- Email: [email protected]
Mevalonate phosphorylation in the neonatal chick liver
Vol. 72, No. 1, 1976
BIOCHEMICAL
MEVALONATE
AND BIOPHYSICAL
PHOSPHORYLATION NEONATAL
J.
Garcia-Martinez,
J. L.
Department
of
Received SUMMARY. extracts studied.
2,
9.5.
attention
hatching,
Ovarian
of shows
of
into
feeding
of
mevalonic
the
of
other
*Abbreviations: MVAPP, glutaryl-CoA.
known
Gallus
by
soybean
acid
(MVA)* the
in
and
E.
Granada,
Garcia-Peregrfn Granada,
Spain
acid by cell-free development was profiles for phospho-
phosphorylation difference
after
increase hatching.
in
the
chick
that
it
differs
in
the
has
chick (2).
biosynthesis
slices
Few
studied to
from
the
from
hog
little
that
of 14’
l[1 and
in from
liver
rabbit
1-14C 1:
3
the
conducted
avian
MVA
kinase (3,4),
acetate
by
been
cholesterol
I
mammals.
estradiol-17@
enhanced
have
mevalonate
C
of
was
cholesterol
of
received
incorporation
liver
of where
The
the 1-6
amount
incorporate
(1).
observed. at pH maximum
during between
testosterone
incorporation
purified
of
formation has been is quite similar shows a clear
domesticus
meal
mammals,
partially
is (EC
on
livers. well 2.4.
liver
1.36) (5)
sources. The
liver
significant at day 7
gonadotrophin
raw
Nevertheless,
and
well
cholesterol
established
a
androstenedione,
presence
acetate
rat
is
progesterone,
been
of
mevalonate no significant
metabolism it
slices
has
University
but formed
though
the
Suarez
pyrophosphomevalonate phosphomevalonate formed the pyrophosphomevalonate
Cholesterol
in
M.D.
1976
The pattern development
days after phosphomevalonate
into
Segovia,
THE
LIVER
- The phosphorylation of mevalonic from chick liver in the early steps A clear difference in the pH-activity
mevalonate and The amount of 7.5-9.5 whereas at pH neonatal
IN
CHICK
Biochemistry,
July
RESEARCH COMMUNICATIONS
has
mechanism been
of
elucidated.
MVA, pyrophosphomevalonic
mevalonic
regulation There acid; acid;
of is
cholesterol
general
MVAP, HMG-CoA,
biosynthesis agreement
phosphomevalonic ;!-hydroxy-p-methyl-
in
that acid;
Vol.
72, No.
1, 1976
t-MC-CoA
reductase
cholesterol role
in
the
shown
that
MVA as
regulation
of
geranyl-PP
plants,
of
French-beans
isoenzymes
of
in
the
chloroplasts
of
7.5
and
kinase
5.5
being
from
active
at As
regulation MVA
of
the
Pinus
pH an
We
7.9
(6)
have
inhibitors
both
terpenyl
of
pyrophosphates
seems
within
the to
seedlings
(9)
the
one
outside
them,
and
Agave
elucidate
the
role
specific
compounds
(7).
of
two
one with
two
pinaster
controlled
presence
demonstrated,
isolated
be
and
certain
been
have
to
cell
have
other
respectively.
activity
that
in
important
Porter
powerful
membranes
kinase
an
and
biosynthesis
pumpkin
mevalonate
enzyme play
Dorsey
segregation
and
and
may
COMMUNICATIONS
MVA.
intracellular
(8)
kinase
are
terpenoid
enzyme
the
rate-limiting
farnesyl-PP
after
RESEARCH
the
suggesting
the of
is
process.
controls
a combination
In
this
liver,
physiological
impermeability
of
pig
BIOPHYSICAL
MVA
and
from
AND
1. 1. 1. 34) However
In by
(EC
synthesis.
kinase
act
BIOCHEMICAL
an
located optimum
fractions
pH
with
americana,
both
MVA fractions
(10).
intent
to
cholesterol
biosynthesis,
phosphorylation
in
the
we
chick
liver
of have in
MVA
kinase
in
the
studied
some
aspects
early
steps
of
the
development. MATERIALS New-born
White
AND
Leghorn
male
METHODS chicks
were
obtai_nv4from
commercial hatchery and maintained on a commercial diet. was supplied as the lactone by the Radiochemical Centre, England. The potassium salt was prepared by treating the 360
for
30
min with Cell-free
in a Potter-Elvehjem pH 7.5, so that was centrifuged supernatant was Unless
an excess extracts
of were
KOH. obtained
by
homogenizing
a
12C]MVA Amershan, lactone at the
liver
homogenizer with 0.05 M Tris-maleate buffer, the final liver/buffer ratio is l/10. The homogenate at 15000 2 for 30 min at 49. Protein content in the determined by the Lowry method (11). otherwise specified, the reaction systyq contained
6 rmol es of MgCl2, 12 umoles of ATP, 75 nmoles 150 pmol-es of Trts-maleate buffer and 0. 2-0.5 mg final volume of 1.5 ml. The reaction mixture was for 30 min. Reactions were stopped by heating the 5 min. Aliquots (25 ,ul) of supernatants were applied paper strips and developed by the ascending technique formic acid-water (77:13:10). Radioactive spots on were detected and measured in a Nuclear-Chicago
203
of ]2C] MVA, of protein in a incubated at 372 tubes to 908 for to
Whatman No 1 in butanolthe dried strips Actigraph III system.
Vol. 72, No. 1, 1976
MVA liver
has
MVAP
whereas
MVAPP
prolonged
to 50
adding
the to
90
It
is
10
-3
of
of
cysteine
has
been
the
Fig.
l.-
1).
The
maximum
we
has
over
increases in of
chick
O-180
min,
O-30
the
min,
reactions
amount
of
are
MVAPP
found
studied
the mixture,
the
MVAP
the
range
the
protector
of
showing
O-O.5 to
case
phosphorylation
formation
linearly
any
MVA
MVA -SH
is mg,
1.5
that
mg
in
proportional whereas
protein
the (Fig.
phosphorylation groups.
1). is
Addition
of
effect.
incorporation studied
as
reaction
Methods
that
no
for
between
far
have
the
added
presence
time
as
from
out
linearly
linear
in in
note
extract
carried
is
protein
formed to
on
reaction
hand,
protein
a cell-free
formed.
other
MVAPP
important
M
MVAP
mg
DISCUSSION
occurs
described
The liver
the
amount
dependent
in
(Fig.
that
O-2.5
amount
by
formation
min
%
conditions the
AND
formation
On by
RESULTS
investigated
that
about
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
phosphorylation
been
showing
is
BIOCHEMICAL
of over
the
MVA
by
range
cell-free pH
6.5-9.5
extracts in
0.05
from M
chick Tris-
(min)
Influence of incubation on MVA phosphorylation The results chick I iver. experiments ? S. E.M. in the text. 0, MVAP;
204
time and protein concentration by cell-free extracts from are given as means of six Experimental details are given 0, MVAPP.
not
BIOCHEMICAL
Vol. 72, No. 1, 1976
Fig.
2.-
Effect
maleate
buffer.
profiles
for
is
similar
quite
maximum
has is
at
the
optimum
surroundings
The development found amount Wrking shown
between of
l-6
a clear
in days
formed with
These for
the
pH MVAP
of
phosphorylation
Fig.
3.
after has
the
and
No
to
the
at
cholesterol
205
from
of
control
which
yeast
its
this
which observed
intracellular
enzyme
the
significant 7 after
activity.
neonatal have
increase
at
those
kinase,
differences
day
almost
difference
pH
Gomez-Capilla total
is
from
during
A
formation
MVAP
The
significant
found
domesticus in
(13).
system
hatching. been
liver
pH-activity
value
enzyme
5.3-10.0
a
pH
means are
a clear
differ
pig
similar
kinase
MVA
this
as
MVAP
shows
results
the
suggest
Gallus
decrease
(12)
at
the
The
MVAPP
MVAPP
unlike
of
the
in
2).
cell-free
given details
observed
(Fig.
7. 5.
range
to
shown
MVAP also
7. 3,
pattern
is
pH
Popjak
pH
seems
of
by
results are Experimental 0, MVAPP.
been
whereas
amount
the
phosphorylation
formation
at
of in
MVA
has
7.5-9.5
and pH
the
MVAPP
The
Hellig
on
chick liver. The six experiments. text. l , MVAP;
obtained
active
between
pH
difference
pH
9.5.
optimum
equally
clear and
that by
an
A
pH
than
reported
the from of the
MVAP
at
4-fold
of
extracts ? S.E.M. given in
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
been in
the
hatching. et
al.
day
5
(14) and
have 10
after
Vol.
72, No.
BIOCHEMICAL
1,1976
1
2
3
AND
4
BIOPHYSICAL
5
6
7
RESEARCH
8
9
COMMUNlCATlONS
lo
days
Fig.
3.-
MVA liver
phosphorylation during the
by
cell-free extracts from chick development. The results are with the number of observations ? S. E.M., Experimental details are given in the text. neonatal
given as means in parentheses. 0,
eclosion,
MVAP;
a decrease
phosphorylation the
Only
Barnes
fluctuation of
in
development
MVA
Sarcophaga
that
kinase
On
the
have
be
parallel
other
from
to
hand,
kinase determined
activity
and of
livers
decarboxylation
in
MVA
little
is
animal
some
larva,
the
known
sources.
developmental
pharate
adult
and
of is
one
Shah
(16)
mevalonate
suckling
have by
and
rate-limiting
studied 105000
weaned
the 9
rats,
step
in
the
used
in
all
showing hepatic
synthesis.
experiments,
the fraction
l-MG-CoA system
to
mevalonate
(15)
from
Although
soluble
of
decarboxylation
fraction
cholesterol
seem
period.
Ramachandran and
MVAPP
not
bullata.
Recently,
supernatant
this
Goodfellow
the
phosphorylation
does
pattern
and in
MVAPP.
which
ability
about
adult
0,
ceil-free MVA (Table
reductase of
the
cholesterol
extracts
activating 1).
The
(unpublished
have
enzymes
are
microsomal
suggests in
206
essentially tocation
results)
metabolism
been
this
source.
the
located of
another
the
chick regulation
in liver
the
Vol.
72, No.
BIOCHEMICAL
1, 1976
AND
Table Intracellular
distribution
of
BIOPHYSICAL
RESEARCH
1
MVA-activating
enzymes
Specific
activity
from
(dpm
/
homogenate
15000
2
31.99
t 1.21
6.24
_+ 0.48
pellet
105 000
2
supernatant
75.22
t 2.03
105000
9
pellet
13.79
? 0.61
Results
are
given
as
means
chick
mg
liver.
protein)
MVAPP
MVAP Crude
COMMUNICATIONS
? S. E.M.
of
3.32
Z 0.30
9.21’_
three
0.66
experiments.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Boucek, R. J. and 15, 6-11. Sklan, D., Ascarelli,
Savard,
K. I.
(1970)
and
53, 1239-1241. Levy, H. R. and Popjak, G. Beytia, E., Dorsey, J.K., Porter, J. W. (1970) J. Biol. Markely, K. and Smallamn, 47, 327-335. Dorsey, J. K. and Porter, 4667-4670.
Gen.
Bubowski, (1960) Marr, Chem. E.
J. W.
Comp. P.
Endocrinol.
(1974)
Poultry
Biochem. J. 75, 417-428. J., Cleland, W. W. and 245, 5450-5458.
(1961)
Biochim.
(1968)
J.
Biophys. Biol.
Chem.
Rogers, L. J., Shah, S. P. J. and Goodwin, T. W. (1968) Photosynthetica 2, 184-207. Rogers, L. J., Shah, S. P. J. and Goodwin, T. W. (1966) Biochem. J. 100, 14C-17c. Loomis, W.D. and Battaille, J. (1963) Biochim. Biophys. 67, 54-63. Garcia-Peregrfn, E., Suarez, M.D. and Mayor, F. (1973) FEBS Lett. 30, 15-17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, (1951) J. Biol. Chem. 193, 265-275. Hellig,
H.
and
Popjak,
J.
(1961)
207
Sci.
J.
Lipid
Res.
2,
235-243.
Acta 243,
Acta
R. J.
BIOCHEMICAL
Vol. 72, No. 1,1976
13. 14. 15. 16.
Bloch, K., Chaykin, (1959) J. Biol. Chem. Gomez-Capilla, J.A., Osorio, C. (1975) Barnes, F. J. and 17, 1415-1427. Ramachandran, Res. Comm.
C. 69,
AND BIOPHYSICAL
S.,
Phillips, A.H. 234, 2595-2604. Macarulla, J.M.,
Rev. Esp. Goodfellow, K. and 42-47.
Fisiol. R.D.
Shah,
208
S.N.
RESEARCH COMMUNICATIONS
and
de
Waard,
Martin-And&s,
31, (1971) (1976)
177-182. J. Insect Biochem.
A. A. Physiol. Biophys.
and
BIOCHEMICAL
MEVALONATE
AND BIOPHYSICAL
PHOSPHORYLATION NEONATAL
J.
Garcia-Martinez,
J. L.
Department
of
Received SUMMARY. extracts studied.
2,
9.5.
attention
hatching,
Ovarian
of shows
of
into
feeding
of
mevalonic
the
of
other
*Abbreviations: MVAPP, glutaryl-CoA.
known
Gallus
by
soybean
acid
(MVA)* the
in
and
E.
Granada,
Garcia-Peregrfn Granada,
Spain
acid by cell-free development was profiles for phospho-
phosphorylation difference
after
increase hatching.
in
the
chick
that
it
differs
in
the
has
chick (2).
biosynthesis
slices
Few
studied to
from
the
from
hog
little
that
of 14’
l[1 and
in from
liver
rabbit
1-14C 1:
3
the
conducted
avian
MVA
kinase (3,4),
acetate
by
been
cholesterol
I
mammals.
estradiol-17@
enhanced
have
mevalonate
C
of
was
cholesterol
of
received
incorporation
liver
of where
The
the 1-6
amount
incorporate
(1).
observed. at pH maximum
during between
testosterone
incorporation
purified
of
formation has been is quite similar shows a clear
domesticus
meal
mammals,
partially
is (EC
on
livers. well 2.4.
liver
1.36) (5)
sources. The
liver
significant at day 7
gonadotrophin
raw
Nevertheless,
and
well
cholesterol
established
a
androstenedione,
presence
acetate
rat
is
progesterone,
been
of
mevalonate no significant
metabolism it
slices
has
University
but formed
though
the
Suarez
pyrophosphomevalonate phosphomevalonate formed the pyrophosphomevalonate
Cholesterol
in
M.D.
1976
The pattern development
days after phosphomevalonate
into
Segovia,
THE
LIVER
- The phosphorylation of mevalonic from chick liver in the early steps A clear difference in the pH-activity
mevalonate and The amount of 7.5-9.5 whereas at pH neonatal
IN
CHICK
Biochemistry,
July
RESEARCH COMMUNICATIONS
has
mechanism been
of
elucidated.
MVA, pyrophosphomevalonic
mevalonic
regulation There acid; acid;
of is
cholesterol
general
MVAP, HMG-CoA,
biosynthesis agreement
phosphomevalonic ;!-hydroxy-p-methyl-
in
that acid;
Vol.
72, No.
1, 1976
t-MC-CoA
reductase
cholesterol role
in
the
shown
that
MVA as
regulation
of
geranyl-PP
plants,
of
French-beans
isoenzymes
of
in
the
chloroplasts
of
7.5
and
kinase
5.5
being
from
active
at As
regulation MVA
of
the
Pinus
pH an
We
7.9
(6)
have
inhibitors
both
terpenyl
of
pyrophosphates
seems
within
the to
seedlings
(9)
the
one
outside
them,
and
Agave
elucidate
the
role
specific
compounds
(7).
of
two
one with
two
pinaster
controlled
presence
demonstrated,
isolated
be
and
certain
been
have
to
cell
have
other
respectively.
activity
that
in
important
Porter
powerful
membranes
kinase
an
and
biosynthesis
pumpkin
mevalonate
enzyme play
Dorsey
segregation
and
and
may
COMMUNICATIONS
MVA.
intracellular
(8)
kinase
are
terpenoid
enzyme
the
rate-limiting
farnesyl-PP
after
RESEARCH
the
suggesting
the of
is
process.
controls
a combination
In
this
liver,
physiological
impermeability
of
pig
BIOPHYSICAL
MVA
and
from
AND
1. 1. 1. 34) However
In by
(EC
synthesis.
kinase
act
BIOCHEMICAL
an
located optimum
fractions
pH
with
americana,
both
MVA fractions
(10).
intent
to
cholesterol
biosynthesis,
phosphorylation
in
the
we
chick
liver
of have in
MVA
kinase
in
the
studied
some
aspects
early
steps
of
the
development. MATERIALS New-born
White
AND
Leghorn
male
METHODS chicks
were
obtai_nv4from
commercial hatchery and maintained on a commercial diet. was supplied as the lactone by the Radiochemical Centre, England. The potassium salt was prepared by treating the 360
for
30
min with Cell-free
in a Potter-Elvehjem pH 7.5, so that was centrifuged supernatant was Unless
an excess extracts
of were
KOH. obtained
by
homogenizing
a
12C]MVA Amershan, lactone at the
liver
homogenizer with 0.05 M Tris-maleate buffer, the final liver/buffer ratio is l/10. The homogenate at 15000 2 for 30 min at 49. Protein content in the determined by the Lowry method (11). otherwise specified, the reaction systyq contained
6 rmol es of MgCl2, 12 umoles of ATP, 75 nmoles 150 pmol-es of Trts-maleate buffer and 0. 2-0.5 mg final volume of 1.5 ml. The reaction mixture was for 30 min. Reactions were stopped by heating the 5 min. Aliquots (25 ,ul) of supernatants were applied paper strips and developed by the ascending technique formic acid-water (77:13:10). Radioactive spots on were detected and measured in a Nuclear-Chicago
203
of ]2C] MVA, of protein in a incubated at 372 tubes to 908 for to
Whatman No 1 in butanolthe dried strips Actigraph III system.
Vol. 72, No. 1, 1976
MVA liver
has
MVAP
whereas
MVAPP
prolonged
to 50
adding
the to
90
It
is
10
-3
of
of
cysteine
has
been
the
Fig.
l.-
1).
The
maximum
we
has
over
increases in of
chick
O-180
min,
O-30
the
min,
reactions
amount
of
are
MVAPP
found
studied
the mixture,
the
MVAP
the
range
the
protector
of
showing
O-O.5 to
case
phosphorylation
formation
linearly
any
MVA
MVA -SH
is mg,
1.5
that
mg
in
proportional whereas
protein
the (Fig.
phosphorylation groups.
1). is
Addition
of
effect.
incorporation studied
as
reaction
Methods
that
no
for
between
far
have
the
added
presence
time
as
from
out
linearly
linear
in in
note
extract
carried
is
protein
formed to
on
reaction
hand,
protein
a cell-free
formed.
other
MVAPP
important
M
MVAP
mg
DISCUSSION
occurs
described
The liver
the
amount
dependent
in
(Fig.
that
O-2.5
amount
by
formation
min
%
conditions the
AND
formation
On by
RESULTS
investigated
that
about
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
phosphorylation
been
showing
is
BIOCHEMICAL
of over
the
MVA
by
range
cell-free pH
6.5-9.5
extracts in
0.05
from M
chick Tris-
(min)
Influence of incubation on MVA phosphorylation The results chick I iver. experiments ? S. E.M. in the text. 0, MVAP;
204
time and protein concentration by cell-free extracts from are given as means of six Experimental details are given 0, MVAPP.
not
BIOCHEMICAL
Vol. 72, No. 1, 1976
Fig.
2.-
Effect
maleate
buffer.
profiles
for
is
similar
quite
maximum
has is
at
the
optimum
surroundings
The development found amount Wrking shown
between of
l-6
a clear
in days
formed with
These for
the
pH MVAP
of
phosphorylation
Fig.
3.
after has
the
and
No
to
the
at
cholesterol
205
from
of
control
which
yeast
its
this
which observed
intracellular
enzyme
the
significant 7 after
activity.
neonatal have
increase
at
those
kinase,
differences
day
almost
difference
pH
Gomez-Capilla total
is
from
during
A
formation
MVAP
The
significant
found
domesticus in
(13).
system
hatching. been
liver
pH-activity
value
enzyme
5.3-10.0
a
pH
means are
a clear
differ
pig
similar
kinase
MVA
this
as
MVAP
shows
results
the
suggest
Gallus
decrease
(12)
at
the
The
MVAPP
MVAPP
unlike
of
the
in
2).
cell-free
given details
observed
(Fig.
7. 5.
range
to
shown
MVAP also
7. 3,
pattern
is
pH
Popjak
pH
seems
of
by
results are Experimental 0, MVAPP.
been
whereas
amount
the
phosphorylation
formation
at
of in
MVA
has
7.5-9.5
and pH
the
MVAPP
The
Hellig
on
chick liver. The six experiments. text. l , MVAP;
obtained
active
between
pH
difference
pH
9.5.
optimum
equally
clear and
that by
an
A
pH
than
reported
the from of the
MVAP
at
4-fold
of
extracts ? S.E.M. given in
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
been in
the
hatching. et
al.
day
5
(14) and
have 10
after
Vol.
72, No.
BIOCHEMICAL
1,1976
1
2
3
AND
4
BIOPHYSICAL
5
6
7
RESEARCH
8
9
COMMUNlCATlONS
lo
days
Fig.
3.-
MVA liver
phosphorylation during the
by
cell-free extracts from chick development. The results are with the number of observations ? S. E.M., Experimental details are given in the text. neonatal
given as means in parentheses. 0,
eclosion,
MVAP;
a decrease
phosphorylation the
Only
Barnes
fluctuation of
in
development
MVA
Sarcophaga
that
kinase
On
the
have
be
parallel
other
from
to
hand,
kinase determined
activity
and of
livers
decarboxylation
in
MVA
little
is
animal
some
larva,
the
known
sources.
developmental
pharate
adult
and
of is
one
Shah
(16)
mevalonate
suckling
have by
and
rate-limiting
studied 105000
weaned
the 9
rats,
step
in
the
used
in
all
showing hepatic
synthesis.
experiments,
the fraction
l-MG-CoA system
to
mevalonate
(15)
from
Although
soluble
of
decarboxylation
fraction
cholesterol
seem
period.
Ramachandran and
MVAPP
not
bullata.
Recently,
supernatant
this
Goodfellow
the
phosphorylation
does
pattern
and in
MVAPP.
which
ability
about
adult
0,
ceil-free MVA (Table
reductase of
the
cholesterol
extracts
activating 1).
The
(unpublished
have
enzymes
are
microsomal
suggests in
206
essentially tocation
results)
metabolism
been
this
source.
the
located of
another
the
chick regulation
in liver
the
Vol.
72, No.
BIOCHEMICAL
1, 1976
AND
Table Intracellular
distribution
of
BIOPHYSICAL
RESEARCH
1
MVA-activating
enzymes
Specific
activity
from
(dpm
/
homogenate
15000
2
31.99
t 1.21
6.24
_+ 0.48
pellet
105 000
2
supernatant
75.22
t 2.03
105000
9
pellet
13.79
? 0.61
Results
are
given
as
means
chick
mg
liver.
protein)
MVAPP
MVAP Crude
COMMUNICATIONS
? S. E.M.
of
3.32
Z 0.30
9.21’_
three
0.66
experiments.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Boucek, R. J. and 15, 6-11. Sklan, D., Ascarelli,
Savard,
K. I.
(1970)
and
53, 1239-1241. Levy, H. R. and Popjak, G. Beytia, E., Dorsey, J.K., Porter, J. W. (1970) J. Biol. Markely, K. and Smallamn, 47, 327-335. Dorsey, J. K. and Porter, 4667-4670.
Gen.
Bubowski, (1960) Marr, Chem. E.
J. W.
Comp. P.
Endocrinol.
(1974)
Poultry
Biochem. J. 75, 417-428. J., Cleland, W. W. and 245, 5450-5458.
(1961)
Biochim.
(1968)
J.
Biophys. Biol.
Chem.
Rogers, L. J., Shah, S. P. J. and Goodwin, T. W. (1968) Photosynthetica 2, 184-207. Rogers, L. J., Shah, S. P. J. and Goodwin, T. W. (1966) Biochem. J. 100, 14C-17c. Loomis, W.D. and Battaille, J. (1963) Biochim. Biophys. 67, 54-63. Garcia-Peregrfn, E., Suarez, M.D. and Mayor, F. (1973) FEBS Lett. 30, 15-17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, (1951) J. Biol. Chem. 193, 265-275. Hellig,
H.
and
Popjak,
J.
(1961)
207
Sci.
J.
Lipid
Res.
2,
235-243.
Acta 243,
Acta
R. J.
BIOCHEMICAL
Vol. 72, No. 1,1976
13. 14. 15. 16.
Bloch, K., Chaykin, (1959) J. Biol. Chem. Gomez-Capilla, J.A., Osorio, C. (1975) Barnes, F. J. and 17, 1415-1427. Ramachandran, Res. Comm.
C. 69,
AND BIOPHYSICAL
S.,
Phillips, A.H. 234, 2595-2604. Macarulla, J.M.,
Rev. Esp. Goodfellow, K. and 42-47.
Fisiol. R.D.
Shah,
208
S.N.
RESEARCH COMMUNICATIONS
and
de
Waard,
Martin-And&s,
31, (1971) (1976)
177-182. J. Insect Biochem.
A. A. Physiol. Biophys.
and