BIOCHEMICAL
Vol. 75, No. 3, 1977
Peptides
Derived
from Pyruvate
Pyruvate Paul Clayton
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Dehydrogenase
Dehydrogenase
F. Davis,
Flora
Kinase
H. Pettit,
as Substrates
for
and Phosphatase and Lester
J.
Reed
Foundation Biochemical Institute and the Department of Chemistry The University of Texas at Austin, Austin, Texas 78712
Received
February
14,1977
is presented that phosphopeptides produced by tryptic diSUMMARY. Evidence gestion of phosphorylated pyruvate dehydrogenase are effective substrates for pyruvate dehydrogenase phosphatase and that the dephosphopeptides can serve as substrates for pyruvate dehydrogenase kinase. These findings indicate that the phosphatase and the kinase do not require an intact tertiary structure in pyruvate dehydrogenase, but apparently recognize components of the local primary sequence around the phosphorylation sites.
Activity
of the mammalian
by phosphorylation
molar (2-9).
various
the
alternate
and ATP.
peptides
to the
catalyzed,
respectively,
substrates
by ATP/ADP,
Previous
on three possess
these
to the
seryl
residues
the amino
acid
showed
is a protein,
be due to binding
regulatory
are phosphorylated that
phosphorylation of PDH.
shown below.
Abbreviations used: PDHa, active, nonphosphorylated phosphorylated form. genase; PDHb, inactive,
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
and I@ ions
enzyme, sought
of the or to
and have Phosphopeptides
PDHb are dephosphorylated
in the O-chain sequences
and NADH/NAD
Ca2+,
we have
of 32P-labeled
(10,ll)
Intercon-
and the phosphatase.
and the dephosphopeptides studies
(1,2).
Mg2+,
could
possibilities
regulated by a
and the phosphatase
activities
the kinase
is
acetyl-CoA/CoA,
of pyruvate,
substrate,
for
digestion
phosphatase
the kinase
protein
between
by tryptic
PDHb phosphatase,
for
and phosphatase
To distinguish
produced
tially
substrate
of kinase effecters
both.
complex
and a Mg2+ -requiring
and by the concentrations
Since
modulation
found
kinase
of PDHa and PDHb is modulated ratios
dehydrogenase
and dephosphorylation
MgATP2- -requiring version
pyruvate
by
by PDH, kinase occurs
The tryptic
sequen-
phospho-
Phosphorylation form of pyruvate
at dehydro-
541 ISSN
0006291
X
BIOCHEMICAL
Vol. 75, No. 3, 1977
Site Reed,
1 correlates unpublished
closely
with
AND BIOPHYSICAL
inactivation
RESEARCH COMMUNICATIONS
of PDH (10,
T.E.
Roche and L.J.
observations).
T-l
Site 1 Tyr-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser-Tyr-Arg
T-2
5r-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser(P)-Sr-Arg
T-3
Site 3 Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg
Site
MATERIALS
2
AND METHODS
Highly purified preparations of the bovine kidney pyruvate dehydrogenase complex and its component enzymes were obtained by procedures described previously (12) or by modifications thereof. Trypsin was purchased from Worthington, soybean trypsin inhibitor from Sigma, and [u-32PlATP from ICN Pharmaceuticals. PDHb was prepared by incubating 100 mg of PDHa for 2 hr at 23O with 1.5 mg of kinase-transacetylase subcomplex (12), 0.05 mM [V-32P]ATP (36,000 cpm/nmol), 2 mM dithiothreitol, 1 mM MgC12, 0.1 mM EDTA and 20 mM potassium phosphate buffer (pH 7.0) in a final volume of 20 ml. The incubation mixture was dialyzed for 48 hr at 4' to remove ATP. PDHb preparations contained 7.1-10.4 nmol of 32P per mg of protein. Digestion with trypsin. PDH, (20 mg/ml) or PDHb (5 mg/ml) in 0.2 M NH4HC03 was incubated with trypsin (5O:l w/w) for 6 hr at 23O. A three-fold excess of soybean trypsin inhibitor was added, and the digest was lyophilized. Purification of phosphopeptides. A tryptic digest of radioactive PDHb was chromatographed on a column of Sephadex G-25 (fine) with 0.2 M NH4HC03. The radioactive-fractions were pooled, concentrated, and then subjected to paper (Whatman 3MM) efectrophoresis for 45 min at pH 6.5 and 3000 V (11). The radioactive bands, located with Kodak No Screen X-ray film, were electrophoresed for 45 min at pH 1.9 and 3000 V (11). The bands were eluted with 1 M NH4OH. In a typical purification, 0.29 pm01 of peptide T-l, 0.44 pm01 of T-2, and 0.09 umol of T-3 were obtained from 1.5 umol of PDHb. Dephosphorylation of phosphopeptides. The-standard assay mixture contained, peptide, 50 mM potassium in a final volume of 0.35 ml, 12 nmol of 32P-labeled 2-(N-morpholino)propanesulfonate buffer (pH 7.0), 2 mM dithiothreitol, 0.1 mM (12) of kidney PDHb phosphatase. The reaction was started, CaC12, and 990 units of MgC12 (final concentration, after 2 min preincubation at 30°, by the addition (50 pl) were removed at I-, 2-, and 4-min intervals and added 10 mM). Aliquots to 150 pl of 1 M acetic acid. The samples were chromatographed on Dowex 50X2 to separate 32Pi and unreacted phosphopeptide as described by Nolan --et al. (13). Radioactivity was determined in a Beckman LS-230 scintillation counter with ACS cocktail (Amersham/Searle). Phosphorylation of peptides. The standard assay mixture contained, in a final volume of 0.16 ml, 20 mM phosphate buffer (pH 7.0), 1 mM MgC12, 10 mM dithiothreitol, 0.1 mM EDTA, uncomplexed kinase or kinase-transacetylase subThe recomplex, and amounts of tryptic digest of PDH, indicated in the text. action was started, after 2 min preincubation at 300, by the addition of [I-32Pl ATP (final concentration, 0.1 mM). Aliquots (50 pl) were removed at l-, 2-, and 3-min intervals, acidified with 2 pl of 1 M HCl, and subjected to paper electrophoresis for 45 min at pH 1.9 and 3000 V. The radioactive peptides were located by radioautography, cut out, and counted as described above. Formation of phosphorylated peptide was also followed with the assay described by Aliquots (25 pl) of the reaction mixtures were apCarlson and Graves (14). plied to 2.3 cm disks of Whatman P81 phosphocellulose paper. The papers were washed four times (20 min each) with 1 N acetic acid to remove ATP. The papers, which contained the phosphopeptide, were washed twice with ethanol and ether, dried, and counted. 542
BIOCHEMICAL
Vol. 75, No. 3, 1977
Fig. 1. Radioautograph PDHb phosphatase. A bated at 300 with 186 volume of 0.14 ml as right) 30-pl aliquots pH 1.9 and 3000 V for
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
illustrating de hosphorylation of phosphopeptides by tryptic digest of !2P-labeled PDHb (0.46 mg) was incuunits of PDHb phosphatase and 10 mM MgC12 in a final described in Methods. At 0, 5, and 10 min (left to were removed and subjected to paper electrophoresis at 35 min.
RESULTS AND DISCUSSION of PDHb. When 32 P-labeled
Proteolysis (5O:l
w/w)
material 1.9)
6 hr in 0.2 M NH4HC03 at 23',
was soluble
of the digest
portions ation
for
in 10% trichloroacetic showed
of the three of PDH.
of lightly
PDHb was incubated
Peptide
phosphorylated
acid.
three
radioactive
radioactive
peptides
T-l
is
the major
essentially
spots varied radioactive
PDH (up to about
543
with
all
trypsin
of the radioactive
An electrophoretogram (Fig. with
1). the
The relative extent
component
50% inactivation).
(pH pro-
of phosphoryl-
in tryptic As the
digests extent
of
Vol. 75, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
-G 2 > at5 F: -0
0
0
2
4 TIM
6 E (min)
Fig. 2. Time course of dephosphorylation Components and conditions are described
phosphorylation
increased,
and the
of T-2
Dr.
amounts
Dennis
to monitor trypsin,
Bleile,
dodecyl
with
these
lyzed
by paper
of the
to 32Pi
the radioactive phosphatase peptides
with
not
time
observed varied shown). from course
The apparent
whereas
1).
with
the
tryptic of the Km values
544
was depro-
digests
in the
of a radioactive
spot
of ana-
amounts corre-
of dephosphorylation time
and the
experiments,
the
of PDHb as described
dephosphorylation of the
p-chain
by
to PDHb by limited
a decrease
incubation
digests
rapidly
and Mg*+ and then
The extent
For subsequent
the
by
was used
When tryptic
PDHb phosphatase
(Fig.
performed
up to 7OO:l.
PDHb phosphatase.
and the appearance
decreased,
was attacked
was restored ratios
digest
electrophoresis
The a-chain
and radioautography,
peptides
isolated
2 shows the
phosphatase.
incubated
peptides (data
were
for
by PDHb phosphatase.
experiments
gel
and subtilisin,
at weight
substrates
were
In recent
No activity
electrophoresis
radioactive
sponding
rate.
T-l
in the tryptic
sulfate-polyacrylamide
proteases
PDHb were
of T-l
increased.
as by chymotrypsin
Phosphopeptide 32P-labeled
amount
IO
of peptide in Methods.
of PDHb and PDHa.
at a much slower
teolysis
Fig.
and T-3
the digestion as well
graded
the
8
phosphatase
of peptide for
of
amount
three
of PDHb
radioactive
in Methods. T-l
peptides
by PDHb T-l,
T-2,
Vol. 75, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Effects
of Transacetylase, Activity toward
I
Ca2+ and Mg2+ on PDHb Phosphatase Tryptic Phosphopeptide T-l
Additions
Phosphatase
activity
None
(%)
100
Transacetylase
+ EGTA
Transacetylase
+ Ca2+
103 88
No Mg2+
1
The incubation mixtures contained, in a final volume of 0.35 ml, 12 nmol of 32P-labeled peptide T-l, 50 mM potassium 2-(N-morpholino)propanesulfonate buffer (pH 7.0), 2 mM dithiothreitol, 990 units of kidney PDHb phosphatase and, where indicated, 0.82 mg of transacetylase, 0.1 mM CaC12, and 2 mM ethylene glycol bis(@a.minoethyl ether) N,N'-tetraacetate (EGTA). The reaction was started by the addition of MgC12 (10 mM), and the initial rate was determined as described in Methods. In the complete incubation mixture, 0.9 nmol of 32Pi was releasedlmin.
and T-3 were
were
found
estimated
to be about
to be about
mol of phosphatase. PDHb is
phatase.
These
for
the protein
strates
are Previous
affect
the
indicate
that
the
substrates
are
only
studies
and that
(5)
from
showed
about
transacetylase,
bind
the phosphatase
tase
activity
hibited
and 1.2 mol of 32Pi apparent
The V values
released
per min per
Km of the phosphatase is
apparent
about
the V values
phos-
of the phosphatase
higher
for
un-
6 mol min ml mol-l
Km values
2- to 12-fold
for
than
for
the apparent
the peptide
and protein
B,,, sub-
similar.
phosphatase
In the present
9.0,
53 pM, and the V value
substrate
PDHb is decreased dihydrolipoyl
about
data
phosphopeptide
6.4,
220 and 106 pM, respectively.
By comparison,
complexed
the
625,
with
activity
the
apparent
58 pM to about with toward
little
both
uncomplexed
effect,
change,
uncomplexed
if
rather
545
the
a slight
in V.
to the
transacetylase inhibitory
for
of Ca2+ and Ca2+ does not
Ca2+ is
required
PDHb and PDHb bound that
presence
any,
PDHb, but Mg2+ is
we observed but
Km of the phosphatase
2.9 uM in the
to the transacetylase.
investigation
no stimulatory
that
for
required
to
PDHb phospha-
transacetylase. and Ca2+ exeffect,
on phos-
Vol. 75, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 3. Radioautograph showing time course of phosphorylation by PDH, kinase and [Y-32P]ATP of tryptic peptides derived from PDH,. A tryptic digest of PDH, (0.64 mg) was incubated at 30° with 92 pg of kinase-transacetylase subcomplex, 0.13 mM [I-32P]ATP, 0.13 mM MgC12, and 0.14 M NH4HC03 in a final volume of 40 pl. At 0, 2, 5, 10, 20, 30, and 60 min (left to right) 5-pl aliquots were removed and subjected to paper electrophoresis at pH 1.9 and 3000 V for 45 min. Spots in ascending order represent [7-32P]ATP, peptide T-3, peptide T-2, and peptide T-l.
phatase lute
activity
toward
requirement
binding acetylase
for
peptide
Mg2+.
T-l
(Table
We interpret
these
of PDHb and of the phosphatase results
PDHb and the
in
favorable
catalytic
center
(in
orientation of the
I).
The phosphatase results
the presence
to indicate
that
of Ca2+)
to the
of the phosphoseryl
phosphatase,
546
thereby
showed
residues
facilitating
an absospecific transin the
BIOCHEMICAL
Vol. 75, No. 3, 1977
Mg2+-dependent
dephosphorylation.
peptide
T-l,
apparently
latter
protein
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Since
dephosphopeptide
do not
bind
to the
affect
the
orientation
does not
T-l
transacetylase
and,
(see
of the
by inference,
below),
phosphoseryl
the
residue
in
the peptide. Peptide
substrates
radioactive
peptides
PDHb phosphatase moved,
for T-l
and T-2 were
and 10 mM MgC12 for
and the mixtures
phosphatase.
At l-
subcomplex together
were
ed by radioautography. T-l
and T-2,
and ATP showed mobility
as peptide
phosphorylated the kinase
than
at Site
the
samples
radioactive
exhibits
digest
specificity
for
the
the
amounts
incubated
migrated
as a substrate
peptides
were
followed detected
(data
not
incubation
followof
with
with
the
kinase
same
indicate
that
for
PDHa kinase
seryl
residue
the deand
at Site
1
shown)
were
identical
those
of the
three
PDHb (Fig.
1).
corresponds
increasing
incubation
peptides The tryptic
the
same three
and in the protein digest
and two minor pep-
peptide
that
a major
of the three
radioactive
indicate
by paper
The mobilities
in tryptic
of kinase-transacetylase
was analyzed
3).
The major
with
mixture
the kinase-transacetylase
(Fig.
detected
increased
with
by radioautography,
peptides
tryptic
that
re-
and subjected,
trace
had been
can serve
radioactive
data
that
were
at pH 1.9,
only
observations
and the
at pH 1.9,
at pH 1.9 and 6.5
amounts
acidified,
showed
of PDHa was incubated
and [V-32P]ATP,
radioactive
spot
samples
of kinase-transacetylase
and other
greater
with
electrophoresis
component
These
by incubation
2.
electrophoresis
tides
samples
purified,
to NaF to inhibit
addition
removed,
The control
T-l.
When a tryptic subcomplex
were
experiments
Control
respect
after
to paper
tetradecapeptide
that
45 min at 30°.
samples,
whereas
a major
dephosphorylated
intervals
aliquots
the control
In preliminary
made 10 mM with
and 2-min
and [^/-32P]ATP, with
peptides
PDH, kinase.
digests
of 32P-labeled
to peptide
T-l.
time
3) and with
subcomplex seryl
(Fig.
(data
residues
substrate,
of PDH, apparently
547
with
not
The intensity
shown).
of this
increasing These
are phosphorylated
and other in the
PDH,. did
not
contain
inhibitory
peptfdes,
BIOCHEMICAL
Vol. 75, No. 3, 1977
since
the digest
had little
substrate.
The apparent
decapeptide
were
respectively. for
effect,
PDH, are
buffer,
markedly
stimulates
specific
binding
acetylase.
0.6
Previous
in the
apparent
acetylase,
the V value
the It
dephosphopeptide transacetylase should
affects
be noted the
rate
of its that
PDHb can serve consistent
phatases
recognize
phorylation facilitate fiers
the
site
components (16,17).
elucidation of PDH, kinase
that
that
protein
of the
local
site
primary
and mechanism
presence
from
of the trans-
observation It
indicates appears
that
on the kinase. of PDH, markedly
and ATP (15). of PDH, and respectively,
and phosphoprotein sequence
around
peptide
of action
phosthe phos-
substrates
should
of the known modi-
activities.
in part ACKNOWLEDGMENTS. This work was supported We thank Cynthia Hook and Karen Hobson for skilled
by USPHS Grant GMO6590. technical assistance.
REFERENCES 1. Linn, T.C., Pettit, F.H., and Reed, L.J. (1969) Proc. Nat. U.S.A. 62, 234-241. 2. Linn, T.C., Pettit, F.H., Hucho, F., and Reed, L.J. (1969) Acad. Sci. U.S.A. 64, 227-234.
548
from about
and PDHb phosphatase,
of these
and PDHb phosphatase
by the trans-
derived
digestion
kinases
to
and the V value
the conformation
PDH, kinase
The availability of the
or both,
transacetylase.
by tryptic
20 mM
due apparently
This
by the kinase
obtained
proposal
2.5-fold.
kinase
transacetylase
PDH, decreased
in the
tetrakinase,
(in
the
substrate
to the
phosphorylation
for
that
on PDH, and a V effect
connection
as substrates with
about
mol-1
kinase
transacetylase,
Km was observed
a Km effect
peptides
showed
tetradecapeptide
increased
in this
mol-1
for
the
of uncomplexed
PDH, as substrate,
of the
does not bind
exerts
The finding
is
T-l
(3)
Km of the kinase
pM in the presence
PDHa no change
values
PDH, as
for
250 @I and 1.1 mol min'l
with
the
with
kinase
of PDH, or the kinase,
apparent
With
but
of uncomplexed
studies
activity
P-fold.
that
about
activity
20 pM and 5 mol min-1
and orientation
20 PM to about increased
about
kinase
the
on kinase
the corresponding
pH 7.0).
Thus,
any,
to be about
By comparison,
phosphate
if
Km and V values
estimated
uncomplexed
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acad.
Sci.
Proc.
Nat.
Vol. 75, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3. Hucho, F., Randall, D.D., Roche, T.E., Burgett, M.W., Pelley, J.W., and Reed, L.J. (1972) Arch. Biochem. Biophys. 151, 328-340. 4. Denton, R.M., Randle, P.J., and Martin, B-R. (1972) Biochem. J. 128, 161-163. 5. Pettit, F.H., Roche, T.E., and Reed, L.J. (1972) Biochem. Biophys. Res. Commun. 49, 563-571. 6. Roche, T.E., and Reed, L.J. (1974) Biochem. Biophys. Res. Commun. 59, 1341-1348. 7. Wieland, O.H., and Portenhauser, R. (1974) Eur. J. Biochem. 45, 577-588. 8. Pettit, F.H., Pelley, J.N., and Reed, L.J. (1975) Biochem. Biophys. Res. Commun. 65, 575-582. 9. Kerbey, A.L., Randle, P.J., Cooper, R.H., Whitehouse, S., Pask, H.T., and Denton, R.M. (1976) Biochem. J. 2, 327-348. 10. Hutcheson, E.T. (1971) Ph.D. Dissertation, University of Texas at Austin. C.R., Namihira, G., Hamilton, L., Munk, P., Eley, M.H., Linn, 11. Barrera, and Reed, L.J. (1972) Arch. Biochem. Biophys. l&S, 343-358. T.C., J.W., Pettit, F.H., Hucho, F., Randall, D.D., and 12. Linn, T.C., Pelley, Reed, L.J. (1972) Arch. Biochem. Biophys. 148, 327-342. E.H. (1964) Biochemistry 13. Nolan, C., Novoa, W.B., Krebs, E.G., and Fischer, Ii, 542-551. G.M., and Graves, D.J. (1976) Biochemistry l5, 4476-4481. 14. Carlson, 15. Butler, J.R., Pettit, F.H., Davis, P.F., and Reed, L.J. (1977) Biochem. Biophys. Res. Commun., in press. E., and Krebs, E.G. (1976) Proc. Nat. Acad. Sci. 16. Kemp, B.E., Benjamini, U.S.A. 73, 1038-1042. 17. Daile, P., Carnegie, P.R., and Young, J.D. (1975) Nature 257, 416-418.
549
Vol. 75, No. 3, 1977
Peptides
Derived
from Pyruvate
Pyruvate Paul Clayton
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Dehydrogenase
Dehydrogenase
F. Davis,
Flora
Kinase
H. Pettit,
as Substrates
for
and Phosphatase and Lester
J.
Reed
Foundation Biochemical Institute and the Department of Chemistry The University of Texas at Austin, Austin, Texas 78712
Received
February
14,1977
is presented that phosphopeptides produced by tryptic diSUMMARY. Evidence gestion of phosphorylated pyruvate dehydrogenase are effective substrates for pyruvate dehydrogenase phosphatase and that the dephosphopeptides can serve as substrates for pyruvate dehydrogenase kinase. These findings indicate that the phosphatase and the kinase do not require an intact tertiary structure in pyruvate dehydrogenase, but apparently recognize components of the local primary sequence around the phosphorylation sites.
Activity
of the mammalian
by phosphorylation
molar (2-9).
various
the
alternate
and ATP.
peptides
to the
catalyzed,
respectively,
substrates
by ATP/ADP,
Previous
on three possess
these
to the
seryl
residues
the amino
acid
showed
is a protein,
be due to binding
regulatory
are phosphorylated that
phosphorylation of PDH.
shown below.
Abbreviations used: PDHa, active, nonphosphorylated phosphorylated form. genase; PDHb, inactive,
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
and I@ ions
enzyme, sought
of the or to
and have Phosphopeptides
PDHb are dephosphorylated
in the O-chain sequences
and NADH/NAD
Ca2+,
we have
of 32P-labeled
(10,ll)
Intercon-
and the phosphatase.
and the dephosphopeptides studies
(1,2).
Mg2+,
could
possibilities
regulated by a
and the phosphatase
activities
the kinase
is
acetyl-CoA/CoA,
of pyruvate,
substrate,
for
digestion
phosphatase
the kinase
protein
between
by tryptic
PDHb phosphatase,
for
and phosphatase
To distinguish
produced
tially
substrate
of kinase effecters
both.
complex
and a Mg2+ -requiring
and by the concentrations
Since
modulation
found
kinase
of PDHa and PDHb is modulated ratios
dehydrogenase
and dephosphorylation
MgATP2- -requiring version
pyruvate
by
by PDH, kinase occurs
The tryptic
sequen-
phospho-
Phosphorylation form of pyruvate
at dehydro-
541 ISSN
0006291
X
BIOCHEMICAL
Vol. 75, No. 3, 1977
Site Reed,
1 correlates unpublished
closely
with
AND BIOPHYSICAL
inactivation
RESEARCH COMMUNICATIONS
of PDH (10,
T.E.
Roche and L.J.
observations).
T-l
Site 1 Tyr-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser-Tyr-Arg
T-2
5r-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser(P)-Sr-Arg
T-3
Site 3 Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg
Site
MATERIALS
2
AND METHODS
Highly purified preparations of the bovine kidney pyruvate dehydrogenase complex and its component enzymes were obtained by procedures described previously (12) or by modifications thereof. Trypsin was purchased from Worthington, soybean trypsin inhibitor from Sigma, and [u-32PlATP from ICN Pharmaceuticals. PDHb was prepared by incubating 100 mg of PDHa for 2 hr at 23O with 1.5 mg of kinase-transacetylase subcomplex (12), 0.05 mM [V-32P]ATP (36,000 cpm/nmol), 2 mM dithiothreitol, 1 mM MgC12, 0.1 mM EDTA and 20 mM potassium phosphate buffer (pH 7.0) in a final volume of 20 ml. The incubation mixture was dialyzed for 48 hr at 4' to remove ATP. PDHb preparations contained 7.1-10.4 nmol of 32P per mg of protein. Digestion with trypsin. PDH, (20 mg/ml) or PDHb (5 mg/ml) in 0.2 M NH4HC03 was incubated with trypsin (5O:l w/w) for 6 hr at 23O. A three-fold excess of soybean trypsin inhibitor was added, and the digest was lyophilized. Purification of phosphopeptides. A tryptic digest of radioactive PDHb was chromatographed on a column of Sephadex G-25 (fine) with 0.2 M NH4HC03. The radioactive-fractions were pooled, concentrated, and then subjected to paper (Whatman 3MM) efectrophoresis for 45 min at pH 6.5 and 3000 V (11). The radioactive bands, located with Kodak No Screen X-ray film, were electrophoresed for 45 min at pH 1.9 and 3000 V (11). The bands were eluted with 1 M NH4OH. In a typical purification, 0.29 pm01 of peptide T-l, 0.44 pm01 of T-2, and 0.09 umol of T-3 were obtained from 1.5 umol of PDHb. Dephosphorylation of phosphopeptides. The-standard assay mixture contained, peptide, 50 mM potassium in a final volume of 0.35 ml, 12 nmol of 32P-labeled 2-(N-morpholino)propanesulfonate buffer (pH 7.0), 2 mM dithiothreitol, 0.1 mM (12) of kidney PDHb phosphatase. The reaction was started, CaC12, and 990 units of MgC12 (final concentration, after 2 min preincubation at 30°, by the addition (50 pl) were removed at I-, 2-, and 4-min intervals and added 10 mM). Aliquots to 150 pl of 1 M acetic acid. The samples were chromatographed on Dowex 50X2 to separate 32Pi and unreacted phosphopeptide as described by Nolan --et al. (13). Radioactivity was determined in a Beckman LS-230 scintillation counter with ACS cocktail (Amersham/Searle). Phosphorylation of peptides. The standard assay mixture contained, in a final volume of 0.16 ml, 20 mM phosphate buffer (pH 7.0), 1 mM MgC12, 10 mM dithiothreitol, 0.1 mM EDTA, uncomplexed kinase or kinase-transacetylase subThe recomplex, and amounts of tryptic digest of PDH, indicated in the text. action was started, after 2 min preincubation at 300, by the addition of [I-32Pl ATP (final concentration, 0.1 mM). Aliquots (50 pl) were removed at l-, 2-, and 3-min intervals, acidified with 2 pl of 1 M HCl, and subjected to paper electrophoresis for 45 min at pH 1.9 and 3000 V. The radioactive peptides were located by radioautography, cut out, and counted as described above. Formation of phosphorylated peptide was also followed with the assay described by Aliquots (25 pl) of the reaction mixtures were apCarlson and Graves (14). plied to 2.3 cm disks of Whatman P81 phosphocellulose paper. The papers were washed four times (20 min each) with 1 N acetic acid to remove ATP. The papers, which contained the phosphopeptide, were washed twice with ethanol and ether, dried, and counted. 542
BIOCHEMICAL
Vol. 75, No. 3, 1977
Fig. 1. Radioautograph PDHb phosphatase. A bated at 300 with 186 volume of 0.14 ml as right) 30-pl aliquots pH 1.9 and 3000 V for
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
illustrating de hosphorylation of phosphopeptides by tryptic digest of !2P-labeled PDHb (0.46 mg) was incuunits of PDHb phosphatase and 10 mM MgC12 in a final described in Methods. At 0, 5, and 10 min (left to were removed and subjected to paper electrophoresis at 35 min.
RESULTS AND DISCUSSION of PDHb. When 32 P-labeled
Proteolysis (5O:l
w/w)
material 1.9)
6 hr in 0.2 M NH4HC03 at 23',
was soluble
of the digest
portions ation
for
in 10% trichloroacetic showed
of the three of PDH.
of lightly
PDHb was incubated
Peptide
phosphorylated
acid.
three
radioactive
radioactive
peptides
T-l
is
the major
essentially
spots varied radioactive
PDH (up to about
543
with
all
trypsin
of the radioactive
An electrophoretogram (Fig. with
1). the
The relative extent
component
50% inactivation).
(pH pro-
of phosphoryl-
in tryptic As the
digests extent
of
Vol. 75, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
-G 2 > at5 F: -0
0
0
2
4 TIM
6 E (min)
Fig. 2. Time course of dephosphorylation Components and conditions are described
phosphorylation
increased,
and the
of T-2
Dr.
amounts
Dennis
to monitor trypsin,
Bleile,
dodecyl
with
these
lyzed
by paper
of the
to 32Pi
the radioactive phosphatase peptides
with
not
time
observed varied shown). from course
The apparent
whereas
1).
with
the
tryptic of the Km values
544
was depro-
digests
in the
of a radioactive
spot
of ana-
amounts corre-
of dephosphorylation time
and the
experiments,
the
of PDHb as described
dephosphorylation of the
p-chain
by
to PDHb by limited
a decrease
incubation
digests
rapidly
and Mg*+ and then
The extent
For subsequent
the
by
was used
When tryptic
PDHb phosphatase
(Fig.
performed
up to 7OO:l.
PDHb phosphatase.
and the appearance
decreased,
was attacked
was restored ratios
digest
electrophoresis
The a-chain
and radioautography,
peptides
isolated
2 shows the
phosphatase.
incubated
peptides (data
were
for
by PDHb phosphatase.
experiments
gel
and subtilisin,
at weight
substrates
were
In recent
No activity
electrophoresis
radioactive
sponding
rate.
T-l
in the tryptic
sulfate-polyacrylamide
proteases
PDHb were
of T-l
increased.
as by chymotrypsin
Phosphopeptide 32P-labeled
amount
IO
of peptide in Methods.
of PDHb and PDHa.
at a much slower
teolysis
Fig.
and T-3
the digestion as well
graded
the
8
phosphatase
of peptide for
of
amount
three
of PDHb
radioactive
in Methods. T-l
peptides
by PDHb T-l,
T-2,
Vol. 75, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Effects
of Transacetylase, Activity toward
I
Ca2+ and Mg2+ on PDHb Phosphatase Tryptic Phosphopeptide T-l
Additions
Phosphatase
activity
None
(%)
100
Transacetylase
+ EGTA
Transacetylase
+ Ca2+
103 88
No Mg2+
1
The incubation mixtures contained, in a final volume of 0.35 ml, 12 nmol of 32P-labeled peptide T-l, 50 mM potassium 2-(N-morpholino)propanesulfonate buffer (pH 7.0), 2 mM dithiothreitol, 990 units of kidney PDHb phosphatase and, where indicated, 0.82 mg of transacetylase, 0.1 mM CaC12, and 2 mM ethylene glycol bis(@a.minoethyl ether) N,N'-tetraacetate (EGTA). The reaction was started by the addition of MgC12 (10 mM), and the initial rate was determined as described in Methods. In the complete incubation mixture, 0.9 nmol of 32Pi was releasedlmin.
and T-3 were
were
found
estimated
to be about
to be about
mol of phosphatase. PDHb is
phatase.
These
for
the protein
strates
are Previous
affect
the
indicate
that
the
substrates
are
only
studies
and that
(5)
from
showed
about
transacetylase,
bind
the phosphatase
tase
activity
hibited
and 1.2 mol of 32Pi apparent
The V values
released
per min per
Km of the phosphatase is
apparent
about
the V values
phos-
of the phosphatase
higher
for
un-
6 mol min ml mol-l
Km values
2- to 12-fold
for
than
for
the apparent
the peptide
and protein
B,,, sub-
similar.
phosphatase
In the present
9.0,
53 pM, and the V value
substrate
PDHb is decreased dihydrolipoyl
about
data
phosphopeptide
6.4,
220 and 106 pM, respectively.
By comparison,
complexed
the
625,
with
activity
the
apparent
58 pM to about with toward
little
both
uncomplexed
effect,
change,
uncomplexed
if
rather
545
the
a slight
in V.
to the
transacetylase inhibitory
for
of Ca2+ and Ca2+ does not
Ca2+ is
required
PDHb and PDHb bound that
presence
any,
PDHb, but Mg2+ is
we observed but
Km of the phosphatase
2.9 uM in the
to the transacetylase.
investigation
no stimulatory
that
for
required
to
PDHb phospha-
transacetylase. and Ca2+ exeffect,
on phos-
Vol. 75, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 3. Radioautograph showing time course of phosphorylation by PDH, kinase and [Y-32P]ATP of tryptic peptides derived from PDH,. A tryptic digest of PDH, (0.64 mg) was incubated at 30° with 92 pg of kinase-transacetylase subcomplex, 0.13 mM [I-32P]ATP, 0.13 mM MgC12, and 0.14 M NH4HC03 in a final volume of 40 pl. At 0, 2, 5, 10, 20, 30, and 60 min (left to right) 5-pl aliquots were removed and subjected to paper electrophoresis at pH 1.9 and 3000 V for 45 min. Spots in ascending order represent [7-32P]ATP, peptide T-3, peptide T-2, and peptide T-l.
phatase lute
activity
toward
requirement
binding acetylase
for
peptide
Mg2+.
T-l
(Table
We interpret
these
of PDHb and of the phosphatase results
PDHb and the
in
favorable
catalytic
center
(in
orientation of the
I).
The phosphatase results
the presence
to indicate
that
of Ca2+)
to the
of the phosphoseryl
phosphatase,
546
thereby
showed
residues
facilitating
an absospecific transin the
BIOCHEMICAL
Vol. 75, No. 3, 1977
Mg2+-dependent
dephosphorylation.
peptide
T-l,
apparently
latter
protein
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Since
dephosphopeptide
do not
bind
to the
affect
the
orientation
does not
T-l
transacetylase
and,
(see
of the
by inference,
below),
phosphoseryl
the
residue
in
the peptide. Peptide
substrates
radioactive
peptides
PDHb phosphatase moved,
for T-l
and T-2 were
and 10 mM MgC12 for
and the mixtures
phosphatase.
At l-
subcomplex together
were
ed by radioautography. T-l
and T-2,
and ATP showed mobility
as peptide
phosphorylated the kinase
than
at Site
the
samples
radioactive
exhibits
digest
specificity
for
the
the
amounts
incubated
migrated
as a substrate
peptides
were
followed detected
(data
not
incubation
followof
with
with
the
kinase
same
indicate
that
for
PDHa kinase
seryl
residue
the deand
at Site
1
shown)
were
identical
those
of the
three
PDHb (Fig.
1).
corresponds
increasing
incubation
peptides The tryptic
the
same three
and in the protein digest
and two minor pep-
peptide
that
a major
of the three
radioactive
indicate
by paper
The mobilities
in tryptic
of kinase-transacetylase
was analyzed
3).
The major
with
mixture
the kinase-transacetylase
(Fig.
detected
increased
with
by radioautography,
peptides
tryptic
that
re-
and subjected,
trace
had been
can serve
radioactive
data
that
were
at pH 1.9,
only
observations
and the
at pH 1.9,
at pH 1.9 and 6.5
amounts
acidified,
showed
of PDHa was incubated
and [V-32P]ATP,
radioactive
spot
samples
of kinase-transacetylase
and other
greater
with
electrophoresis
component
These
by incubation
2.
electrophoresis
tides
samples
purified,
to NaF to inhibit
addition
removed,
The control
T-l.
When a tryptic subcomplex
were
experiments
Control
respect
after
to paper
tetradecapeptide
that
45 min at 30°.
samples,
whereas
a major
dephosphorylated
intervals
aliquots
the control
In preliminary
made 10 mM with
and 2-min
and [^/-32P]ATP, with
peptides
PDH, kinase.
digests
of 32P-labeled
to peptide
T-l.
time
3) and with
subcomplex seryl
(Fig.
(data
residues
substrate,
of PDH, apparently
547
with
not
The intensity
shown).
of this
increasing These
are phosphorylated
and other in the
PDH,. did
not
contain
inhibitory
peptfdes,
BIOCHEMICAL
Vol. 75, No. 3, 1977
since
the digest
had little
substrate.
The apparent
decapeptide
were
respectively. for
effect,
PDH, are
buffer,
markedly
stimulates
specific
binding
acetylase.
0.6
Previous
in the
apparent
acetylase,
the V value
the It
dephosphopeptide transacetylase should
affects
be noted the
rate
of its that
PDHb can serve consistent
phatases
recognize
phorylation facilitate fiers
the
site
components (16,17).
elucidation of PDH, kinase
that
that
protein
of the
local
site
primary
and mechanism
presence
from
of the trans-
observation It
indicates appears
that
on the kinase. of PDH, markedly
and ATP (15). of PDH, and respectively,
and phosphoprotein sequence
around
peptide
of action
phosthe phos-
substrates
should
of the known modi-
activities.
in part ACKNOWLEDGMENTS. This work was supported We thank Cynthia Hook and Karen Hobson for skilled
by USPHS Grant GMO6590. technical assistance.
REFERENCES 1. Linn, T.C., Pettit, F.H., and Reed, L.J. (1969) Proc. Nat. U.S.A. 62, 234-241. 2. Linn, T.C., Pettit, F.H., Hucho, F., and Reed, L.J. (1969) Acad. Sci. U.S.A. 64, 227-234.
548
from about
and PDHb phosphatase,
of these
and PDHb phosphatase
by the trans-
derived
digestion
kinases
to
and the V value
the conformation
PDH, kinase
The availability of the
or both,
transacetylase.
by tryptic
20 mM
due apparently
This
by the kinase
obtained
proposal
2.5-fold.
kinase
transacetylase
PDH, decreased
in the
tetrakinase,
(in
the
substrate
to the
phosphorylation
for
that
on PDH, and a V effect
connection
as substrates with
about
mol-1
kinase
transacetylase,
Km was observed
a Km effect
peptides
showed
tetradecapeptide
increased
in this
mol-1
for
the
of uncomplexed
PDH, as substrate,
of the
does not bind
exerts
The finding
is
T-l
(3)
Km of the kinase
pM in the presence
PDHa no change
values
PDH, as
for
250 @I and 1.1 mol min'l
with
the
with
kinase
of PDH, or the kinase,
apparent
With
but
of uncomplexed
studies
activity
P-fold.
that
about
activity
20 pM and 5 mol min-1
and orientation
20 PM to about increased
about
kinase
the
on kinase
the corresponding
pH 7.0).
Thus,
any,
to be about
By comparison,
phosphate
if
Km and V values
estimated
uncomplexed
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acad.
Sci.
Proc.
Nat.
Vol. 75, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3. Hucho, F., Randall, D.D., Roche, T.E., Burgett, M.W., Pelley, J.W., and Reed, L.J. (1972) Arch. Biochem. Biophys. 151, 328-340. 4. Denton, R.M., Randle, P.J., and Martin, B-R. (1972) Biochem. J. 128, 161-163. 5. Pettit, F.H., Roche, T.E., and Reed, L.J. (1972) Biochem. Biophys. Res. Commun. 49, 563-571. 6. Roche, T.E., and Reed, L.J. (1974) Biochem. Biophys. Res. Commun. 59, 1341-1348. 7. Wieland, O.H., and Portenhauser, R. (1974) Eur. J. Biochem. 45, 577-588. 8. Pettit, F.H., Pelley, J.N., and Reed, L.J. (1975) Biochem. Biophys. Res. Commun. 65, 575-582. 9. Kerbey, A.L., Randle, P.J., Cooper, R.H., Whitehouse, S., Pask, H.T., and Denton, R.M. (1976) Biochem. J. 2, 327-348. 10. Hutcheson, E.T. (1971) Ph.D. Dissertation, University of Texas at Austin. C.R., Namihira, G., Hamilton, L., Munk, P., Eley, M.H., Linn, 11. Barrera, and Reed, L.J. (1972) Arch. Biochem. Biophys. l&S, 343-358. T.C., J.W., Pettit, F.H., Hucho, F., Randall, D.D., and 12. Linn, T.C., Pelley, Reed, L.J. (1972) Arch. Biochem. Biophys. 148, 327-342. E.H. (1964) Biochemistry 13. Nolan, C., Novoa, W.B., Krebs, E.G., and Fischer, Ii, 542-551. G.M., and Graves, D.J. (1976) Biochemistry l5, 4476-4481. 14. Carlson, 15. Butler, J.R., Pettit, F.H., Davis, P.F., and Reed, L.J. (1977) Biochem. Biophys. Res. Commun., in press. E., and Krebs, E.G. (1976) Proc. Nat. Acad. Sci. 16. Kemp, B.E., Benjamini, U.S.A. 73, 1038-1042. 17. Daile, P., Carnegie, P.R., and Young, J.D. (1975) Nature 257, 416-418.
549
