Vol. 79, No. 3, 1977
BlOCHEMlCAL
Studies
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on the Biosynthesis Evidence
for
of Dolichyl
the --In Vitro
2,3-Dehydrodolichyl Dorothy
Received
October
Phosphate:
Formation
of
Phosphate
K. Grange and W. Lee Adair, Department of Biochemistry Medical Center University of South Florida Tampa, Florida 33612
Jr.
17,1977
SUMMARY: A particulate enzyme preparation from hen oviduct is shown to carry out the biosynthesis of a long chain polyprenyl phosphate from isopentenyl pyrophosphate and farnesyl pyrophosphate. The compound has the physical and chemical properties of 2,3-dehydrodolichyl phosphate. The enzyme system is inhibited by EDTA and stimulated by Triton X-100 and dithiothreitol. If the product of the reaction is 2,3-dehydrodolichyl phosphate, it may be derived from 2,3-dehydrodolichyl pyrophosphate, a likely intermediate in the biosynthesis of dolichyl phosphate.
Dolichol tributed
is a long
in both
logical
source,
prene
units,
higher
with
the
of dolichol
synthesis,
functioning
for
In spite the
of its
biosynthesis
mevalonic of pigs
and rabbits. Based on the
ichol
shares at the
on its
bio-
of 16-21
iso-
The phosphorylated in glycoprotein
(3,4).
bio-
analogous
Indirect
glycosylation
to its
evidence
of ovalbumin
could
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
little
et al.
(6)
be incorporated
into
dolichol
results
known structure
of farnesyl
function,
Butterworth,
a common biosynthetic level
(2).
in a manner
in cellular
Similar
(7).
off
importance
--in vivo
polymer
dis-
has
(5).
of dolichol.
acid
in the
is widely
Depending
to participate
phosphate
phosphate
recently
saturated
"carrier"
undecaprenyl
which
(I).
of a linear
unit
as a lipid
of dolichyl
been demonstrated
consists
u-isoprene
alcohol
eucaryotes
has been shown
counterpart
the role
polyisoprenoid
and lower
the molecule
monoester
bacterial
chain
were
later
known of
demonstrated
obtained
of dolichol,
it
pathway
with
cholesterol,
(8).
Dolichol
pyrophosphate
is
that
in the livers with
rat
was proposed
liver
that
dol-
branching biosynthesis
734 ISSN
0006291
X
Vol.79,
BIOCHEMICAL
No. 3, 1977
would
continue
followed
This
a single
bacterial
system
report with
as a model the
and farnesyl the --in vitro
the physical
to that
we decided
and chemical
worked
precursor
properties
in bacteria
(9).
in
to With
pyro-
we wish
polyisoprenyl
of dehydrodolichyl
the
biosynthesis
isopentenyl
communication
chain
units
dolichol
substrates
In this
of a long
out
units,
and dephos-
pyrophosphate
to investigate
pyrophosphate. biosynthesis
unit
of isoprene
undecaprenyl
probable
of isoprene
of the a-isoprene
the cis-addition
to produce
using
cis-addition
is analogous
enzyme catalyzes
pyrophosphate
phosphate
reduction
pathway
geranyl
in hen oviduct,
by sequential
point
by stereospecific
phorylation. which
at this
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to
phosphate phosphate.
MATERIALS AND METHODS: Pig liver dolichol and dolichyl phosphate were from Sigma. Dolichol was isolated from hen oviduct as described previDolichyl phosphate was prepared from hen oviduct dolichol ously (10). by the method of Wedgwood, --et al. (11). [1-3Hl dolichol (14.8 Ci/mmole) was obtained from New England Nuclear. [1-14C] isopentenyl pyrophosphate (54 mCi/mnole) was from Amersham Searle. Al 1 trans farnesyl pyrophosphate was prepared chemically by the method of Popjak (12). Thin layer chromatography was carried out on 20 cm plates (EM Labs) in the following solvents: A, 30% ether-petroleum ether; B, chloroformmethanol-water (60:25:4); C, chloroform-methanol-15 M NHs-water (65:35:4:4). Hen oviduct (Pel Freeze) was homogenized in five volumes of buffer (10 mM Tris-Cl, pH 7.4, 50 mM KCl, 5 mM MgC12, 6 mM 6-mercaptoethanol, 0.25 m sucrose, 0.1% Triton X-100). The suspension was centrifuged at 1000 g for 5 min, washed once with homogenizing buffer, and suspended in 0.1 M Tris-Cl, pH 7.4 (1.5 ml/g original tissue). Incubations were performed at 37" with gentle shaking. Reaction.conditions are given under the figures. Reactions were stopped by the addition of 2.5 volumes of chloroform-methanol (2:1), vortexing, and separating the layers by centrifugation. The upper layer was extracted with an equal volume of chloroform and the organic layers were combined and washed with methanolwater (1:l). The resulting organic phase was passed through a 1x2 column of DEAE (acetate), pre-equilibrated with chloroform-methanol (2:l) and eluted with 8 ml 0.3 M ammonium acetate in chloroform-methanol (2:l). The column eluates were washed once with 2 ml water to remove ammonium acetate prior to subsequent chromatography or liquid scintillation counting. Radioisotope counting was performed with a Packard Tri Carb Model 3390 liquid scintillation counter using a Triton X-100 based scintillation cocktail. Radiochromatograms were scanned with a Packard Model 7200 Radiochromatogram Scanner. RESULTS AND DISCUSSION: tities
of dolichol
to investigate phosphate culate
has been shown to contain
(10) and therefore the biosynthesis
and farnesyl enzymes,
Hen oviduct
of this
pyrophosphate
a substance
seemed an appropriate
was formed
molecule.
were with
735
incubated
large tissue
When isopentenyl with
oviduct
the chromatographic
quanin which pyro-
partiproperties
Vol.79,No.3,
Fig.
1977
BIOCHEMICAL
1. Reaction mixtures contained: Tris-Cl, pH 7.4 (0.1 M); KF (0.05 M), ATP (2.5 mM); dithiothreitol (1 mM); farnesyl pyrophosphate (7 PM); [14C]-isopentenyl pyrophosphate (24 !.IM, 2~10~ cpm); Triton X-100 (2%); and oviduct enzyme (4-6 mg) in a final volume of 0.8 ml. The mixtures were incubated at 37“ with shaking and at the indicated times were terminated by the addition of 2.5 volumes of chloroform:methanol (2:l). The product was isolated as described in Materials and Methods.
of dolichyl
phosphate.
chloroform-methanol is
approximately
ATP were tases
linear
required,
which
a 25-fold tration cation
further
degrade
in Fig.
are
the action
the reaction
substrates.
Although
the strong
inhibition
preparation
other
was unstable
at 4" led
1.
prenyl
The reaction
time. I.
KF and
of endogenous Triton
of 2%; increasing
effect.
for
of 14~ into
shown in Table
to inhibit
as has been shown
storage
shown
at a concentration
was not necessary,
was suspended;
is
incorporation
was without
The particulate
of incorporation
up to 30 min of incubation for
stimulation
course
material
probably
might
required,
after
The time soluble
The requirements
is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the addition
phospha-
X-100
the concenof a divalent
by EDTA suggests transferase
in the Tris
to a 50% decrease
provided
that
reactions
buffer
in which
in enzymic
activity
16 hours. After
extraction
of the
reaction
mixture
736
as described
one
in Methods,
(13). it
Vol.79,
No. 3, 1977
Table
I.
BIOCHEMICAL
Requirements
for
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the biosynthesis
of oviduct
polyprenyl
cpm incorporated Complete system* -KF -ATP -Triton X-100 -dithiothreitol -farnesyl-PP +EDTA oiled
the
product
C.
A single
was observed
0.51)
standard
phate
320
1.2
2).
(Rf = 0.12), results
When subjected
to gel
filtration
limit
of 6000, [1-3H]
molecular
weight
clearly
acetate
are
in this material
resin
with
product [prepared
given
in solvent dolichyl
faster
of dolichyl with
B.
of OR PVA-6000
by the method
of lower
Therefore,
the molecular
(MW 387), weight
as dolichyl
(Mw 1400). Although
the
biosynthetic
product
behaved
737
identically
au-
of Wedgwood,
Substances
same range
excluwith
and cholesterol
the
(EM Labora-
weight
(MW 849),
within
and pyrophos-
solvent
a molecular
3.
(Rf =
(Rf = 0.27)
co-chromatographed
in Fig.
system. falls
obtained
The incubation in Materials and
gel
migrated
as that
of columns
such as ubiquinone-50
resolved
the biosynthesized
1.
authentic
monophosphate
were
phosphate
The results
with
as well
the biosynthetic
dolichyl
(ll)].
et -- al.
polyvinyl
of silica
The compound
of farnesyl
Similar
a porous
layers
coinciding
(Rf = 0.33).
thentic
were
(Fig.
markers
pyrophosphate
tories), sion
2:.A 3:6 31.6 5.5 7.7
on thin
peak of radioactivity
phosphate,
farnesyl
100
of the complete system is given in Fig. and the product was isolated as described
was chromatographed
than
% control
27,000 300 7,250 960 8,520 1,480 2,100
enzyme control
* The composition time was 2 hours Methods.
phosphate
to dolichyl
of
phosphate
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-origin
-solvent
Fig.
2. Radioscan of biosynthesized material chromatographed layers of silica gel G in solvent C. Authentic dolichyl migrated as shown at the top.
phosphate its
with
chemical
terial
respect
to chromatographic
properties
were
quite
in chloroform:methanol:0.8
lowed
by thin
verted the
.
front
front
In the
less
Rf values
polar
dolichol
authentic
hen oviduct
results
for
(14,15),
a-isoprene
unit
both
on pig
liver
of the compound group.
Previous
hydrolysis both
in the
of which cis
hydrolysis for
at 80",
compound
phosphate,
properties, of the ma-
1 hour
of the
fol-
to be con-
migrating
of radioactivity
migrating
phosphate
the acid
phosphate
A, two peaks
dolichyl
lability
weight
with
C.
Under
The acid
all
dolichyl
(Rf = 0.32).
observations
phosphate
than
and 0.66,
previous
an allylic
showed
polar
solvent
of 0.52
Acid
M HCl (10:10:3)
in solvent
authentic
with
distinct.
chromatography
to a form much less
solvent
with
layer
and molecular
on thin phosphate
substantially
identical
than
was not degraded,
in accordance
of undecaprenyl
(11).
phosphate suggests
investigators
the presence
have obtained pyrophosphate
phosphates The degradation
738
faster
of hydrolysis
strongly
configuration.
observed,
conditions
dolichyl
are allylic
were
of
similar
and neryl
containing products
an of
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
. ‘0
.
x ’
% u ,I
.*
0 5 8
.L
Fig.
material was chromatographed on a 1x30 column 3. The biosynthesized of OR PVA-6000 equilibrated with chloroform methanol (2:l) containing 0.2 M annnonium acetate. 0.25 ml fractions were collected.fl--I, 14C-labeled biosynthetic product; Ophosphate; 0, 3H-dolichyl 3H-cholesn-n , ubiquinone (measured at 420 nm); l 0, terol.
these
two compounds
and hydrocarbons than
the
which
parent
for is
dolichyl
phosphate
molecule
could
pyrophosphate,
prene
unit
Addition
the
that
mobility
biosynthesized
arise
material,
phosphate,
by pyrophosphatase logical
Attempts
product on our
of the biosynthesized
or subsequent
products.
layer
alcohols
chromatography
One possible
prene
reductase
contains
ester
and that
a potent
compatible
of an unsaturated cleavage
part
to effect
material
to incubation explanation
distinguished
addition the
viologen
far
the
pyrophosphatase
degrades
739
of the
to the
is
ester
unit.
This
substrate rather
a-iso-
been unsuccessful.
to no decrease
a pyrophosphate
from
to farnesyl
reduction
has led that
the above
of 2,3-dehydrodolichyl
have thus
methyl
with
cr-isoprene
of cis-isoprene
of NADPH, NADH, or reduced.
mixture
of tertiary
on thin
of 2,3-dehydrodolichyl
by the presence
the
pyrophosphate.
have a faster
as a mixture
alcohols.
A structure observations,
have been identified
incubation in acid for
than
the molecule
labile
the a-iso-
a monophosphate to the mono-
BIOCHEMICAL
Vol. 79, No. 3, 1977
phosphate this
before
reduction
hypothesis While
are
these
is accomplished.
investigations of dolichyl
preparation
that
was utilized
authentic
dolichyl
phosphate
product.
The marked in this
Further
investigations
on
in progress.
the biosynthesis
sented
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were phosphate
probably
as judged
and Lezica (16).
lack
their
of acid
observations
the difference
reported
The enzyme
one and apparently
by the
between reflect
Daleo
in pea seedlings
was a soluble
differences
paper
in progress,
synthesized lability
of the
and those
in the source
pre-
of en-
zyme employed. ACKNOWLEDGEMENTS: Society
Grant
This
investigation
was supported
by American
Cancer
BC-234.
REFERENCES:
1. 2. i: 5. 6. i: 9.
Hemming, F. W. (1974) in MTP International Reviews in Science, Biochemistry, Series-i. Biochemistry of Lipids (Goodwin, T. W., ed.), Vol 4, pp. 39-98, Butterworth, London. Dunphy, P. J., Kerr, J. D., Pennock, J. F., Whittle, K. J., and Feeney, J. (1967) Biochim. Biophys. Acta 136, 136-147. Waechter, C. J. and Lennarz, W. J. (1976) Ann. Rev. Biochem. 45, 95-112. Wright, A., Dankert, M., Fennessey, P., and Robbins, P. W. (1967) Proc. Nat. Acad. Sci. 57, 1798-1803. Struck, D. K. and Lennarz, W. J. (1977) J. Biol. Chem. 252, 1007-1013. Butter-worth, P. H. W., Draper, H. H., Hemning, F. W., and Morton, R. A. (1966) Arch. Biochem. Biophys. 113, 646-653. Martin, H. G. and Thorne, K. J. 1.1974) Biochem. J. 138, 277-280. Gough, D. P. and Hemning, F. W. (1970) Biochem. J. ll& 163-166. Allen, C. M., Keenan, M. V., and Sack, J. (1976) Arch. Biochem. Biophys.
175, 236-248.
13.
Keller, R. K. and Adair, W. L. (1977) Biochim. Biophys. Acta, in press. Wedgwood, J. F., Strominger, J. L., and Warren, C. D. (1976) J. Biol. Chem. 249, 6316-6324. Popjak, G., in Methods in Enzymology (R. B. Clayton, ed.), Vol s, pp. 393-45x Academic Press, New York. J. W. (1966) J. Biol. Chem. Dorsey, J. K., Dorsey, J. A. , and Porter,
14.
Keenan,
::: 12.
24J, 5353-5360.
M. V. and Allen,
C. M. (1974) Arch.
Biochem.
Biophys.
161,
375-383. E:
Valenzuela, P. and Cori, Daleo, G. R. and Lezica,
0. (1967) Tetrahedron Letts. 132, 3089-3094. R. P. (1977) FEBS Letts. 74, 247-250.
740
BlOCHEMlCAL
Studies
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on the Biosynthesis Evidence
for
of Dolichyl
the --In Vitro
2,3-Dehydrodolichyl Dorothy
Received
October
Phosphate:
Formation
of
Phosphate
K. Grange and W. Lee Adair, Department of Biochemistry Medical Center University of South Florida Tampa, Florida 33612
Jr.
17,1977
SUMMARY: A particulate enzyme preparation from hen oviduct is shown to carry out the biosynthesis of a long chain polyprenyl phosphate from isopentenyl pyrophosphate and farnesyl pyrophosphate. The compound has the physical and chemical properties of 2,3-dehydrodolichyl phosphate. The enzyme system is inhibited by EDTA and stimulated by Triton X-100 and dithiothreitol. If the product of the reaction is 2,3-dehydrodolichyl phosphate, it may be derived from 2,3-dehydrodolichyl pyrophosphate, a likely intermediate in the biosynthesis of dolichyl phosphate.
Dolichol tributed
is a long
in both
logical
source,
prene
units,
higher
with
the
of dolichol
synthesis,
functioning
for
In spite the
of its
biosynthesis
mevalonic of pigs
and rabbits. Based on the
ichol
shares at the
on its
bio-
of 16-21
iso-
The phosphorylated in glycoprotein
(3,4).
bio-
analogous
Indirect
glycosylation
to its
evidence
of ovalbumin
could
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
little
et al.
(6)
be incorporated
into
dolichol
results
known structure
of farnesyl
function,
Butterworth,
a common biosynthetic level
(2).
in a manner
in cellular
Similar
(7).
off
importance
--in vivo
polymer
dis-
has
(5).
of dolichol.
acid
in the
is widely
Depending
to participate
phosphate
phosphate
recently
saturated
"carrier"
undecaprenyl
which
(I).
of a linear
unit
as a lipid
of dolichyl
been demonstrated
consists
u-isoprene
alcohol
eucaryotes
has been shown
counterpart
the role
polyisoprenoid
and lower
the molecule
monoester
bacterial
chain
were
later
known of
demonstrated
obtained
of dolichol,
it
pathway
with
cholesterol,
(8).
Dolichol
pyrophosphate
is
that
in the livers with
rat
was proposed
liver
that
dol-
branching biosynthesis
734 ISSN
0006291
X
Vol.79,
BIOCHEMICAL
No. 3, 1977
would
continue
followed
This
a single
bacterial
system
report with
as a model the
and farnesyl the --in vitro
the physical
to that
we decided
and chemical
worked
precursor
properties
in bacteria
(9).
in
to With
pyro-
we wish
polyisoprenyl
of dehydrodolichyl
the
biosynthesis
isopentenyl
communication
chain
units
dolichol
substrates
In this
of a long
out
units,
and dephos-
pyrophosphate
to investigate
pyrophosphate. biosynthesis
unit
of isoprene
undecaprenyl
probable
of isoprene
of the a-isoprene
the cis-addition
to produce
using
cis-addition
is analogous
enzyme catalyzes
pyrophosphate
phosphate
reduction
pathway
geranyl
in hen oviduct,
by sequential
point
by stereospecific
phorylation. which
at this
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to
phosphate phosphate.
MATERIALS AND METHODS: Pig liver dolichol and dolichyl phosphate were from Sigma. Dolichol was isolated from hen oviduct as described previDolichyl phosphate was prepared from hen oviduct dolichol ously (10). by the method of Wedgwood, --et al. (11). [1-3Hl dolichol (14.8 Ci/mmole) was obtained from New England Nuclear. [1-14C] isopentenyl pyrophosphate (54 mCi/mnole) was from Amersham Searle. Al 1 trans farnesyl pyrophosphate was prepared chemically by the method of Popjak (12). Thin layer chromatography was carried out on 20 cm plates (EM Labs) in the following solvents: A, 30% ether-petroleum ether; B, chloroformmethanol-water (60:25:4); C, chloroform-methanol-15 M NHs-water (65:35:4:4). Hen oviduct (Pel Freeze) was homogenized in five volumes of buffer (10 mM Tris-Cl, pH 7.4, 50 mM KCl, 5 mM MgC12, 6 mM 6-mercaptoethanol, 0.25 m sucrose, 0.1% Triton X-100). The suspension was centrifuged at 1000 g for 5 min, washed once with homogenizing buffer, and suspended in 0.1 M Tris-Cl, pH 7.4 (1.5 ml/g original tissue). Incubations were performed at 37" with gentle shaking. Reaction.conditions are given under the figures. Reactions were stopped by the addition of 2.5 volumes of chloroform-methanol (2:1), vortexing, and separating the layers by centrifugation. The upper layer was extracted with an equal volume of chloroform and the organic layers were combined and washed with methanolwater (1:l). The resulting organic phase was passed through a 1x2 column of DEAE (acetate), pre-equilibrated with chloroform-methanol (2:l) and eluted with 8 ml 0.3 M ammonium acetate in chloroform-methanol (2:l). The column eluates were washed once with 2 ml water to remove ammonium acetate prior to subsequent chromatography or liquid scintillation counting. Radioisotope counting was performed with a Packard Tri Carb Model 3390 liquid scintillation counter using a Triton X-100 based scintillation cocktail. Radiochromatograms were scanned with a Packard Model 7200 Radiochromatogram Scanner. RESULTS AND DISCUSSION: tities
of dolichol
to investigate phosphate culate
has been shown to contain
(10) and therefore the biosynthesis
and farnesyl enzymes,
Hen oviduct
of this
pyrophosphate
a substance
seemed an appropriate
was formed
molecule.
were with
735
incubated
large tissue
When isopentenyl with
oviduct
the chromatographic
quanin which pyro-
partiproperties
Vol.79,No.3,
Fig.
1977
BIOCHEMICAL
1. Reaction mixtures contained: Tris-Cl, pH 7.4 (0.1 M); KF (0.05 M), ATP (2.5 mM); dithiothreitol (1 mM); farnesyl pyrophosphate (7 PM); [14C]-isopentenyl pyrophosphate (24 !.IM, 2~10~ cpm); Triton X-100 (2%); and oviduct enzyme (4-6 mg) in a final volume of 0.8 ml. The mixtures were incubated at 37“ with shaking and at the indicated times were terminated by the addition of 2.5 volumes of chloroform:methanol (2:l). The product was isolated as described in Materials and Methods.
of dolichyl
phosphate.
chloroform-methanol is
approximately
ATP were tases
linear
required,
which
a 25-fold tration cation
further
degrade
in Fig.
are
the action
the reaction
substrates.
Although
the strong
inhibition
preparation
other
was unstable
at 4" led
1.
prenyl
The reaction
time. I.
KF and
of endogenous Triton
of 2%; increasing
effect.
for
of 14~ into
shown in Table
to inhibit
as has been shown
storage
shown
at a concentration
was not necessary,
was suspended;
is
incorporation
was without
The particulate
of incorporation
up to 30 min of incubation for
stimulation
course
material
probably
might
required,
after
The time soluble
The requirements
is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the addition
phospha-
X-100
the concenof a divalent
by EDTA suggests transferase
in the Tris
to a 50% decrease
provided
that
reactions
buffer
in which
in enzymic
activity
16 hours. After
extraction
of the
reaction
mixture
736
as described
one
in Methods,
(13). it
Vol.79,
No. 3, 1977
Table
I.
BIOCHEMICAL
Requirements
for
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the biosynthesis
of oviduct
polyprenyl
cpm incorporated Complete system* -KF -ATP -Triton X-100 -dithiothreitol -farnesyl-PP +EDTA oiled
the
product
C.
A single
was observed
0.51)
standard
phate
320
1.2
2).
(Rf = 0.12), results
When subjected
to gel
filtration
limit
of 6000, [1-3H]
molecular
weight
clearly
acetate
are
in this material
resin
with
product [prepared
given
in solvent dolichyl
faster
of dolichyl with
B.
of OR PVA-6000
by the method
of lower
Therefore,
the molecular
(MW 387), weight
as dolichyl
(Mw 1400). Although
the
biosynthetic
product
behaved
737
identically
au-
of Wedgwood,
Substances
same range
excluwith
and cholesterol
the
(EM Labora-
weight
(MW 849),
within
and pyrophos-
solvent
a molecular
3.
(Rf =
(Rf = 0.27)
co-chromatographed
in Fig.
system. falls
obtained
The incubation in Materials and
gel
migrated
as that
of columns
such as ubiquinone-50
resolved
the biosynthesized
1.
authentic
monophosphate
were
phosphate
The results
with
as well
the biosynthetic
dolichyl
(ll)].
et -- al.
polyvinyl
of silica
The compound
of farnesyl
Similar
a porous
layers
coinciding
(Rf = 0.33).
thentic
were
(Fig.
markers
pyrophosphate
tories), sion
2:.A 3:6 31.6 5.5 7.7
on thin
peak of radioactivity
phosphate,
farnesyl
100
of the complete system is given in Fig. and the product was isolated as described
was chromatographed
than
% control
27,000 300 7,250 960 8,520 1,480 2,100
enzyme control
* The composition time was 2 hours Methods.
phosphate
to dolichyl
of
phosphate
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-origin
-solvent
Fig.
2. Radioscan of biosynthesized material chromatographed layers of silica gel G in solvent C. Authentic dolichyl migrated as shown at the top.
phosphate its
with
chemical
terial
respect
to chromatographic
properties
were
quite
in chloroform:methanol:0.8
lowed
by thin
verted the
.
front
front
In the
less
Rf values
polar
dolichol
authentic
hen oviduct
results
for
(14,15),
a-isoprene
unit
both
on pig
liver
of the compound group.
Previous
hydrolysis both
in the
of which cis
hydrolysis for
at 80",
compound
phosphate,
properties, of the ma-
1 hour
of the
fol-
to be con-
migrating
of radioactivity
migrating
phosphate
the acid
phosphate
A, two peaks
dolichyl
lability
weight
with
C.
Under
The acid
all
dolichyl
(Rf = 0.32).
observations
phosphate
than
and 0.66,
previous
an allylic
showed
polar
solvent
of 0.52
Acid
M HCl (10:10:3)
in solvent
authentic
with
distinct.
chromatography
to a form much less
solvent
with
layer
and molecular
on thin phosphate
substantially
identical
than
was not degraded,
in accordance
of undecaprenyl
(11).
phosphate suggests
investigators
the presence
have obtained pyrophosphate
phosphates The degradation
738
faster
of hydrolysis
strongly
configuration.
observed,
conditions
dolichyl
are allylic
were
of
similar
and neryl
containing products
an of
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
. ‘0
.
x ’
% u ,I
.*
0 5 8
.L
Fig.
material was chromatographed on a 1x30 column 3. The biosynthesized of OR PVA-6000 equilibrated with chloroform methanol (2:l) containing 0.2 M annnonium acetate. 0.25 ml fractions were collected.fl--I, 14C-labeled biosynthetic product; Ophosphate; 0, 3H-dolichyl 3H-cholesn-n , ubiquinone (measured at 420 nm); l 0, terol.
these
two compounds
and hydrocarbons than
the
which
parent
for is
dolichyl
phosphate
molecule
could
pyrophosphate,
prene
unit
Addition
the
that
mobility
biosynthesized
arise
material,
phosphate,
by pyrophosphatase logical
Attempts
product on our
of the biosynthesized
or subsequent
products.
layer
alcohols
chromatography
One possible
prene
reductase
contains
ester
and that
a potent
compatible
of an unsaturated cleavage
part
to effect
material
to incubation explanation
distinguished
addition the
viologen
far
the
pyrophosphatase
degrades
739
of the
to the
is
ester
unit.
This
substrate rather
a-iso-
been unsuccessful.
to no decrease
a pyrophosphate
from
to farnesyl
reduction
has led that
the above
of 2,3-dehydrodolichyl
have thus
methyl
with
cr-isoprene
of cis-isoprene
of NADPH, NADH, or reduced.
mixture
of tertiary
on thin
of 2,3-dehydrodolichyl
by the presence
the
pyrophosphate.
have a faster
as a mixture
alcohols.
A structure observations,
have been identified
incubation in acid for
than
the molecule
labile
the a-iso-
a monophosphate to the mono-
BIOCHEMICAL
Vol. 79, No. 3, 1977
phosphate this
before
reduction
hypothesis While
are
these
is accomplished.
investigations of dolichyl
preparation
that
was utilized
authentic
dolichyl
phosphate
product.
The marked in this
Further
investigations
on
in progress.
the biosynthesis
sented
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were phosphate
probably
as judged
and Lezica (16).
lack
their
of acid
observations
the difference
reported
The enzyme
one and apparently
by the
between reflect
Daleo
in pea seedlings
was a soluble
differences
paper
in progress,
synthesized lability
of the
and those
in the source
pre-
of en-
zyme employed. ACKNOWLEDGEMENTS: Society
Grant
This
investigation
was supported
by American
Cancer
BC-234.
REFERENCES:
1. 2. i: 5. 6. i: 9.
Hemming, F. W. (1974) in MTP International Reviews in Science, Biochemistry, Series-i. Biochemistry of Lipids (Goodwin, T. W., ed.), Vol 4, pp. 39-98, Butterworth, London. Dunphy, P. J., Kerr, J. D., Pennock, J. F., Whittle, K. J., and Feeney, J. (1967) Biochim. Biophys. Acta 136, 136-147. Waechter, C. J. and Lennarz, W. J. (1976) Ann. Rev. Biochem. 45, 95-112. Wright, A., Dankert, M., Fennessey, P., and Robbins, P. W. (1967) Proc. Nat. Acad. Sci. 57, 1798-1803. Struck, D. K. and Lennarz, W. J. (1977) J. Biol. Chem. 252, 1007-1013. Butter-worth, P. H. W., Draper, H. H., Hemning, F. W., and Morton, R. A. (1966) Arch. Biochem. Biophys. 113, 646-653. Martin, H. G. and Thorne, K. J. 1.1974) Biochem. J. 138, 277-280. Gough, D. P. and Hemning, F. W. (1970) Biochem. J. ll& 163-166. Allen, C. M., Keenan, M. V., and Sack, J. (1976) Arch. Biochem. Biophys.
175, 236-248.
13.
Keller, R. K. and Adair, W. L. (1977) Biochim. Biophys. Acta, in press. Wedgwood, J. F., Strominger, J. L., and Warren, C. D. (1976) J. Biol. Chem. 249, 6316-6324. Popjak, G., in Methods in Enzymology (R. B. Clayton, ed.), Vol s, pp. 393-45x Academic Press, New York. J. W. (1966) J. Biol. Chem. Dorsey, J. K., Dorsey, J. A. , and Porter,
14.
Keenan,
::: 12.
24J, 5353-5360.
M. V. and Allen,
C. M. (1974) Arch.
Biochem.
Biophys.
161,
375-383. E:
Valenzuela, P. and Cori, Daleo, G. R. and Lezica,
0. (1967) Tetrahedron Letts. 132, 3089-3094. R. P. (1977) FEBS Letts. 74, 247-250.
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