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Alterations in the sensitivity to effectors of rat liver acetyl-CoA carboxylase following trypsin treatment
P~rint~
in~`rsreat Brvitâiâ ' Part II, pp . 1157-1185,
1989 .
Pergamon Press
ALT~RàTIONS IN THE SSNSITIPITY TO EFFECTORS OF RAT LIVBR ACETYh-CoA CARBOZYhASE FCLhOW1NG TRYP3IN TREATM$NT Nobuko Iritani, Shigetada Nakanishi~ and Shosaku Numa Department oß Medical Chemistry Syoto IIniversity Faculty oß Medicine, Byoto, Japan (Received 30 May 1989 ; in final form 4 August 1989) Swanaon, Curry and Anker (1) have recently found that acetyl-CoA carbo~rlase (acetyl-CoA : C0 2 ligase (ADP), EC 6 .4 .1 .2) Brom rat liver is activated by incubation with trypsin .
Although trypsin treatment stimulated the enzyme
during preincubation as citrate did, it was not decided whether or not trypsin-treated carbo~ylase was active when citrate was omit+ed during assay itselß .
Moreover, the question, whether
the activation was due solely to the action of trypsin, remained to be answered, since a crude or partially purified enzyme preparation was employed by these investigators . The present study has demonstrated that highly purified acetyl-CoA csrbo~ylase from rat liver, when treated with trypsin, exhibits considerable activity even in the assay mixture without citrate .
Furthermore, the trypsin-treated enzyme has been found
to be lees sensitive to an inhibitor, palmityl-CoA, than control enzyme .
Sucrose density gradient centrißugation oß partially
*Supported in part by research grants from the Miniatxq oß Rducation oß Japan, the Wakaman Foundation oß Japan and the Japanese Medical Association. **Recipient oß Sigma Chemical Postgraduate Fellowship 1157
115 8
TRYP3IN AND ACETYL-CoA DECARBOXYLASE Vol. 8, No. 22
purified carboxylase has shown that the sedimentation coefficient of the enzyme is alao altered by trypsin treatment in correlation with the changes in the sensitivity to effectors . Materials and Methods "Purified" acetyl-CoA carbo~ylase was prepared from livers of Wistar strain rats, which had been fasted for 48 hours and subsequently refed a fat-free diet for 48 hours, by a modification of the procedure employed by Goto et al . (2) .
The enzyme preparation
thus obtained sedimented as a single boundary in the analytical ultracentrifuge and possessed a specific activity of 1 .5 - 1 .7 un.ita per milligram of protein (for definition of unit, see (3)) . Protein was determined by the method of Warburg and Christian (4) . "Partial.ly purified" acetyl-CoA carboxylase was obtained from livers of similarly treated rats by carrying out the same purification procedure through the second ammonium sulfate fractionation step and exhibited a specific activity of 0 .03 - 0 .05 unit per milligram of protein. Bovine pancreas trypsin and soybean trypain inhibitor were purchased from Sigma Chemical Compar~p . Sucrose density gradient centrifugation was carried out by the method of Martin and Ames (5) with a gradient from 5 to 20 96 (w/v) sucrose.
The preparation of samples for centrifugation is
described in the legend to Fig. 2 .
The gradient tubes were cen-
trifuged for 80 min at 38,000 rpm and 25 ° in a SW 39 rotor in a Spinco model Z ultracentrifuge .
After centrifugation, 20 frac-
tions were collected from each gradient .
Potassium citrate in a
final concentration of 10 mM was added to the fractions derived from the gradients containing no citrate .
The method of
Vol. 8, No. 22
TRYPSIN AND ACETYL-CoA DECARBORYLA3E
115 9
carboxylaae assay was essentially the same as described previous],y (3) .
Fatty acid synthetase from yeast, which was kid provided
by Dra . F . Itiynen and D . Oeaterhelt, was employed as standard enzyme with a known sedimentation coefficient ( a20,w = 43 8) . Results and Diacussi2n In the experiment represented in Fig . l, "purified"
Z
E
M
â .
O
0 .8 0 .T 0 .6 0.5 0.4
CONTROL ENZ NO CITRATE
A
I ~
~9
~ NO CSTRATE
C p
CONTROL ENZ + CITRATE
TRYPSIN ENZ
~+ CITRATE
0
4
8
12
TIME (MIN FIG . 1 Effects of trypsin and citrate on rat liver acetyl-CoA carboxylase . The préincubation mixture (0 .125 ml) contained approximately 130 U.g/ml "purified" carbôxylase, 40 mM Tris-C1buffer, pH 7 .5, and the following component(s) : A, none ; B, 4 ug/ml trypsin ; C, 10 mM potassium citrate ; D, 4 leg/ml trypain and 10 mM potassium citrate . After preincubation at 25 ° for 10 min, either 0 .025 ml of 80 ug/ml soybean trypain inhibitor (B, D) or 0 .025 ml of water (A, C) was added, and the reaction was initiated by add ing 0 .02 ml of the mixture to an assay medium (0 .78 ml) containing 50 mM Tris-C1- buffer, pH 7 .5, 3 .75 mM ATP, 10 mM MgCl2, 25 ~nM ~IC03, 0 .125 mM acetyl-CoA, 3 .75 mM GSH, 0 .75 mg ml bovine serum albumin, 0 .50 mM potassium phgaphoenolpyruvate, 0 .125 mM NADIR, 12 .5 U,g/ml pyruvate kinase, 37 .5 ug/~ lactate dehydrogenase and for C and D, 10 mM potassium citrate ; no citrate was added for A and B . The oxidation of NADH was fol lowed at 25 ° with a Gilford recording spectrophotometer (wave length = 340 mlt ) .
VoI . 8, No. 22
TRYPSIN AND ACETYL-CoA DECARHORYLA3E
118 0
carbo~lase was preincubated either with citrate, with trypsin or xith both, and the reaction was initiated by adding the preincubated enzyme to the assay mixture.
The enzyme activity was
determined by coupling the carboxylase reaction with the pyruvate kinase-lactate del~ydrogena,se system and measuring the rate of NADH oxidation .
Citrate is known to be required both during
preincubation and during assay itself for full activation of rat liver carbo~lase (6) .
Both these effects of citrate were re-
placed by trypain treatment, although the extent ,of activation attained by trypain was no more than 30 to 50 96 of that provoked by citrate. additive .
The effects of trypain and of citrate were nearly Neither trypain inhibitor itself nor a mixtare of TABLE I
Effect of Palmityl-CoA on Trypsin-treated Carboxylase Trypain treatment of carboxylase was carried out as described in the legend to Fig. l . Control or trypsin-treated "purified" carboxylase (approximately 3 Kg) was preincubated at 25 ° for 10 min in a cuvette containing palmityl-CoA in the indicated concentrations (final concentration after addition of gHC03) and all the ingredients of the assay medium includ ing citrate except H~iC03 (see the legend to Fig. l ; volume = 0.76 ml) . The reaction was then started by addition of 0 .04 ml of 500 mM SHC03, and the initial rate of NADH oxidation was determined at 25 ° as described in the legend to Fig . l.
Palmityl-CoA
0 2
5 10 20
Relative Activity Trypain-treated Carboxylase
Control Carboxylase
100
100 68
94 86 71 68
44 39 34
Vol. 8, No: 22
TRYPSIN AND ACETYL-CoA DECARBOXYLASE
1181
trypsin and trypsin inhibitor affected carboxylase activity . Since "purified" carbo~ylase proved to be homogeneous in the analytical ultracentrifuge, these results indicate that the activation of carboxylase is due to the action of trypsin alone. Zong-chain fatty acyl-CoA derivatives are known to inhibit rat liver acetyl-CoA carboxylase (3, 7) .
Table I shows the re-
sults of an experiment in which trypsin-treated enzyme and control enzyme were assayed in the presence of palmityl-CoA in varying concentrations .
Trypsin-treated enzyme was leas sensi-
tive toward inhibition by palmityl-CoA than control enzyme . A correlation between activity and sedimentation coefficient of mp~A~ ian acetyl-CoA carboxylase was observed under a variety of conditions affecting the enzyme activity (3, 8, 9) .
Subsequent
studies on pure chicken liver carboxylase revealed some molecular properties of the active "large" form and the inactive "small" form (10, 11) .
Hence, the sedimentation behavior of trypsin-
treated enzyme was studied under different conditions by means of sucrose density gradient centrifugation .
In these experiments,
"partially purified" carboxylase was employed, and the results are represented in Fig. 2A - C .
Trypain-treated carboxylase sediment* ed as a large form even in a medium without citrate , whereas, as demonstrated previously (3), control carboxylase sedimented as a small form in the absence of citrate and as a large form in its ** presence (Fig . 2A) . In the presence of palmityl-CoA (Fig . 2B)
**
This finding disagrees with the report of Swanson et al . (12) that trypsin treatment fails to increase the sedime~~ion coefficient of carboxylaee . IInder the conditions employed, trypsin-treated enzyme and control enzyme exhibited 56 q6 and 10 96 respectively of the activity measured after preincubation with citrate but no inhibitor .
1182
TRYP3IN AND ACETYL-CaA DECARHORYLA3E
Vol. 8, No. 22
DIRECTION OF SEDIMENTATION .. z
0 Û Q
N H 2
r U Q Q r
k O
m Q U
5 10 15 FRACTION NUMBER
20
Fza. 2 Sucrose density gradient centriuugation oY trypsin-treated carboaylase . A. "Partia]~y parried" carbo~ylase (approzimate],y 3 .8 mg/ml) was incubated at 25 ° for 10 min in the follow ing
Vol . 8, No. 22
TRYPSIN AND ACETYL-CoA DECARHOXYLA3E
1183
medium : I (~-~), 50 mM Tris-Cl- buffer, pH 7 .5, 5 mM 2mercaptoethanol 1 mM EDTA and 3 mg/ml bovine serum alb»min (control enzymej ; II (o-o), medium for I plus 10 mM potassium citrate ; III (i-i), medium for I plus 4 ug/m7- trypain (trypsintreated enzyme) . After the incubation, soybean trypsin inhibitor (4 times as much as trypain) was added to the trypain-treated enzyme solution (III) . To the other two incubation mixtures (I, II), trypain inhibitor and then trypsin were added in equivalent concentrations . Two-tenth ml of each enzyme solution was applied on sucrose density gradients cont a~ the same additions as the respective incubation medium except trypsin and trypsin inhibitor. B C . Control enzyme (o-o) and trypsin-treated enzyme (~-ij, which were obtained by incubation as described for I and III under A, were sûbjected to a second incubation carried out at 25° for 10 min with 10 mM potassium citrate and either 39 ~iM palmityl-CoA (B) or 3 .75 mM ATP plus 10 mM MgCl2~(C) . The enzyme concentration during the second incubation was approximately 2 .5 mg/ml . Two-tenth ml of each enzyme solution was applied on sucrose density gradients containing the same additions as the respective first and second incubation media except trypsin and trypsin inhibitor . For other details, see "Materials and Methods" . or of ATP p lus Mgt (Fig . 2C) , trypsin-treated enzyme assumed a large form, whereas control enzyme a small form .
Thus, trypain
treatment appears to result in conformational changes of enzyme protein similar to those induced by citrate and to lead to an aggregation of enzyme molecules .
The conformational changes
occurring in both cases might not be identical, however, as sugL Bested by the fact that trypsin-treated carboxylase sedimented faster than citrate-treated carboxylase .
In addition, trypsin-
treated enzyme, in contrast to control enzyme, failed to dissociate in the presence of the inhibitors .
This fact was reflected
by the reduced sensitivity of trypsin-treated enzyme to the Preincubation with ATP plus Mgt is known to inhibit carboxylase (6) Under the conditions employed, trypain-treated enzyme and control enzyme exhibited 50 and 24 96 respectively of the activity measured after preincubation with citrate but no inhibitor . Likewise, Swanson et al . (1) reported earlier that the activation of carboxylase in rat liver cell sap treated with trypsin was not reversed by preincubation with ATP.
TRYPSIN AND ACETYL-CaA DECARBOXYLASE
1184
Vol . 8, No. 22
inhi bitors . The present results are consistent with the assumption that the site s) specific for citrate and palmityl-CoA might be modified by trypsin treatment, whereas the catalytic site might re main intact .
The presence of specific site s) for citrate and
long-chain fatty acyl-CoA derivatives would support the hypothesis that these effectora represent physiologic regulators of the activity of acetyl-CoA carboxylase, a key enzyme for control of long-chain fatty acid synthesis (see (13)) .
Highly purified acetyl-CoA carboxylase from rat liver, when treated with trypain, exhibited considerable activity even in the absence of citrate .
2.
The trypain-treated enzyme was less sensitive to an in-
hibitor, palmityl-CoA, than control enzyme .
3.
Sucrose density gradient centrifugation~of partially
purified carbozylase revealed that the sedimentation coefficient of the enzyme was also altered by trypain treatment in correla tion with the changes in the sensitivity to effectors . References 1.
R.F . 3fiANSOH, W .M . CURRY and H.s . AHgER, Proc . Natl . Aced . ici. II . s . ~8, 1243 (1967) .
2.
T . QOTO, $ . RINaLLMANN, B . RI~DEL and S . AIIMA, >;ife sci . 6,
3.
3 . N[JMA, l3 . RI1QaRLMANA and F. L~TSiT, Biochem. Z . ~, 243
4.
o . rARBQRa and w . c~sTiAx, Biochem. z . ~lo, 384 (1942) .
5.
R.a . MARTIA and B.N . AMES~ J . Biol . Chem . ~, 1372 (1961) .
785 (1967) . (1965) .
-
Vol . 8, No. 22
TRYPSIN AND ACETYL-CoA DECARBOXYLASE
1185
6.
M . GREENSPAN and J .M . LOWENSTEIN, Arch . Biochem, Biophys. 118, 260 (1967),
7.
W .M . BORTZ and F . LYNlfii, Biochem, Z , ~, 505 (1963) .
8.
P .R . VAGELOS, A,W, ALBERTS and D .B . MARTIN, J . Biol, Chem . 533 (1963),
9.
S . NUMA and E . RINGELMANN, Biochem. Z , ~, 258 (1965) .
10 .
C . GREGOLIN, E . RYDER, A.S . %LEIASCHI+LCDT, R.C . WARNER and M .D . LANE, Proc, Natl . Acad . Sci . U . S . ~6, 148 (1966),
11 .
S . NUMA, E . RINGELMANN and B. R2EDEL, Biochem. Biophys . Res . Commun . 2~, 750 (1966) .
12 .
R .F . SWANSON, W .M . CURRY and H.S, ANgER, Biochem. Biophss . Acta lam, 390 (1968) .
13,
S . NOMA, W,M. BORTZ and F, LYNEN, in G. Weber (Editor), Advances in Enz e Re ation Vol III, Pergamon Press, Oxîord and New York, p, 7 1 5 .
in~`rsreat Brvitâiâ ' Part II, pp . 1157-1185,
1989 .
Pergamon Press
ALT~RàTIONS IN THE SSNSITIPITY TO EFFECTORS OF RAT LIVBR ACETYh-CoA CARBOZYhASE FCLhOW1NG TRYP3IN TREATM$NT Nobuko Iritani, Shigetada Nakanishi~ and Shosaku Numa Department oß Medical Chemistry Syoto IIniversity Faculty oß Medicine, Byoto, Japan (Received 30 May 1989 ; in final form 4 August 1989) Swanaon, Curry and Anker (1) have recently found that acetyl-CoA carbo~rlase (acetyl-CoA : C0 2 ligase (ADP), EC 6 .4 .1 .2) Brom rat liver is activated by incubation with trypsin .
Although trypsin treatment stimulated the enzyme
during preincubation as citrate did, it was not decided whether or not trypsin-treated carbo~ylase was active when citrate was omit+ed during assay itselß .
Moreover, the question, whether
the activation was due solely to the action of trypsin, remained to be answered, since a crude or partially purified enzyme preparation was employed by these investigators . The present study has demonstrated that highly purified acetyl-CoA csrbo~ylase from rat liver, when treated with trypsin, exhibits considerable activity even in the assay mixture without citrate .
Furthermore, the trypsin-treated enzyme has been found
to be lees sensitive to an inhibitor, palmityl-CoA, than control enzyme .
Sucrose density gradient centrißugation oß partially
*Supported in part by research grants from the Miniatxq oß Rducation oß Japan, the Wakaman Foundation oß Japan and the Japanese Medical Association. **Recipient oß Sigma Chemical Postgraduate Fellowship 1157
115 8
TRYP3IN AND ACETYL-CoA DECARBOXYLASE Vol. 8, No. 22
purified carboxylase has shown that the sedimentation coefficient of the enzyme is alao altered by trypsin treatment in correlation with the changes in the sensitivity to effectors . Materials and Methods "Purified" acetyl-CoA carbo~ylase was prepared from livers of Wistar strain rats, which had been fasted for 48 hours and subsequently refed a fat-free diet for 48 hours, by a modification of the procedure employed by Goto et al . (2) .
The enzyme preparation
thus obtained sedimented as a single boundary in the analytical ultracentrifuge and possessed a specific activity of 1 .5 - 1 .7 un.ita per milligram of protein (for definition of unit, see (3)) . Protein was determined by the method of Warburg and Christian (4) . "Partial.ly purified" acetyl-CoA carboxylase was obtained from livers of similarly treated rats by carrying out the same purification procedure through the second ammonium sulfate fractionation step and exhibited a specific activity of 0 .03 - 0 .05 unit per milligram of protein. Bovine pancreas trypsin and soybean trypain inhibitor were purchased from Sigma Chemical Compar~p . Sucrose density gradient centrifugation was carried out by the method of Martin and Ames (5) with a gradient from 5 to 20 96 (w/v) sucrose.
The preparation of samples for centrifugation is
described in the legend to Fig. 2 .
The gradient tubes were cen-
trifuged for 80 min at 38,000 rpm and 25 ° in a SW 39 rotor in a Spinco model Z ultracentrifuge .
After centrifugation, 20 frac-
tions were collected from each gradient .
Potassium citrate in a
final concentration of 10 mM was added to the fractions derived from the gradients containing no citrate .
The method of
Vol. 8, No. 22
TRYPSIN AND ACETYL-CoA DECARBORYLA3E
115 9
carboxylaae assay was essentially the same as described previous],y (3) .
Fatty acid synthetase from yeast, which was kid provided
by Dra . F . Itiynen and D . Oeaterhelt, was employed as standard enzyme with a known sedimentation coefficient ( a20,w = 43 8) . Results and Diacussi2n In the experiment represented in Fig . l, "purified"
Z
E
M
â .
O
0 .8 0 .T 0 .6 0.5 0.4
CONTROL ENZ NO CITRATE
A
I ~
~9
~ NO CSTRATE
C p
CONTROL ENZ + CITRATE
TRYPSIN ENZ
~+ CITRATE
0
4
8
12
TIME (MIN FIG . 1 Effects of trypsin and citrate on rat liver acetyl-CoA carboxylase . The préincubation mixture (0 .125 ml) contained approximately 130 U.g/ml "purified" carbôxylase, 40 mM Tris-C1buffer, pH 7 .5, and the following component(s) : A, none ; B, 4 ug/ml trypsin ; C, 10 mM potassium citrate ; D, 4 leg/ml trypain and 10 mM potassium citrate . After preincubation at 25 ° for 10 min, either 0 .025 ml of 80 ug/ml soybean trypain inhibitor (B, D) or 0 .025 ml of water (A, C) was added, and the reaction was initiated by add ing 0 .02 ml of the mixture to an assay medium (0 .78 ml) containing 50 mM Tris-C1- buffer, pH 7 .5, 3 .75 mM ATP, 10 mM MgCl2, 25 ~nM ~IC03, 0 .125 mM acetyl-CoA, 3 .75 mM GSH, 0 .75 mg ml bovine serum albumin, 0 .50 mM potassium phgaphoenolpyruvate, 0 .125 mM NADIR, 12 .5 U,g/ml pyruvate kinase, 37 .5 ug/~ lactate dehydrogenase and for C and D, 10 mM potassium citrate ; no citrate was added for A and B . The oxidation of NADH was fol lowed at 25 ° with a Gilford recording spectrophotometer (wave length = 340 mlt ) .
VoI . 8, No. 22
TRYPSIN AND ACETYL-CoA DECARHORYLA3E
118 0
carbo~lase was preincubated either with citrate, with trypsin or xith both, and the reaction was initiated by adding the preincubated enzyme to the assay mixture.
The enzyme activity was
determined by coupling the carboxylase reaction with the pyruvate kinase-lactate del~ydrogena,se system and measuring the rate of NADH oxidation .
Citrate is known to be required both during
preincubation and during assay itself for full activation of rat liver carbo~lase (6) .
Both these effects of citrate were re-
placed by trypain treatment, although the extent ,of activation attained by trypain was no more than 30 to 50 96 of that provoked by citrate. additive .
The effects of trypain and of citrate were nearly Neither trypain inhibitor itself nor a mixtare of TABLE I
Effect of Palmityl-CoA on Trypsin-treated Carboxylase Trypain treatment of carboxylase was carried out as described in the legend to Fig. l . Control or trypsin-treated "purified" carboxylase (approximately 3 Kg) was preincubated at 25 ° for 10 min in a cuvette containing palmityl-CoA in the indicated concentrations (final concentration after addition of gHC03) and all the ingredients of the assay medium includ ing citrate except H~iC03 (see the legend to Fig. l ; volume = 0.76 ml) . The reaction was then started by addition of 0 .04 ml of 500 mM SHC03, and the initial rate of NADH oxidation was determined at 25 ° as described in the legend to Fig . l.
Palmityl-CoA
0 2
5 10 20
Relative Activity Trypain-treated Carboxylase
Control Carboxylase
100
100 68
94 86 71 68
44 39 34
Vol. 8, No: 22
TRYPSIN AND ACETYL-CoA DECARBOXYLASE
1181
trypsin and trypsin inhibitor affected carboxylase activity . Since "purified" carbo~ylase proved to be homogeneous in the analytical ultracentrifuge, these results indicate that the activation of carboxylase is due to the action of trypsin alone. Zong-chain fatty acyl-CoA derivatives are known to inhibit rat liver acetyl-CoA carboxylase (3, 7) .
Table I shows the re-
sults of an experiment in which trypsin-treated enzyme and control enzyme were assayed in the presence of palmityl-CoA in varying concentrations .
Trypsin-treated enzyme was leas sensi-
tive toward inhibition by palmityl-CoA than control enzyme . A correlation between activity and sedimentation coefficient of mp~A~ ian acetyl-CoA carboxylase was observed under a variety of conditions affecting the enzyme activity (3, 8, 9) .
Subsequent
studies on pure chicken liver carboxylase revealed some molecular properties of the active "large" form and the inactive "small" form (10, 11) .
Hence, the sedimentation behavior of trypsin-
treated enzyme was studied under different conditions by means of sucrose density gradient centrifugation .
In these experiments,
"partially purified" carboxylase was employed, and the results are represented in Fig. 2A - C .
Trypain-treated carboxylase sediment* ed as a large form even in a medium without citrate , whereas, as demonstrated previously (3), control carboxylase sedimented as a small form in the absence of citrate and as a large form in its ** presence (Fig . 2A) . In the presence of palmityl-CoA (Fig . 2B)
**
This finding disagrees with the report of Swanson et al . (12) that trypsin treatment fails to increase the sedime~~ion coefficient of carboxylaee . IInder the conditions employed, trypsin-treated enzyme and control enzyme exhibited 56 q6 and 10 96 respectively of the activity measured after preincubation with citrate but no inhibitor .
1182
TRYP3IN AND ACETYL-CaA DECARHORYLA3E
Vol. 8, No. 22
DIRECTION OF SEDIMENTATION .. z
0 Û Q
N H 2
r U Q Q r
k O
m Q U
5 10 15 FRACTION NUMBER
20
Fza. 2 Sucrose density gradient centriuugation oY trypsin-treated carboaylase . A. "Partia]~y parried" carbo~ylase (approzimate],y 3 .8 mg/ml) was incubated at 25 ° for 10 min in the follow ing
Vol . 8, No. 22
TRYPSIN AND ACETYL-CoA DECARHOXYLA3E
1183
medium : I (~-~), 50 mM Tris-Cl- buffer, pH 7 .5, 5 mM 2mercaptoethanol 1 mM EDTA and 3 mg/ml bovine serum alb»min (control enzymej ; II (o-o), medium for I plus 10 mM potassium citrate ; III (i-i), medium for I plus 4 ug/m7- trypain (trypsintreated enzyme) . After the incubation, soybean trypsin inhibitor (4 times as much as trypain) was added to the trypain-treated enzyme solution (III) . To the other two incubation mixtures (I, II), trypain inhibitor and then trypsin were added in equivalent concentrations . Two-tenth ml of each enzyme solution was applied on sucrose density gradients cont a~ the same additions as the respective incubation medium except trypsin and trypsin inhibitor. B C . Control enzyme (o-o) and trypsin-treated enzyme (~-ij, which were obtained by incubation as described for I and III under A, were sûbjected to a second incubation carried out at 25° for 10 min with 10 mM potassium citrate and either 39 ~iM palmityl-CoA (B) or 3 .75 mM ATP plus 10 mM MgCl2~(C) . The enzyme concentration during the second incubation was approximately 2 .5 mg/ml . Two-tenth ml of each enzyme solution was applied on sucrose density gradients containing the same additions as the respective first and second incubation media except trypsin and trypsin inhibitor . For other details, see "Materials and Methods" . or of ATP p lus Mgt (Fig . 2C) , trypsin-treated enzyme assumed a large form, whereas control enzyme a small form .
Thus, trypain
treatment appears to result in conformational changes of enzyme protein similar to those induced by citrate and to lead to an aggregation of enzyme molecules .
The conformational changes
occurring in both cases might not be identical, however, as sugL Bested by the fact that trypsin-treated carboxylase sedimented faster than citrate-treated carboxylase .
In addition, trypsin-
treated enzyme, in contrast to control enzyme, failed to dissociate in the presence of the inhibitors .
This fact was reflected
by the reduced sensitivity of trypsin-treated enzyme to the Preincubation with ATP plus Mgt is known to inhibit carboxylase (6) Under the conditions employed, trypain-treated enzyme and control enzyme exhibited 50 and 24 96 respectively of the activity measured after preincubation with citrate but no inhibitor . Likewise, Swanson et al . (1) reported earlier that the activation of carboxylase in rat liver cell sap treated with trypsin was not reversed by preincubation with ATP.
TRYPSIN AND ACETYL-CaA DECARBOXYLASE
1184
Vol . 8, No. 22
inhi bitors . The present results are consistent with the assumption that the site s) specific for citrate and palmityl-CoA might be modified by trypsin treatment, whereas the catalytic site might re main intact .
The presence of specific site s) for citrate and
long-chain fatty acyl-CoA derivatives would support the hypothesis that these effectora represent physiologic regulators of the activity of acetyl-CoA carboxylase, a key enzyme for control of long-chain fatty acid synthesis (see (13)) .
Highly purified acetyl-CoA carboxylase from rat liver, when treated with trypain, exhibited considerable activity even in the absence of citrate .
2.
The trypain-treated enzyme was less sensitive to an in-
hibitor, palmityl-CoA, than control enzyme .
3.
Sucrose density gradient centrifugation~of partially
purified carbozylase revealed that the sedimentation coefficient of the enzyme was also altered by trypain treatment in correla tion with the changes in the sensitivity to effectors . References 1.
R.F . 3fiANSOH, W .M . CURRY and H.s . AHgER, Proc . Natl . Aced . ici. II . s . ~8, 1243 (1967) .
2.
T . QOTO, $ . RINaLLMANN, B . RI~DEL and S . AIIMA, >;ife sci . 6,
3.
3 . N[JMA, l3 . RI1QaRLMANA and F. L~TSiT, Biochem. Z . ~, 243
4.
o . rARBQRa and w . c~sTiAx, Biochem. z . ~lo, 384 (1942) .
5.
R.a . MARTIA and B.N . AMES~ J . Biol . Chem . ~, 1372 (1961) .
785 (1967) . (1965) .
-
Vol . 8, No. 22
TRYPSIN AND ACETYL-CoA DECARBOXYLASE
1185
6.
M . GREENSPAN and J .M . LOWENSTEIN, Arch . Biochem, Biophys. 118, 260 (1967),
7.
W .M . BORTZ and F . LYNlfii, Biochem, Z , ~, 505 (1963) .
8.
P .R . VAGELOS, A,W, ALBERTS and D .B . MARTIN, J . Biol, Chem . 533 (1963),
9.
S . NUMA and E . RINGELMANN, Biochem. Z , ~, 258 (1965) .
10 .
C . GREGOLIN, E . RYDER, A.S . %LEIASCHI+LCDT, R.C . WARNER and M .D . LANE, Proc, Natl . Acad . Sci . U . S . ~6, 148 (1966),
11 .
S . NUMA, E . RINGELMANN and B. R2EDEL, Biochem. Biophys . Res . Commun . 2~, 750 (1966) .
12 .
R .F . SWANSON, W .M . CURRY and H.S, ANgER, Biochem. Biophss . Acta lam, 390 (1968) .
13,
S . NOMA, W,M. BORTZ and F, LYNEN, in G. Weber (Editor), Advances in Enz e Re ation Vol III, Pergamon Press, Oxîord and New York, p, 7 1 5 .