Differences in the amino acid compositions of the subunits of succinate dehydrogenase

Differences in the amino acid compositions of the subunits of succinate dehydrogenase

BIOCHEMICAL Vol. 48, No. 4, 1972 AND BIOPHYSICAL RESEARCH COMMUNICATIONS DIFFERENCES IN THE AMINO ACID COMPOSITIONS OF THB SUBUNITS OF SUCCINATE DE...

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BIOCHEMICAL

Vol. 48, No. 4, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DIFFERENCES IN THE AMINO ACID COMPOSITIONS OF THB SUBUNITS OF SUCCINATE DEHYDROGENASE Norman T. Felberg Graduate

Received

Department

July

Summary:

The subunits

the larger

The subunits

subunit

probably

EC 1.3.99.1)

succinate

differ

could

succinate and their

so widely

not be derived

dehydrogenase

has been prepared

has a subunit

structure

dehydrogenase amino acid com-

in composition

that

from a dimerization

of the

and labile

subunit,

and labile

with

in a highly

sulfide

sulfide

purified

70,000,

in a mole reported

weight

contains

ratio

(1,2).

The enzyme

of 4:4

subunit,

peptide-bound

of 1:4:4.

of about

to contain

form

The FP or flavoprotein

in a mole ratio

a molecular

of the enzyme are

(succinate:(acceptor)oxidoreductase,

of two peptides.

weight of about

a molecular

heme iron, tein

University

one. Recently,

with

of highly purified by SDS electrophoresis

isolated

determined.

smaller

of Biochemistry, Brandeis Waltham, Mass. 02154

11, 1972

preparationswere positions

and Thomas C. Hollocher

27,000,

(1,3,4).

flavin,

The IP or iron contains

pro-

non-heme

Homogeneous

10.3 _+ 0.4 nmoles

noniron

preparations

flavin

x (mg protein)

(1). lies

We wish

to report

in their

amino

Methods to Baginsky

and Hatefi were

enzyme according 10.3

the separated contaminants phoresis

(2) and Davis determined

difference

between

dehydrogenase and Hatefi

after

tryptic

and Stemple

Protein

the subunits

bovine

(1)

was prepared from beef

digestion

(5)

was determined

crystalline

and by ultraviolet

major

Succinate

to Hatefi

1 xmmole-l). using

a further

compositions.

and Materials:

concentrations

method

that acid

(oxidized (Sigma)

heart.

of the heat minus

by dry weight,

serum albumin

according Flavin denatured

reduced&450

=

by the Folin-Lowry as the standard

(6),

absorption

via the method of Edelhoch (7) in the case of The enzyme preparations contained no more than 10%

subunits. which

in sodium

were

separated

dodecyl

from the

sulfate

two subunits

(SDS) by the method

on disc

electro-

of Weber and Osborn

03). Following fluorescence

electrophoretic of its

covalently

Copyright @ I972 by Academic Press, Inc. All rights of reproduction in any form reserved.

separation, bound

933

flavin

FP was located by virtue of the and IP was located by the fluo-

-1

BIOCHEMICAL

Vol. 48, No. 4, 1972

rescent

stain,

according

8-anilino-1-naphthalene

to Hartman

homogenized obtained

The subunits

for

were

guanidinium

precipitation 4 hrs

also

chloride

Cysteine methionine through

performic cysteic

the use of oxidized was determined

according

column

chromatography

on agarose

were

performed

sensitivity

and Discussion:

Assuming

as independently

determined

IP/FP

compares

in the

with

viously

in the

tropic

and (13).

were

calculated

type

I-A).

Sigma,

TrYPby

separated

chloride.

analyzer,

c

acid

to Hirs

sulfone in subunits

Amino

employing

are presented

acid

the enhanced

II

for

in

acid

appears

to occur.

in TABLE I.

FP and IP, normalized several significant

not

included

deviation

5) to be about

for

acids

to 100,000 differences.

standard

deviations

of the subunits

amino

isoleucine,

and phenylalanine show smaller

in the

of the

While

rather,

that Chao-

subunits,

of which

extraction it

has been

more recent of succinate

of IP

suggested

studies

in that

934

below.

(columns the above

A comparison

4 and 5), show five amino acids,

ratios

(column

Lysine,

differences; seems likely

histidine,

of we

4 divided

from 1 of + 15% or about

significant. It

dehydrogenase

serine, cysteine and methionine are considered

comparisons

composition

show major

differences.

con-

procedure. into

threonine, and for

5%, and a deviation

and proline

a pre-

of IP is an artifact

differential

(15))

g of protein Except for

to be probably

This

(14).

The values

labile

(4),

mole

a major

We conclude,

dehydrogenase

findings

and with

to detect

the purification A slight

compositions

dimer

the excess

(1).

(FP) and

the apparent

to be 1.20-1.25.

a 1:l

success.

and that

of 70,000

laboratory,

Procedures

without pure

succinate

our

and are

the

(1) (4).

of IP is due to contaminants

(partially

semiquantative

standard

were

complex

substantiate

The amino

by column

1.3

can dissociate

the excess

laboratory

consider

of 1.0

to be the more soluble to FP from

tyrosine

ratio

nearly

weights

in this

was determined

the use of perchlorate

anions

relative

very

molecular

fractions

of about

IP fraction is

frola

IP appears that

value

IP fraction

resulting

separated

an expected

published

taminant the

(7)

in 6 M

circuit.

(IP),

ratio

according

(Bovine,

(10).

SDS (12).

as cysteic

in 6 M guanidinium

Results 27,000

oxidation

on a Beckman 120

Pellets

on agarose

in 0.15%

and methionine

to Edelhoch

(8).

in 6 N HCl in sealed,

estimated

Ribonuclease-A

tophan analyses

acid

salt),

and then

to Roach and Gehrke

G-200

were

acid

magnesium

cut out

chromatography

and on Sephadex

after for

hydrolyzed

by column

contents

(Eastman were

to Weber and Csborn

_+ 2O according

separated (11)

constants

acid

The bands

were

at 145'

and methionine sulfone

Analyzer

(9).

in 0.2% SDS according

from acetone tubes

sulfonic

and Udenfriend

and eluted

evacuated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

three valine,

aspartic acid, glycine, that the values for

Vol. 48, No. 4, 1972

cysteine

BIOCHEMICAL

may also

tryptophan,

prove

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Arginine,

to be different.

and leucine

fail

to show significant

g lutamic

acid,

alanine,

differences.

TABLE I Amino Amino

acid

composition

acid

of FPa

Lysine Histidine Arginine Aspartic Acid Threonine Serine Glutamic Acid Proline Glycine Alanine Cysteinee Valine Methioninee Isoleucine Leucine Tyrosine Phenylalanine Tryptophanf

27.0 21.3 41.1 57.6 36.9 31.4 68.9 27.2 69.9 54.3 21.7 42.0 14.1 25.8 52.1 22.3 23.3 3.8

the subunits IPb 21.7 3.8 13.4 28.8 11.3 10.0 25.6 13.1 20.1 17.9 16.9 5.7 7.9 14.5 19.1 6.2 5.3 1.7

a

Residues

per

70,000

g protein.

b-

Residues

per 27,000

g protein.

C

Addition

of FP and IP (columns

de

Residues

per

f

100,000

of succinate FPd

FP + IPc

38.5 30.4 58.6 82.1 52.6 44.8 98.3 38.8 99.7 77.4 30.9 59.9 20.1 36.8 74.3 31.8 33.2 5.4

48.7 25.1 54.5 86.4 48.2 41.4 94.5 40.3 90.0 72.2 38.6 47.7 22.0 40.3 71.2 28.5 28.6 5.5

1 plus

dehydrogenase I$

80.4 14.1 49.7 106.8 41.9 37.1 94.9 48.6 74.5 66.4 62.6 21.2 29.3 53.8 70.8 23.0 19.6 6.3

2).

g protein.

Determined

after

performic

Determined

spectrophotometrically

acid

oxidation. according

to Edelhoch

(7).

Acknowledgements: We acknowledge the technical assistance of L. M. Buckley. This work was supported by grants from the National Science Foundation (T.C.H.) and Training Grant NB-5241 from the National Institutes of Neurological Diseases and Stroke, National Institutes of Health (N.T.F.). References 1. 2. 3. 4. 5. 6.

Davis, K. A. and Hatefi, Y., Biochem., IO, 2509 (1971). Baginsky, M. L. and Hatefi, Y., J. Biol. Chem., 244, 5313 (1969). Reghetti, P. and Cerletti, P., FEBS Letters l3, 181 (1971). Coles, C. J., Tisdale, H. D., Kenney, W. C., and Singer, T. P., Biochem. Biophys. Res. Commun., 46, 1843 (1972). Hatefi, Y. and Stempel, K. E., J. Biol. Chem., 244, 2350 (1969). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem., 193, 265 (1951).

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Vol. 48, No. 4, 1972

7. 8. 9. 10. 11. 12. 13. 14. 15.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Edelhoch, I-I., Biochemistry, 5, 1948 (1967). Weber, K. and Osborn, M., J. Biol. Chem., 244, 4406 (1969). Hartman, B. K. and Udenfriend, S., Anal. Biochem., 30, 391 (1969). Roach, D. and Gehrke, C. W., Abstracts, 160th Meeting Amer. Chem. Sot., Chicago, September (1970). Fish, W. W., Mann, K. G., and Tanford, C., J. Biol. Chem., 244, 4989 (1969). yurewicz, Es C., Ghalsmbor, M. A., Duckworth, D. H., and Heath, E. C., J. Biol. Chem.,, 246 5607 (1971). Hirs, C. H. W., Metd in Enzymology, 11, 59 (1967). Singer, T. P., personal communication of 23 June 1972. Moore, S. and Stein, W. H., Methods in Enzymology, 5, 819 (1963).

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