Vol. 68, No. 3, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SPECIFIC MODIFICATION LACTATING
OF FATTY ACID SYNTHETASE FROM RAT MAMMARY GLAND BY CHYMOTRYPSIN AND TRYPSIN'
Elisabetta
Agradi',
Louis
Libertini
and Stuart
Smith3
Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center, Oakland, California 94609
Received
December
8,1975 SUMMARY
Fatty acid synthetase from lactating rat mammary gland after limited proteolysis with chymotrypsin or trypsin synthesizes longer chain fatty acids than those produced by the native enzyme. Of the seven partial reactions of the multienzyme complex, only the thioesterase activity was decreased. The results suggest that modification of the fatty acid synthetase product specificity by chymotrypsin and trypsin results from a specific action of these proteases on the thioesterase component. Trypsin, but not chymotrypsin, cleaved a catalytically active thioesterase from the complex; it thus appears that limited trypsinization will be a useful tool for the isolation of the thioesterase component of the multienzyme.
In higher from
organisms,
acetyl-
the
and malonyl-CoA
and most
microorganisms
tional
proteins
(1).
isolation
Limited
enzymes we report
regarding
under the
fact
from
and study
proteolysis
information
the
that
numerous
of the
fatty
consist
of large
polypeptide
the
results
of experiments
which
complex,
synthetase
little
properties (2-4
demonstrate
1.
multienzyme has been complex. useful
of a number n this
the potential
in
monofunc-
of the
has yielded
acids
whereas
progress
components
conditions
chains
of fatty
as discrete,
acid
sources,
and functional
which
synthesis
are present
individual
nondenaturing
structure
in the
in a multienzyme
the enzymes
Despite
have been purified
made in the
involved
are arranged
plants
complexes
enzymes
of
communication of
'This investigation was supported in part by grants AM 16073, AM 17489 and General Research Support 5 SO1 RR05467 from the National Institutes of Health and grant BMS 7412723 from the National Science Foundation. 'Present address National Heart Institutes of Health, Bethesda,
and Lung Institute, Maryland 20014.
3Established Investigator of the American pondence should be addressed.
Copyright Alt rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
894
Heart
Building Association,
10, National to whom corres-
Vol.68,No.3,
1976
BIOCHEMICAL
proteolytic
enzymes
multienzyme
complex.
in unraveling
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
structure
of the fatty
acid
synthetase
MATERIALS AND METHODS Fatty acid synthetase was purified from lactating rat mammary gland (5). Assay mixtures contained 0.1 M potassium phosphate buffer (pH 6.6), 1.5 x low4 M NADPH, 5 x lo-' M acetyl-CoA and 5.4 x 10s5 M [2- "+C] malonyl-CoA (50 nCi) in a volume of 0.5 ml and were monitored spectrophotometrically. A unit of activity is defined as the amount of enzyme catalyzing the malonyl-CoA dependent oxidation of 1 nmole NADPH per min at 30". The products were identified by gas-liquid radiochromatography (6). The partial reactions of the fatty acid synthetase (acetyl transferase, malonyl transferase, condensation-CO exchange reaction, S-acetoacetyl-N-acetyl cysteamine reductase, S-B-hydroxybut&yl-N-acetyl cysteamine dehydrase and S-crotonyl-N-acetyl cysteamine reductase) were assayed with model substrates by the procedures described by Kumar --et al (7) with only minor modifications. All assays were performed under optimal cofactor concentrations; reaction rates were a linear function of protein concentration and time. The assay system for the thioesterase reaction contained 0.2 M otassium phosphate buffer (pH 6.6), 50 ug bovine serum albumin and 5 UM [l-'"C 7 palmityl-CoA (10 nCi) in a final volume of 1.0 ml. Reaction mixtures were preincubated at 30" for 2 min, substrate was added and 1 min later the reaction was stopped with 0.1 ml of 21% perchloric acid and 1.0 ml ethanol. The Cl-'"Cl palmitic acid released was extracted with petroleum ether (2 x 3 ml), the solvent evaporated and the radio-
TABLE I THE EFFECT OF CHYMOTRYPSIN AND TRYPSIN ON ACTIVITY AND PRODUCT SPECIFICITY OF FATTY ACID SYNTHETASE % Inhibition fatty acid
Protease None Chymotrypsin,
0.6 ug
of synthetase
Products
(moles '18
X)
c14
'16
c20
0
4.7
90.2
5.1
15
4.4
84.6
11.0
0
61.3
34.2
0.6
0
Chymotrypsin,
2.1
pg
47
3.9
Chymotrypsin,
4.2
ug
61
2.9
30.1
61.2
5.8
Trypsin,
2.1 1-19
60
4.7
70.5
21.7
3.0
Trypsin,
4.2
94
0
46.8
46.1
7.1
pg
Rat mammary gland fatty acid synthetase (100 units, 0.1 mg protein) was incubated at 30" with protease in a final volume of 0.15 ml containing 0.1 M potassium phosphate buffer (pH 7.0), 1 mM dithiothreitol and 1 mM EDTA. Incubations with chymotrypsin were for 10 mins, those with trypsin were for 20 mins. At the end of the incubation 20 ul portions were removed and assayed immediately for fatty acid synthetase activity and the chain length of the fatty acids produced was determined. Calculations of moles % assume one unlabeled acetyl group per fatty acid molecule. 895
Vol. 68, No. 3, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activity determined by liquid scintillation is defined as the amount of enzyme catalyzing CoA per min at 30".
spectrometry. the hydrolysis
A unit of activity of 1 nmole palmityl-
Bovine pancreatic trypsin (210 U/mg) and chymotrypsin A (55 U/mg) were obtained from Worthington. Each enzyme was found to be free of contamination with the other by potentiometric assay (8,9). Soybean trypsin inhibitor was obtained from Calbiochem. RESULTS Preliminary proteases
altered
Whereas
palmitic
of stearic extent
experiments the product acid
and arachidic
of proteolysis.
with
chymotrypsin
specificity
was the main acids
of the
product
synthesized
The shift
and trypsin fatty
acid
of the native increased
in product
specificity
revealed
that
synthetase
enzyme,
both (Table
I).
the proportion
in proportion
to the
was accompanied
by a
TABLE II THE EFFECT OF CHYMOTRYPSIN AND TRYPSIN ON THE PARTIAL ACTIVITIES OF FATTY ACID SYNTHETASE % of Control Enzyme Activity Fatty Acetyl
acid
synthetase
transferase
Malonyl
transferase
Condensing Keto-reductase Dehydrase Enoyl-reductase Thioesterase
enzyme
Chymotrypsinized
Activity Trypsinized
13
60
106
105
100
100
90
94
90
97
98
95
100
111
29
60
For chymotrypsinization, rat mammary gland fatty acid synthetase (5,670 units, 4.65 mg protein) was incubated for 10 min at 30" with 30 pg chymotrypsin in 9.0 ml of 0.1 M potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and 1 mM EDTA. Fatty acid synthetase was precipitated with ammonium sulfate (33% saturation) and redissolved in 1.0 ml of 0.25 M potassium phosphate buffer containing 1 mM dithiothreitol and 1 mM EDTA. Samples were dialyzed for 3 hours against the same buffer prior to assay of enzyme activities. For trypsinization, rat mammary gland fatty acid synthetase (10,100 units, 10.0 mg protein) was incubated for 1 hr at 30" in 4.0 ml of potassium phosphate buffer, 0.1 (pH 7.0) containing 1 mM dithiothreitol 1 mM EDTA and 5 pg trypsin. Digestion was arrested by the addition of 15 ug trypsin inhibitor and the enzyme activities measured directly.
896
M
Vol.68,
BIOCHEMICAL
No. 3, 1976
progressive reaction
decrease of fatty
centration
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in the capacity
acid
of protease
synthesis.
of the complex Similar
was kept
results
constant
to catalyze were
the overall
obtained
and the duration
when the con-
of incubation
increased. To determine
which
or chymotrypsin,
we examined
these
proteases.
which
were
acid
synthetase native
capacity
for effect
are
identically
of these
enzyme.
Proteolysis
acid
synthesis
on the other
partial
reaction
given
except
activity
fatty
of the multienzyme
each partial
The results
treated
original
ficant
components
in Table that
control
reactions
60 TIME
II
as a percentage
preparations
and in the
affected
of the complex
the protease
resulted
40
were
was omitted. was identical
in a decrease thioesterase was observed.
120
by trypsin treated
with
of controls The fatty to the
in the overall activity, It
but no signi-
seems likely,
160 ”
(MIN)
Fig. 1. Time course for inactivation of fatty acid synthetase and release of thioesterase by trypsin. Rat mammary gland fatty acid synthetase (0.95 mg, 1180 units of fatty acid synthetase, 560 units of thioesterase) was incubated at 30" in 0.1 W potassium phosphate buffer pH 7.0 containing 1 mM dithiothreitol, 1 mM EOTA and 0.7 ug trypsin. Final volume was 1.0 ml. Reactions were terminated by the addition of 1.4 ug trypsin inhibitor. A portion of each sample was kept for determination of fatty acid synthetase activity, the remainder was adjusted to 33% saturation with respect to ammonium sulfate, left at 0" for 30 min and then the precipitated, protein was removed by centrifugation. Portions of the 33% saturated ammonium sulfate supernatant fraction were taken for determination of thioesterase activity; no fatty acid synthetase activity was present in this fraction..
897
Vol.68,No.3,1976
therefore,
that
the
decrease
for
the
change
responsible is
BIOCHEMICAL
in agreement
probability
with
for
likelihood
acid
synthetase
tase
activity
remained
precipitated experiments
showed
trypsinization)
complex.
Similar
The time
decrease
was accompanied
scale activities
for
acid of the
80 min,
fraction. in this
The amount type
of experiment
and has no effect
in Fig.
1, the amount
supernatant experiments of over
was too
is small,
of protein
remaining
small
to be measured isolated
per mg protein.
898
less
in Fig.
1.
the complex. activity
ammonium sulfate to the 0.02
reliably.
thioesterase con-
In the experiment ammonium
In subsequent
thioesterase represents
super-
M final
in the 33% saturated
This
The
thioesterase
than
of
on trypsinization
from
activity.
the
fraction.
and release
transferred
usually
thioesterase
we have consistently 1000 units
33% saturated
on the
supernatant
component
of ammonium sulfate
the thioester-
observed
38% of the
to stop
significant
shown
activity
approximately
Control
the multienzyme
synthetase is
thioesterase
in the
from
be
(used
that
sulfate
acid
reaction synthetase
could
not yield
ammonium
of fatty
enzyme was recovered
centration,
sulfate
in fatty
did
with
of thioesterase
inhibitor
originated
synthe-
keto-reductase,
activity
confirming
fatty
along
amount
trypsin
chymotrypsin
trypsinization
the
acid
to 80% saturation.
nor
33% supernatant with
fatty
thioesterase
activity,
inactivation
by a release
trypsinization
of the native
shown
for
in a typical
progressive
assay
in the
a decreased
trypsinized
enzyme,
a considerable
in the 33% saturated
course
thioesterase
thioesterase
result
increase
ammonium sulfate condensing
trypsin
This
in which
residual
concentration
the
experiments
activity
salt
neither
recovered
thioesterase
natant
the
(10)
to precipitate of the
This
supernatant.
complex.
was
acids.
transferase, However,
possessed
ase activity
All
by proteolysis
the enzyme would
fatty
sulfate.
malonyl
that
off
by 33% saturated
by increasing
induced of the
we had occasion
ammonium
in the
group
chain
and enoyl-reductase.
activity
After
of longer
transferase,
specificity
acyl
experiments with
activity
scheme of Sumper --et al
was precipitated
the acetyl dehydrase
in product
of the
of synthesis
In subsequent
in thioesterase
the model
transfer
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with
large
specific
a significant
BIOCHEMICAL
Vol. 68, No. 3, 1976
increase
over
the
multienzyme
specific
complex,
activity
usually
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the
about
thioesterase
500 units
associated
with
the
per mg protein.
DISCUSSION Under
conditions
of limited
selectively
attack
multienzyme
complex.
arginyl
bonds
the thioesterase
(8)
Trypsin,
actually active
with
a discrete,
chymotrypsin,
the thioesterase, that
trypsinization this
is
is also
the reason
thioesterase Lornitzo acid
complex
contains
inhibitor
(12).
activity
activity
is
homogeneous
with
the complex
from
as judged 32,000.
This
represents
study
partial
activities
detailed
study
activity. which
of the fatty of the properties
treated Presumably
inactivation from
the multienzyme
proteolysis
and
the complex
is
cleavage
of
the complex.
complex
and it
more than
subunits
is
about
of the
The theory
by
likely
that
40% of the
species,
less
all
50% of the does
thioesterase
675 as one component
which
appears
The molecular
attempt
synthetase
multienzyme
899
is further
thioesterase
the
successful
separately.
than
The released
thioesterase
fatty
the multienzyme
be identical,
ultracentrifugation.
of the
that
liver
one or more thioesterases
since
since
pigeon
a thioesterase
say whether
of Sephadex
acid
from
in rapid
fluoride,
form.
molecular
the first
lysyl
not obtained.
may or may not
by trypsinization
by analytical
is approximately
from
both
we cannot
a column
for
form.
in the dissociated
of a single
eluted
results
phenylmethylsulfonyl point,
is
to recover
that
thioesterase
recovered
seem to consist
have found
At this
from
is
(11)
two thioesterases,
by studies
released
to further
contain
supported
are
susceptible
in the dissociated
--et al
synthetase
released
synthetase
synthetase
or after
thioesterase
we have been unable
activity
acid
thioesterase (9)
acid
component
When the fatty active
and trypsin
specificity
the thioesterase
on the multienzyme,
the
chymotrypsin
of the fatty
has a narrow
of chymotrypsin
either
We have found
which
form.
specificity
both
component
cleaves
in a catalytically
the broader
proteolysis,
to isolate complex.
component
will
weight
one of the A be reported
Vol.68,No.3,
1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGEMENTS We are substrates and trypsin
grateful
to Dr.
used in this
study
K. N. Dileepan and to Dr.
for
synthesizing
G. Green
for
assaying
some of the model the
chymotrypsin
preparations. REFERENCES
:: :: 2: 7. 8. 9. 10. 11. 12.
Brindley, D. N., Matsumura, S., and Bloch, K. (1969) Nature, 224, 666-669. Setlow, P., Brutlag, D., and Kornberg, A. (1972) J. Biol. Chem, 247, 224-232. Piszkiewicz, D., and Goitein, R. K. (1974) Biochemistry, 13, 2505-2511. Skar, D. C., Rohrbach, M. S., and Bodley, J. W. (1975) Biochemistry, 14, 3922-3926. Smith, S., and Abraham, S. (1970) J. Biol. Chem, 245, 3209-3217. Abraham, S., Matthes, K. J., and Chaikoff, I. L. (1961) Biochim. Biophys. Acta, 49, 268-285. Kumar, S., Dorsey, J. A., Muesing, R. A., and Porter, J. W. (1970) J. Biol. Chem, 245, 4732-4744. Walsh, K. A., and Wilcox, P. E. (1970) in Methods in Enzymology (Perlmann, G. E. and Lorand, L., eds.), Vol. 19, pp. 31-41, Academic Press, New York. Wilcox, P. E. (1970) in Methods in Enzymology (Perlmann, G. E. and Lorand, L., eds.), Vol. 19, pp. 64-108, Academic Press, New York. Sumper, M., Oesterhelt, D., Riepertinger, C., and Lynen, F. (1969) Eur. J. Biochem, 10, 377-387. Lornitzo, F. A., Qureshi, A. A., and Porter, J. W. (1975) J. Biol. Chem, 250, 4520-4529. Kumar, S. (1973) Biochim. Biophys. Res. Cosun, 53, 334-341.
900
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SPECIFIC MODIFICATION LACTATING
OF FATTY ACID SYNTHETASE FROM RAT MAMMARY GLAND BY CHYMOTRYPSIN AND TRYPSIN'
Elisabetta
Agradi',
Louis
Libertini
and Stuart
Smith3
Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center, Oakland, California 94609
Received
December
8,1975 SUMMARY
Fatty acid synthetase from lactating rat mammary gland after limited proteolysis with chymotrypsin or trypsin synthesizes longer chain fatty acids than those produced by the native enzyme. Of the seven partial reactions of the multienzyme complex, only the thioesterase activity was decreased. The results suggest that modification of the fatty acid synthetase product specificity by chymotrypsin and trypsin results from a specific action of these proteases on the thioesterase component. Trypsin, but not chymotrypsin, cleaved a catalytically active thioesterase from the complex; it thus appears that limited trypsinization will be a useful tool for the isolation of the thioesterase component of the multienzyme.
In higher from
organisms,
acetyl-
the
and malonyl-CoA
and most
microorganisms
tional
proteins
(1).
isolation
Limited
enzymes we report
regarding
under the
fact
from
and study
proteolysis
information
the
that
numerous
of the
fatty
consist
of large
polypeptide
the
results
of experiments
which
complex,
synthetase
little
properties (2-4
demonstrate
1.
multienzyme has been complex. useful
of a number n this
the potential
in
monofunc-
of the
has yielded
acids
whereas
progress
components
conditions
chains
of fatty
as discrete,
acid
sources,
and functional
which
synthesis
are present
individual
nondenaturing
structure
in the
in a multienzyme
the enzymes
Despite
have been purified
made in the
involved
are arranged
plants
complexes
enzymes
of
communication of
'This investigation was supported in part by grants AM 16073, AM 17489 and General Research Support 5 SO1 RR05467 from the National Institutes of Health and grant BMS 7412723 from the National Science Foundation. 'Present address National Heart Institutes of Health, Bethesda,
and Lung Institute, Maryland 20014.
3Established Investigator of the American pondence should be addressed.
Copyright Alt rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
894
Heart
Building Association,
10, National to whom corres-
Vol.68,No.3,
1976
BIOCHEMICAL
proteolytic
enzymes
multienzyme
complex.
in unraveling
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
structure
of the fatty
acid
synthetase
MATERIALS AND METHODS Fatty acid synthetase was purified from lactating rat mammary gland (5). Assay mixtures contained 0.1 M potassium phosphate buffer (pH 6.6), 1.5 x low4 M NADPH, 5 x lo-' M acetyl-CoA and 5.4 x 10s5 M [2- "+C] malonyl-CoA (50 nCi) in a volume of 0.5 ml and were monitored spectrophotometrically. A unit of activity is defined as the amount of enzyme catalyzing the malonyl-CoA dependent oxidation of 1 nmole NADPH per min at 30". The products were identified by gas-liquid radiochromatography (6). The partial reactions of the fatty acid synthetase (acetyl transferase, malonyl transferase, condensation-CO exchange reaction, S-acetoacetyl-N-acetyl cysteamine reductase, S-B-hydroxybut&yl-N-acetyl cysteamine dehydrase and S-crotonyl-N-acetyl cysteamine reductase) were assayed with model substrates by the procedures described by Kumar --et al (7) with only minor modifications. All assays were performed under optimal cofactor concentrations; reaction rates were a linear function of protein concentration and time. The assay system for the thioesterase reaction contained 0.2 M otassium phosphate buffer (pH 6.6), 50 ug bovine serum albumin and 5 UM [l-'"C 7 palmityl-CoA (10 nCi) in a final volume of 1.0 ml. Reaction mixtures were preincubated at 30" for 2 min, substrate was added and 1 min later the reaction was stopped with 0.1 ml of 21% perchloric acid and 1.0 ml ethanol. The Cl-'"Cl palmitic acid released was extracted with petroleum ether (2 x 3 ml), the solvent evaporated and the radio-
TABLE I THE EFFECT OF CHYMOTRYPSIN AND TRYPSIN ON ACTIVITY AND PRODUCT SPECIFICITY OF FATTY ACID SYNTHETASE % Inhibition fatty acid
Protease None Chymotrypsin,
0.6 ug
of synthetase
Products
(moles '18
X)
c14
'16
c20
0
4.7
90.2
5.1
15
4.4
84.6
11.0
0
61.3
34.2
0.6
0
Chymotrypsin,
2.1
pg
47
3.9
Chymotrypsin,
4.2
ug
61
2.9
30.1
61.2
5.8
Trypsin,
2.1 1-19
60
4.7
70.5
21.7
3.0
Trypsin,
4.2
94
0
46.8
46.1
7.1
pg
Rat mammary gland fatty acid synthetase (100 units, 0.1 mg protein) was incubated at 30" with protease in a final volume of 0.15 ml containing 0.1 M potassium phosphate buffer (pH 7.0), 1 mM dithiothreitol and 1 mM EDTA. Incubations with chymotrypsin were for 10 mins, those with trypsin were for 20 mins. At the end of the incubation 20 ul portions were removed and assayed immediately for fatty acid synthetase activity and the chain length of the fatty acids produced was determined. Calculations of moles % assume one unlabeled acetyl group per fatty acid molecule. 895
Vol. 68, No. 3, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activity determined by liquid scintillation is defined as the amount of enzyme catalyzing CoA per min at 30".
spectrometry. the hydrolysis
A unit of activity of 1 nmole palmityl-
Bovine pancreatic trypsin (210 U/mg) and chymotrypsin A (55 U/mg) were obtained from Worthington. Each enzyme was found to be free of contamination with the other by potentiometric assay (8,9). Soybean trypsin inhibitor was obtained from Calbiochem. RESULTS Preliminary proteases
altered
Whereas
palmitic
of stearic extent
experiments the product acid
and arachidic
of proteolysis.
with
chymotrypsin
specificity
was the main acids
of the
product
synthesized
The shift
and trypsin fatty
acid
of the native increased
in product
specificity
revealed
that
synthetase
enzyme,
both (Table
I).
the proportion
in proportion
to the
was accompanied
by a
TABLE II THE EFFECT OF CHYMOTRYPSIN AND TRYPSIN ON THE PARTIAL ACTIVITIES OF FATTY ACID SYNTHETASE % of Control Enzyme Activity Fatty Acetyl
acid
synthetase
transferase
Malonyl
transferase
Condensing Keto-reductase Dehydrase Enoyl-reductase Thioesterase
enzyme
Chymotrypsinized
Activity Trypsinized
13
60
106
105
100
100
90
94
90
97
98
95
100
111
29
60
For chymotrypsinization, rat mammary gland fatty acid synthetase (5,670 units, 4.65 mg protein) was incubated for 10 min at 30" with 30 pg chymotrypsin in 9.0 ml of 0.1 M potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and 1 mM EDTA. Fatty acid synthetase was precipitated with ammonium sulfate (33% saturation) and redissolved in 1.0 ml of 0.25 M potassium phosphate buffer containing 1 mM dithiothreitol and 1 mM EDTA. Samples were dialyzed for 3 hours against the same buffer prior to assay of enzyme activities. For trypsinization, rat mammary gland fatty acid synthetase (10,100 units, 10.0 mg protein) was incubated for 1 hr at 30" in 4.0 ml of potassium phosphate buffer, 0.1 (pH 7.0) containing 1 mM dithiothreitol 1 mM EDTA and 5 pg trypsin. Digestion was arrested by the addition of 15 ug trypsin inhibitor and the enzyme activities measured directly.
896
M
Vol.68,
BIOCHEMICAL
No. 3, 1976
progressive reaction
decrease of fatty
centration
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in the capacity
acid
of protease
synthesis.
of the complex Similar
was kept
results
constant
to catalyze were
the overall
obtained
and the duration
when the con-
of incubation
increased. To determine
which
or chymotrypsin,
we examined
these
proteases.
which
were
acid
synthetase native
capacity
for effect
are
identically
of these
enzyme.
Proteolysis
acid
synthesis
on the other
partial
reaction
given
except
activity
fatty
of the multienzyme
each partial
The results
treated
original
ficant
components
in Table that
control
reactions
60 TIME
II
as a percentage
preparations
and in the
affected
of the complex
the protease
resulted
40
were
was omitted. was identical
in a decrease thioesterase was observed.
120
by trypsin treated
with
of controls The fatty to the
in the overall activity, It
but no signi-
seems likely,
160 ”
(MIN)
Fig. 1. Time course for inactivation of fatty acid synthetase and release of thioesterase by trypsin. Rat mammary gland fatty acid synthetase (0.95 mg, 1180 units of fatty acid synthetase, 560 units of thioesterase) was incubated at 30" in 0.1 W potassium phosphate buffer pH 7.0 containing 1 mM dithiothreitol, 1 mM EOTA and 0.7 ug trypsin. Final volume was 1.0 ml. Reactions were terminated by the addition of 1.4 ug trypsin inhibitor. A portion of each sample was kept for determination of fatty acid synthetase activity, the remainder was adjusted to 33% saturation with respect to ammonium sulfate, left at 0" for 30 min and then the precipitated, protein was removed by centrifugation. Portions of the 33% saturated ammonium sulfate supernatant fraction were taken for determination of thioesterase activity; no fatty acid synthetase activity was present in this fraction..
897
Vol.68,No.3,1976
therefore,
that
the
decrease
for
the
change
responsible is
BIOCHEMICAL
in agreement
probability
with
for
likelihood
acid
synthetase
tase
activity
remained
precipitated experiments
showed
trypsinization)
complex.
Similar
The time
decrease
was accompanied
scale activities
for
acid of the
80 min,
fraction. in this
The amount type
of experiment
and has no effect
in Fig.
1, the amount
supernatant experiments of over
was too
is small,
of protein
remaining
small
to be measured isolated
per mg protein.
898
less
in Fig.
1.
the complex. activity
ammonium sulfate to the 0.02
reliably.
thioesterase con-
In the experiment ammonium
In subsequent
thioesterase represents
super-
M final
in the 33% saturated
This
The
thioesterase
than
of
on trypsinization
from
activity.
the
fraction.
and release
transferred
usually
thioesterase
we have consistently 1000 units
33% saturated
on the
supernatant
component
of ammonium sulfate
the thioester-
observed
38% of the
to stop
significant
shown
activity
approximately
Control
the multienzyme
synthetase is
thioesterase
in the
from
be
(used
that
sulfate
acid
reaction synthetase
could
not yield
ammonium
of fatty
enzyme was recovered
centration,
sulfate
in fatty
did
with
of thioesterase
inhibitor
originated
synthe-
keto-reductase,
activity
confirming
fatty
along
amount
trypsin
chymotrypsin
trypsinization
the
acid
to 80% saturation.
nor
33% supernatant with
fatty
thioesterase
activity,
inactivation
by a release
trypsinization
of the native
shown
for
in a typical
progressive
assay
in the
a decreased
trypsinized
enzyme,
a considerable
in the 33% saturated
course
thioesterase
thioesterase
result
increase
ammonium sulfate condensing
trypsin
This
in which
residual
concentration
the
experiments
activity
salt
neither
recovered
thioesterase
natant
the
(10)
to precipitate of the
This
supernatant.
complex.
was
acids.
transferase, However,
possessed
ase activity
All
by proteolysis
the enzyme would
fatty
sulfate.
malonyl
that
off
by 33% saturated
by increasing
induced of the
we had occasion
ammonium
in the
group
chain
and enoyl-reductase.
activity
After
of longer
transferase,
specificity
acyl
experiments with
activity
scheme of Sumper --et al
was precipitated
the acetyl dehydrase
in product
of the
of synthesis
In subsequent
in thioesterase
the model
transfer
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with
large
specific
a significant
BIOCHEMICAL
Vol. 68, No. 3, 1976
increase
over
the
multienzyme
specific
complex,
activity
usually
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the
about
thioesterase
500 units
associated
with
the
per mg protein.
DISCUSSION Under
conditions
of limited
selectively
attack
multienzyme
complex.
arginyl
bonds
the thioesterase
(8)
Trypsin,
actually active
with
a discrete,
chymotrypsin,
the thioesterase, that
trypsinization this
is
is also
the reason
thioesterase Lornitzo acid
complex
contains
inhibitor
(12).
activity
activity
is
homogeneous
with
the complex
from
as judged 32,000.
This
represents
study
partial
activities
detailed
study
activity. which
of the fatty of the properties
treated Presumably
inactivation from
the multienzyme
proteolysis
and
the complex
is
cleavage
of
the complex.
complex
and it
more than
subunits
is
about
of the
The theory
by
likely
that
40% of the
species,
less
all
50% of the does
thioesterase
675 as one component
which
appears
The molecular
attempt
synthetase
multienzyme
899
is further
thioesterase
the
successful
separately.
than
The released
thioesterase
fatty
the multienzyme
be identical,
ultracentrifugation.
of the
that
liver
one or more thioesterases
since
since
pigeon
a thioesterase
say whether
of Sephadex
acid
from
in rapid
fluoride,
form.
molecular
the first
lysyl
not obtained.
may or may not
by trypsinization
by analytical
is approximately
from
both
we cannot
a column
for
form.
in the dissociated
of a single
eluted
results
phenylmethylsulfonyl point,
is
to recover
that
thioesterase
recovered
seem to consist
have found
At this
from
is
(11)
two thioesterases,
by studies
released
to further
contain
supported
are
susceptible
in the dissociated
--et al
synthetase
released
synthetase
synthetase
or after
thioesterase
we have been unable
activity
acid
thioesterase (9)
acid
component
When the fatty active
and trypsin
specificity
the thioesterase
on the multienzyme,
the
chymotrypsin
of the fatty
has a narrow
of chymotrypsin
either
We have found
which
form.
specificity
both
component
cleaves
in a catalytically
the broader
proteolysis,
to isolate complex.
component
will
weight
one of the A be reported
Vol.68,No.3,
1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGEMENTS We are substrates and trypsin
grateful
to Dr.
used in this
study
K. N. Dileepan and to Dr.
for
synthesizing
G. Green
for
assaying
some of the model the
chymotrypsin
preparations. REFERENCES
:: :: 2: 7. 8. 9. 10. 11. 12.
Brindley, D. N., Matsumura, S., and Bloch, K. (1969) Nature, 224, 666-669. Setlow, P., Brutlag, D., and Kornberg, A. (1972) J. Biol. Chem, 247, 224-232. Piszkiewicz, D., and Goitein, R. K. (1974) Biochemistry, 13, 2505-2511. Skar, D. C., Rohrbach, M. S., and Bodley, J. W. (1975) Biochemistry, 14, 3922-3926. Smith, S., and Abraham, S. (1970) J. Biol. Chem, 245, 3209-3217. Abraham, S., Matthes, K. J., and Chaikoff, I. L. (1961) Biochim. Biophys. Acta, 49, 268-285. Kumar, S., Dorsey, J. A., Muesing, R. A., and Porter, J. W. (1970) J. Biol. Chem, 245, 4732-4744. Walsh, K. A., and Wilcox, P. E. (1970) in Methods in Enzymology (Perlmann, G. E. and Lorand, L., eds.), Vol. 19, pp. 31-41, Academic Press, New York. Wilcox, P. E. (1970) in Methods in Enzymology (Perlmann, G. E. and Lorand, L., eds.), Vol. 19, pp. 64-108, Academic Press, New York. Sumper, M., Oesterhelt, D., Riepertinger, C., and Lynen, F. (1969) Eur. J. Biochem, 10, 377-387. Lornitzo, F. A., Qureshi, A. A., and Porter, J. W. (1975) J. Biol. Chem, 250, 4520-4529. Kumar, S. (1973) Biochim. Biophys. Res. Cosun, 53, 334-341.
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