On the identification and nomenclature of the polypeptide subunits of bovine cytochrome C oxidase

Vol. 99, No. 1,198l March

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

16, 1981

ON THE IDENTIFICATION AND NOMENCLATURE OF THE POLYPEPTIDE OF BOVINE CYTOCHROME C OXIDASE

Victor Institute

M. Darley-Usmar*

of Molecular

Biology,

and Michael

University

51-57

SUBUNITS

T. Wilson"

of Oregon,

Eugene,

OR 97403

U.S.A.

t Dept. of Chemistry, University of Essex, Wivenhoe Park, Colchester CO4 3SQ U.K.

Received

December

31,

1980

SUMMARY: The polypeptide subunits of cytochrome c oxidase (E.C. 1.9.3.1.) have been identified. isolated from beef heart which react with ""[Hg]Cl rapid and simple method for identifying and number1 -6 g the subunits is described which is independent of the type of sodium dodecyl sulphate-polyacrylamide gel system employed to separate them. Cytochrome

c oxidase

inner

mitochondrial

atory

chain,

membrane

is a complex

The number

of polypeptides

techniques

and their

these

gel

systems

conditions leads

used,

In this groups

peptide.

method

reaction

of the oxidase

SDS gels

is

of

protein

electron

acceptor

of several

based

by SDS1 gel

(1,Z).

of migration

is dependent

individual

respir-

electrophoretic

order

of migration

of the

of the

polypeptides

upon their

identifying

component

in on the

polypeptides

naturally

(3,6,7).

and present

c) mercury

terminal

As the order

we compare

a) gross

treatment;

ing

this

a major

enzyme consisting

nomenclature

Assignment

trophoresis:

and the

(2,3,4,5).

paper

1.9.3.1.),

has been determined

to some confusion

search

(E.C.

A

the

polypeptide

a scheme for

rests

on three

numbering

unequivocally

simple

criteria

apparent

molecular

weight;

binding

properties.

This

with

'03[Hg]

followed

systems identifying

determined b) sensitivity latter

criterion

by autoradiography

of various a given from

repoly-

SDS elec-

to Trypsin involves of the result

(8).

IAbbreviations used in this paper are following: SDS, sodium phate, PGGE, polyacrylamide gradient gel electrophoresis. * Author to whom correspondence should be directed.

dodecyl

sul-

0006-291X/81/050051-07$01.00/0 51

Copyright 0 1981 by Academic Press, Inc. All rights of reproduciion in any form reserved.

Vol. 99, No. 1,198l

BIOCHEMICAL

METHODS: Cytochrome c oxidase the method of Yonetani (9).

AND

BIOPHYSICAL

was prepared

from

RESEARCH

beef

heart

COMMUNICATIONS

mitochondria

by

Preparation of protease treated cytochrome oxidase. Trypsin treated oxidase was prepared by incubating 0.5 ml of 280 uM oxidase (total haem) in 0.1 M Nsphos pH 7.4, 1% Tween 80 with 0.5 mg of trypsin for thirty minutes at room temperature. Trypsin and proteolytic fragments were separated from oxidase on a Sephadex 6.75 column (1 x 30 cm) equilibrated with 0.1 M sodium phosphate 1% Tween 80 pH 7.4. Control samples of oxidase were treated identically but without trypsin. Reaction with mercuric chloride and SDS-PGGE*. Samples of oxidase after gel filtration were treated with mercuric chloride (203[Hg]C1 ) by incubation In a typical experim ii nt 100 ul of at room temperature for thirty minutes. cytochrome c oxidase (40 uM total haem) was mixed with 5-40 ul of Z03[HglC12 (1.2 mM in 1N HCl,0.18 Ci/M). After incubation 25 ul of oxidase samples (s 100 ug of protein) were mixed with denaturing solutions containing 5% SDS, 0.25 M Tris/HCl pH 6.2, 8 M urea with or without P-mercaptoethanol (57 mM). SDS-PGGE and autoradiography was conducted as described previously (2,8). Autoradiograms and gels were scanned at 600 nm using a Perkin-Elmer 575 spectrophotometer.

RESULTS: The effect shows

the

pared

and Fig.

of proteolysis

polypeptide

banding

lb after

among the low molecular polypeptides al.

which

(6).

other

polypeptides

(Fig.

la).

al.

weight

polypeptides.

less

these

c oxidase are

Digestion

agreement intensely

with with

as a broad

observations

cytochrome

Differences

evident

has removed

the

results

coomassie

band on SDS gel

have been noted

Figure

pattern.

as preonly only

of Ludwig

blue

la

than

two et

the

electrophoresis

previously

by Downer

et

(3).

In these

of the

experiments

subunits

Each aliquot

in a denaturing

(57 mM) was present. radioactive

mercury

of cytochrome

two conditions

at room temperature

two aliquots. bation

treatment.

stains

banding

of bovine

trypsin

in reasonable

III

polypeptide

pattern

and migrates

Both

Labellinq

bation

is

Subunit

on the

with

labelling

203[Hg]C12

was prepared

medium These

for

in only treatments

among the

c oxidase

the for

were enzyme

SDS gel

one of which, result

subunits.

52

with

mercuric

chloride.

used.

Following

sample

was divided

electrophoresis however,

in different

incuinto

by incu-

2 mercaptoethanol distributions

of

BIOCHEMICAL

Vol. 99, No. 1,198l

Fiq.

1.

Protein and 2"3[Hg] Gel Electrophoresis.

AND

Banding

BIOPHYSICAL

Patterns

after

RESEARCH

COMMUNICATIONS

SDS-Polyacrylamide

Gradient

Figs. a and b show the protein banding pattern of coomassie blue stained samples. Fig. a shows beef cytochrome c oxidase as prepared and Fiq. b after proteolytic treatment. Figs. c-f show traces of autoradiograms of "[Hg] labelled oxidase (Hg:haem a = 4:l). Other conditions were as follows: Figs. c and d show autoradiographs of samples labelled in the absence of E-mercaptoethanol and Figs. e and f in its presence. Figs. d and f show autoradiograms of trypsin treated samples. Three levels of coomassie blue staining or '03[Hg] labelling are shown in Fig. 1. These are:

Figs. the

lc

trypsin

treated

this

reacts

specifically

tide

a,

high

0

moderate

::I::

low

and Id compare

cussed

of Ludwig

D

enzyme

labelling

et al.

and subunit(s)

three

VII.

enzyme show no significant its

stain or

stain

labelling

'03[Hg]

labelling

pattern

of the control

residues labelled

(8). polypeptides

The trypsin differences

publication

treated in either

We have dis-

and shown

Using

that

the numbering are

oxidase

and

mercury system

subunit

II,

and the

untreated

the amount

polypep-

of label

bound

or

distribution. In contrast

bated lc

labelling

in a previous

cysteine

the

or

in the absence of mercaptoethanol.

pattern with

(6)

the

labelling

with

the labelling

mercaptoethanol

and le which

compare

pattern

of the same protein

do show some differences. labelling

of the control

53

samples

These enzyme under

are

when incu-

shown the

in Figs.

two condi-

Vol. 99, No. 1,198l

BIOCHEMICAL

AND

BIOPHYSICAL

Table Comparison Wilson

of

et

the

al.

polypeptide

Downer

(2) No.

Mr

No.

35,700

IV

16,900

I II III IV

a

12,500

a

11,700

V

10,500

b

21,100

VI

7,600

VI

C

7,000

C

VII

4,500

tions.

- not

The

low

label

on

Figure

If

on

addition

molecular by

trypsin

bovine

peptides them workers tides in

of use.

subunits notably

No.

Mr

(t-f)

I

40,000

II

21 ,000

III

14,800

0

(12)

13,500

3

(17)

11,600

1

(13)

4

(14)

16,000 11 ,600

VI

of

24,000

IV

12,500

2 (11) 2 (1’3)

V

nr

8,200

4,400 arranged in the

VI

nr

v11 VIII

nr

9,500

9,500 VII

6,000

so that the same horizontal

is

now is

of

7,600

same polypeptide, row.

of the

whatever

its

the

generally

and

1.

in

the

also

ld).

The

newly

labelling

order

not

numbered

of

by

the

and

subunits

II

et

al.

(6)

designated

and

same

III

in

a

a mobilisa-

labelled

is

lower

substantially

re-

activity

functional (2,5,6,10).

convention from

the

resolution

(3,7).

In

The of

behaviour all

shows

full

and

some

54

and

less.

Examples the

migration

VI

have

the

weight.

the

le)

correspondingly

polypeptides

Unfortunately is

(le) is

that

6-10

molecular

Table

which

sample

accepted with

(Fig.

sample

control of

displaced

labelled.

(Cf

extent

decreasing

been

become

mercaptoethanol

oxidase

Changes

has

treated

associated

in

VII

and has

identified

shown

V and

II

protease

so

cytochrome

Ludwig

Number Cysteine residues

36,000

nr

the

and

been

order are

Yu & Yu

19,000

V

12,400

of

oxidase have

in

nr

polypeptide

DISCUSSION: It of

16,800

polypeptide

shows

weight

moved

oxidase

(4)

Mr

I II III IV

21,000

subunit

weight

the

cytochrome

reported

molecular

tion

& Buse

No.

24,100

are is

of

(11)

35,400

VII

The polypeptides designated number nr

systems

Steffens

Mr

22,500

b

numbering al.

COMMUNICATIONS

1

(3)

II III

V

et

RESEARCH

gel

the

systems have

addition

polypeptides

labelling

a number of

been some

as

poly-

of

polypepcurrently noted workers,

contaminants

for

BIOCHEMICAL

Vol. 99, No. 1,198l

since

they

of the

can be removed

AND

by limited

is

experimental

results.

not satisfactory

below

16000

particularly

basis

to proteolysis

of

for

concentrations

mercury

the small next

stains

at high

in the activity

groups

to correlate

reacts

groups.

extent three

of molecular with

that copper

weight

alone

molecular

weight

concentration

(8).

For this has only with

be very

but only two are

blue.

by various

that

II

are

(13).

at high

203[Hg]C12

by limited

et al.

(6).

polypeptide

tide numbered VII by Buse, which has four

5.5

cysteine

and

1) which

ligand labelled

present

in the sequence

polypeptide (see

from

Subunit

Table number

subunit V is

has been 1).

This

of free V which

labelled

proteolysis

Of the

and have

been

we have learned

5 corresponds residues

from to a

concentrations.

Recently

(11).

of the molecule

a large

residue

(Table

copper

The next

it

group)

has two cysteine

surface

groups

of

at low

preferentially

the

suggesting

removed

b and c by Ludwig communication)

near

we can discriminate

one cysteine

III

is a putative

[no cysteines

mercury

mercury

subunit

Subunit

following weight

1 Hg atom/haem

is clearly

coomassie

with

reason

mercury

polypeptides

- personal

subunit

mercury

sensitivity

molecular

II with

than

one of which

IV (4) and V (5) strongly

high

of subunit

weight

must

or

on SDS-PGGE in the

by the

molecular

with

resistance

to haem 5 e.g.,

as this

can be discrimi-

mercury. found

reaction

site

strongly

weight,

discriminated

196 and 200)

(6),

named contaminants (Buse

change

each polypeptide

pattern

(relative

apparent

the

as 'a'

sequence

are

IV does not bind

polypeptide thiol

banding

Daltons),the

therefore

(12)1, and numbered

our

II and III

(positions

Subunit

by which

radioactive

only

by the mercury

data

with

higher

We conclude

(8).

basis

polypeptides

and labelling

of mercury

slightly

residues

those

molecular

I (QJ 35,000

binds

different

on the

apparent

I,

Subunit

subunit

for

its

We have assigned

its

without

COMMUNICATIONS

Daltons.

on the

way:

important To do this

Here we have presented nated

proteolysis

RESEARCH

enzyme (6).

It has become increasingly their

BIOPHYSICAL

to the (14).

polypep-

In view

of

Vol. 99, No. 1,1981

the

fact

that

this

the disulphide teines

VII,

(15).

labelled

shows that

personal

quite

(14).

polypeptides

the other

VI is

The last

polypeptide

is

COMMUNICATIONS

in the

possible

neither

consisting

that

amino

20"[Hg]-labelling

contains

cysteine

acids

of

the cysby trypsin

weight

of three

but

presence

removed

low molecular

(N-terminal

residues

RESEARCH

strongly it

Subunit

component

no cysteine

of cysteine

different

from

literature

subunits

is

Table

1 that

their

is

molecular

fraction, polypeptides

Ileu

and Ser)

does occur residues

laboratories

of

which

(G. Steffens

or,

technique

is not

routine

method

N-terminal for

of different

cytochrome

residues

identifying

groups

(5).

subunits

so that

a variety

known,

cytochrome

the

oxidase.

have adopted

may be referred

The unambiguous

sequence

with

where

on oxidase

polypeptide

acid

made difficult

have blocked

a given

and,

of bovine

working

one number.

amino

weight

polypeptides

such that

The former

available

nomenclature

of the

systems

through

method

polypeptides

numbering,

by more than

analysis. latter

the

residues

labelling

in the

readily

labelled

by 'a' [Hg].

1 compares

It is clear

minal

BIOPHYSICAL

communication). Table

number

AND

mercaptoethanol

is an heterogeneous

three

indicates

the

bonds

The two sequenced

these

is

reagent

disulphide

nor

subunit

polypeptide

reducing

form

treatment

BIOCHEMICAL

failing

to

way to identify that,

in most oxidase

through

N-ter-

laboratories

and

as many of the

Here we offer

a simple

and

and a comparison

between

the

of different

gel

systems

may be employed.

ACKNOWLEDGEMENT: gratefully

This

work

was supported

by SRC Grant

GR/A66390

which

we

acknowledge.

REFERENCES 1. Erecinska, M. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 188, 1-14. 2. Wilson, M.T., Lalla-Maharajh, W., Darley-Usmar, V.M., Boneventura, J., Boneventura, C., and Brunori, M. (1980) J. Biol. Chem. 255, 2722-2728. 3. Downer, N.W., Robinson, N. and Capaldi, R.A. (1976) Bio?'f%mistry 15, 29302936. 4. Yu, C.A. and Yu, L. (1977) Biochim. Biophys. Acta 495, 248-259. 5. Steffens, G. and Buse, G. (1976) Hoppe-Seylers Z. Physiol. Chem. ---) 357 1125-1137.

56

Vol. 99, No. 1,198l

6. 7. a. 9. F: 12. 13. 14. 15. 16. 17.

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Ludwig, B., Downer, N.W. and Capaldi, R.A. (1979) Biochemistry 3, 14011407. Capaldi, R.A., Bell, R.L. and Branchek, T. (1977) Biochem. Biophys. Res. Comm. 74, 425-433. Darley-Usmar, V.M., Alizai, N., Al-Ayash, A.I., Jones, G.D., A. Sharpe, and Wilson, M.T. Comp. Biochem. and Physiol. (In Press). Yonetani, T. (1960) J. Biol. Chem. 235, 845-852. Penttila, T., Saraste, M. and Wikstrom, M. (1979) FEBS Lett. 101, 295-300. Steffens, G.J. and Buse, G. (1979) Hoppe-Seylers Z. Physiol. Chem. 360, 613-619. Sacher, R., Steffens, G.J. and Buse, G. (1979) Hoppe-Seylers Z. Physiol. Chem. 360, 1385-1392. Tanaka, M., Haniu, M., Yasunobu, K.T., Yu, C.A., Yu, L., Yau-Huei, Wei and King, T.E. (1979) J. Biol. Chem. 254, 3879-3885. G.J. and Buse, G. (1979) Hoppe-Seylers Z. Steffens, G.C.M., Steffens, Physiol. Chem. 360, 1641-1650. Buse, G. and Steffens, 6.3. (1978) Hoppe-Seylers Z. Physiol. Chem. 359, 1005-1009. Barell, G. (personal communication). Yasunobu, K.T., Tanaka, M., Haniu, M., Sameshima, M., Reimer, N., Tetsuo, E. King, T.E., Yu, C.A. and Yu, L. (1979) in Developments in Biochemistry 5 King, T.E., Orii, Y., Chance, B. and Okunuki, K. eds. Elsevier/North Fiolland.

57