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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 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).
935
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).
936
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).
935
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).
936