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Purification and crystallization of acetyl CoA carboxylase
LIFE SCIENCES Vol, 6, pp . 785-790, 1967 . Printed in Great Britain.
Pergamon Presa Ltd.
PURIFICATION AND CRYSTALLIZATION OF ACETYL CoA CARHO%YLASE Tamotsu Goto, Erika Ringelmann, Barbara Riedel and Shosaku Numa Max-Planck-Institut für Zellchemie, München, Germany (Received 18 January 1967) Acetyl CoA carboxylase (E .C . 6 .4 .1 .2 .), the biotin enzyme which catalyzes the formation of malonyl CoA, was discovered by Wakil (1) and partially purified from various sources (2-8 ), There is evidence indicating that this enzyme plays a critical role in the regulation of fatty acid synthesis (9),
Recently,
some molecular properties of the highly purified chicken liver enzyme have been reported by Gregolin . et al . (10) as well as from this laboratory (11) . the enzyme .
We have now been able to crystallize
The present communication concerns the procedure
for purification and crystallization of acetyl CoA carboxylase . Table I represents a summary of typical purification data . All operations were carried out at 0 - 4°C, unless other~rise stated .
Freshly-frozen chicken livers (3 .3 kg) were thawed and
homogenized with 4,950 ml of 0 .1 M phosphate buffer* in a blaring blendor for 90 sec . x g for 40 min .
The homogenate was centrifuged at 16,300
The supernatant fluid (4,900 ml) was further
centrifuged at 73,000 x g for 55 min,
The crude extract thus
obtained (3,945 ml) was diluted 2-fold with 0 .1 M phosphate buffer . added,
To the diluted extract, 1,295 g of (NH4) 2504 were The resulting precipitate was collected by centrifugation
*All phosphate buffers employed were potassium phosphate buffer, pH 7 .5, containing 2 mM 2-mercaptoethanol and 5 mM potassium ethylenediaminetetraacetate (EDTA), unless otherwise specified . 785
786
ACETYL CoA CARBOXYLASE
Vol . 6, Vo . 8
TABLE I Purification of Acetyl CoA Carboxylase Fraction
Volume (ml)
Protein (mg)
Crude Extract
3.945
141,000
282
0 .002
1st (NH4)2S04
380
12,300
434
0 .035
Ca 3 (P04 ) 2 Ge.l
2,020
2,830
242
0 .086
2nd (NH4)2504
51
1,260
195
0 .155
DEAF-cellulose
214
107
106
0 .995
Gradient Centrifugation
58
14,5
Total Activity (units*)
51 .3
Specific Activity**
3 "54
*One unit of enzyme is defined as that amount which catalyzes the carboxylation of 1 4tmole of ace~yl CoA per minute under the standard assay conditions at 25 C described previously (12) . The activity in the crude extract was assayed by the isotopic method (Assay A, see (7)) with some modifications . **Specific activity is defined as units per mg of protein . Protein was determined by the method of Lowry et al . (13) with bovine serum albumin as standard . at 16,300 x g for 35 min and dissolved in 0 .02 M phosphate buffer .
The insoluble material was removed by centrifugation
at 37,000 x g for 15 min .
The first (NH4)2504 fraction (380 ml),
which had been stored at -15°C, was diluted with distilled water to give a protein concentration of 11 mg/ml .
The diluted enzyme
solution was added to 900 ml of Ca 3 (P04 ) 2 gel (16 .5 mg/ml) (protein : gel = 1 : 1 .2) .
After stirring for 5 min, the gel
was collected by centrifugation at 3,000 x g for 5 min and eluted 3 times, each time with 675 ml of 0 .033 M phosphate buffer, and then 3 times, each time with 675 ml of 0 .2 M phosphate buffer .
The latter eluates (0 .2 M) were combined
Vol . 6, No . 8 (2,020 ml) .
ACETYL CoA CARBOXYLASE
787
To this solution, after addition of 106 ml of
0 .1 M MgK2EDTA, 285 g of (NH4 )2S0 4 were added .
The resulting
precipitate was collected by centrifugation at 16,300 x g for 35 min and dissolved in 0 .02 M phosphate buffer .
The enzyme
solution was freed from the insoluble material by centrifugation at 37,000 x g for 15 min.
The second (NH4 )250 4 fraction
(51 ml), which had been stored at -15° C, was dialyzed against 2 liters of 0 .01 M phosphate buffer for ~ hours with a change of buffer .
The dialyzed solution (60 ml) was diluted 3-fold
with 0 .01 M phosphate buffer, and the insoluble material was removed by centrifugation at 37,000 x g for 25 min .
The super-
natant solution was applied to a DEAE-cellulose column (6 x 25 cm) previously equilibrated with 0 .01 M phosphate buffer . Elution was carried out with a linear concentration gradient established between 1 liter of 0 .02 M phosphate buffer and 1 liter of 0 .75 M phosphate buffer . single peak .
Enzyme emerged as a
The fractions possessing a specific activity
above 0 .6 (between approximately 760 and 974 ml effluent volume) were combined .
To the pooled eluate (214 ml) were
added 92 ml of saturated (0 °C)(NH4 )2S0 4 solution .
The re-
sulting precipitate was collected by centrifugation at 16,300 x g for 35 min, dissolved in approximately 6 ml of 0 .05 M phosphate buffer and dialyzed against 400 ml of the same buffer for 3 hours with a change of buffer .
The insoluble material was
removed by centrifugation at 37,000 x g for 10 min .
The dia-
lyzed solution (6 .4 ml) was layered on 6 linear density gradients made from 15 ml of 5 ~ (w/v) sucrose and 15 ml of 20 (w/v) sucrose .
The sucrose gradients contained, in addition,
0 .05 M phosphate buffer .
The gradient tubes were centrifuged
788
ACETYL CoA CARBO%YLASE
Vol .
6, No . 8
at 24,000 rpm for 11 hours in the SW-25 .1 swinging bucket rotor in the Spinco Model L ultracentrifuge .
Twenty-two fractions
xere collected from each gradient tube .
Protein sedimented in
two peaks, and the fractions corresponding to the smaller and faster-sedimenting peak xere combined (58 ml) .
The specific
activity of 10 enzyme preparations made by the procedure described above ranged from 3 to 5 . Crystallization xas achieved as folloxs : The enzyme of the last purification step (14 mg) xas precipitated by addition of an equal volume of saturated (0 °C)(NH4 )2504 solution .
The pre
cipitate xaa collected by centriflxgation at 16,300 x g for 35 min and suspended in 10 ml of 30
;6
saturated (NH4)230 4 solu-
tion, pH 7 .5, containing 0 .01 M phosphate buffer, 5 mM 2mereaptoethanol and 1 mM EDTA .
After occasional gentle stir-
ring for 10 min, the suspension xaa centrifuged at 37,000 x g for 5 min .
The precipitate xas treated in a similar manner suc-
cessively xith the folloxing solutions : 10 ml of 20 (NH4 )2304 solution, 4 ml of 10
y6
;6
saturated
saturated (NH4)250 4 solution
and 1 ml of 10 % saturated (NH4)2304 solution ; all solutions contained the same ingredients as the 30 solution .
;6
saturated (NH4 ) 2304
The txo extracts xith the 10 ~ saturated (NH4)250 4
solution, xhich contained nearly all protein (protein concentration, approximately 3 and 1 mg/ml, respectively), xere warmed up to room temperature (22 - 25 °C) during a period of about 6 hours and alloxed to stand at this temperature . days, crystallization began in both extracts . a photomicrograph of enzyme crystals .
After several Fig. 1 represents
Crystals were obtained
also by a similar extraction procedure with 10 % saturated (NH4 )250 4 solution, pH 8 .0, containing 0 .01 M Na 4P207 -i~Cl
Vol . 6, No . 8
ACETYL CoA CARBOJCYLASE
789
FIG. 1 Phase contrast photomicrograph of crystalline acetyl CoA carboxylase . Magnification, 1,000 x. buffer, 0 .01 M potassium citrate, 1 mM EDTA .
5 mM 2-mercaptoethanol and
The specific activity of the crystalline enzyme xas
approximately the same as that of the enzyme of the last purification step . The crystalline enzyme contained 0.70 ~g of (+)-biotin per mg of protein dry weight (for biotin determination, see (14)) . The sedimentation behavior of the enzyme has recently been re ported (11) ; in these experiments, amorphous preparations whose specific activity and biotin content were comparable to those of the crystalline enzyme were employed . Açknowled~nents We are indebted to Professor F. Lynen for his encouragement during the course of this work .
A research grant from the
790
ACETYL CoA CARBOXYLASE
Vol . 6,
No .
Deutsche Forschungsgemeinschaft is gratefully acknowledged . One of the authors (T .G .) thanks the Alexander von HumboldtStiftung for a stipend . References 1 . S .J . Wakil, J . Am . Chem . Soc . 80, 6465 (1958) . 2 . S .J . Wakil, J .W . Porter and D .M . Gibson, Biochem, et Biophys . Acta 24, 453 (1957) " 3 . M .D . Hatch and P .K . Stumpf, J . Biol . Chem . ~, 2879 (1961) . 4 . M. Waite and S .J . Wakil, J . Biol . Chem . ~, 2750 (1962) . 5 . P .R . Yagelos, A .W . Alberts and D .B . Martin, J . Biol . Chem . 533 (1963) " 6 . M . Matsuhashi, S . Matsuhashi, S . Numa and F . Lynen, Biochem . z . ~. 243 (1964) . 7 . M. Matsuhashi, S . Matsuhashi and F . Lynen, Biochem . Z . ~40, 263 (1964) . 8 . S . Numa, E . Ringelmann and F. Lynen, Biochem . Z . ~, 228 (1964) . 9 . S . Numa, W .M . Bortz and F . Lynen, in G . Weber (Editor), Advances in Enz e Re lation VoL III, Pergamon Press, Ox or sn New Yor , p . 7 10 . C . Gregolin, E . Ryder, A .K . Kleinschmidt, R.C . Warner and M .D . Lane, Proc . Natl . Acad . Sci ., II .S ., ~, 148 (1966) . 11 . S . Numa, E . Ringelmann and B. Riedel, Biochem . and Biophys . Research Communs . 24, 750 (1866) . 12 . S . Numa, E . Ringelmann and F . Lynen, Biochem . Z . ~,, 243 (1965) . 13 . O .H . Lowry, N .J . Rosebrough, A .L . Farr and R .J . Randall, J . Biol . Chem . ~, 265 (1951) " 14 . F. Lynen, J . Knappe, E . Lorch, G . Jütting, E . Ringelmann and J .P . Lachance, Biochem . Z . ~, 123 (1961) .
8
Pergamon Presa Ltd.
PURIFICATION AND CRYSTALLIZATION OF ACETYL CoA CARHO%YLASE Tamotsu Goto, Erika Ringelmann, Barbara Riedel and Shosaku Numa Max-Planck-Institut für Zellchemie, München, Germany (Received 18 January 1967) Acetyl CoA carboxylase (E .C . 6 .4 .1 .2 .), the biotin enzyme which catalyzes the formation of malonyl CoA, was discovered by Wakil (1) and partially purified from various sources (2-8 ), There is evidence indicating that this enzyme plays a critical role in the regulation of fatty acid synthesis (9),
Recently,
some molecular properties of the highly purified chicken liver enzyme have been reported by Gregolin . et al . (10) as well as from this laboratory (11) . the enzyme .
We have now been able to crystallize
The present communication concerns the procedure
for purification and crystallization of acetyl CoA carboxylase . Table I represents a summary of typical purification data . All operations were carried out at 0 - 4°C, unless other~rise stated .
Freshly-frozen chicken livers (3 .3 kg) were thawed and
homogenized with 4,950 ml of 0 .1 M phosphate buffer* in a blaring blendor for 90 sec . x g for 40 min .
The homogenate was centrifuged at 16,300
The supernatant fluid (4,900 ml) was further
centrifuged at 73,000 x g for 55 min,
The crude extract thus
obtained (3,945 ml) was diluted 2-fold with 0 .1 M phosphate buffer . added,
To the diluted extract, 1,295 g of (NH4) 2504 were The resulting precipitate was collected by centrifugation
*All phosphate buffers employed were potassium phosphate buffer, pH 7 .5, containing 2 mM 2-mercaptoethanol and 5 mM potassium ethylenediaminetetraacetate (EDTA), unless otherwise specified . 785
786
ACETYL CoA CARBOXYLASE
Vol . 6, Vo . 8
TABLE I Purification of Acetyl CoA Carboxylase Fraction
Volume (ml)
Protein (mg)
Crude Extract
3.945
141,000
282
0 .002
1st (NH4)2S04
380
12,300
434
0 .035
Ca 3 (P04 ) 2 Ge.l
2,020
2,830
242
0 .086
2nd (NH4)2504
51
1,260
195
0 .155
DEAF-cellulose
214
107
106
0 .995
Gradient Centrifugation
58
14,5
Total Activity (units*)
51 .3
Specific Activity**
3 "54
*One unit of enzyme is defined as that amount which catalyzes the carboxylation of 1 4tmole of ace~yl CoA per minute under the standard assay conditions at 25 C described previously (12) . The activity in the crude extract was assayed by the isotopic method (Assay A, see (7)) with some modifications . **Specific activity is defined as units per mg of protein . Protein was determined by the method of Lowry et al . (13) with bovine serum albumin as standard . at 16,300 x g for 35 min and dissolved in 0 .02 M phosphate buffer .
The insoluble material was removed by centrifugation
at 37,000 x g for 15 min .
The first (NH4)2504 fraction (380 ml),
which had been stored at -15°C, was diluted with distilled water to give a protein concentration of 11 mg/ml .
The diluted enzyme
solution was added to 900 ml of Ca 3 (P04 ) 2 gel (16 .5 mg/ml) (protein : gel = 1 : 1 .2) .
After stirring for 5 min, the gel
was collected by centrifugation at 3,000 x g for 5 min and eluted 3 times, each time with 675 ml of 0 .033 M phosphate buffer, and then 3 times, each time with 675 ml of 0 .2 M phosphate buffer .
The latter eluates (0 .2 M) were combined
Vol . 6, No . 8 (2,020 ml) .
ACETYL CoA CARBOXYLASE
787
To this solution, after addition of 106 ml of
0 .1 M MgK2EDTA, 285 g of (NH4 )2S0 4 were added .
The resulting
precipitate was collected by centrifugation at 16,300 x g for 35 min and dissolved in 0 .02 M phosphate buffer .
The enzyme
solution was freed from the insoluble material by centrifugation at 37,000 x g for 15 min.
The second (NH4 )250 4 fraction
(51 ml), which had been stored at -15° C, was dialyzed against 2 liters of 0 .01 M phosphate buffer for ~ hours with a change of buffer .
The dialyzed solution (60 ml) was diluted 3-fold
with 0 .01 M phosphate buffer, and the insoluble material was removed by centrifugation at 37,000 x g for 25 min .
The super-
natant solution was applied to a DEAE-cellulose column (6 x 25 cm) previously equilibrated with 0 .01 M phosphate buffer . Elution was carried out with a linear concentration gradient established between 1 liter of 0 .02 M phosphate buffer and 1 liter of 0 .75 M phosphate buffer . single peak .
Enzyme emerged as a
The fractions possessing a specific activity
above 0 .6 (between approximately 760 and 974 ml effluent volume) were combined .
To the pooled eluate (214 ml) were
added 92 ml of saturated (0 °C)(NH4 )2S0 4 solution .
The re-
sulting precipitate was collected by centrifugation at 16,300 x g for 35 min, dissolved in approximately 6 ml of 0 .05 M phosphate buffer and dialyzed against 400 ml of the same buffer for 3 hours with a change of buffer .
The insoluble material was
removed by centrifugation at 37,000 x g for 10 min .
The dia-
lyzed solution (6 .4 ml) was layered on 6 linear density gradients made from 15 ml of 5 ~ (w/v) sucrose and 15 ml of 20 (w/v) sucrose .
The sucrose gradients contained, in addition,
0 .05 M phosphate buffer .
The gradient tubes were centrifuged
788
ACETYL CoA CARBO%YLASE
Vol .
6, No . 8
at 24,000 rpm for 11 hours in the SW-25 .1 swinging bucket rotor in the Spinco Model L ultracentrifuge .
Twenty-two fractions
xere collected from each gradient tube .
Protein sedimented in
two peaks, and the fractions corresponding to the smaller and faster-sedimenting peak xere combined (58 ml) .
The specific
activity of 10 enzyme preparations made by the procedure described above ranged from 3 to 5 . Crystallization xas achieved as folloxs : The enzyme of the last purification step (14 mg) xas precipitated by addition of an equal volume of saturated (0 °C)(NH4 )2504 solution .
The pre
cipitate xaa collected by centriflxgation at 16,300 x g for 35 min and suspended in 10 ml of 30
;6
saturated (NH4)230 4 solu-
tion, pH 7 .5, containing 0 .01 M phosphate buffer, 5 mM 2mereaptoethanol and 1 mM EDTA .
After occasional gentle stir-
ring for 10 min, the suspension xaa centrifuged at 37,000 x g for 5 min .
The precipitate xas treated in a similar manner suc-
cessively xith the folloxing solutions : 10 ml of 20 (NH4 )2304 solution, 4 ml of 10
y6
;6
saturated
saturated (NH4)250 4 solution
and 1 ml of 10 % saturated (NH4)2304 solution ; all solutions contained the same ingredients as the 30 solution .
;6
saturated (NH4 ) 2304
The txo extracts xith the 10 ~ saturated (NH4)250 4
solution, xhich contained nearly all protein (protein concentration, approximately 3 and 1 mg/ml, respectively), xere warmed up to room temperature (22 - 25 °C) during a period of about 6 hours and alloxed to stand at this temperature . days, crystallization began in both extracts . a photomicrograph of enzyme crystals .
After several Fig. 1 represents
Crystals were obtained
also by a similar extraction procedure with 10 % saturated (NH4 )250 4 solution, pH 8 .0, containing 0 .01 M Na 4P207 -i~Cl
Vol . 6, No . 8
ACETYL CoA CARBOJCYLASE
789
FIG. 1 Phase contrast photomicrograph of crystalline acetyl CoA carboxylase . Magnification, 1,000 x. buffer, 0 .01 M potassium citrate, 1 mM EDTA .
5 mM 2-mercaptoethanol and
The specific activity of the crystalline enzyme xas
approximately the same as that of the enzyme of the last purification step . The crystalline enzyme contained 0.70 ~g of (+)-biotin per mg of protein dry weight (for biotin determination, see (14)) . The sedimentation behavior of the enzyme has recently been re ported (11) ; in these experiments, amorphous preparations whose specific activity and biotin content were comparable to those of the crystalline enzyme were employed . Açknowled~nents We are indebted to Professor F. Lynen for his encouragement during the course of this work .
A research grant from the
790
ACETYL CoA CARBOXYLASE
Vol . 6,
No .
Deutsche Forschungsgemeinschaft is gratefully acknowledged . One of the authors (T .G .) thanks the Alexander von HumboldtStiftung for a stipend . References 1 . S .J . Wakil, J . Am . Chem . Soc . 80, 6465 (1958) . 2 . S .J . Wakil, J .W . Porter and D .M . Gibson, Biochem, et Biophys . Acta 24, 453 (1957) " 3 . M .D . Hatch and P .K . Stumpf, J . Biol . Chem . ~, 2879 (1961) . 4 . M. Waite and S .J . Wakil, J . Biol . Chem . ~, 2750 (1962) . 5 . P .R . Yagelos, A .W . Alberts and D .B . Martin, J . Biol . Chem . 533 (1963) " 6 . M . Matsuhashi, S . Matsuhashi, S . Numa and F . Lynen, Biochem . z . ~. 243 (1964) . 7 . M. Matsuhashi, S . Matsuhashi and F . Lynen, Biochem . Z . ~40, 263 (1964) . 8 . S . Numa, E . Ringelmann and F. Lynen, Biochem . Z . ~, 228 (1964) . 9 . S . Numa, W .M . Bortz and F . Lynen, in G . Weber (Editor), Advances in Enz e Re lation VoL III, Pergamon Press, Ox or sn New Yor , p . 7 10 . C . Gregolin, E . Ryder, A .K . Kleinschmidt, R.C . Warner and M .D . Lane, Proc . Natl . Acad . Sci ., II .S ., ~, 148 (1966) . 11 . S . Numa, E . Ringelmann and B. Riedel, Biochem . and Biophys . Research Communs . 24, 750 (1866) . 12 . S . Numa, E . Ringelmann and F . Lynen, Biochem . Z . ~,, 243 (1965) . 13 . O .H . Lowry, N .J . Rosebrough, A .L . Farr and R .J . Randall, J . Biol . Chem . ~, 265 (1951) " 14 . F. Lynen, J . Knappe, E . Lorch, G . Jütting, E . Ringelmann and J .P . Lachance, Biochem . Z . ~, 123 (1961) .
8