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A possible error during assays for the enzymic phosphorylation of proteins and nucleic acids
Vol. 47, No. 3, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A POSSIBLE ERROR DURING ASSAYS FOR THE ENZYMIC PHOSPHORYLATION OF PROTEINS AND NUCLEIC ACIDS" P. J. Greenaway
Received
Cold
Spring
Harbor
April
10, 1972
Laboratory,
Cold
Spring
Harbor,
New York
Summary: The formation of an insoluble complex between Mg*, sodium fluoride and ATP was studied. The precipitation of ATP was dependent on the time of incubation and on the concentration of ATP, Mg" and sodium fluoride. The requirement for sodium fluoride was not replaced by sodium or potassium chloride but the requirement for Mgf+ was replaced by La+++ and Sn+'. Precipitation was stimulated by CAMP but was inhibited at high ionic strength and by EDTA, dATP, ADP and sodium pyrophosphate. The complex was slightly soluble in acid and completely soluble in alkali. Formation of this complex could result in errors in --in vitro assays for enzymic phosphorylation of proteins and nucleic acids by acid precipitation. Several nucleic lated
acids
mally reports possible eters attempt
of organisms
or nucleic
% -phosphate
during acids
an absolute
phosphatase involved
phosphorylations
*This CA 11432-02
this
fluoride
the
by acid
care
assayed
reaction in --in vitro
(7,8).
in the precipitation
to emphasize
synthesis
This
required
by following
iso-
many functions (6)
the
and the
and there assays
incorporation
Mgu
product.
is nor-
have been occasional as an inhibitor
describes
when undertaking
and
The phosphorylation
of
some of the param-
of ATP by Mg++ and sodium assays
fluoride
in an
of enzymic
precipitation.
investigation was supported and CA 11432-03.
by U.S.
639 Copyrigbt@1972,by
paper
fulfill
(7).
proteins have been
enzymes
an acid-precipitable for
activity
enzymes
hormone-sensitive
is readily
requirement
on the use of sodium
These
protein
of ATP into
of ATP onto
phosphotransferases)
(l-5).
of certain
of ribosomes
of proteins
the 1 -phosphate
and nucleotidyl
the activation
inactivation
transfer
(ATP:protein
from a variety
including
of the
enzymes which
AcademicPress,Inc.
Public
Health
Service
Grants
Vol. 47, No. 3,1972
MATERIALS England
BIOCHEMICAL
AND METHODS. Nuclear,
Boston,
(CAMP) was obtained chemicals
were Typical
ATP-3H(G)
Calbiochem,
analytical
grade for
5 umole
of Tris-HCl,
2 pmole
of NaP and 10 nmole
tions
were
1% (W/V)
discs
of Whatman GF/C glass
washed phate
with
After
of dithiothreitol
for
other
of ATP contained (DTT),
30 min and were
(50 ~1)
fiber
(2.4
ethanol.
The discs
and 0.005%
(W/V)
benzene
in an Intertechnique
addition
trichloroacetic
cm diameter);
dried
of MgC12, The reac-
by the
material
were
2 pmole
in 0.3 ml.
terminated
and 100% (W/V)
paper
2,5-Diphenyloxazole in toluene]
All
the precipitation
30 min at 0°C the precipitated
absolute
monophosphate
reagents.
was collected
0.2 M sodium and counted
scintillation
pyrophoa
C0.4% (W/V)
counter.
TABLE1 THE RECOVERY OF ATP AS AN ACID INSOLUBLE COMPLEX
nmole Full
reaction
mix
Full
reaction
mix
Full
reaction
mix
Full
reaction
Full
3H-ATP
nmole
32P-v-ATP
0.80
0.95
- DTT
0.65
0.71
- MgC12
0.007
0.008
mix - NaF
0.005
0.004
reaction
mix
0.33
0.29
Full
reaction
mix + 0.1 M NaCl
0.008
0.006
Full
reaction
mix + 10 nmole
dATP
0.048
0.019
Full
reaction
mix + 10 nmole
ADP
0.28
0.22
Full
reaction
mix + 10 nmole sodium pyrophoaphate
0.048
0.023
+ 1 mM ELYTA
640
on
was
1,4,bis-2-(4-methyl-S-phenyloxaeolyl)liquid
of
acid
each disc
20 ml of 20% TCA, 20 ml of 5% TCA containing
and with
from New
California.
of ATP (3H and 32P labeled)
serum albumin
(50 pi).
obtained
Adenosine-3',5'-cyclic
investigating
at 37'C
(TCA)
were
Los Angeles,
pH 8, 2 umole
incubated bovine
and ATP-v-32P
Massachusetts.
from
assays
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 47, No. 3, 1972
BIOCHEMICAL
RESULTS AND DISCUSSION. investigating
Table
1 shows
the precipitation
recovered
as an acid-precipitable
dependent
on the time
the presence
AND BIOPHYSICAL RESEARCH CCWvWNICATIONS
of ATP. complex.
of incubation
of DTT (Table
the results Almost
of a typical 10% of the
1) and totally
input
The precipitation
at 37'12 (Fig. dependent
la),
assay
for
ATP was
of ATP was
slightly
dependent
on the concentration
on of
(bl
Time
1
of lncubotion
2 pmole
3 Mg**
Fig. 1. The effect of of ATP, (c) concentration precipitation of ATP. 0 -0 A-A
20 nmole
(min)
4
%--TYT-
5
pmole
(a)
Xl ATP
time of incubation at 37'C, of Mg&++ and (d) concentration
Precipitation
of ATP-%
Precipitation
of ATP-3H(G).
-32P.
641
40
50
10 NoF
(b) concentration of NaF, on the
Vol. 47, No. 3,1972
BIOCHEMICAL
ATP, Mg++ and NaF (Table used; the
magnesium lower
1 and Fig.
acetate
ionization
did
The requirement
for
of
1).
enzymes
which
CAMP, which (9),
dependent was almost
also
replace
compound.
replaced
the
on the concentration a fivefold
stimulation
of the concentration
A-A
Precipitation
of ATP-3S(C).
on DEAE paper
(11)
on
during
these
polyethyleneimine-cellulose
to ensure
that
were
pyrophosphate also
slightly
This
stimulation
was
at low concentrations
there
4
5
CAMP
of CAMP on the precipitation
of ATP- 1 -32P.
chromatography
of ATP. 2);
ionic
some phosphorylating
3
Precipitation
layer
Mg++.
of precipitation.
2
precipitated
the excepfor
ADP and sodium activity
precipitation
o-0
The material
with
by EDTA and at high
shown to stimulate
mole
The effect
due to
the requirement
by dATP,
of CAMP (Fig.
1
Fig. 2. ATP.
Other ‘metals,
had no phosphorylating
stimulated
MgC12 was
by NaCl or KCl.
inhibited
has been
reactions
MgC12 presumably
replace
of ATP was prevented
Proteins
inhibitory.
this
RESEARCH COMMUNICATIONS
In these
not efficiently
and was competitively
(Table
lb-ld).
efficiently
NaF was not
The precipitation strength
not
constant
of La +++ and So ++ , did
tion
AND BIOPHYSICAL
the results
642
reactions
was analyzed (10)
of these
of
by thin-
and by ionophoresis assays
were
not
due to
BIOCHEMICAL
Vol. 47, No. 3,1972
the breakdown breakdown
of ATP.
of ATP under
precipitated
insoluble
ATP was acid
without
the ATP.
prior
reported
in the precipitation p,$
inhibition
this
interaction
an absolute
rylating errors
results
enzymes
assays
for indicate
give
by acid
may be avoided
if
emphasizes
for
ATP:protein
was repeated
and nucleotidyl
involved
complex
explain
with
and the competiindicates
of ATP described these
that
in this
results
as NaF is
reaction. may occur
during
assays
of the phosphorylated
the acid-precipitated
the absolute
then
Mgg++ is kngwrf to interact
fully
errors
this
25% of
some of the parameters
the precipitation
precipitation
reprecipitated;
paper
cannot
that
was insol-
10% of the ATP originally
in the precipitation
interaction
requirement
NaOH and then This
this
reactions
by dATP, ADP and pyrophosphate
a part
24 hr.
in 0.1 N NaOH, addition
of ATP to form an insoluble
plays
for
of the
soluble.
paper
of precipitation
However,
These
in this
cent
ATP became
and reprecipitation
acid
per
TCA solubilized
soluble
of ATP by Mgg++ and NaF.
-pyrophosphate
tive
paper.
dissolution
this
with
negligible
complex
these
TCA; treatment
ATP was completely
The results
during
approximately
If
Ninety
but upon re-reaction
with
was only
of the insoluble
ATP was completely
in the complex.
there
described.
The ATP precipitated treatment
that
conditions
by dialysis
TCA reprecipitated
the remaining
indicated
soluble
The precipitated
of 20% (W/V)
the
the assay
once more.
present
results
ATP was removed
The remaining
uble
These
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
product
procedure
should
necessity
for
is
phosphotransferases
phospho-
products. dissolved
be repeated
this
for
procedure
These in 0.1 N
at least during
by acid
twice. all
precipita-
tion. ACKNOWLEDGMENTS Levine ship.
I wish to acknowledge the excellent technical assistance of Mrs. D. and to thank the Damon Runyon Memorial Fund for a postdoctoral fellow-
REFERENCES 1. 2.
Jergil, B., and Dixon, G. H., J. Biol. Hatanaka, M., Twiddy, E., and Gilden, 643
Chem., 245, 425 (1970). R. V., Virology, 47, 536 (1972).
Vol. 47, No. 3,1972
3. 4. 5. 6. 7. 8. 9. 10. 11.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Richardson, C. C., Proc. Natl. Aced. Sci. U.S., 54, 158 (1965). Novogrodsky, A., and Hurwitz, J., J. Biol. Chem., 241, 2923 (1966). Majumder, G. C., and Turkington, R. W., J. Biol. Chem., 246, 2650 (1971). liuttunen, J. K., Steinberg, D., and Mayer, S. E., Biochem. Biophys. Res. comm., 4l, 1350 (1970). Kabat, D., Biochemistry, 2, 4160 (1970). Strand, M., and August, J. T., Nature New Biol., 233, 137 (1971). Kuo, J. F., and Greengard, P., Proc. Natl. Aced. Sci. U.S., 64, 1349 (1969). Randerath, K., and Randerath, E., in "Methods in Enzymology" (L. Grossman and K. Moldave, eds.), vol. 12, part A, 323, Academic Press, New York, 1967. Sanger, F., Brownlee, G. G., and Barrell, B. G., J. Mol. Biol., l3, 373 (1965).
644
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A POSSIBLE ERROR DURING ASSAYS FOR THE ENZYMIC PHOSPHORYLATION OF PROTEINS AND NUCLEIC ACIDS" P. J. Greenaway
Received
Cold
Spring
Harbor
April
10, 1972
Laboratory,
Cold
Spring
Harbor,
New York
Summary: The formation of an insoluble complex between Mg*, sodium fluoride and ATP was studied. The precipitation of ATP was dependent on the time of incubation and on the concentration of ATP, Mg" and sodium fluoride. The requirement for sodium fluoride was not replaced by sodium or potassium chloride but the requirement for Mgf+ was replaced by La+++ and Sn+'. Precipitation was stimulated by CAMP but was inhibited at high ionic strength and by EDTA, dATP, ADP and sodium pyrophosphate. The complex was slightly soluble in acid and completely soluble in alkali. Formation of this complex could result in errors in --in vitro assays for enzymic phosphorylation of proteins and nucleic acids by acid precipitation. Several nucleic lated
acids
mally reports possible eters attempt
of organisms
or nucleic
% -phosphate
during acids
an absolute
phosphatase involved
phosphorylations
*This CA 11432-02
this
fluoride
the
by acid
care
assayed
reaction in --in vitro
(7,8).
in the precipitation
to emphasize
synthesis
This
required
by following
iso-
many functions (6)
the
and the
and there assays
incorporation
Mgu
product.
is nor-
have been occasional as an inhibitor
describes
when undertaking
and
The phosphorylation
of
some of the param-
of ATP by Mg++ and sodium assays
fluoride
in an
of enzymic
precipitation.
investigation was supported and CA 11432-03.
by U.S.
639 Copyrigbt@1972,by
paper
fulfill
(7).
proteins have been
enzymes
an acid-precipitable for
activity
enzymes
hormone-sensitive
is readily
requirement
on the use of sodium
These
protein
of ATP into
of ATP onto
phosphotransferases)
(l-5).
of certain
of ribosomes
of proteins
the 1 -phosphate
and nucleotidyl
the activation
inactivation
transfer
(ATP:protein
from a variety
including
of the
enzymes which
AcademicPress,Inc.
Public
Health
Service
Grants
Vol. 47, No. 3,1972
MATERIALS England
BIOCHEMICAL
AND METHODS. Nuclear,
Boston,
(CAMP) was obtained chemicals
were Typical
ATP-3H(G)
Calbiochem,
analytical
grade for
5 umole
of Tris-HCl,
2 pmole
of NaP and 10 nmole
tions
were
1% (W/V)
discs
of Whatman GF/C glass
washed phate
with
After
of dithiothreitol
for
other
of ATP contained (DTT),
30 min and were
(50 ~1)
fiber
(2.4
ethanol.
The discs
and 0.005%
(W/V)
benzene
in an Intertechnique
addition
trichloroacetic
cm diameter);
dried
of MgC12, The reac-
by the
material
were
2 pmole
in 0.3 ml.
terminated
and 100% (W/V)
paper
2,5-Diphenyloxazole in toluene]
All
the precipitation
30 min at 0°C the precipitated
absolute
monophosphate
reagents.
was collected
0.2 M sodium and counted
scintillation
pyrophoa
C0.4% (W/V)
counter.
TABLE1 THE RECOVERY OF ATP AS AN ACID INSOLUBLE COMPLEX
nmole Full
reaction
mix
Full
reaction
mix
Full
reaction
mix
Full
reaction
Full
3H-ATP
nmole
32P-v-ATP
0.80
0.95
- DTT
0.65
0.71
- MgC12
0.007
0.008
mix - NaF
0.005
0.004
reaction
mix
0.33
0.29
Full
reaction
mix + 0.1 M NaCl
0.008
0.006
Full
reaction
mix + 10 nmole
dATP
0.048
0.019
Full
reaction
mix + 10 nmole
ADP
0.28
0.22
Full
reaction
mix + 10 nmole sodium pyrophoaphate
0.048
0.023
+ 1 mM ELYTA
640
on
was
1,4,bis-2-(4-methyl-S-phenyloxaeolyl)liquid
of
acid
each disc
20 ml of 20% TCA, 20 ml of 5% TCA containing
and with
from New
California.
of ATP (3H and 32P labeled)
serum albumin
(50 pi).
obtained
Adenosine-3',5'-cyclic
investigating
at 37'C
(TCA)
were
Los Angeles,
pH 8, 2 umole
incubated bovine
and ATP-v-32P
Massachusetts.
from
assays
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 47, No. 3, 1972
BIOCHEMICAL
RESULTS AND DISCUSSION. investigating
Table
1 shows
the precipitation
recovered
as an acid-precipitable
dependent
on the time
the presence
AND BIOPHYSICAL RESEARCH CCWvWNICATIONS
of ATP. complex.
of incubation
of DTT (Table
the results Almost
of a typical 10% of the
1) and totally
input
The precipitation
at 37'12 (Fig. dependent
la),
assay
for
ATP was
of ATP was
slightly
dependent
on the concentration
on of
(bl
Time
1
of lncubotion
2 pmole
3 Mg**
Fig. 1. The effect of of ATP, (c) concentration precipitation of ATP. 0 -0 A-A
20 nmole
(min)
4
%--TYT-
5
pmole
(a)
Xl ATP
time of incubation at 37'C, of Mg&++ and (d) concentration
Precipitation
of ATP-%
Precipitation
of ATP-3H(G).
-32P.
641
40
50
10 NoF
(b) concentration of NaF, on the
Vol. 47, No. 3,1972
BIOCHEMICAL
ATP, Mg++ and NaF (Table used; the
magnesium lower
1 and Fig.
acetate
ionization
did
The requirement
for
of
1).
enzymes
which
CAMP, which (9),
dependent was almost
also
replace
compound.
replaced
the
on the concentration a fivefold
stimulation
of the concentration
A-A
Precipitation
of ATP-3S(C).
on DEAE paper
(11)
on
during
these
polyethyleneimine-cellulose
to ensure
that
were
pyrophosphate also
slightly
This
stimulation
was
at low concentrations
there
4
5
CAMP
of CAMP on the precipitation
of ATP- 1 -32P.
chromatography
of ATP. 2);
ionic
some phosphorylating
3
Precipitation
layer
Mg++.
of precipitation.
2
precipitated
the excepfor
ADP and sodium activity
precipitation
o-0
The material
with
by EDTA and at high
shown to stimulate
mole
The effect
due to
the requirement
by dATP,
of CAMP (Fig.
1
Fig. 2. ATP.
Other ‘metals,
had no phosphorylating
stimulated
MgC12 was
by NaCl or KCl.
inhibited
has been
reactions
MgC12 presumably
replace
of ATP was prevented
Proteins
inhibitory.
this
RESEARCH COMMUNICATIONS
In these
not efficiently
and was competitively
(Table
lb-ld).
efficiently
NaF was not
The precipitation strength
not
constant
of La +++ and So ++ , did
tion
AND BIOPHYSICAL
the results
642
reactions
was analyzed (10)
of these
of
by thin-
and by ionophoresis assays
were
not
due to
BIOCHEMICAL
Vol. 47, No. 3,1972
the breakdown breakdown
of ATP.
of ATP under
precipitated
insoluble
ATP was acid
without
the ATP.
prior
reported
in the precipitation p,$
inhibition
this
interaction
an absolute
rylating errors
results
enzymes
assays
for indicate
give
by acid
may be avoided
if
emphasizes
for
ATP:protein
was repeated
and nucleotidyl
involved
complex
explain
with
and the competiindicates
of ATP described these
that
in this
results
as NaF is
reaction. may occur
during
assays
of the phosphorylated
the acid-precipitated
the absolute
then
Mgg++ is kngwrf to interact
fully
errors
this
25% of
some of the parameters
the precipitation
precipitation
reprecipitated;
paper
cannot
that
was insol-
10% of the ATP originally
in the precipitation
interaction
requirement
NaOH and then This
this
reactions
by dATP, ADP and pyrophosphate
a part
24 hr.
in 0.1 N NaOH, addition
of ATP to form an insoluble
plays
for
of the
soluble.
paper
of precipitation
However,
These
in this
cent
ATP became
and reprecipitation
acid
per
TCA solubilized
soluble
of ATP by Mgg++ and NaF.
-pyrophosphate
tive
paper.
dissolution
this
with
negligible
complex
these
TCA; treatment
ATP was completely
The results
during
approximately
If
Ninety
but upon re-reaction
with
was only
of the insoluble
ATP was completely
in the complex.
there
described.
The ATP precipitated treatment
that
conditions
by dialysis
TCA reprecipitated
the remaining
indicated
soluble
The precipitated
of 20% (W/V)
the
the assay
once more.
present
results
ATP was removed
The remaining
uble
These
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
product
procedure
should
necessity
for
is
phosphotransferases
phospho-
products. dissolved
be repeated
this
for
procedure
These in 0.1 N
at least during
by acid
twice. all
precipita-
tion. ACKNOWLEDGMENTS Levine ship.
I wish to acknowledge the excellent technical assistance of Mrs. D. and to thank the Damon Runyon Memorial Fund for a postdoctoral fellow-
REFERENCES 1. 2.
Jergil, B., and Dixon, G. H., J. Biol. Hatanaka, M., Twiddy, E., and Gilden, 643
Chem., 245, 425 (1970). R. V., Virology, 47, 536 (1972).
Vol. 47, No. 3,1972
3. 4. 5. 6. 7. 8. 9. 10. 11.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Richardson, C. C., Proc. Natl. Aced. Sci. U.S., 54, 158 (1965). Novogrodsky, A., and Hurwitz, J., J. Biol. Chem., 241, 2923 (1966). Majumder, G. C., and Turkington, R. W., J. Biol. Chem., 246, 2650 (1971). liuttunen, J. K., Steinberg, D., and Mayer, S. E., Biochem. Biophys. Res. comm., 4l, 1350 (1970). Kabat, D., Biochemistry, 2, 4160 (1970). Strand, M., and August, J. T., Nature New Biol., 233, 137 (1971). Kuo, J. F., and Greengard, P., Proc. Natl. Aced. Sci. U.S., 64, 1349 (1969). Randerath, K., and Randerath, E., in "Methods in Enzymology" (L. Grossman and K. Moldave, eds.), vol. 12, part A, 323, Academic Press, New York, 1967. Sanger, F., Brownlee, G. G., and Barrell, B. G., J. Mol. Biol., l3, 373 (1965).
644