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Noncovalent complexes of diadenosine 5′,5‴-P1,P4-tetraphosphate with divalent metal ions, biogenic amines, proteins and poly(dT)
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1037-1 043
Vol. 120, No. 3, 1984 May 16, 1984
NCNCOVALENT COMPLEXES OF DIADENOSINE 5',5'''-P j P4-TETRAPHOSPHATE WITH DIVALENT METAL IONS, BIOGENIC AMINES, PROTEINS ANDPOLY(dT) Eggehard Holler Institut fHr Biophysik und physikalische Biochemie, Universit~t Regensburg, Universititsstra~e 3~, D-8400 Regensburg, GFR Received April 10, 1984 SUMMARY: Diadenosine 5',5'''-P q ,P~-tetraphosphate was shown by circular dichroic measurements to bind to metal ions (Mg 2+, Ca2+, INn2+, 0o 2+ , Zn2+), to biogenic amines (cadaverine, putrescine, spermidine, spermine), to L-arginine, to proteins (lysozyme, bovine serum albumin, Arg-rich histone f3, Lysrich histone), and to poly(dT). Most cations effect destacking of the intramolecular adenine rings. Poly(dT) bound to the dinucleotide with a stoichiometry of 2 residues TFiP per molecule of adenosine 5',5'''-PI,P4-tetraphosphate. INTRODUCTION: nucleoside
Evidence
is accumulating
oligophosphates
tetraphosphate
such as diadenosine
(AppppA ~) are ubiquitous
(4,2). AppppA accumulates boundary
that certain bis-
in living organisms
in proliferating
cells at the G~/S
in the cell cycle (3), or in organisms
stress (2). In its role as a pleiotypic involved in a variety of biological grounds,
5',5'''-PI,P 4-
exposed to
effector,
processes
AppppA is
(~). On chemical
it may be predicted that this class of compounds may
reversibly bind ligands by virtue of both the highly charged oligophosphate
chain and the purine bases. Binding could be
controlled by the mode of intramolecular
stacking of the pu-
rine rings (4). We report here such interactions a variety of metal ions, with biogenic with proteins, Abbreviations
and with poly(dT). used:
AppppA,
of AppppA with
amines, with L-arginine,
We have measured th~ effect
diadenosine
5',5'''-P~,P4-tetra-
pho sphat e. 0006-291X/84 $1.50 1037
Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of these ligands on the intramolecular stacking of the adenine rings. MATERIALS AND METHODS: Europium(Ill) nitrate was obtained from Merck/Darmstadt, terbium(lll) chloride from Ventron/Karlsruhe, and tris(hydroxymethyl)-aminomethane, EDTA, Arg-rich histone (fraction f3), Lys-rich histone, hen egg white lysozyme, bovine serum albumin, calcium acetate and all other cations (in the chloride form) from Sigma/Munich. Quartz-distilled water was used throughout the work. Reaction solutions contained 10 mM Tris-HCl buffer pH 7.5. In the absence of metal ions, solutions contained I mM EDTA. Proteins were dialysed twice against buffer containing 10 mM EDTA. Poly(dT) was obtained from Sigma as sodium salt and was dialysed before use. Laboratory plastic ware was used in order to avoid contamination by metal ions. Circular dichroism was measured at 19 + 0.5 °C using quartz cells of 2.0 or 40.0 cm optical path l~ngth with a Jasco J-5OOA spectropolarimeter equipped with a Jasco DP-5OON data processor. Values of dissociation constants and of stoichiometries were calculated from experimental data as described (4). Spectra were corrected by subtraction of contributions from material other than AppppA. RESULTS DIVALENT METAL IONS:
Alkali earth metal ions, Mg 2+ and Ca 2+,
and the heavy metal ions Co2+, Mn 2+ and Zn 2+, bound to AppppA with similar values of KDISS(app.)
(10 - 40 ~M; Table I), and
with similar decreases in ellipticity at 277 nm. The rare earth metal ions Eu 3+ and Tb 3+ reacted with AppppA to form tight com-
TABLE I Divalent metal ion complexes of AppppA a)
AOZAx/Oo
~oiss(app.)
at 277 nm Mg2+ b) Ca 2+ Mn 2+ Co 2+ Zn2+ b)
0.33 0.53 0.45 0.42 0.57
28 36 11 29 15
a) Conditions were 2 - 20 ~M 10 mM Tris-HC1 pH 7.5, 19 + 0.5 C. Symbols A~M~X AppppA, and Oo refer to magnitude of decre~se in ellipticity at saturating concentration of ligand and to magnitude of ellipticity in the absence of ligand. b) Values refer to 22°C and are taken from (4). 1038
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 2 Biogenic amine complexes of AppppA
AOZAXI@o
KDISs(app.)
at 277 nm
L-Arginine Cadaverine Putrescine Spermidine Spermine
~M
0.46 0.19 0.21 0.35 0.40
27 O00 I 900 910 120 19
Conditions were those of Table I except that q mM EDTA had been added.
plexes, which precipitated (~E-con~entrations, BIOGENIC AMINES:
shortly after mixing of the reactants
not shown).
L-Arginine and biogenic amines formed com-
plexes with AppppA (Table 2). Increasing positive charges favoured complex formation,
indicating electrostatic attraction
to be the driving force. Spermine and divalent metal ions had comparable affinities for AppppA. PROTEINS:
The results for L-srginine and biogenic amines
suggested the possibility of (unspecific) between AppppA and proteins. for several proteins,
complex formation
This is born out from the results
shown in Figure q and in Table 3. In each
case binding was observed as a decrease in ellipticity at 277 nm. In the case of lysozyme and of bovine serum albumin,
concen-
trations were not sufficiently high as to yield a hyperbolic saturation curve. The observed linear dependence is explained by assuming values of KDISS(app.)
that are above the highest
concentrations used (25 ~M for bovine serum albumin, Mr = 68 000, and 48 ~M for lysozyme, Mr = J4 300). Histones,
on the contrary,
showed very strong binding of AppppA, presumably because of their high basicity.
Their titration curves in Figure J are typi1039
¢ol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
°~I /'~" °,~ !
//
j
.A.0ri...,s.0.E,.~,. 7/ • /~.s0~.E /~v,.~s~.u.A~0.,. ./
0.2
0.1
k~ /,/11 /'/ 17 / / " n / / II !/J . /
~ o - " " " ~ o......~-""~ININE
/
rng/ml Protein
FIGURE I Titration of AppppA with protein Fixed concentrations of AppppA were 53 ~ for histones, 15 pM for bovine serum albumin and 20 ~M for lysozyme. Conditions were otherwise the same as for Table 2. cal for cases where the (fixed) technique
values
concentration
of AppppA.
did not allow determination
for histones.
The concentrations
are given in Table
3 as upper
nation of stoichiometry and high
of KDISS(app.)
(53 ~N)
The resolution
concentrations
constants
(the fixed reactant)
for KDISS(app.).
respectively
below
of the p r e s e n
of dissociation
of AppppA
limits
values
are considerably
Determi-
at low (d - ~O ~M)
of AppppA according
to (4) re-
TABLE 3 Protein complexes of AppppA Stoichiometry mol AppppA : mol ligand at ~ AppppA
KDISS(app') ~M
Arg-rich histone (H3) a)
I at 10 ~ # at 53 ~M
10 53
Lys-rich histone (HI) b)
I at I ~M 3 at 53 ~
I 53
Bovine serum albumin
n . d . c)
25
Lysozyme
n.d.
48
Conditions were those of Table 2. a) Arg-rich histone consisted of H3, ) Lys-rich histonecof HI. Molecular weights were 15 300 and 21 000, respectively. ) n. d. = not determined. Parameters were estimated, in part, from titration curves in Figure I. 1040
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ARfiININE EADAVERINE PUTRESCI NE (-)..- ~(+) SPERMIDINE SPERMI NE LYS-rkh HISTONE(HI) [ARfi-richHISTONE(H3)] Ca2÷ [Mg2+] [C°2÷] a ~
LYSOZYME BOVINE SERUMALBUMIN (-) ~- O-~('+)
a( b
27Ohm
B
A
300nm
FIGURE 2
Representative circular dichroic spectra of free AppppA (trace a) and of AppppA in the presence of subsaturating amounts of ligands (trace b) Experimental conditions were the same as for Table ~ and Table 2, respectively. Traces (a) were corrected for contribution from buffer, traces (b) for contributions from added ligands. Ellipticities in the case of Mg + and Co 2+, respectively, did not decrease in a completely symmetrical fashion (not shown).
vealed
a single tightly bound AppppA,
with lower affinity per molecule Circular
dichroic
stacking
of the adenine
symmetrical Figure
rings
in AppppA
in ellipticities
2B as a consequence
as a (partial)
ration.
and bovine
ceivable
cities
of the metal POLY(dT): a small
opening
in the wavelength
with various
of the stacked
in these properties
This probably
ions with the adenine
Association
increase
of AppppA
in ellipticity
1041
of
in ellipti-
ligation
(4).
and poly(dT) (Fig.
It is con-
was also seen for
reflected
ring(s)
configu-
as a result
decrease
decrease
ligands
display
range of interest.
2A. An asymmetrical
Mn 2+ and Zn 2+ (not shown).
to intramolecular
at 277 nm and 254 nm in
is seen as the asymmetrical
of Figure
(Table 3).
serum albumin themselves
that a perturbation
AppppA binding
others bound
(5). Hence the observed
of interaction
can be explained
strong dichroism
of histone
spectra have been attributed
decrease
Lysozyme
and several
was monitored
3A). As in the case of
as
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
( - ) ~ 0-'.'{+)
B ~61
,
/ ~1 I~MPOL¥Idrl
4~
,
|
o
=__~
E t o a ~ o ' ~ i °°° ~2 111aMAppppA oI.~~
~
i
2'0
pM AppppA
FIGURE 3 Titration of poly(dT) with AppppA Panel A: Representative circular dichroic spectra for free AppppA (Ref.) and for 30 ~M AppppA plus 31 ~M poly(dT). Panel B: Increase in ellipticity at 277 nm as a functibn of concentration of AppppA.
histones,
the titration curve was not hyperbolic and reflected
KDISS(app.) ~ 3 J
~N (= the concentration of poly(dT)
of TFLP residues,
as the fixed reactant).
in terms
The stoichiometry was
calculated from the position of the bend of the curve (Fig. 3B) to be 2 residues TNP per molecule AppppA.
The small effect on
circular dichroism seemed to exclude the occurrence of larger changes in conformations of poly(dT)
and AppppA.
DISCUSSION The proposed role of AppppA as a pleiotypic
effector is stressed
by the present findings that the dinucleotide can bind to various different
ligands. Enzymatic reactions with AppppA that require
its open conformation, may require prior binding to such ligands. The diversity of ligands that are able to bind AppppA will have to be taken into account when intracellular concentrations of AppppA are calculated.
Tight binding of AppppA to histones may
be of importance during nucleosome assembly. ciation with poly(dT)
The observed asso-
lends support for the proposed role of
AppppA as primer of DNA synthesis in vivo (6). Experiments are in progress to establish dissociation constants, significance of reactions described here.
1042
specificity and
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES I 2 3 4 5 6
Zamecnik, P. (1983) Anal. Bioehem. I_3_#, 1-10. Lee, P.C., Bochner, B. and Ames, B. (1983) Proc. Natl. Acad. Sci. USA 80, 7496-7500. Weinmann-Dorsch, C., Hedl, A., Grummt, I., Albert, W., Ferdinand, F.-J., Friis, R.R., Pierron, G., Moll, W. and Grummt, F. (1984) Eur. J. Biochem. 138, 479-185. Holler, E., Holmquist, B., Vallee, B.L., Taneja, K. and Zamecnik, P. (1983) Biochemistry 22, 4924-4933. Scott, J.F. and Zamecnik, P. (1969-7 Proc. Natl. Aoad. Sci. USA 64, 1308-1314. Zamecnik, P., Rapaport, E. and Baril, E.F. (1982) Proc. Natl. Acad. Sci. USA 79, 1791-1794.
1043
Vol. 120, No. 3, 1984 May 16, 1984
NCNCOVALENT COMPLEXES OF DIADENOSINE 5',5'''-P j P4-TETRAPHOSPHATE WITH DIVALENT METAL IONS, BIOGENIC AMINES, PROTEINS ANDPOLY(dT) Eggehard Holler Institut fHr Biophysik und physikalische Biochemie, Universit~t Regensburg, Universititsstra~e 3~, D-8400 Regensburg, GFR Received April 10, 1984 SUMMARY: Diadenosine 5',5'''-P q ,P~-tetraphosphate was shown by circular dichroic measurements to bind to metal ions (Mg 2+, Ca2+, INn2+, 0o 2+ , Zn2+), to biogenic amines (cadaverine, putrescine, spermidine, spermine), to L-arginine, to proteins (lysozyme, bovine serum albumin, Arg-rich histone f3, Lysrich histone), and to poly(dT). Most cations effect destacking of the intramolecular adenine rings. Poly(dT) bound to the dinucleotide with a stoichiometry of 2 residues TFiP per molecule of adenosine 5',5'''-PI,P4-tetraphosphate. INTRODUCTION: nucleoside
Evidence
is accumulating
oligophosphates
tetraphosphate
such as diadenosine
(AppppA ~) are ubiquitous
(4,2). AppppA accumulates boundary
that certain bis-
in living organisms
in proliferating
cells at the G~/S
in the cell cycle (3), or in organisms
stress (2). In its role as a pleiotypic involved in a variety of biological grounds,
5',5'''-PI,P 4-
exposed to
effector,
processes
AppppA is
(~). On chemical
it may be predicted that this class of compounds may
reversibly bind ligands by virtue of both the highly charged oligophosphate
chain and the purine bases. Binding could be
controlled by the mode of intramolecular
stacking of the pu-
rine rings (4). We report here such interactions a variety of metal ions, with biogenic with proteins, Abbreviations
and with poly(dT). used:
AppppA,
of AppppA with
amines, with L-arginine,
We have measured th~ effect
diadenosine
5',5'''-P~,P4-tetra-
pho sphat e. 0006-291X/84 $1.50 1037
Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of these ligands on the intramolecular stacking of the adenine rings. MATERIALS AND METHODS: Europium(Ill) nitrate was obtained from Merck/Darmstadt, terbium(lll) chloride from Ventron/Karlsruhe, and tris(hydroxymethyl)-aminomethane, EDTA, Arg-rich histone (fraction f3), Lys-rich histone, hen egg white lysozyme, bovine serum albumin, calcium acetate and all other cations (in the chloride form) from Sigma/Munich. Quartz-distilled water was used throughout the work. Reaction solutions contained 10 mM Tris-HCl buffer pH 7.5. In the absence of metal ions, solutions contained I mM EDTA. Proteins were dialysed twice against buffer containing 10 mM EDTA. Poly(dT) was obtained from Sigma as sodium salt and was dialysed before use. Laboratory plastic ware was used in order to avoid contamination by metal ions. Circular dichroism was measured at 19 + 0.5 °C using quartz cells of 2.0 or 40.0 cm optical path l~ngth with a Jasco J-5OOA spectropolarimeter equipped with a Jasco DP-5OON data processor. Values of dissociation constants and of stoichiometries were calculated from experimental data as described (4). Spectra were corrected by subtraction of contributions from material other than AppppA. RESULTS DIVALENT METAL IONS:
Alkali earth metal ions, Mg 2+ and Ca 2+,
and the heavy metal ions Co2+, Mn 2+ and Zn 2+, bound to AppppA with similar values of KDISS(app.)
(10 - 40 ~M; Table I), and
with similar decreases in ellipticity at 277 nm. The rare earth metal ions Eu 3+ and Tb 3+ reacted with AppppA to form tight com-
TABLE I Divalent metal ion complexes of AppppA a)
AOZAx/Oo
~oiss(app.)
at 277 nm Mg2+ b) Ca 2+ Mn 2+ Co 2+ Zn2+ b)
0.33 0.53 0.45 0.42 0.57
28 36 11 29 15
a) Conditions were 2 - 20 ~M 10 mM Tris-HC1 pH 7.5, 19 + 0.5 C. Symbols A~M~X AppppA, and Oo refer to magnitude of decre~se in ellipticity at saturating concentration of ligand and to magnitude of ellipticity in the absence of ligand. b) Values refer to 22°C and are taken from (4). 1038
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 2 Biogenic amine complexes of AppppA
AOZAXI@o
KDISs(app.)
at 277 nm
L-Arginine Cadaverine Putrescine Spermidine Spermine
~M
0.46 0.19 0.21 0.35 0.40
27 O00 I 900 910 120 19
Conditions were those of Table I except that q mM EDTA had been added.
plexes, which precipitated (~E-con~entrations, BIOGENIC AMINES:
shortly after mixing of the reactants
not shown).
L-Arginine and biogenic amines formed com-
plexes with AppppA (Table 2). Increasing positive charges favoured complex formation,
indicating electrostatic attraction
to be the driving force. Spermine and divalent metal ions had comparable affinities for AppppA. PROTEINS:
The results for L-srginine and biogenic amines
suggested the possibility of (unspecific) between AppppA and proteins. for several proteins,
complex formation
This is born out from the results
shown in Figure q and in Table 3. In each
case binding was observed as a decrease in ellipticity at 277 nm. In the case of lysozyme and of bovine serum albumin,
concen-
trations were not sufficiently high as to yield a hyperbolic saturation curve. The observed linear dependence is explained by assuming values of KDISS(app.)
that are above the highest
concentrations used (25 ~M for bovine serum albumin, Mr = 68 000, and 48 ~M for lysozyme, Mr = J4 300). Histones,
on the contrary,
showed very strong binding of AppppA, presumably because of their high basicity.
Their titration curves in Figure J are typi1039
¢ol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
°~I /'~" °,~ !
//
j
.A.0ri...,s.0.E,.~,. 7/ • /~.s0~.E /~v,.~s~.u.A~0.,. ./
0.2
0.1
k~ /,/11 /'/ 17 / / " n / / II !/J . /
~ o - " " " ~ o......~-""~ININE
/
rng/ml Protein
FIGURE I Titration of AppppA with protein Fixed concentrations of AppppA were 53 ~ for histones, 15 pM for bovine serum albumin and 20 ~M for lysozyme. Conditions were otherwise the same as for Table 2. cal for cases where the (fixed) technique
values
concentration
of AppppA.
did not allow determination
for histones.
The concentrations
are given in Table
3 as upper
nation of stoichiometry and high
of KDISS(app.)
(53 ~N)
The resolution
concentrations
constants
(the fixed reactant)
for KDISS(app.).
respectively
below
of the p r e s e n
of dissociation
of AppppA
limits
values
are considerably
Determi-
at low (d - ~O ~M)
of AppppA according
to (4) re-
TABLE 3 Protein complexes of AppppA Stoichiometry mol AppppA : mol ligand at ~ AppppA
KDISS(app') ~M
Arg-rich histone (H3) a)
I at 10 ~ # at 53 ~M
10 53
Lys-rich histone (HI) b)
I at I ~M 3 at 53 ~
I 53
Bovine serum albumin
n . d . c)
25
Lysozyme
n.d.
48
Conditions were those of Table 2. a) Arg-rich histone consisted of H3, ) Lys-rich histonecof HI. Molecular weights were 15 300 and 21 000, respectively. ) n. d. = not determined. Parameters were estimated, in part, from titration curves in Figure I. 1040
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ARfiININE EADAVERINE PUTRESCI NE (-)..- ~(+) SPERMIDINE SPERMI NE LYS-rkh HISTONE(HI) [ARfi-richHISTONE(H3)] Ca2÷ [Mg2+] [C°2÷] a ~
LYSOZYME BOVINE SERUMALBUMIN (-) ~- O-~('+)
a( b
27Ohm
B
A
300nm
FIGURE 2
Representative circular dichroic spectra of free AppppA (trace a) and of AppppA in the presence of subsaturating amounts of ligands (trace b) Experimental conditions were the same as for Table ~ and Table 2, respectively. Traces (a) were corrected for contribution from buffer, traces (b) for contributions from added ligands. Ellipticities in the case of Mg + and Co 2+, respectively, did not decrease in a completely symmetrical fashion (not shown).
vealed
a single tightly bound AppppA,
with lower affinity per molecule Circular
dichroic
stacking
of the adenine
symmetrical Figure
rings
in AppppA
in ellipticities
2B as a consequence
as a (partial)
ration.
and bovine
ceivable
cities
of the metal POLY(dT): a small
opening
in the wavelength
with various
of the stacked
in these properties
This probably
ions with the adenine
Association
increase
of AppppA
in ellipticity
1041
of
in ellipti-
ligation
(4).
and poly(dT) (Fig.
It is con-
was also seen for
reflected
ring(s)
configu-
as a result
decrease
decrease
ligands
display
range of interest.
2A. An asymmetrical
Mn 2+ and Zn 2+ (not shown).
to intramolecular
at 277 nm and 254 nm in
is seen as the asymmetrical
of Figure
(Table 3).
serum albumin themselves
that a perturbation
AppppA binding
others bound
(5). Hence the observed
of interaction
can be explained
strong dichroism
of histone
spectra have been attributed
decrease
Lysozyme
and several
was monitored
3A). As in the case of
as
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
( - ) ~ 0-'.'{+)
B ~61
,
/ ~1 I~MPOL¥Idrl
4~
,
|
o
=__~
E t o a ~ o ' ~ i °°° ~2 111aMAppppA oI.~~
~
i
2'0
pM AppppA
FIGURE 3 Titration of poly(dT) with AppppA Panel A: Representative circular dichroic spectra for free AppppA (Ref.) and for 30 ~M AppppA plus 31 ~M poly(dT). Panel B: Increase in ellipticity at 277 nm as a functibn of concentration of AppppA.
histones,
the titration curve was not hyperbolic and reflected
KDISS(app.) ~ 3 J
~N (= the concentration of poly(dT)
of TFLP residues,
as the fixed reactant).
in terms
The stoichiometry was
calculated from the position of the bend of the curve (Fig. 3B) to be 2 residues TNP per molecule AppppA.
The small effect on
circular dichroism seemed to exclude the occurrence of larger changes in conformations of poly(dT)
and AppppA.
DISCUSSION The proposed role of AppppA as a pleiotypic
effector is stressed
by the present findings that the dinucleotide can bind to various different
ligands. Enzymatic reactions with AppppA that require
its open conformation, may require prior binding to such ligands. The diversity of ligands that are able to bind AppppA will have to be taken into account when intracellular concentrations of AppppA are calculated.
Tight binding of AppppA to histones may
be of importance during nucleosome assembly. ciation with poly(dT)
The observed asso-
lends support for the proposed role of
AppppA as primer of DNA synthesis in vivo (6). Experiments are in progress to establish dissociation constants, significance of reactions described here.
1042
specificity and
Vol. 120, No. 3, 1984
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES I 2 3 4 5 6
Zamecnik, P. (1983) Anal. Bioehem. I_3_#, 1-10. Lee, P.C., Bochner, B. and Ames, B. (1983) Proc. Natl. Acad. Sci. USA 80, 7496-7500. Weinmann-Dorsch, C., Hedl, A., Grummt, I., Albert, W., Ferdinand, F.-J., Friis, R.R., Pierron, G., Moll, W. and Grummt, F. (1984) Eur. J. Biochem. 138, 479-185. Holler, E., Holmquist, B., Vallee, B.L., Taneja, K. and Zamecnik, P. (1983) Biochemistry 22, 4924-4933. Scott, J.F. and Zamecnik, P. (1969-7 Proc. Natl. Aoad. Sci. USA 64, 1308-1314. Zamecnik, P., Rapaport, E. and Baril, E.F. (1982) Proc. Natl. Acad. Sci. USA 79, 1791-1794.
1043