Vol.

64, No.

3, 1975

ON

BIOCHEMICAL

THE

John

SIZE

AND

Russell,

Received

April

CHEMICAL OF BOVINE

Litai

The

AND

Weng,

Department

BIOPHYSICAL

RESEARCH

NATURE OF THE LIVER RHODANESE

Pamela

S. Keim,

of Biochemistry, Chicago, Illinois

COMMUNICATIONS

POLYPEPTIDE

and

Robert

University 60637

CHAIN

L.

Heinrikson

of Chicago

24,1975

Evidence from molecular weight studies and sequence analysis of bovine liver rhodanese that the enzyme is a single polypeptide of molecular weight 35,200, and not a dimer of identical subunits half this size. The rhodanese molecule contains 317 amino acids including 5 methionines, 4 cysteines, and 5 tryptophans. As expected, six fragments were produced by cleavage with cyanogen bromide and these have been aligned in the enzyme with the aid of overlapping tryptic peptides isolated from a [ 148 carboxymethylmethionyl rhodanesederivative. The cyanogen bromide fragments account for all of the amino acid residues of the parent rhodanese molecule. Methionine residues are located at positions 72, 112, 214, 217, and 235 in the polypeptide chain and the active site cysteine is at position 251, in the carboxyl-terminal segment of the molecule.

%== rn rcates

S&bo

In his early

characterization

(1) reported

a molecular

measurements.

is a dimer

18,000.

studies

spectroscopy appear

(2), (5).

to be two

objection

to the

to demonstrate treated results difficult based peptide

of this

k inetic Further

sites

proposed dissociation

still

yield

obtained

thus

to reconcile primarily containing

far

the

enzyme

dimer

values

structure

in the

x-ray

crystallographic

the

dimer

hypothesis

covalent

structural

amino

acid

residues

EXPERIMENTAL Molecular

weight

molecular

weights

determination. of 3-5

mg samples

Rhodanese of the

was native

under

the

study This

from

the

(4,9,10).

a molecular

presents

rhodanese

investigators preparations

so

Moreover, are

definitive

is a single-chain

weight

there

serious

at 4 Al resolution

communication

sedimentatic

that

by numerous

of 35,000

having

fact One

conditions;

that

and

(6,7,8,4).

of rhodanese

native

polarization

denaturing

analysis,

l),

weight

filtration

fluorescence

failure

that

of molecular gel

derived

neighborhood

(11).

and

and

36,000

has been

monomers

from

(2,4),

weight

conception

monomers

obtained

EC 2.8.1.

velocity-diffusion

generally-held

has been

of molecular

into

sedimentation

or similar

mapping

in the

partial 317

identical

peptide

enzyme

sulfurtransferase,

led to the

(3),

rhodanese

weight

with

upon

with

per enzyme

of the

molecular

have

upon

has been

for

dimeric

b ased

interpretation

analysis support

active

however,

equilibrium

in favor

(thiosulfate:cyanide

of 37,500

studies,

in rapid

Evidence

velocity

weight

Subsequent

rhodanese

of rhodanese

evidence, poly-

of 35,200.

PROCEDURES prepared enzyme

as described and

derivatives

earlier

(4,12)

obtained

and

the

by reduction

and

Vol.

64, No.

3,1975

carboxymethylation

(13,14)

chromatography

under

calibrated

with

Isolation

obtained eluted

were

with

50%

isolated

cleaved

Three

this

single

CNBr-II

was

hydrochloride

(92 mg;

0.5

mmol;

dark,

the

of the

product

was was

(NEMAC), trypsin into

methionine. taining fragments

To-Ill one

of their

of Sephadex

purified

WCS

from

(RCM-

(CNBr)

CNBr-IV,

as described

order

of elution,

G-50

equilibrated

and

CNBr-V

a mixture

of partially-

of SP-Sephadex

by a similar

procedure

(1%

developed

into

two

with

fractions,

indicating

= 35,000).

The

and

two

accounted

and

TA-XIII

were

was

further

resolved

residues, for

Analytical

procedures.

rhodanese,

CNBr

fragments,

all

pure into

Amino

acid and

peptides two of [

sequences

tryptic

peptides

acid

24 hours

specific

in the

radioactivity

moles

4 hrs,

filtration was

of substituent

37’)

recovered

generated

and G-50

of residues were

1091

by

peptides in 0.1

TA-I,

residue

each

TA-lllb

and

14 C] -carboxymethylmethionine. residues

the

in fractions

a single peptides,

N-ethylmorpholine

were

on Sephadex

containing radioactive

1 mM

residues

Worthington; by gel

5 methionine

against

at arginyl

radioactivity

respectively, of the

dialyzed

TRTPCK-lFA,

of th e original

6 fi

-carboxymethyI-methionyl-RCM-rhodanese

Cleavages

to TA-XIII)

for

of 4.5

and

iodoacetic

The

incorporation

hydrochloride,

lyophilized.

(TA-I

All

[ 14C]

C]

reaction

lyophilized.

To 10 ml

pH 4,0,

14

of [2After

and

the

acetate,

solution

cpm/pmole). water

RCM-rhodanese.

&sodium

of an aqueous

distilled

by weight;

citraconylated

in 0.1

cpm/pmole,

and

from

40,000

against

13 fractions

TA-I

ml

in 6 J1 guanidine

bicarbonate.

TA-XIII.

0.5

radioactivity,

pH 8.5,

with

ammonium

added

(MW (18)

resolved

was

dialyzed

of rhodanese

hydrolysis

basis

CNBr-III, was

column

(13).

bromide

on a column

peptides mg of RCM-rhodanese

180,000

citraconylated

acetate

The

permeation

rhodanese

on the

fractions,

resolved

(16).

weight

of cyanogen

CNBr-I

200

specific

solution

mole

et al.

by gel

CNBr-IIB.

containing

guanidine

determined

on a column

chromatography

COMMUNICATIONS

carboxymethylated

mixture

step.

ion-exchange

Fish

to CNBr-V

of the

were

molecular

by weight

reaction

of ]4C-carboxymethylmethionyl

of a solution

and

by

8 11. urea.

and

Isolation

were

by

containing

CNBr-IIA

was

form

components

buffers

acid.

by

and

CNBr-I

of the

acetic

in pure

excess

RESEARCH

(15)

of known

Reduced

designated

filtration

oxidation

standards

a two-fold

fractions,

BIOPHYSICAL

as described

fragments.

with

by gel

acid

conditions

bromide

Five

AND

performic

derivatized

treated

(17).

and

per

similarly

was

earlier

and

denaturing

of cyanogen

rhodanese)

were

BIOCHEMICAL

-M TA-III,

of labeled TA-llla,

These

tryptic

in rhodanese. at the derived

NH2-termini by automated

of intact Edman

RCMdegradation

con-

Vol.

(19)

64, No. 3, 1975

in a Beckman

peptide

protein-peptide

programs

quantitated

BIOCHEMICAL

supplied

Sequencer

by the

conversion

(23),

were

to amino

employed

(20),

acids

for

(21).

purposes

weight.

weight

high Thin

pressure

layer

gel

electrophoresis

35,000.

determined

by gel

in sodium

modified

sulfate

the same

result

is obtained

cysteinyl

residues

presence

Table

of 6 -M guanidine

1, together

employing

with

these

independent

approaches

that

subunits,

rule

out

this

as well indicates

if present,

must

under (SDS)

and

when

estimations

the

be linked

were

and

identified and

or without were

ninhydrin

conditions

mercaptoethanol

(25),

native

methods.

molecular

(4) the

nondenaturing

rhodanese

weight

by unconventional

of rhodanese

and was

on columns These made

The of the

evidence enzyme

covalent

are

by various

Filtration 6% agarose; 0.1 M sodium acetate 6 M guanidine HCI; 0.1 M meXaptoethanol (cf ref 16)

2.)

Sephadex G-75; 0.01 -M sodium

3.) B.

C.

Sephadex

Sedimentation

0.1 M thiosuTfte,

Tris

* S04, pH 8.0

G-100

Polyacrylamide gel in SDS-mercaptoethanol

by various

is about Data

35,000 19,000;

electrophcresis (cf ref

velocity

1092

25)

Weight (native) (RCM) (oxidized)

(4) 37,000

35,400 (4) 35,000 (9) 32,600 (10) 37,500 (1) 19,000; 38,000

agarose

in

summarized

in

by these 35,000 presented

procedures

35,500 36,000 36,000

to be

invesgiators

provided

bonds.

Molecular

Gel 1.)

reported

of 6%

data

polyacrylamide

thereof

1

as determined

Procedure A.

(24).

molecular

or derivatives

filtration

of rhodanese

Table weights

staining

manually

possibility.

Molecular

and

hydrolytic

sequenced

Heinrikson

mercaptoethanol.

as sedimentotion that

and

to gel

and

weight

protein

DISCUSSION

subjected

hydrochloride

molecular

techniques

are

dimethylallylamine

with

peptides

by Blumenthal filtration

COMMUNICATIONS

chromatography,

(22),

Small AND

dodecyl

Essentially

containing the

publication

with

liquid

methods

of confirmation.

In an earlier

of rhodanese,

890-C)

RESEARCH

Phenylthiohydantoins

RESULTS Molecular

BIOPHYSICAL

(model

manufacturer.

by gas chromatography

back

AND

(2)

(2)

and below

Vol.

64, No.

3,1975

BIOCHEMICAL

AND

BIOPHYSICAL

Table

Amino -w

acid

composition

of--m reduced

1 Amino

and

RESEARCH

COMMUNICATIONS

2

carboxymethylated

rhodanese

Recoverya ielative quanti

acid 24 hrs

48 hrs

molar tiesb

No. of esidues per molecule

72 hrs

ii+zlF Lysine

44.2

43.1

44.0

16.9

17

Histidine

21.3

20.8

19.2

7.9

8

Arginine

56.3

56.0

55.7

21.1

21

S-carboxymethyl

Cysteine

9.3

8.5

8.0

3.5

4

+ Asparagine

67.1

66.2

65.5

25.8

26

31.2

28.0

22.2

13.9

14

50.0

31.7

20.8

20.7

21

86.3

81.3

82.0

32.2

32

Proline

52.5

51.1

52.4

20.2

20

Aspartic

Acid

Threonine Serine Glutamic

Acid+

Glutamine

Glycine

73.8

71.5

70.7

27.9

28

Alanine

66.9

65.9

67.8

26.0

26

Valine

69.4

69.5

72.1

27.3

27

Methionine

12.6

12.0

11.2

4.8

5

lsoleucine

20.6

19.8

20.8

7.9

8

Leucine

69.9

69.9

69.2

27.1

27

Tyrosine

24.9

18.3

13.9

11.7

12

Phenylalanine

41.2

40.4

40.8

15.7

16

Tryptophanc

5

Total

a b

’

317

Samples

of 90 ug were

Calculated

by dividing the corrected compositions 1 residue of amino acid. Based on Ala

Determined

by hydrolysis

hydrolyzed

--in vacua

in methanesulfonic

in 6 N HCI

acid

1093

at

110’

for the

by 2.58 mumoles, = 26.0 residues. (27)

and

by peptide

times the

indicated.

equivalent

analysis.

of

BlOCHEMlCAL

Vol.&i,No.3,1975

running

time

Molecular by

being

AND BIOPHYSICAL

extended

weight

to

determinations

of

SDS-polyacrylamidefelectrophoresis The standards (15).

catalase,

= 60,000,

of

bands

610

nm.

staining

was

measured

Results: minent

fraction results

P-450

frac

(NH4)2S04, lab

of

richest

failed

components chrome

I and

type

these

two

vitro

fraction

I II

recombined

all

Separation

of

figure

the

3 revealed:

weights

ranging

tative

and

tions

within

treated

animals.

treated

groups

feel on

seven

major to

same The

was

largest

an

I

in

cent in

other and

known re-

have

shown

present

if

is

used

40-50,

studies

detailed

characterifractions.

these with

as large

quanti-

from in

the

P-450

after

frac-

differences

fraction

in

variously variously

content

at

phenobarbital

treat-

ment. By comparing figure tide

2, to

signment

with

each of

in

Fig.

ly

determined

'SDS

quantitative of

= sodium

band

intensity,

we

the

4 known

components

activities

4 was

functional

supported components,

to by

have

measurements, assigned (Fig.

electrophoretic a comparison e.g.

reductases

dodecylsulfate

1101

one

seen major

3-right). components

of

the or

in

molecular (2)

quantitative

cytochrome

for

shown

ammoniumsulfate

seen

that a of

in

daltons;

our

NADPH-cyto-

gels

between

concentrations

per

useful

components 100,000

difference

increase

ammoniumsulfate

and

a more

(3)

the

are

that

ammoniumsulfate

that

prominently

SDS-polyacrylamide

preparation;

the

most

contained

differences

a given for

on

10,000

qualitative

components

elevated

(1) from

we

pro-

with

results

30-35

most

three

NADH-

are

at

microsomes

The

II

lipids

proteins

liver

activities

dependent and

is

40-50

Preliminary

however, are

reported

b5

hydroxylase

peptides

who

transport

40-50.

density

agreement

findings.

fractions,

activity of

in

rat

cytochrome

measurements;

zation

with

electron

and

=

optical

P-450

not between

these

microsomal

in

in

(3),

previously = 68,000,

photometer

that

is

peptides

hemoglobin

the

scanning

obtained

confirm

both

of

was

and

apparent

Levin

microsomal

blue,

This

experiments

presented mixture

is

and

c reductase, type

2 it

tion

to

of

a Gillford

Lu

however,

have

with

30-35.

separation.

was also as serum albumine

Comassie

II

better

major

= 43,000,

with

F ig.

From

in

earlier

ovalbumine

After

for

the

were

described 15,500.

7 hours

RESEARCH COMMUNICATIONS

ratio cytochromes,

in pep-

The

as-

as

described

of

similarwith

Vol.

64,

BIOCHEMICAL

No. 3, 1975

NH2-Vol-His-Gin-Vol-Leu-Tyr-Arg

AND

(CM

Cys,

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Asx~,T~~~,S~~~,GIX~,P~O~,G~~~,AI~~,

5 It------TAVa15,

Lys3,Arg)

lle5,Tyr2,Phe2,His,Trp2,

I

Arg-Asp-Lys-Ala-Ser-Pro-Tyr-Glu-Val65 TA-I

Leu-Pro-Ser-Glu-Ala-Gly-Phe 75 __._ Tyr3,

. Phe,His)

+--

(Asx7,Thr,Ser3,Pro,Gly4,Alo2,Val3,lle,Leu2,

..--TA-XI,I-

Arg-Val-Trp

Phe-Arg-Val-Phe-Gly-His-Arg-Thr-Val-Ser-Val115

Leu-Asp-Gly-Gly-Phe-Arg 125

Tyr3 r Phe2,His2,

Pi-07, GIy6,Ala5,Va15,

(Asx 6,Thr4,Ser5,Gly2,

Lys5,Arg5,Trp)

120

(Asx, Glx4,Gly,

TA-lllb-

Pro, Leu, Ph e,Lys,Ser)

Arg-Ala cl

TA-lllb

Ile3,

Met 235

Phe-Glu-Ala-Lys-Lys240

__I___c(

Val-Asp-Leu-Thr-Lys-Pro-Leu-lie-Ala-Thr-Cys*-Arg 245

Gly6,Ala7,Va14,

Leu9,

Arg-Gly-Ser-Val-Asn 210

TA-Illa.-Leu-Thr220

Ile2,

Leu4,Tyr2,

(Asx5,

Cys2,Thr2,Ser2,Glx4,

Pro*,

250

Lys3, Arg)

Phe3,His2,

Arg-Ala-Pro-Pro-Glu-Thr-Trp305

‘/al-Ser-Gln-;;;-Lys-Gly-COOH

Fig. 1. Partial sequence analysis of bovine liver rhodanese indicating alignment of the six C Br fragments. Arrows over each methionine residue indicate tryptic peptides from citraconylated [ cl C] -carboxymethylmethionyl-RCM-rhodanese. Amino acids included within parentheses are based upon compositional analyses of each fragment minus residues placed in sequence. The latter assignments were made from automated Edman degradation of the fragments and carboxypeptidase Y digestion (see text).

TA-I: residues CNBr-III. CNBr-III: with t-sequence analysis gave the

TA-XIII: sequence:

(residues 64-109). Sequence through 79 and provided the (residues in TA-I the sequence:

analysis of this overlap between

73-112). Edman degradation and established its location -Val-Trp-Hse.

(residues 110-l Val-Trp-Met-Phe-Arg.

14).

The

overlapping This is the

peptide CNBr-IIB

allowed for the placement of and the next CNBr fragment,

proved the as the second

equivalence of positions CNBr fragment. COOH-

73-79

peptide between CNBr-III and CNBr-I gave fifth tryptophan peptide in rhodanese (cf. 28).

1095

Vol.64,No.3,

the

CNBr-I: Cwminal

1975

(residues

BIOCHEMICAL

AND

113-214). Residues 113 sequence of this 102-residue

BIGPHYSICAL

through fragment

RESEARCH

COMMUNICATIONS

129 were placed by Edman degradation was shown to be: -Val-Asn-Hse.

TA-llla: (residues 210-233). Amino acid analysis of this peptide revealed the residueseled methionine. Automated Edman degradation allowed the placement 210-221, thus locating the fourth methionine at 217. This peptide contains CNBr-V, peptide Pro-Phe-Hse, and provides the overlap into CNBr-IV, the penultimate CNBr in rhodanese. CNBr-IV: cation=CNBr-V Hse was identified

(residues

218-235). Sequence and the COOH-terminal at its COOH-terminus.

analysis of this fragment peptide CNBr-IIA.

and

presence of 2 of residues the trifragment

was in accord with its The sequence of -Arg-Ala-

lo-

TA-lllb: (residues 234-252). Automated Edman degradation proved that this fragment is that whichcontoins the active site cysteine residue sequenced previously (14). The earlier analysis was in error and the corrected sequence in the vicinity of the active site sulfhydryl reported herein in agreement with the structure found by Bossa et al. (30) for the tryptic peptide from bovine kidney rhodanese. CNBr-IIA: (residues 236-317). The only CNBr fragment terminal fragment in rhodanese. Its NH2-terminal sequence 252) in Ta-lllb. The COOH-terminal sequence was obtained citraconylated material, the only peptide lacking in arginine. earlier (28) in a study of the tryptophyl peptide sequences.

is

lacking Hse, this peptide is the COOHa is equivalent to residues 3-19 (236by analysis of a tryptic peptide from Most of this structure was reported

Thus, the total number of amino acids observed in the six cyanogen bromide fragments from rhodanese is equal to 317 residues, the number calculated from its composition (Table 2). One cysteine is located in the NH2-terminal segment of the molecule and the remaining three cysteines, including the catalytically essential residue, are near the COOH-terminus. These findings constitute the first chemical proof for the existence of rhodanese as a single polypeptide chain the size of which is in accord with the majority of molecular weight studies of the enzyme. Moreover, they provide a structural framework for interpreting the results of x-ray crystallographic analysis and mechanistic studies of rhodanese currently underway in other laboratories. ACKNOWLEDGMENTS The authors gratefully acknowledge the many helpful suggestions of Dr. John Westley and the expert technical assistance of Ms. Elaine Krueger. throughout the course of these studies, This work was supported by grant GB-29098 from the National Science Foundation REFERENCES S&bo,

B. H.,

Acta

Chem.

Stand.,

7,

1129,1137

(1953).

:: Volini, M., DeToma, F., an d West&y, J., J. Biol. Chem., 242, 5220 and Westley, J., J. Biol. Chem., 241, 5168 (19m Volini, M., 2430 i: Blumenthal, K. M., and Heinrikson, R. L., J BirChem., 246, 5. Horowitz, P., and Westley, J., J. Biol. Chem., 245, 986 VO). Green, J.R., and Westley, J., J. Biol. Chem., 236, 3047 (1961). 547 (1962). F: Westley, J., and Nakamoto, T., J. Biol. Chem.,T7,

8. 9. 10. 11.

E: 14. 15. 16.

(1967). (1971).

J., Biochim. Biophys. a, 207, 144 (1970). DeToma, F., and Westley, and Woodward, C.K., Biochim. Biophys. Acta, 379, 385 (1975). Ellis, L. M., Trumpower, B. L., Katki, A., and Horowitz, P., Biochem. Biophys. Res. Communs., 57, 532 (1974). Smit, J.D.G., Ploegman, J.H., Pierrot, M., Kalk, K.H., Jansonius, J. N., and Drenth, Isr. J. Chem., 12, 287 (1974). F., J. Biol. Chem., 245, 984 (1970). Horowitz, P., anmeToma, Heinrikson, R. L., Sterner, R., Noyes, C., Cooperman, B.S., and Bruckmann, R. H., J. Biol . Chem., 248, 252 1 (1973). Biophys. Acta, 278, 530 (1972). Blumenthal, K. M., and Heinrikson, R. L., Biochim. Hirs, C. H.W., Methods enz G??&&ii~d~“c.(,T’,4”B;:,,. Chem., 244, 4989 (1969). Fish, W.W., Max ., an

1096

J.,

Vol.

17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

64, No.

3, 1975

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Sterner, R., Noyes, C., and Heinrikson, R.L., Biochemistry, 13, 91 (1974). Sterner, R., and Heinrikson, R. L., Arch. Biochem. Biophys., rpress (1975). Edman, P., and Begg, G., Eur. J. Biochem. 1, 80 (1967). Pisano, J.J., and Bronzert, T. J., J. Biol. Cf?em., 244, 5597 (1969). Smithies, O., Gibson, D., Fanning, E.M., Goodflix, R.M., Gilman, J.G., Baliantyne, D. L., Biochemistry lo, 4912 (1971). Jeppsson, J.-O., ond Sihuist, J., Anal. Biochem., 18, 264 (1967). Inagami, T., and Murakami, K., Anal. Biochem., 47301 (1972). Peterson, J.D., Nehrlich, S., Oyer, P.E. ,and Steiner, D.F., J. Bioi. Chem., (1972). Weber, K., and Osborn, M., J. Biol. Chem., 244, 4406 (1969). S%bo, B. H., Acta Chem. Stand., 17, 2205 (lm Liu, T-Y., and Chang, Y.H., J. t%I. Chem., 246, 2842 (1971). Heinrikson, R.L., and Russell J., Biochim. Biophys. Acta, 278, 546 (1972). Hayaski, R., Moore, S., and Stein, W.H., J. Biol. Chem.748, 2296 (1973). Bossa, F., Cannello, C., Federici, G., Barra, D., and Peccix, FEBS Letters, 170 (1973).

1097

and

247,

29,

4866