutive component which is active in the --in vitro formation of NADPH-nitrate reductase when mixed with extracts of N. crassa The constitutive component in these extracts is-dialyzabie --nit-l. and is insensitive to trypsin and protease. The constitutive component which substitutes for the absence of the --nit-l gene product in the --in vitro formation of NADPH-nitrate reductase is postulated to be a low molecular weight cofactor or polypeptide and is shown to be present in a number of unrelated bacteria. INTRODUCTION The Neurospora

assimilatory

oxidoreductase,

E.C.1.6.6.2.)

heteromultimeric

protein,

chrome 557 fied

(l-6).

evidence

flavin suggests

of NADPH-nitrate the

flavin

(9),

have

by mixing contains wild

type

at least

reductase

stimulated

the

four

in -*N

crassa,

The component

contributed

from

associated

with

-N. -3crassa

mutants

of 2.

uninduced

reductase

in the

one of which (7,8).

of NADPH-nitrate

-c reductase,

the

involved

at least

reductase which

with

extracts

extracts

other

and

Genetic formation codes

for

Nason et al.,

nit-l --2

crassa

puri-

NADPH-(FAD)-cyto-

reductase.

c reductase

induced

reductase

and cyto-

(MVH) nitrate

are

dalton,

molybdenum

reductase,

genes

formation

of nitrate

230,000

(FADH2) nitrate

the NADPH-(FAD)-cytochrome or nitrate

are

viologen

NADPH-cytochrome

demonstrated

flavin,

activities

dinucleotide

that

extracts

contains

methyl

(NADPH-nitrate:

inducible

NADPH-nitrate

reduced

adenine

reductase

a nitrate

enzymatic

reductase:

chrome -c reductase, reduced

is which

Four

NADPH-nitrate

NADPII-nitrate

--in vitro specifically of uninduced

than -nit-l --

to the --in vitro

BIOCHEMICAL

Vol. 52, No. 4, 1973

formation

of NADPH-nitrate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

reductase

is

called

the

constitutive

compon-

ent. Pateman

et al.,

"common

cofactor"

in Aspergillus

nidulans

nitrate

reductase

and xanthine

dehydrogenase

(11)

(10)

demonstrated

that

enzymes

xanthine

tribute

the

nitrate

reductase

oxidase,

nitrate

enzymes

with

the

constitutive

Nason et al.,

(11,12). that

Acid

the

oxidase,

formation

have been

In explaining

can also of NADPH-

containing

shown

these

constitutive

treated

reductase

the known molybdenum

component

nitrate,

similar the

sensitive

to the --in vitro

con-

of NADPH-

sulfite

nitrate

(13)

oxidase

formation treated.

both

containing

and aldehyde

acid

a

et al.,

of the molybdenum

respiratory

all

for

Ketchum

nitrogenase),

of hydrogenase

demonstrates

can reduce

that

Therefore

is

necessary

activity.

first

(from

component

postulated

is

there

to contain

observations,

component

is

a moly-

cofactor.

pap'er

ments

are

exception

(12)

which

that

to the --in vitro

and --E. coli

(12).

evidence

dehydrogenase

enzymes

the constitutive reductase

This

these

reductase

nitrate

bdenum

xanthine component

if

genetic

preparations

of the Fe-MO protein

contribute

the

from

purified

constitutive

preparations plant

proposed

that contain

to those

constitutive to trypsin

organisms

which

can fix

the constitutive

reported component

component.

by Ketchum in

these

dinitrogen

and Sevilla cells

is

or which

Dialysis (14)

dialyzable

experi-

demonstrate and is

in-

and protease. MATERIALS

AND METHODS

Micrococcus denitrificans ATCC 19367 and Pseudomonas denitrificans ATCC -13867 were grown aerobically in peptone-ammonium medium which contained 0.1 gm peptone, 0.5 gm NH4C1, 0.36 gm Na2HP04, 0.1 gm KH2PO4, 3 mg M&04. 7H20, 0.6 mg FeS04 and 2.0 mg CaC12 per 100 ml of distilled water. Bacillus cereus ATCC 11950, Bacillus licheniformis ATCC 94458, BacillusGbtilis -and Bacillus thurengiensis ATCC 10792 were grown aerobica-on NYB medium (0.8 gm nutrient broth, 0.3 gm yeast extract per 100 ml of distilled water). Rhizobium japonicum ATCC 10324 was grown aerobically on NH4-arabinose-glycerol medium (15). Azotobacter vinelandii was grown Cells were aerobically on Burks medium containing 0.5% casamino acids.

1451

Vol. 52, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

washed twice and then resuspended in 0.02M Na2HP04/NaH2P04 buffer pH 6.5 containing 10m4M Na2MoO4 in a ratio of three volumes of buffer to one gm wet weight of cell. These cell suspensions were passed through a French Pressure Cell at 20,000 lb/in2. The supernatant obtained by centrifugation at 20,000 3 for 20 minutes is designated the crude extract. Crude extracts of nitrate induced --nit-l were prepared as previously described (14). Nitrate reductase and MVH-nitrate reductase were assayed according to the procedures of Garrett and Nason (6). One unit of nitrate reductase equals one nmole nitrite formed per 10 minutes at room temperature. The specific activity of the constitutive component in bacterial extracts was determined as previously described (14). Dialysis tubing (flatwidth of 0.75 inches) was obtained from Fisher Scientific Co. and was prepared by boiling it in sodium bicarbonate, EDTA and then twice in distilled water. Dialysis experiments were performed by adding bacterial crude extracts to 13 x 150 mm test tubes which contained enough glass beads to bring the level of the crude extract outside the dialysis sac to the level of the --nit-l extract inside the dialysis sac. The dialysis sac containing a crude extract of --nit-l was inserted into the tube containing the bacterial extract and the experiment was incubated at room temperature. Aliquots were removed from inside the dialysis Trichlorsac at timed intervals and assayed for NADPH-nitrate reductase. oacetic acid (TCA) soluble protein was determined by measuring the amount of protein by (OD28O/GD260) after precipitating the insoluble protein by making the preparation 5% TCA. NADPH-nitrate reductase is reported as units/ml of the --nit-l extract. RESULTS Extracts

of all

component

which

reductase. extracts crude

participates

in

of Bacillus

2.

component

treatment.

activity

hour.

This

wild

type 2.

Cell

free

MVH-nitrate

is

wild

extracts reductase respectively contained

type g.

in these

contrast

crassa

the --in vitro

extracts

which

(not

more than

after

centrifugation

constitutive

is particulate

specific shown).

hydrogenase

crassa

constitutive

of NADPH-nitrate component

than (9).

that

in crude

observed

The activity

was observed

without

70 per

of the

prior

cent

acid

of the

at 144,000 component

in

a

confor

one

in uninduced

(9).

of g. denitrificans with

greater

extract

to the

the

formation

of the constitutive

was soluble in

1) contained

was 4 to 7 times

In each bacterial

stitutive

(Table

activity

of uninduced

constitutive

vinelandii

tested

The specific

extracts

protein

the bacteria

and -Ps. activities Extracts

as measured

1452

denitrificans of 159.0

of RJ&.

contained and 5.8 units/mg

japonicum

by the methyl

and Azoto.

viologen

re-

Vol. 52, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 52, No. 4, 1973

duction

method

was observed in place

of Kleiner in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and Burris

(13).

the above hydrogenase

of hydrogen

NADPH-nitrate of --nit-l

BIOCHEMICAL

assay

viologen

when nitrogen

reduction

gas was used

gas.

reductase

against

No methyl

was formed

during

of -Ps.

denitrificans

extracts

the dialysis (Fig..

of crude 1).

extracts

Protease

and

Time (minutes) FIGURE 1: Time course of the --in vitro formation of NADPH-nitrate reductase during the dialysis of --nit-l against extracts of -*Ps -de* .-me. , control nitrificans. Three experiments are shown: with no additions; B x--x, 1.0 mg protease/l.5 ml of Ps. denitrificans extract added at time zero; C o-o, 1.0 mg trypzn/l.5 ml of Ps. denitrificans added at time zero. The amount of TCA soluble protein increased from 0 to 14.1% of the total protein present in the Ps. denitrificans extract after 120 minutes i-evueriment B and from 0 to 24.6% in experiment C, (Table 1).

trypsin ulated

digestion

of the --Ps

the formation

constitutive

component

contradiction due to the fact

of NADPH-nitrate is

to a previous that

denitrificans

the

extract reductase

insensitive

to these

report

(9).

complementing

1454

This activity

during

dialysis

demonstrating

that

proteolytic

enzymes

contradiction in -nit-l -)

stim-

is is

the in

probably extremely

Vol. 52, No. 4, 1973

sensitive when

BIOCHEMICAL

to trypsin,

the trypsin

When trypsin

added

reaction,

nitrate

formed

for

The formation against

the

bacterial

is

shown

in Fig.

dialysis

1.

integrity

Trypsin

except stimulated

exception

of protease

ment of extracts amount

(Table

of trypsin

experiment

dialysis

values

the formation

of NADPH-nitrate

reductase

of w.

of proteolytic

was used.

digestion

occured

dialysis

experiment,

protease with

the

treat-

A significant

the bacterial

Table

in all

Protease

performed.

in

during

reductase

of E. denitrificans. was not

report-

Likewise,

of NADPH-nitrate

iaponicum

serves

to

are

extracts

treatment

also

similar

of the bacterial

formation

(23,000

of nit-l

in curves

120 minute

denitrificans

of NADPH-

membrane.

during

the

the amount

dialysis

(9)

the --in vitro

treatment

the

each 120 minute

the

performed

was determined.

The presence

1) resulted only

not

during

mg/ml,

of the dialysis

when g.

treatment

extract

by 95%.

reductase

were component

of 0.01

during

1; however,

stimulated

experiments,

constitutive

reduced extract

extracts

controls

to the --nit-l

of NADPH-nitrate

ed in Table

ing

directly

in the bacterial

as a control

those

of the

at a concentration

reductase

daltons)

appropriate

sensitivity

is

formation

and that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

extracts

dur-

1.

DISCUSSION The in vitro extracts nit-l rate

of NADPH-nitrate

of the nitrate (9).

The other

uninduced containing experiments

i.e. activities

reductase wild

g.

reported

here

reductase

mutant

of -N. crassa,

formation,

during

in the

four

the

stimulated

flavin

crassa,

(11,12)

specifically

results

appear

enzymes

reductase

one of the

when extracts type

nitrate

in --nit-l

of only

reductase,

nitrate

induced

The mutation

induction,

nitrate (8).

formation

activities

during

(9)

demonstrate

incubated

treated

preparations

of Rhodospirillum that

1455

the

loss

NADPH-

2 reductase

formation with

nit-

with

NADPH-cytochrome

are

acid

or extracts

associated

the --in vitro

of --nit-l

requires

of NADPH-

extracts

of

of molybdenum rubrum

of the

(14).

gene product

The

BIOCHEMICAL

Vol. 52, No. 4, 1973

brought

about

trypsin

and protease

in extracts bolyzing

by the mutation

small

denitrogen

(16)

of

protein is

polypeptide

in E. crassa , nit-l dialyzable

insensitive,

of a selected

sumed to contain

AND BIOPHYSICAL

group

and/or molybdenum

the purification of nitrogenase

which

postulated

our

which

component

be viewed

polypeptide

view

that

the

is

capable

by a present

of metais pre-

as a cofactor

(10,12,14).

of a molybdenum supports

are

The constitutive

and can probably

as previously

can be replaced

component

of bacteria

nitrate.

RESEARCH COMMUNICATIONS

The recent from

or report

the Fe-MO

constitutive

component

a molybdo-polypeptide. ACKNOWLEDGMENT

This work was supported by the National from the Molecular Biology Section.

Science

Foundation

grant

GE-27490

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Nason, A., and H.J. Evans. 1953. J. Biol. Chem., 202:655-673. Nicholas, D.J.D., A. Nason, and W.D. McElroy, 1954. --J. Biol. Chem. 207:341-351. Nicholas, D.J.D., and A. Nason. 1954. -J. Biol. Chem., 207:353-360. Ibid. , 1954. Z:183-197. Garrett, R.H., and A. Nason. 1967. Proc. Natl. Acad. m. U.S., J&:1603-1610. Garrett, R-H., and A. Nason. 1969. J. Biol. Chem., 244:2870-2882. Sorger, G.J., and N.H. Giles. 1965. Genetics, 52~777-788. Sorger, G.J. 1966. Biochim. Biophys. -.Acta ) U:484-494. Nason, A., A.D. Antoine, P.A. Ketchum, W-A. Frazier III, and D.K. Lee. 1970. Proc. Natl. Acad. u. U.S., 65:137-144. Pateman, J.A., D.J. Cove, B.M. Rever and D.B. Roberts. 1964. Nature -3 -*201.58-60. Ketchum, P.A., H.Y. Cambier, W.A. Frazier III, C.H. Madansky and A. Nason. 1970. Proc. m. Sci -U.S-* 1 E:1016-1023. --Acad -a Nason, A., K.Y. Lee, S.S. Pan, P.A. Ketchum, A. Lamberti and J. DeVries. 1971. Proc. Natl. Acad. Sci. U.S., 68:2242-2246. Kleiner, D. and R.H. Burris. 1970xiochim. zophvs. --Acta 3 212:417-427. Ketchurn, P.A. and C.L. Sevilla. 1973. 2. Bacterial., (in press). Lowe, R. and H. Evans. 1964. Biochim. Biophys. =., 85:377-389. Ganelin, V.L., N.N. L'vov, I.S. Sergeev, G.L. Shanoshnzov and V.L. Kretovich. 1972. DAN SSSR, %:1236-1238.

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