BIOCHEMICAL
Vol. 68, No. 2, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AFFECT OF SPLEEN AND SNAKE VENOM PEOSPHODIESTERASES ON NUCLEOTIDES CONTAINING >WCLEOSIDES IN THE SYN CONFORllATION Kelvin K. Oqilvie Department of Chemistry McGill University Montreal, Quebec H3C 3Gl Frank H. Hruska Department of Chemistry University of Manitoba ?7innipeq, Manitoba Received
October
28,197s
SUMMARY: Dinucleotides containing 6-methyldeoxyuridine and inosine have been prepared and subjected to spleen and snake venom phosSpleen enzyme degrades all nucleotides tested phodiesterases. while snake venom causes very little degradation of nucleotides having 6-methyldeoxyuridine in the 3'-terminal position. The clear implication is that snake venom will not recognize nucleoside units in the syn conformation.
Recently catalyzing
several
authors
polynucleotide
phorylase
synthesis,
and RNA polymerase,
side
di-
This
has caused considerable
mation (4,5)
or triphosphates
of nucleosides
"wobble"
will
having
in the
not
the bases in the
calculations
indicate
the barrier
paper we show that
little uridine(A)
degradation but
nucleo-
have been In addition
state
to rotation
confornucleosides
has been shown to exist
in the crystalline
this
(6,7)
in solution.
conformations that
phos-
the
such as 8-halopurine
-svn and anti
enzymes
syn conformation.
in determining
syn conformation inosine
certain
use as substrates
and 6-methyluridine
nucleoside
that
such as polynucleotide
interest
and several,
and 6-methylcytidine
shown to exist the
have shown (l-3)
in both
(8) although is
snake venom phosphodiesterase
low
(9).
In
causes very
of deoxyadenylyl-(3'-5')-6-methyl-2'-deoxy-
readily
degrades
thymidylyl-(3'-5')-inosine(D)
and
BIOCHEMICAL
Vol. 68, No. 2, 1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
thymidylyl-(3'-5')-isopropylidineinosine(C). esterase
degrades
all
Spleen
of the above nucleotides
phosphodi-
and also
6-methyl-
2'-deoxyuridylyl-(3'-5')-thymidine(B). MATERIALS AND METHODS All priately
nucleotides
described
protected
were synthesized
nucleoside
units
using
method of Letsinger
and Ogilvie
(10).
used to protect
5'- hydroxyl
positions
synthesis protect
the
3'-hydroxyl
was used during removed prior
the
Spleen including Corp., of
synthesis
was obtained
the Department the
Biochemicals
were carried (12,13).
out
using
reactions
groups
were
and electrophoresis.
Corp.,
from several Worthington donated
of McGill
gave the
standard
procedures
were carried
out
sources Biochemicals
by Dr.
University.
same results.
Snake venon phosphodiesterase
Enzymatic
was used to
Protecting
sample kindly
of Biochemistry
above sources
during
enzymes and the pure nucleotides
phosphodiesterase
purified
was
Isopropylidineinosine
of C and D.
with
group
nucleosides
group(II)
chromatography
Nutritional
from all
of all
by paper
and a highly
phosphotriester
of deoxynucleosides.
to treatment
were characterized
the
appro-
The methoxytrityl
and the -t-butyldimethylsilyl the
from the
J.
Spencer
Enzyme
Degradations
at pH 6.5 and 37°C
was obtained using
standard
from Calbiochem. procedures
at
pH 9 and 37°C. RESULTS AND DISCUSSION We have recently possess side it
the
syn-conformation
and when incorporated
was clearly
this rapid
shown (14)
of interest
nucleotide. degradation
caused only
Spleen
by PMR that
in solution, into
both
nucleotides
to determine
after
such as A.
376
free
hours
nucleo-
As a result
of enzymes on
caused complete
However nine
in the
the effect
phosphodiesterase
of the molecule.
14% degradation
6-methyl-2'-deoxyuridine
and
snake venom enzyme at
37°C and 54% after
Vol.
68, No.
2, 1976
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
HO
0 ‘C’
B
c
CH:
‘CH,
I-I
H
D
24
We therefore
hrs.
synthesized
thymidine
(B)
and found
molecule
as did
snake
Because
of
conformation
the
venom
test
conformation
solution. in
and have
-syn
barrier very venom
and anti less A
X-ray
fact
completely
that
snake
the
the
terminal
of
we decided
studies
have
shown
in
to
the
the
the
nucleoside
that
the
nucleotides
the
enzyme
inosine
since shown
C-N glycoside
bond
in
the
C and D show
barrier is
a period in
377
of the
exist (8,151
inosine
is
that
the
inosine that
is snake
nucleotides.
some degradation to
high
can
in
state
have
cause
to
a nucleotide
cr!ystalline
these
Asvn-
of
studies
venom did
and over
to
observation
degrades
that
effect
to use
attached
6-methyldeoxyuridine
an equilibrium
having
idea
the
position
on nucleotides
and
with in
3' -terminal
Theoretical
studies
degraded
when
about
and our
completely
inosine
importance
nucleoside.
consistent
the
of
or -anti-conformations
rapidly The
in
of
to rotation low
implication
phosphodiesterase,
implied
a "wobble"
enzyme
phosphodiesterase.
obvious
Several
either
6-methyl-2'-deoxyuridylyl-(3'-5')
spleen
venom
nucleoside
on snake the
that
0
but time
rotation that
of A is between
it
those
anti-conformation
is
syn
neverthe-
molecules will
of be
Vol. 68, No. 2, 1976
degraded.
BIOCHEMICAL
Thus this
observation
venom phosphodiesterase has shown here has freedom
that
phosphodiesterase
will inosine,
to exist
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of an anti
have many important at
least
appear
for
snake
implications
and
in the dinucleotides
in the anti-conformation
does not
requirement
in
to have a similar
solution.
studied, Spleen
conformational
requirement.
ACKNOWLEDGEMENTS We thank Upjohn
the National
Company (KKO) for
Research financial
Council
of Canada and the
assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15.
Kapuler, A.M., Monny, C., and Michelson, A.M. (1970) Biochem. Biophys. Acta, 217, 18-29. Kapuler, A.M., and Reich, E. (1971) Biochemistry, 10, 4050-4061. Ikehara, M., Tazawa, I., and Fukui, T. (1969) ibid, 8, 736-743. Tavale, S.S., and Sobell, H.M. (1970) J. Mol. m., 48, 109-123. Ikehara, M., Uesugi, S., and Yoshida, K. (1972) Biochemistry, 11, 830-836. Suck, D., and Saenger, W. (1972) Nature, 235, 333-334. Schweizer, M.P., Banta, E.B., Witkowski, J.T., and Robins, R.K. (1973) J. Amer. Chem. Sot., 95, 3770-3778. Subramanian, E., Madden, J.J., and Bugg, C.E. (1973) Biochem. Biophys. Res. Comm., 50, 691-696. Jordan, F. (1973) Biopolymers, 12, 243-255. Letsinger, R.L., and Ogilvie, K.K. (1967) J. Amer. Chem. Sot., 89, 4801-4803. Ogilvz, K.K. (1973) Can. J. Chem., 2, 3799-3807. Ralph, R.K., Connors, W.J., Schaller, H., and Khorana, H.G. (1963) J. Amer. Chem. Sot., 85, 1988-1997. Razzell, W.F., and Khorana, H.G.,71961) J. Biol. Chem., 236, 1144-1149. Manuscript in preparation. Munns, A.R.I., and Tollin, P. (1970) Acta. Cryst., E, 1101-1113.
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