BIOCHEMICAL
Vol. 69, No. 2, 1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
METAL ION COMPLEXES OF CYTIDINFDIPHOSPHOCHOLINE G.A.
Cook,*
W.D. Perry,
and H.H.
Daron
Department of Animal and Dairy Sciences, Agricultural Experiment Station and Department of Chemistry Auburn University, Auburn, Alabama 36830 Received
December
19,1975 SUMMARY
Cytidine-5'-diphosphocholine and Mn2+ ions as indicated by in the presence of these ions. is the decrease in absorption solutions of 8-hydroxyquinoline the Mg-CDPcholine complex was All metal
ion
phosphate
known for
phosphotransferases, activity
and the
by certain
complex.
(1).
complex
kinases.
NMR spectroscopy (4,5).
This This
(CDPcholine) forms a complex with Mg2+ the broadened 3lP NMR peaks of CDPcholine Additional evidence for the complex at 360 nm when CDPcholine is added to and Mg2+. The stability constant of found to be 20 M-1.
is
might
ions
one exception, form
the actual
and the
provides
complex
1,2-diacylglycerol
Metal
The formation (2,3)
report
with
stability for
be the
substrate
cholinephosphotransferase
the
for
with
a divalent
adenosine
reactions
of the complex
formation for
tri-
catalyzed
has been demonstrated
constant
evidence
EXPERIMENTAL
complexes
substrate
of Mg-ATP
require
with determined
of a metal-CDPcholine
cytidine-5'-diphosphocholine:
(EC 2.7.8.2). PROCEDURE
31P NMR spectra (3,6) of 0.5 M CDPcholine solutions were obtained in the presence and absence of divalent metal ions. MgSOq and CaSOq concentrations were 0.5 M and the concentration of MnSO4 ranged from 5 x 10-6 to 1 x 10-4 M. The pH of the solutions was 8.5 and the temperature was 22 f l°C. Spectra were obtained with a Varian Associates HA-601L NMR spectrometer at 24.3 MHz. Sample tubes of 3 mm diameter were used with sample sizes varying from 0.3 ml to 0.5 ml. All chemical shifts were measured relative to 85% phosphoric acid, which was used as an external standard. Spectra were calibrated using standard side band techniques. The stability by a method used
constant to measure
of the Mg-CDPcholine complex was determined the stability constants of the Mg2+ complexes
*Present address: Cardiovascular Research Program, Oklahoma Medical Foundation and the Department of Biochemistry and Molecular Biology, of Medicine, University of Oklahoma, Oklahoma City, Oklahoma 73104.
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
411
Research College
Vol. 69, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of ATP and ADP (4,5,7). The method is based on the competition for magnesium ion between the ligand being investigated and B-hydroxyquinoline, since the concentration of the Mg-B-hydroxyquinoline complex is conveniently determined spectrophotometrically at 360 nm (a360 = 2300 M-l cm-l) (4). In the present experiments, the concentration of S-hydroxyquinoline was 1 mM, CDPcholine was 5, 10, 20 and 40 mM in four independent experiments, and MgC12, which was added by microliter syringe or pipet from a 0.05 M stock solution, varied from zero to 1.2 mM in each experiment. The solution also contained 0.05 M tris-(hydroxymethyl)-aminomethane (Tris) buffer, pH 8.5. All absorption measurements were made at 360 nm with an Hitachi Perkin-Elmer Spectrophotometer Model 139. The temperature of the liquid in the sample cells was maintained at 37.0 + 0.2oC by circulating water through the bottom of the cell holder compartment. NMR data from the study of manganese ion effects on chemical shift and peak width were subjected to analysis of variance and Duncan's multiple range test. Data from the study of Ca2+ and Mg2+ effects were analyzed by Student's t-test. RESULTS AND DISCUSSION The 3lP NMR spectrum whether
metal
ppm upfield expected,
ions from
with
(6 = 11 ppm) of 6, nor also
(4). the
with
which
Since
metal
they
form
were
also
with
increasing
a complex
concentration peak widths data
reported
the
NMR peaks
by increasing
of MnS04 were (Pc.05 greater at all for
change
as
the value These
of ATP but not (Pc.005)
data
ADP (3). change
ion
(Table
times,
Table
1 x 10e5 M were
also
(Pc.01).
A significant
412
change
peak widths
the
formation
Peak widths from 1).
with
in peak widths
1) indicates
and CDPcholine.
concentrations
of compounds
increase
or PC.01 as shown in
ATP (3).
agrees,
1 and 2).
relaxation
different
other
not
of the nuclei
significantly
than
was 11.8
of ATP and ADP
significant
sequential
Mn2+ concentration metal
but
(6)
2).
The observed
this
7 to 12 did
peak
This
a-phosphorus
a-phosphorus
was a small,
broaden
complexes
between
MnS04
for
(Table
measured.
centrations lacked
there
ions
the
shift
1 and 2).
of Mn2+ or Ca2+ (Tables
reported
shift
(Tables for
absorption
The chemical
the pH from
presence
data
chemical
reported
Changing
had a single
or not.
the H3P04 reference
When MgS04 was added in the
present
the value
did
agree
were
of CDPcholine
the
This
is
at all
con-
control,
Peak widths
significantly
of
which at each
different consistent
in the peak width
from with
was
BIOCHEMICAL
Vol. 69, No. 2, 1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 1 31 P - NMR of CDPcholine Ion
Manganese Concentration
with
Chemical
0
Shift
Manganese
Ionsa Width
(ppm)
of Peak
.85 f .lO
(ppm)b (8)'
11.8
+ .1 (9)'
5 x 10-6 M
12.0
f .l
(4)
1.08 +_ .08 (4>d
1 x 1O-5 M
12.0
-t .l
(4)
1.23
f
.07
(5)e
2 x 1O-5 M
12.0
? .2 (3)
1.51
+ .lO
(5>e
5 x 1O-5 M
11.7
k .l
2.07 + .lO
(5>e
1 x 1O-4 M
11.9
-f: .2 (5)
aThe CDPcholine concentration bMeasured
(4)
3.02 k .20 (4)e
was .5 M, pH 8.5, and MnSO4 was present conditions are described in the text.
concentration shown. Other
at the
at 4 height.
CMean + standard parentheses.
The number
deviation.
dSignificantly
different
from
control
eSignificantly
different
from control
of observations
is
given
in
(Pc.05).
(Px.01).
TABLE 2 31 P - NMR of CDPcholine Metal
Chemical
Ion
with
Magnesium
Shift
(ppm)
None
11.8
+ .l
(9>c
Mg2+
12.2
-r .l
(4)d
Ca2+
11.9
f .l
(4)
'Mean
Ionsa
Width
of Peak
(ppm)b
.85 k .lO (8)' 1.02
?r .07 (4>e
.77 i .03 (4)
aThe CDPcholine concentration was .5 M, pH 8.5, or CaS04 was present at .5 M. Other conditions bMeasured
and Calcium
and, where indicated, MgS04 are described in the text.
at 4 height.
+ standard
deviation.
The number
of observations
dSignificantly
different
from
control
(Pc.005).
eSignificantly
different
from
control
(Pc.05).
413
is
given
in parentheses.
Vol. 69, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
FIGURE 1. Absorbancy of the Mg2+ complex of 8-hydroxyquinoline in the presence and absence of CDPcholine. Solutions contained 50 mM Tris buffer, pH 8.5, 1 mM 8-hydroxyquinoline, MgC12 as shown, and either no CDPcholine (0) or 20 mM CDPcholine 01). The other conditions are described in the text. See text for a description of the curves.
also
caused
Table
2),
lution
by the diamagnetic but
there
NMR would
two phosphorus broadening. of the
be needed
two phosphates
measured
of CDPcholine resulted This
is
are
relaxation
and the metal
of a solution
interpreted
absorbance as the
result
as a cause
interaction
resoof the
of the peak
between
each
1
complex,
1 mM 8-hydroxyquinoline of MgC12 from
(circles,
Curve
of 20 mM in the for
separation
of the Mg-CDPcholine
concentrations
shown in Figure
High
ion.
containing
different
(Pc.05,
(Pq.05).
partial
time
unequal
constant
at a concentration
in a lower
between
indicate
the stability
at several
results
to distinguish
would
concentration
when CaS04 was added
and increased
The former
To determine
These
was no change
peaks
the absorption
Mg2+ ion at equimolar
each concentration of the
formation
414
A).
was zero
to 1.2 mM.
The inclusion
8-hydroxyquinoline of MgC12 (Figure of a Mg-CDPcholine
solution 1, squares). complex,
BIOCHEMICAL
Vol. 69, No. 2,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-0
1.2 MgCI,
~4COt4CEN18AN&8
( ml41
of the Mg2+ complex of 8-hydroxyquinoline at FIGURE 2. Absorbancy various concentrations of Mg2+ and CDPcholine. The solutions were the same as described for figure 1 except l = no CDPcholine; A= 5 mM CDPcholine; The m = 10 mM CDPcholine; v= 20 mM CDPcholine; and += 40 mM CDPcholine. curves were calculated assuming a value of KS = 20 M-l (see text).
with
correspondingly
The decrease
curves
in absorbance The curves
complex.
calculated
respectively. 1 indicates obtained for
that
constant
the
curves
1 are
assumed
the
data is near
concentrations agreement
additional
of the Mg-CDPcholine theoretical of 10,
points
Q
20 M-l.
absorption
20, 30, and 100 M-l, and curve Figure
The curves
C in Figure
2 shows data
assuming
a value
calculated -1 for of 20 M
of the
experimental
data
with
the
stability
concentrations.
The general gives
constants
constant
CDPcholine
of the Mg-CDPcholine
The addition
stability
between
stability
Cholinephosphotransferase
(8).
by 8-hydroxyquinoline.
B, C, D, and E in Figure
CDPcholine
constant.
related
binding stability
The agreement
each of the
theoretical
is
for to the
from
at various
stability
Mg2+ available
less
support
for
this
value
were
for
complex. requires
of Ca2+ even in the
415
either presence
Mg2+ or Mn2+ for
activity
of Mg2+ or Mn2+ is
inhibitory
the the
BIOCHEMICAL
Vol. 69, No. 2, 1976
Since
(8).
not with strate
Ca2+, for
complex
is
therefore, than would
CDPcholine
that
complexes
with
both
may be the metal-CDPcholine
the
enzyme.
quite
low,
the
intracellular
of free
allow
tration
it
forms
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Although the
the
regulation
of magnesium
The low
of phospholipid within
the
which
constant
concentration
concentration
CDPcholine.
complex
stability
intracellular
Mg2+ and Mn
is
but
the sub-
of magnesium
constant
biosynthesis
endoplasmic
ions
of the Mg-CDPcholine
of Mg-CDPcholine stability
2+
could for
by changes
is
high;
be higher
the
complex
in concen-
reticulum.
ACKNOWLEDGMENT This
research
was supported
by Public
Health
Service
Grant
HE-02615.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Morrison, J. F. and Heyde, E. (1972) Ann. Rev. Biochem., 3, 29-54. Mildvan, A. S. (1970) in The Enzymes, 3rd ed., Boyer, P. D. (ed.), Vol. 2, pp. 445-536, Academic Press, New York. Cohn, M. and Hughes, T. R., Jr. (1962) J. Biol. Chem., 237, 176-181. Burton, K. (1959) Biochem. J., I_L, 388-395. O'Sullivan, W. J. and Perrin, D. D. (1964) Biochemistry, 2, 18-26. Cohn, M. and Hughes, T. R., Jr. (1960) J. Biol. Chem., 235, 3250-3253. Nasanen, R. (1952) Acta Chem. Stand., 5, 352-356. Ewing, R. D., and Finamore, F. J. (1970) Biochim. Biophys. Acta, 218, 474-481.
416
Vol. 69, No. 2, 1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
METAL ION COMPLEXES OF CYTIDINFDIPHOSPHOCHOLINE G.A.
Cook,*
W.D. Perry,
and H.H.
Daron
Department of Animal and Dairy Sciences, Agricultural Experiment Station and Department of Chemistry Auburn University, Auburn, Alabama 36830 Received
December
19,1975 SUMMARY
Cytidine-5'-diphosphocholine and Mn2+ ions as indicated by in the presence of these ions. is the decrease in absorption solutions of 8-hydroxyquinoline the Mg-CDPcholine complex was All metal
ion
phosphate
known for
phosphotransferases, activity
and the
by certain
complex.
(1).
complex
kinases.
NMR spectroscopy (4,5).
This This
(CDPcholine) forms a complex with Mg2+ the broadened 3lP NMR peaks of CDPcholine Additional evidence for the complex at 360 nm when CDPcholine is added to and Mg2+. The stability constant of found to be 20 M-1.
is
might
ions
one exception, form
the actual
and the
provides
complex
1,2-diacylglycerol
Metal
The formation (2,3)
report
with
stability for
be the
substrate
cholinephosphotransferase
the
for
with
a divalent
adenosine
reactions
of the complex
formation for
tri-
catalyzed
has been demonstrated
constant
evidence
EXPERIMENTAL
complexes
substrate
of Mg-ATP
require
with determined
of a metal-CDPcholine
cytidine-5'-diphosphocholine:
(EC 2.7.8.2). PROCEDURE
31P NMR spectra (3,6) of 0.5 M CDPcholine solutions were obtained in the presence and absence of divalent metal ions. MgSOq and CaSOq concentrations were 0.5 M and the concentration of MnSO4 ranged from 5 x 10-6 to 1 x 10-4 M. The pH of the solutions was 8.5 and the temperature was 22 f l°C. Spectra were obtained with a Varian Associates HA-601L NMR spectrometer at 24.3 MHz. Sample tubes of 3 mm diameter were used with sample sizes varying from 0.3 ml to 0.5 ml. All chemical shifts were measured relative to 85% phosphoric acid, which was used as an external standard. Spectra were calibrated using standard side band techniques. The stability by a method used
constant to measure
of the Mg-CDPcholine complex was determined the stability constants of the Mg2+ complexes
*Present address: Cardiovascular Research Program, Oklahoma Medical Foundation and the Department of Biochemistry and Molecular Biology, of Medicine, University of Oklahoma, Oklahoma City, Oklahoma 73104.
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
411
Research College
Vol. 69, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of ATP and ADP (4,5,7). The method is based on the competition for magnesium ion between the ligand being investigated and B-hydroxyquinoline, since the concentration of the Mg-B-hydroxyquinoline complex is conveniently determined spectrophotometrically at 360 nm (a360 = 2300 M-l cm-l) (4). In the present experiments, the concentration of S-hydroxyquinoline was 1 mM, CDPcholine was 5, 10, 20 and 40 mM in four independent experiments, and MgC12, which was added by microliter syringe or pipet from a 0.05 M stock solution, varied from zero to 1.2 mM in each experiment. The solution also contained 0.05 M tris-(hydroxymethyl)-aminomethane (Tris) buffer, pH 8.5. All absorption measurements were made at 360 nm with an Hitachi Perkin-Elmer Spectrophotometer Model 139. The temperature of the liquid in the sample cells was maintained at 37.0 + 0.2oC by circulating water through the bottom of the cell holder compartment. NMR data from the study of manganese ion effects on chemical shift and peak width were subjected to analysis of variance and Duncan's multiple range test. Data from the study of Ca2+ and Mg2+ effects were analyzed by Student's t-test. RESULTS AND DISCUSSION The 3lP NMR spectrum whether
metal
ppm upfield expected,
ions from
with
(6 = 11 ppm) of 6, nor also
(4). the
with
which
Since
metal
they
form
were
also
with
increasing
a complex
concentration peak widths data
reported
the
NMR peaks
by increasing
of MnS04 were (Pc.05 greater at all for
change
as
the value These
of ATP but not (Pc.005)
data
ADP (3). change
ion
(Table
times,
Table
1 x 10e5 M were
also
(Pc.01).
A significant
412
change
peak widths
the
formation
Peak widths from 1).
with
in peak widths
1) indicates
and CDPcholine.
concentrations
of compounds
increase
or PC.01 as shown in
ATP (3).
agrees,
1 and 2).
relaxation
different
other
not
of the nuclei
significantly
than
was 11.8
of ATP and ADP
significant
sequential
Mn2+ concentration metal
but
(6)
2).
The observed
this
7 to 12 did
peak
This
a-phosphorus
a-phosphorus
was a small,
broaden
complexes
between
MnS04
for
(Table
measured.
centrations lacked
there
ions
the
shift
1 and 2).
of Mn2+ or Ca2+ (Tables
reported
shift
(Tables for
absorption
The chemical
the pH from
presence
data
chemical
reported
Changing
had a single
or not.
the H3P04 reference
When MgS04 was added in the
present
the value
did
agree
were
of CDPcholine
the
This
is
at all
con-
control,
Peak widths
significantly
of
which at each
different consistent
in the peak width
from with
was
BIOCHEMICAL
Vol. 69, No. 2, 1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 1 31 P - NMR of CDPcholine Ion
Manganese Concentration
with
Chemical
0
Shift
Manganese
Ionsa Width
(ppm)
of Peak
.85 f .lO
(ppm)b (8)'
11.8
+ .1 (9)'
5 x 10-6 M
12.0
f .l
(4)
1.08 +_ .08 (4>d
1 x 1O-5 M
12.0
-t .l
(4)
1.23
f
.07
(5)e
2 x 1O-5 M
12.0
? .2 (3)
1.51
+ .lO
(5>e
5 x 1O-5 M
11.7
k .l
2.07 + .lO
(5>e
1 x 1O-4 M
11.9
-f: .2 (5)
aThe CDPcholine concentration bMeasured
(4)
3.02 k .20 (4)e
was .5 M, pH 8.5, and MnSO4 was present conditions are described in the text.
concentration shown. Other
at the
at 4 height.
CMean + standard parentheses.
The number
deviation.
dSignificantly
different
from
control
eSignificantly
different
from control
of observations
is
given
in
(Pc.05).
(Px.01).
TABLE 2 31 P - NMR of CDPcholine Metal
Chemical
Ion
with
Magnesium
Shift
(ppm)
None
11.8
+ .l
(9>c
Mg2+
12.2
-r .l
(4)d
Ca2+
11.9
f .l
(4)
'Mean
Ionsa
Width
of Peak
(ppm)b
.85 k .lO (8)' 1.02
?r .07 (4>e
.77 i .03 (4)
aThe CDPcholine concentration was .5 M, pH 8.5, or CaS04 was present at .5 M. Other conditions bMeasured
and Calcium
and, where indicated, MgS04 are described in the text.
at 4 height.
+ standard
deviation.
The number
of observations
dSignificantly
different
from
control
(Pc.005).
eSignificantly
different
from
control
(Pc.05).
413
is
given
in parentheses.
Vol. 69, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
FIGURE 1. Absorbancy of the Mg2+ complex of 8-hydroxyquinoline in the presence and absence of CDPcholine. Solutions contained 50 mM Tris buffer, pH 8.5, 1 mM 8-hydroxyquinoline, MgC12 as shown, and either no CDPcholine (0) or 20 mM CDPcholine 01). The other conditions are described in the text. See text for a description of the curves.
also
caused
Table
2),
lution
by the diamagnetic but
there
NMR would
two phosphorus broadening. of the
be needed
two phosphates
measured
of CDPcholine resulted This
is
are
relaxation
and the metal
of a solution
interpreted
absorbance as the
result
as a cause
interaction
resoof the
of the peak
between
each
1
complex,
1 mM 8-hydroxyquinoline of MgC12 from
(circles,
Curve
of 20 mM in the for
separation
of the Mg-CDPcholine
concentrations
shown in Figure
High
ion.
containing
different
(Pc.05,
(Pq.05).
partial
time
unequal
constant
at a concentration
in a lower
between
indicate
the stability
at several
results
to distinguish
would
concentration
when CaS04 was added
and increased
The former
To determine
These
was no change
peaks
the absorption
Mg2+ ion at equimolar
each concentration of the
formation
414
A).
was zero
to 1.2 mM.
The inclusion
8-hydroxyquinoline of MgC12 (Figure of a Mg-CDPcholine
solution 1, squares). complex,
BIOCHEMICAL
Vol. 69, No. 2,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-0
1.2 MgCI,
~4COt4CEN18AN&8
( ml41
of the Mg2+ complex of 8-hydroxyquinoline at FIGURE 2. Absorbancy various concentrations of Mg2+ and CDPcholine. The solutions were the same as described for figure 1 except l = no CDPcholine; A= 5 mM CDPcholine; The m = 10 mM CDPcholine; v= 20 mM CDPcholine; and += 40 mM CDPcholine. curves were calculated assuming a value of KS = 20 M-l (see text).
with
correspondingly
The decrease
curves
in absorbance The curves
complex.
calculated
respectively. 1 indicates obtained for
that
constant
the
curves
1 are
assumed
the
data is near
concentrations agreement
additional
of the Mg-CDPcholine theoretical of 10,
points
Q
20 M-l.
absorption
20, 30, and 100 M-l, and curve Figure
The curves
C in Figure
2 shows data
assuming
a value
calculated -1 for of 20 M
of the
experimental
data
with
the
stability
concentrations.
The general gives
constants
constant
CDPcholine
of the Mg-CDPcholine
The addition
stability
between
stability
Cholinephosphotransferase
(8).
by 8-hydroxyquinoline.
B, C, D, and E in Figure
CDPcholine
constant.
related
binding stability
The agreement
each of the
theoretical
is
for to the
from
at various
stability
Mg2+ available
less
support
for
this
value
were
for
complex. requires
of Ca2+ even in the
415
either presence
Mg2+ or Mn2+ for
activity
of Mg2+ or Mn2+ is
inhibitory
the the
BIOCHEMICAL
Vol. 69, No. 2, 1976
Since
(8).
not with strate
Ca2+, for
complex
is
therefore, than would
CDPcholine
that
complexes
with
both
may be the metal-CDPcholine
the
enzyme.
quite
low,
the
intracellular
of free
allow
tration
it
forms
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Although the
the
regulation
of magnesium
The low
of phospholipid within
the
which
constant
concentration
concentration
CDPcholine.
complex
stability
intracellular
Mg2+ and Mn
is
but
the sub-
of magnesium
constant
biosynthesis
endoplasmic
ions
of the Mg-CDPcholine
of Mg-CDPcholine stability
2+
could for
by changes
is
high;
be higher
the
complex
in concen-
reticulum.
ACKNOWLEDGMENT This
research
was supported
by Public
Health
Service
Grant
HE-02615.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Morrison, J. F. and Heyde, E. (1972) Ann. Rev. Biochem., 3, 29-54. Mildvan, A. S. (1970) in The Enzymes, 3rd ed., Boyer, P. D. (ed.), Vol. 2, pp. 445-536, Academic Press, New York. Cohn, M. and Hughes, T. R., Jr. (1962) J. Biol. Chem., 237, 176-181. Burton, K. (1959) Biochem. J., I_L, 388-395. O'Sullivan, W. J. and Perrin, D. D. (1964) Biochemistry, 2, 18-26. Cohn, M. and Hughes, T. R., Jr. (1960) J. Biol. Chem., 235, 3250-3253. Nasanen, R. (1952) Acta Chem. Stand., 5, 352-356. Ewing, R. D., and Finamore, F. J. (1970) Biochim. Biophys. Acta, 218, 474-481.
416
