Ribulose diphosphate carboxylase and CO2 incorporation in extracts of Hydrogenomonas facilis

Vol. 19, No. 6, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNKA~IONS

RIBULOSE DIPHOSPHATE CARBOXYLASE AND CO2 INCORPORATION IN EXTRACTS OF HYDROCENOMONAS FACILIS* Bruce

A. McFadden

work

although

its

presence

oratories.

enzyme,

the reductive relative

which pentose

amounts

We wish

to report

in Hydrogenomonas

tion,

variation

tracts

is

can be inferred

from

autotrophs is

during

the flow heterotrophic

has been published, obtained

such as hydrogen

path by this

for

that

in many lab-

bacteria

uniquely

CO2 fixation,

and other

the

level

functions

in

may regulate

paths

during

the

heterotro-

growth. data

on the variation

facilis

of levels

as a function

The correlation

incorporation

step

of CO2 through

of RuDP carboxy-

of mode of culture.

of these

the RuDP carboxylase-catalyzed

in adjusting

results

one of the catalysts

phosphate

of ribulose-1,5-diphos-

bacteria

in the DPNH-, ATP-dependent

described.

implicates

detection

in chemosynthetic

of CO2 fixed

and autotrophic

lase

the direct

In facultative

of this

cle

describing

(RUDP) carboxylase

phic

Wash.

May 3, 1965

Little phate

L. Tu

Department of Chemistry State University, Pullman,

Washington

Received

and Chang-chu

the reductive

two sets

of

In addi14

of data

as a major

CO2 by exstrongly

control

pentose

point

phosphate

cy-

growth. METHODS

Culture

and Preparation

trophic

culture

*This search Fund gram award Infectious

of II.

of Cells facilis

were

and Extracts. exactly

Autotrophic as described

and heteroby McFadden

work was supported in part by the Medical and Biological of Washington State University and by a research career (no. i-~3-AI-5268) from the National Institute of Allergy Diseases, U. S. Public Health Service.

728

and Reproand

Vol. 19, No. 6, 1965

BIOCHEMICAL

Homann (1965).

Provision

as defined

by McFadden

was washed

in buffer

GSH.

In

pension than

was treated C.

of H2, 02 and CO2 during and Atkinson

a 20 kc Biosonik

was then used

by the method

GSH absorbancy

After

boxylase

had been established,

MgC12, cept

apparently

brought

to 30'

of controls)

preincubated during

Tris,

to initiate

the reaction.

of 0.1 ml 60% (w/v)

convenience.

and subsequent-radioautography

aerobic

and anaerobic Apparently

reducing grown

power

mixture components

(in

0.6;

45.

flasks.

Tris,

conditions

and ATP were ways were

(pH 7.5)

l4 C fixed

for

also

in the

case

the

reaction

acid

of aerobic

uptake assays

paper

the major

ex-

was then

5 min,

Aerobic

(TCA). of

were

sub-

chromatography

acid-stable

was phosphoglycerate

pmoles):

for

assayed

Incubations were

MgC12, were initiated

CO2 fixation

product

for

both

CO2 incorporation in addition

conducted by adding

at 30' H

14

using

to extract

6; NaH 14 CO3, 12.5;

729

in the presence

Then extracts

established.

of 0.5 ml containing

Reactions

that

umoles):

experiments.

optimal

in various

(in

trichloroacetic

fixation.

revealed

95% of the

at pH 7.5

components

water

After

Two-dimensional

~a.

All

Then RuDP (or

sequently

for

ml)

components 15.

the amount

accounting

with

by RuDP car-

30 min at 0' and the temperature

a 5 min interval.

for

con-

(1941)

(0.25

the following

Early studies at pH 8.0 revealed that 14 H CO; was about 8@ that of anaerobic conducted

g for

Protein

catalysis

mixtures

5; GSH, 0.3;

for

by the addition

C) at 20,000

and Christian

for

reaction

extract,

NaHL4C03,

was added

was stopped

conditions assay

to cell

3; RuDP, 0.15;

at less

described.

of Warburg

sus-

as necessary.

optimal

in addition

RuDP were

in the experiments

2 -mM

The cell

oscillator

(2"

mass

containing

20 -ml-f MgC12.

centrifuged

was

the cell

pH 7.2,

with

Assays.

contained,

in 0.1 g Tris,

for

20 set

growth

harvest,

contained

was estimated for

After

also

The suspension

a correction

autotrophic

the buffer

20 min and the supernatant centration

(1957).

and suspended

some cases

10'

AND BIOPHYSICAL RESEARCH COMMUNlCAilONS

from g.

facilis

a reaction the following

DPNH, 2; ATP, under

of

1.5;

N2 in Warburg

CO<, ATP and DPNH simul-

GSH,

Vol. 19, No. 6, 1965

taneously

BIOCHEMICAL

and stopped

after

AND BIOPHYSICAL RESEARCH COMMUNlCAilONS

30 min by the addition

of 0.2 ml 6%

(w/v)

TCA. Just

before

all

experiments

used was determined of incorporation

gion

by the method were

incorporation

the

(or

made using

specific

of Bray the

kinetic

(1960).

same method.

RuDP carboxylase)

of first-order

of the H 14 CO3 Subsequent measurements

radioactivity

activities

dependence

In all

were

cases,

calculated

upon protein

specific in a re-

concentration.

RESULTS AND DISCUSSION Table

1 shows

the dependence

of RuDP carboxylase

activity

upon several

factors. TABLE 1 RuDP Carboxylase Component

varied ?!a2

RuDP

Activity

and its

final

in Autotrophic

Extracts

d . p . m. H14CO-3 fixed

concn.a

1,666 9,600 10,265

none added 0.01 3 0.1 g none 0.4 0.6

14-

added mM IIIM -

74 711 831

H5@ 10 15

aData for different ferent experiments were essentially as described varied.

Table 2 presents 14 RuDP, H CO; fixation hibited

by lo-3

Replacement

data

components are not comparable since difExperimental protocol was conducted. in the text except for the component

which

establish

is markedly

stimulated

that

in the

absence

by DPNH plus

of exogenous

ATP and is

in-

& AMP.

of DPNH by TPNH in the

3% as much incorporation Incubations

2,080 2,800 2,860

timM -

conducted

emphasizing aerobically

otherwise

complete

the marked resulted

730

in --ca

system

specificity 3%

resulted for

in

reductant.

as much fixation

Vol. 19, No. 6, 1965

BIOCHEMICAL

and were

therefore

is

to have

known

routinely

AND BIOPHYSICAL

RESEARCH COMMUNlCAilONS

under

conditions.

run

a potent

anaerobic

DPNH oxidase

(unpublished

g.

facilis

observation,

H. R.

Homann). TABLE 2 Reductive

CO2 Fixation

Incubation

conditions*

by Autotrophic c.p.m.

Extracts H14CO; fixed

Mg+2 varied

10-3 M

1,000

1o-2 g 10-L 5

14,024 15,845

ATP varied none added

183 870 1,170

2mM

3mM DPNH varied none added

163 3,531

3mM 4mM

3,982

H14CO'z

20 InM 25 mM -

96,564

110,454 17,486

Complete 1mMAMP -

5,552

See footnote

3 illustrates

Table

RuDP carboxylase

in Table

the changes

and reductive

1.

which

occur

CO2 fixation

in specific

as a result

activity

of

of different

modes

of culture. Of interest

is

fructose-grown

cells,

of Gottschalk

et al.

however,

is

the

RuDP carboxylase

the high which

specific is

activity

significantly

(1964) for Hydrogenomonas

qualitative activity.

parallel

between

The observed

731

of RuDP carboxylase different

from

m.

Of major

reductive

CO2 fixation

the

in findings

interest,

CO2 fixation

and

has the properties

BIOCHEMICAL

Vol. 19, No. 6, 1965

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 3 Variations

in RuDP Carboxylase

and Reductive

CO2 Fixation RuDP carboxylaseb specific activity urnmoles CO2 fixed/mini mg protein xl d

Reductive, ATP-dependenta CO2 fixation pmoles CO2 fixedjminl mg prot in xl d

Cultured

& Autotrophically On D-fructose " D-glucose " D-ribose " D,L-lactate " L-glutamate " succinate " acetate

II

4.75 4.83 1.65 2.07 1.73d 0.85 0.24

0.41

.8.8 5.5 2.67 3.6

(15.2)'

0.21

(1.5)

0.07 0.04 0.27

(1.3) (0.9) (0.5)

(E-4) (5.7) (6.7)

aAll data are corrected for results with controls which lacked DPNH and ATP. b All data are corrected for results with controls which lacked RuDP. 'Carboxylase activity in these experiments (bracketed numbers} was measured at pH 8.0 under essentially the same conditions as described in All other values (columns A and B) were obtained for a single the text. extract under conditions described in the text. dThis fixation was inhibited about 7% by 10m3 g AMP.

expected (for

of incorporation

a review

stead

see Calvin,

of TPNH.

so this

by extracts

may be a fundamental photosynthetic

the bulk

of the CO2 fixation

and therefore

g AMP (cf. bacillus

thioparus,

(1965a,b)

have

represent

a basic

observed

Chromatium

In conclusion,

involved

proposed

that

mechanism

our data

establish

the

of evidence leaves,

in autotrophic

1963)

and oxygen-

supposition

the pentose

AMP regulation

that

and Nason,

the

is

in-

as a reductant

chemosynthetic

supporting

and spinach

the reductant

(Aleem

between

On the basis

used by plants

specifically

by RuDP carboxylase

2 and 3).

control

serves

Further

cycle

DPNR is

of Nitrobacter

species.

recently

phosphate that

difference

catalysis

Tables

except

DPNH also

evolving

cle

the pentose

15X52),

Apparently

14CO2 fixation

during

-via

that

phosphate

by 10 -3

inhibition obtained

Johnson

cy-

with

Thio-

and coworkers

of CO2 fixation

may

metabolism.

one important

control

point

Vol. 19, No. 6, 1965

for

CO2 fixation

drogen

bacteria

BIOCHEMICAL

by facultative is

the

level

AND BIOPHYSICAL

chemosynthetic

RESEARCH COMMUNICATIONS

organisms

such as the hy-

of RuDP carboxylase. REFERENCES

Aleem, M. I. H., and Nason, A., Bacterial. Proc., p. 103 (1963). Biochem., I., 279 (1960). Bray, G. A., Anal. Calvin, M., Science, B, 879 (1962). Gottschalk, G., Eberhardt, U., and Schlegel, H. G., Arch. Mikrobiol., 48, 95 (1964). Johnson, E. J., and Peck, H. D., Jr., J. Bacterial., @, l&l (1963a). Johnson, E. J., Bacterial Proc., p. 94 (1965b). McFadden, B. A., and Atkinson, D. E., Arch. Biochem. Biophys., 66, 16 (1957). McFadden, B. A., and Homann, H. Warburg, O., and Christian;W.,

733