Heterogeneity of large subunits of ribulose-1,5-bisphosphate carboxylase from Hydrogenomonas eutropha

BIOCHEMICAL

Vol. 71, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

HETEROGENEITY OF LARGE SUBUNITS OF RIBULOSE-1,5BISPHOSPHATE ~ARB~XYLASE FROM HYDR~GENOM~NAS EUTROPHA

Kris Purohit and Bruce A. McFadden Program in Biochemistry and Biophysics in the Department of Chemistry, Washington State University, Pullman, Washington 99163

Received

June 15,1976

SUMMARY: Homogeneous ribulose-1,5-bisphosphate carboxylase purified from autotrophically grown H dro enomonas eutro ha can be dissociated with sodium dode- a ton su units and large 56,000and 52,000cylsulfate into srn*+* dalton subunits (the latter in a mole ratio of 5:3). The overall mole ratio of small to large subunits is 1.08. Considering the molecular weight of the native enzyme (516,000), the simplest quarternary structure of this enzyme consists of 8 large (mixed) and 8 small subunits. Isolation of the enzyme from cells under conditions that should minimize proteolysis has no effect upon the observed heterogeneity of the large subunits.

We have postulated subunits

the

structural

of ribulose-1,5-bisphosphate

carboxy-lyase the

that

small

(dimerizing),

have been isolated

(RuBP)

EC 4.1.1.391,

15,000-dalton

subunits

(1).

in a stable

hexameric

state

from

stable

octomeric

state

from Thiobacillus

large

gested

state,

the ancient

to a descendent i.e.,

ancient

and occurs

than

f.

structural

gene specifying

gene which

specified

in C. limicola

reflected is

through

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

[3-phospho-D-glycerate

including

to that

lacking

small

subunits

rubrum

(2),

(1,6).

gene duplication

higher

(1,6,8,9).

(3),

quadru-

plants

of (for

We have sugpolypeptide

of higher

in R. rubrum One simple

a

and a

consists

the 55,000-dalton

followed

encoding

and Agmenellum

a gene product

gene as reflected

1220

prior

thiosulfatophilum (4)

7) and a few prokaryota

that

55,000-dalton

form of RuBP carboxylase

in eukaryota

of this

large

Rhodospirillum

intermedius

the expression

may have occurred

from

limicola

the dominant

subunits

carboxylase

was established

state

Chlorobium

see 1 and 6; see also that

mutated

Copyright All rights

In contrast,

and small

reviews

this

(5).

the

RuBP carboxylases

dimeric

stable

plicatum

gene for

aggregation

was more

mechanism by mutation

by which (10).

BIOCHEMICAL

Vol.71,No.4,1976

Such a mechanism, these

genes

et al.

(11)

in organisms reported

lase

of Nicotinia

(12).

However

as an explanation large

subunits

eutropha

however,

under

required

the existence

in which

both

heterogeneity tabacum

proteolysis

after

of RuBP carboxylase which

structural

of large

in which

of the observed

conditions

AND BIOPHYSICAL

cell

of two

kinds

genes

persisted.

subunits

chloroplast

RESEARCH COMMUNICATIONS

derived

DNA encodes

these

was not excluded

heterogeneity.

We now report

should

MATERIALS

from

minimize

the bacterium

of

Indeed,

from

breakage

isolated

of products

Kung

RuBP carboxylarge

by these

subunits authors

heterogeneity

of

Hydrogenomonas

proteolysis.

AND METHODS

H. eutro ha RuBP carboxylase was purified by centrifugation of the ribosomefree ccl -extract in a 0.2-0.8 M discontinuous sucrose gradient (13,14), precipitation of the enzyme from the pooled and dialysed fractions at 35% saturation with respect to (NH4)2S04, and, finally, by centrifugation in another sucrose gradient identical to that just mentioned. The homogeneity of the enzymatically active fractions was analyzed by electrophoresis on gels polymerized from 5% acrylamide. The fractions showing a single Coomassie blue-positive band and corresponding RuBP carboxylase activity were pooled, dialysed, and concentrated with Aquacide. Homogeneity of the pooled fractions was assessed by electrophoresis on gels polymerized from 4-7.5% acrylamide. The concentrated and dialysed enzyme with a specific activity of 1.76 pmoles CO2 fixed/min/mg protein, was The buffer used was TEMBD (16) and the sucrose used in all experiments (15). solutions for gradients were prepared in the same buffer.

-l-?--

During purification in the presence of proteinase inhibitors (17),dithiothreitol (DTT) was omitted from the buffer and the concentration of Mgt2 and In addition to the usual purification, the enzyme was EDTA was also changed. purified in the presence of: (i) 1 mM 1-chloro-3-tosylamido-7-amino-L-2heptanone.HCl(TLCK), (ii) 1 mM diisopropyl fluorophosphate (DFP), (iii) 1 mM Mgt2 and 10 mM EDTA without DTT or (iv) in the absence of DTT. RuBP carboxylase was assayed as described by McFadden et al. (18). Sodium dodecyl sulfate (SDS) was recrystallized from 95% ethanol (19). Electrophoresis was done by the method of Laeronli (20) with els olymerized from Eastman electrophoresis-grade acrylamide containing SDS 9 0.1% P and also with gels without SDS. The native enzyme was dissociated with 1% SDS and 10 mM a-mercaptoethanol The subunits were separated by gel filtration on in boiling water for 2-3 min. 140-400 mesh), preequilibrated with 1% a Sephadex G-100 column (particle size: The fractions corresponding to the SDS and 10 mM 6-mercapto-ethanol in water. subunits, as measured by absorbance at 280 nm and also protein determination (21), were pooled and lyophilized. The SDS was extracted two times with 30 ml of chilled 80% acetone and the precipitated protein corresponding to the large and small subunits was solubilized by adding 10 and 3 mg of SDS respectively. For amino acid analysis, aliquots of SDS-solubilized subunits were hydrolysed in 6N HCl under Np for 72 h at 110-113°C in sealed vials. The hydrolysates were analysed with a Beckman 121 Automatic amino acid analyzer. After performic acid oxidation at 0°C for 3 h followed by acid hydrolysis, methionine was determined as methioninesulfoneand cysteine and cystine as cysteic acid (22).

1221

BIOCHEMICAL

Vol.71,No.4,1976

Fig.

1

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Polyacrylamide gel electrophoretogram of 9.5 pg fi. eutropha boxylase. The electrophoresis was done on gels polymerized (left to right) 4, 5, 5.5, 6.5 and 7.5% acrylamide.

RuBP carfrom

RESULTS In Figure phoresis

1, a single

of RuBP carboxylase

7.5% acrylamide. taminating

higher

This

proteins

In Figure isolation

Coomassie

protein

of closely

B-mercapto-ethanol

greatly similar

2, the dissociation

concentration

band

in each of a series

approach

of the enzyme under

blue-positive

raises

of gels

a variety

polymerized

ratio

homogeneous

and SDS and electrophoresis

1222

after

electrofrom

4% to

of detecting

con-

(23). enzyme

of conditions.

was examined

shown after

the probability

charge:mass

of this

is

In all

dissociation in polyacrylamide

is depicted cases in the

after

when a presence

containing

of SDS

BIOCHEMICAL

Vol.71,No.4,1976

A Fig.

2

(O.l%),

instead

blue-positive

electrophoresis

electrophoretic

pattern

sence

similar

5% acrylamide

proteolysis

0.E

the

mobility

was observed

after

Analogous

polymerized

in only

from

two stained

to the left

dissociation of TLCK, these

bands

into

(not

was lowered two components

in each pair).

of the enzyme DFP, or a high four

experiments

10% acrylamide

protein

band was resolved

2, gels

under

obtained.

of dissociated

the slower

presence

of RuBP carboxylase

also

concentration

(Fig.

were

in gels

resulted

analysis,

of DTT or in the

Isolation

bands

was conducted

When, however,

of closely band

C

SDS (0.1%) polyacrylamide (10%) gel electrophoretogram of dissociated H. eutropha RuBP carbox lase purified, (A) in the absence of DTT, or Tn the presence of: (B{ TLCK, (C) DFP, (D) 1 mM Mgt and 10 mM EDTA, two protein (E) TEMBD buffer (the control). -Each-gel pair represents. concentrations; the gels on the left in each pair were subjected to electrophoresis with 2.5 to 6 ug protein per gel with twice as much protein applied to each gel on the imnediate right.

of from

shown). for

B

two Coomassie

in which

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

conditions

isolated

The same in the ab-

concentration should

of EDTA. greatly

restrict

(17).

The molecular

weights

of the three

polypeptides

1223

oberved

after

dissociation

Vol.71,No.4,

were

1976

56,000,

52,000

dissociation stead ious

BIOCHEMICAL

and 15,000,

products

stained

gels

the typified

shoulder.

By extrapolation

estimated;

this

52,000

dalton

subunits

subunits

In the face

ernary

structure

It

is

which

Amino

0.6.

of this that

contain

varying

this

peak with

mole

overall

mole ratio

of gels

showing

of 516,000 of eight

RuBP carboxylase

exists

proportions

Relative Amino Acid of RuBP Carboxylase

of the

two kinds

large

ratio

of small only

Aspartic Threonine Serine Glutamic Proline Glycine Alanine l/2 Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan

was

likely small

molecular

quatsubunits. species

subunits.

Subunits

Small subunits

2.61 1.35 1.05 1.92 1.02 2.39 2.56 0.33 1.56 0.56 0.89 1.98 0.69 '; $1

2.00 1.22 0.98 2.22 1.19 1.39 1.36 1.90 0.30 0.45 1.05 1.25 0.74 (;.;;I

0:53 1.59

0:31 1.48

analyses.

1224

be

to large

2 bands

and eight

of large

defined

of 56,000:

the most

in distinct

of var-

a well

Composition of Mixed Large and of Small from Hydrogenomonas eutropha.

Large (mixed) subunitsa

of duplicate

(25),

scans

in-

each peak could

the average

scans weight

a major

under

of the of pH 9.3

densimetric

enzyme consists

Acid

aAn average

that

buffer

Six

the area

The average

of a molecular

a running

2 revealed

from densimetric

possible

TABLE 1.

established

Electrophoresis

weights.

to the baseline,

is

ascertained

(24).

employing

same molecular in Fig.

approach

1.08.

respectively

and standards

of 8.3 yielded

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Vol. 71, No. 4, 1976

The mole is

given

content

in Table

of amino

1 for

AND BIOPHYSICAL

acids

mixed

normalized

large

and for

RESEARCH COMMUNICATIONS

with

small

respect

to phenylalanine

subunits.

DISCUSSION The present

data

of small

subunits

molecular

weight.

the

The latter products

In contrast,

(Purohit

and McFadden,

of large

subunit

of other

RuBP carboxylases.

this

This

homogeneous

large

erogeneity

subunits

connection

theory would

boxylases

from

sources.

indeed

is

the

each amino

composition

of large similar

primary Values

in two proteins.

structure, of less

structure. been calculated

were than

compared

50 were

The following from our

taken

present

(26). in this

data:

enzyme from

would

subunits

introduced

1225

of RuBP caralgae

from

(30-32).

several

a parameter,

of proteins,

j& eutropha-E.

the

in this

in mole percent

relatedness

het-

of RuBP carboxylases

subunits

98% of unrelated

DNA-

with

interest

green

large

tobacco

tobacco,

be compatible

Of special

of large

In

If the chloroplast

and unicellular

reflecting

of gel-electro-

of the

Over 5000 pairs

SAQ values

structure

subunits

structure

to indicate

variability

quaternary

isolation

differences

and for

of this

of the

conspecies

and small

(33),

of individual

comparable

electrophoretic

quaternary

to that

to gels.

proteolysis.

(mixed)

and Weltman

sum of squares

acid

(28,29)

of

minimize

(27).

of the

in

that

prokaryota

origin

blue-green

In 1971 Marchalonis is

from

under

In light

focusing

of chloroplast

closely

intermedius

heterogeneous

subunits

subtly

concentrations

54,500-dalton

large

consists

electrophoresis

at lower

conditions

of both

differ

after

reexamination

under

be a reinvestigation

acid

from II. eutropha

which

are

certain

The amino

the

by isoelectri

of analogous

endosymbiont

only

shou d be done after

enzymes

heterogeneity

that

observation).

we urge

enzyme has geen detected encoded

a single

unpublished

structure,

connection,

detectable

had been applied

into

RuBP carboxylase

subunits

of the enzyme from L.

dissociation

phoretically

one prokaryotic of large

were

which

examination

reveals

that

and of two kinds

dissociation

ditions

establish

content

SAQ was ~100.

or homology

rubrum,

of

many of known

proteins

subunit

SAQ,

of primary

comparisons 16 (2);

have i.

eutropha-

Vol.71,No.4,

1.

BIOCHEMICAL

1976

intermedius,

,spinach,

catalytic

Moreover, units

the

from

activity close

similarity

large

of the

values

hydrogenemonad

considerable are:

i.

mixed

and L. intermedius will

prove

enzyme will for

small

RESEARCH COMMUNICATIONS

17 (30);

suggest

(l),

and immunological

The SeQ values suggest

the data

between

subunits

fingerprints,

ellipsoidea,

or potential

the fi. eutropha

the mixed

tryptic

H. eutropha-(;. Once again,

18 (30,34).

contain

that

16 (4);

AND BIOPHYSICAL

that

large

For enzymes

eutropha-Chlorella

large

The composition, subunits

interest. still

from

ellipsoidea,

sub-

predicts

of the two large

are

which

in evolution.

enzymes, respectively,

to be homologous.

which

subunits,

and homogeneous

be of considerable

divergence.

large

have been conserved

relationship

subunits,

and !-l-. eutropha-

of unknown

the

following

77 (30);

1.

function, sources

the

eutropha-spinach,

111

(30,34). In closing, subunits

observed

we emphasize in the present

that

the

exact

research

function

remains

of the

two kinds

of large

to be established.

ACKNOWLEDGMENTS This research has been supported in part by research grant GM-19,972 from We gratefully acknowledge the amino acid the National Institutes of Health. analyses of Dr. S. Gurusiddaiah of the Bioanalytical Lab. REFERENCES 1. 2. 3. Z: 6. 7. 8. 9. it 12. 13.

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BIOCHEMICAL

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Biochem.

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Res.

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Commun. 8,