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Citrate synthase of bean seedlings — comparison of activity following in vitro and in vivo treatments of enzyme
Pergamon Press
Vol . 8, Part II, pp . 933-938, 1969 . Life Sciences Printed in Great Britain
CITRATE SYNTHASE OF BEAN SEEDLINGS - COMPARISON OF ACTIVITY FOLLOWING in vitro AND in vivo TREATMENTS OF ENZYME Igor V. Sarkissian and Frank C . Schmalstieg Texas AéM University, College Station,
Texas
(Received 14 April 1969 ; in final form 26 May 1969) Stimulation of activity in vitro of citrate synthase (E .C .4 .1 .3 .7) by concentrations of indole-3-acetic acid (IAA) has been described (2,3) .
low
It was
suggested that this interaction reflected an allosteric property of citrate synthase (CS) with regard to IAA (3,4,5) .
In the present communication we show
that the allosteric property of CS is very labile and that it depends on the conditions of growth of individuals serving as sources of enzyme .
We use the
word "allosteric", a term defined by Monod, Changeux and Jacob (1) to describe a protein whose function is regulated by a metabolite which is not a necessary component of the reaction .
While their definition indicates that the allosteric
effector binds with the protein in question (although reversibly),
in the
present case the binding of IAA is not involved in the reaction proper synthesis of citric acid . in the protein which,
IAA merely causes an apparently irreversible change
once effected, does not require IAA to maintain this
changed state . Materials and Methods Citrate synthase was isolated from protein of root tips of 6-day-old seedlings of bean
(Phaseolus vulgaris , cultivar Burpee's Stringlesa Greenpod) .
the conditions of culture are shown in the tables . 50-gram batches of root
Extraction was as follows :
tips were chilled immediately after harvest and were
cut into very small pieces .
The roots were then homogenized in the grinding
medium for 4 minutes in a Waring blender with the rheostat set at 40 .
The
grinding medium was 400 ml of 0 .02 M phosphate buffer, pH 7.4, containing 100
933
934
CITRATE SYNTHASE
mg of cyateine .
Vol. 8, No. 18
The homogenate was strained through 4 layers of cheesecloth
and then through nylon cloth with mesh of about 50 u .
The volume of the
filtrate was noted and 31 .3 g of ammonium sulfate per 100 ml of liquid were added slowly with stirring .
the mixture was stirred for additional 10 minutes
to insure complete dissolution of ammonium sulfate and was then centrifuged for 15 minutes at 15,000 x g . carded .
The supernatant was saved and the pellet dis-
Additional ammonium sulfate was added to the supernatant (13 .5 g/100 ml)
with stirring as before . 15,000 x g .
The solution was centrifuged for 15 minutes at
The supernatant was discarded and the white pellet was dissolved
in about 20 ml of cold deionized water .
The protein solution was then dialyzed
for 6 hrs against 4 liters of 0 .002 M potassium phosphate buffer, pH 7 .4 .
After
dialysis, the enzyme solution was centrifuged for 10 minutes at 10,000 x g . The small pellet was discarded and the enzyme was ready for use .
All isolation
procedures were carried out at 0-4° and the final enzyme solution was kept on ice . Activity was determined by rate of disappearance of the thiol ester bond of acetyl-coenzyme A at 233 m u
in nmoles/liter/min .
The rate of the reaction
was linear for at least 2 min .
The reaction mixture was composed of 130 umolea
of potassium phosphate buffer, pH 7 .4 ; enzyme protein as indicated ; 0 .75 umolea of freshly prepared oxalacetate at pH 7 .4 ; 0 .012 umolea of acetyl-Co A . used, IAA was at a final concentration of 1 .54 x 10 -11M . 1 .3 ml with pH of 7 .4 .
The temperature was 28 .8 ° .
apectrophotometrically (6) .
When
The final volume was
Protein was determined
The organic reagents were from the Nutritional
Biochemicals Corporation . Results and Discussion One problem confronting us was the variability of response of isolated citrate synthase to IAA .
We were cultivating the bean seedlings in a green-
house with no regulation of temperature and occasionally the isolated enzyme, although fully active, would not be affected at all by IAA .
Since day - and
night - temperatures are quite high here for about 7 months we suspected that
995
CITRATE SYNTHASE
Vol . 8, No . 18
the environment in which the plants were cultured led to the inability of citrate synthase to be stimulated by IAA .
Data summarized in Table 1 show the
effects of environment on the alloeteric property of CS . TABLE 1 Effects of Light and Temperature on the Allosteric Property of Citrate Synthese In Roots of 6-day-old Bean Seedlings mg Protein
Reaction
in Reaction - per mg Protein
Mixture A.
B.
C.
Velocity
Control
0 .336
9,834
1 .54 x 10 -11M IAA
0 .336
13,529
Control
0 .288
10,815
1 .54 x 10 -1 ~ IAA
0 .288
14,070
Control
0 .288
11,725
1 .54 x 10 -11M IAA
0 .288
12,425
Percent Change
+38
+30
+ 6
Reaction mixture and velocity are defined in Materials and Methods . A ~ seedlinge grown in vermiculite at 20 ° , continuous darkness . B ~ seedlinge grown in vermiculite under daily regime of 12 hrs of 20° and light followed by 12 hrs of 30 ° and darkness . C ~ seedlinge grown in vermiculite at 30 ° , continuous darkness . the enzyme isolated from roots of plants grown in the dark, at 20° was stimulated by IAA .
The enzyme from plants exposed to light and 20° during the
day and darkness and 30 ° during the night also responded to IAA . was somewhat reduced, however .
The response
It is of interest that the control reactions
in this case increased somewhat in specific activity (velocity/mg protein) . Enzyme from plants grown constantly at 30 ° in the dark was almost totally "desensitized" to IAA, i .e ., it could not be stimulated by IAA .
The specific
activity of control CS in this case increased again . These experiments reveal that the alloeteric property of CS with respect to IAA is modified by the environment under which the organism develops . is evident that the key factor is temperature .
It
The enzyme from plants grown
CITRATE SYNTHASE
93 6
Vol . 8, No. 18
at 30° , although highly active, does not respond markedly to IAA . mechanism of "desensitization" is not fully understood .
This
It may be due to
alight changes in the native state of the enzyme or, in the present case, heat denaturation .
The possibility is not discounted that it occurs through inter-
vention of a metabolite which "protects" the enzyme from IAA .
The increase in
activity of control reaction (C va A, Table 1), on the other hand, may be the result of increase of endogenous IAA which stimulated activity of CS and at the same time desensitized it .
If, as suggested by ua recently (5), the allosteric
behavior of citrate synthase is a result of conformational changes of the enzyme regulated by IAA, then both increased activity and desensitization may be related to increase of endogenous IAA .
We have noted repeatedly under our
experimental conditions that activity of CS exposed to IAA is no longer affected by changes in IAA concentrations . In Table 2 are shown activities of CS from roots of beans grown at 20° in the presence of exogenous IAA and without IAA .
The enzyme from seedlings
grown in the absence of IAA is stimulated 72% in vitro by IAA .
The enzyme
from seedlings grown in the presence of 10 -6M IAA is not stimulated in vitro by IAA .
It is significant that the specific activity of the desensitized
enzyme is increased .
When the control reactions (activity without IAA in the
reaction mixture) are compared, the activity of CS from seedlings grown with added IAA exceeds that of control seedlings by 74% .
This increase is almost
identical to that of CS of control seedlings reacted in the presence of IAA . When IAA was added to the homogenate during isolation the effect on the isolated citrate synthase was essentially the same as that when the seedlings were grown in the presence of added IAA (Table 3) .
The enzyme was desensitized
to IAA but the specific activity of control CS was increased .
Vol. 8, No. 18
93 7
CITRATE SYNTHASE
TABLE 2 Effects of Exogenous Indoleacetic Acid on Citrate Synthase of Roots of 6-day-old Bean Seedlings mg
Reaction Mixture A.
- -
Protein
in Reaction
Velocity
Percent
-per m~Protein
Change -
Control
0 .360
6,300
1.54 x 10 -1 ~ IAA
0 .360
10,822
0.360
10,948
0 .360
9,800
B. Control 1 .54 x 10 -1-M IAA Control B vs Control A
+72
-12 +74
Velocity is defined in Materials and Methods . A = seedlings grown at 20 ° in vermiculite, irrigated with deionized wgter. B ~ seedlings grown in vermiculite, irrigated with 1 liter of 1 x 10 M IAA.
TABLE 3 Effects of Addition of Indoleacetic Acid lluring Isolation of Citrate S~~nthase on the Allosteric Property of the Enzyme Reaction
mg Protein
Mixture
in Reaction-- ~er~g Protei n -
A.
Control ] . 54 x l0
K.
-10
rf IAA
Control 1 .54 x 10 -11M IAA
li
IAA vs Control A
felocity
0 .480
8,362
0 .480
11,565
0 .480
11,149
0 .480
11,149
Percent Change
+38
0 +33
A,B = differently treated extracts of citrate synthase of one batch of root tips . A was extracted as usual ; in preparing isolate B, the homogenate following the first ammonium sulfate precipitation was made 4 .6 x 10 M IAA, stirred 10 min and then the isolation was continued as in A.
CITRATE 3YNTHA3E
938
Vol. 8, No. 18
These data indicate that IAA stimulates citrate synthase not only in vitro , but also to the same degree during isolation and fully in vivo .
Lack of
reactivity with IAA of CS stimulated in vivo suggests that such enzyme can not be stimulated further and is consistent with the view that stimulation of citrate synthase involves a conformational change of the enzyme .
The possibility
that IAA interacts with SH groups of CS, thereby ultimately desensitizing the enzyme has been suggested (4,5) .
It appears then, that once CS has been
activated by IAA in vivo or in vitro the activity is irreversible . It would seem then, that the allosteric property of citrate synthase with regard to IAA is extremely labile .
The sensitivity of the enzyme to the
hormone thus appears to be regulated by the hormone itself .
It is suggested
that investigations of detailed mechanism of the citrate synthase : IAA interactions be carried out with an enzyme isolated from tissues where it is not desensitized by heat or activated by the hormone itself or possible by other metabolites . Acknowledgement This work was supported by National Science Foundation Grant GB - 7618 . References 1.
J . MONOD, J . P . CHANCEUX and F . JACOB, J . Mol . Biol ., 6, 306 (1963) .
2.
I .V . SARKISSLAN, Physiol . Plantaru~ 19, 328 (1966) .
3.
I . V . SARKISSIAN, Biochemistry of Plant Growth Hormones , Runge Press, Ottawa, in press .
4.
I . V . SARKISSIAN and F .C . SCHI9ALSTIEG, Federation Proc ., 28, 414 (1969) .
5.
I .V . SARKISSIAN and F .C . SCHMALSTIEG, Naturwissenschaften , 56, 1969, in press .
6.
0 . WARBURG and W . CHRISTIAN, Biochem Z . , 310, 384 (1941-2) .
Vol . 8, Part II, pp . 933-938, 1969 . Life Sciences Printed in Great Britain
CITRATE SYNTHASE OF BEAN SEEDLINGS - COMPARISON OF ACTIVITY FOLLOWING in vitro AND in vivo TREATMENTS OF ENZYME Igor V. Sarkissian and Frank C . Schmalstieg Texas AéM University, College Station,
Texas
(Received 14 April 1969 ; in final form 26 May 1969) Stimulation of activity in vitro of citrate synthase (E .C .4 .1 .3 .7) by concentrations of indole-3-acetic acid (IAA) has been described (2,3) .
low
It was
suggested that this interaction reflected an allosteric property of citrate synthase (CS) with regard to IAA (3,4,5) .
In the present communication we show
that the allosteric property of CS is very labile and that it depends on the conditions of growth of individuals serving as sources of enzyme .
We use the
word "allosteric", a term defined by Monod, Changeux and Jacob (1) to describe a protein whose function is regulated by a metabolite which is not a necessary component of the reaction .
While their definition indicates that the allosteric
effector binds with the protein in question (although reversibly),
in the
present case the binding of IAA is not involved in the reaction proper synthesis of citric acid . in the protein which,
IAA merely causes an apparently irreversible change
once effected, does not require IAA to maintain this
changed state . Materials and Methods Citrate synthase was isolated from protein of root tips of 6-day-old seedlings of bean
(Phaseolus vulgaris , cultivar Burpee's Stringlesa Greenpod) .
the conditions of culture are shown in the tables . 50-gram batches of root
Extraction was as follows :
tips were chilled immediately after harvest and were
cut into very small pieces .
The roots were then homogenized in the grinding
medium for 4 minutes in a Waring blender with the rheostat set at 40 .
The
grinding medium was 400 ml of 0 .02 M phosphate buffer, pH 7.4, containing 100
933
934
CITRATE SYNTHASE
mg of cyateine .
Vol. 8, No. 18
The homogenate was strained through 4 layers of cheesecloth
and then through nylon cloth with mesh of about 50 u .
The volume of the
filtrate was noted and 31 .3 g of ammonium sulfate per 100 ml of liquid were added slowly with stirring .
the mixture was stirred for additional 10 minutes
to insure complete dissolution of ammonium sulfate and was then centrifuged for 15 minutes at 15,000 x g . carded .
The supernatant was saved and the pellet dis-
Additional ammonium sulfate was added to the supernatant (13 .5 g/100 ml)
with stirring as before . 15,000 x g .
The solution was centrifuged for 15 minutes at
The supernatant was discarded and the white pellet was dissolved
in about 20 ml of cold deionized water .
The protein solution was then dialyzed
for 6 hrs against 4 liters of 0 .002 M potassium phosphate buffer, pH 7 .4 .
After
dialysis, the enzyme solution was centrifuged for 10 minutes at 10,000 x g . The small pellet was discarded and the enzyme was ready for use .
All isolation
procedures were carried out at 0-4° and the final enzyme solution was kept on ice . Activity was determined by rate of disappearance of the thiol ester bond of acetyl-coenzyme A at 233 m u
in nmoles/liter/min .
The rate of the reaction
was linear for at least 2 min .
The reaction mixture was composed of 130 umolea
of potassium phosphate buffer, pH 7 .4 ; enzyme protein as indicated ; 0 .75 umolea of freshly prepared oxalacetate at pH 7 .4 ; 0 .012 umolea of acetyl-Co A . used, IAA was at a final concentration of 1 .54 x 10 -11M . 1 .3 ml with pH of 7 .4 .
The temperature was 28 .8 ° .
apectrophotometrically (6) .
When
The final volume was
Protein was determined
The organic reagents were from the Nutritional
Biochemicals Corporation . Results and Discussion One problem confronting us was the variability of response of isolated citrate synthase to IAA .
We were cultivating the bean seedlings in a green-
house with no regulation of temperature and occasionally the isolated enzyme, although fully active, would not be affected at all by IAA .
Since day - and
night - temperatures are quite high here for about 7 months we suspected that
995
CITRATE SYNTHASE
Vol . 8, No . 18
the environment in which the plants were cultured led to the inability of citrate synthase to be stimulated by IAA .
Data summarized in Table 1 show the
effects of environment on the alloeteric property of CS . TABLE 1 Effects of Light and Temperature on the Allosteric Property of Citrate Synthese In Roots of 6-day-old Bean Seedlings mg Protein
Reaction
in Reaction - per mg Protein
Mixture A.
B.
C.
Velocity
Control
0 .336
9,834
1 .54 x 10 -11M IAA
0 .336
13,529
Control
0 .288
10,815
1 .54 x 10 -1 ~ IAA
0 .288
14,070
Control
0 .288
11,725
1 .54 x 10 -11M IAA
0 .288
12,425
Percent Change
+38
+30
+ 6
Reaction mixture and velocity are defined in Materials and Methods . A ~ seedlinge grown in vermiculite at 20 ° , continuous darkness . B ~ seedlinge grown in vermiculite under daily regime of 12 hrs of 20° and light followed by 12 hrs of 30 ° and darkness . C ~ seedlinge grown in vermiculite at 30 ° , continuous darkness . the enzyme isolated from roots of plants grown in the dark, at 20° was stimulated by IAA .
The enzyme from plants exposed to light and 20° during the
day and darkness and 30 ° during the night also responded to IAA . was somewhat reduced, however .
The response
It is of interest that the control reactions
in this case increased somewhat in specific activity (velocity/mg protein) . Enzyme from plants grown constantly at 30 ° in the dark was almost totally "desensitized" to IAA, i .e ., it could not be stimulated by IAA .
The specific
activity of control CS in this case increased again . These experiments reveal that the alloeteric property of CS with respect to IAA is modified by the environment under which the organism develops . is evident that the key factor is temperature .
It
The enzyme from plants grown
CITRATE SYNTHASE
93 6
Vol . 8, No. 18
at 30° , although highly active, does not respond markedly to IAA . mechanism of "desensitization" is not fully understood .
This
It may be due to
alight changes in the native state of the enzyme or, in the present case, heat denaturation .
The possibility is not discounted that it occurs through inter-
vention of a metabolite which "protects" the enzyme from IAA .
The increase in
activity of control reaction (C va A, Table 1), on the other hand, may be the result of increase of endogenous IAA which stimulated activity of CS and at the same time desensitized it .
If, as suggested by ua recently (5), the allosteric
behavior of citrate synthase is a result of conformational changes of the enzyme regulated by IAA, then both increased activity and desensitization may be related to increase of endogenous IAA .
We have noted repeatedly under our
experimental conditions that activity of CS exposed to IAA is no longer affected by changes in IAA concentrations . In Table 2 are shown activities of CS from roots of beans grown at 20° in the presence of exogenous IAA and without IAA .
The enzyme from seedlings
grown in the absence of IAA is stimulated 72% in vitro by IAA .
The enzyme
from seedlings grown in the presence of 10 -6M IAA is not stimulated in vitro by IAA .
It is significant that the specific activity of the desensitized
enzyme is increased .
When the control reactions (activity without IAA in the
reaction mixture) are compared, the activity of CS from seedlings grown with added IAA exceeds that of control seedlings by 74% .
This increase is almost
identical to that of CS of control seedlings reacted in the presence of IAA . When IAA was added to the homogenate during isolation the effect on the isolated citrate synthase was essentially the same as that when the seedlings were grown in the presence of added IAA (Table 3) .
The enzyme was desensitized
to IAA but the specific activity of control CS was increased .
Vol. 8, No. 18
93 7
CITRATE SYNTHASE
TABLE 2 Effects of Exogenous Indoleacetic Acid on Citrate Synthase of Roots of 6-day-old Bean Seedlings mg
Reaction Mixture A.
- -
Protein
in Reaction
Velocity
Percent
-per m~Protein
Change -
Control
0 .360
6,300
1.54 x 10 -1 ~ IAA
0 .360
10,822
0.360
10,948
0 .360
9,800
B. Control 1 .54 x 10 -1-M IAA Control B vs Control A
+72
-12 +74
Velocity is defined in Materials and Methods . A = seedlings grown at 20 ° in vermiculite, irrigated with deionized wgter. B ~ seedlings grown in vermiculite, irrigated with 1 liter of 1 x 10 M IAA.
TABLE 3 Effects of Addition of Indoleacetic Acid lluring Isolation of Citrate S~~nthase on the Allosteric Property of the Enzyme Reaction
mg Protein
Mixture
in Reaction-- ~er~g Protei n -
A.
Control ] . 54 x l0
K.
-10
rf IAA
Control 1 .54 x 10 -11M IAA
li
IAA vs Control A
felocity
0 .480
8,362
0 .480
11,565
0 .480
11,149
0 .480
11,149
Percent Change
+38
0 +33
A,B = differently treated extracts of citrate synthase of one batch of root tips . A was extracted as usual ; in preparing isolate B, the homogenate following the first ammonium sulfate precipitation was made 4 .6 x 10 M IAA, stirred 10 min and then the isolation was continued as in A.
CITRATE 3YNTHA3E
938
Vol. 8, No. 18
These data indicate that IAA stimulates citrate synthase not only in vitro , but also to the same degree during isolation and fully in vivo .
Lack of
reactivity with IAA of CS stimulated in vivo suggests that such enzyme can not be stimulated further and is consistent with the view that stimulation of citrate synthase involves a conformational change of the enzyme .
The possibility
that IAA interacts with SH groups of CS, thereby ultimately desensitizing the enzyme has been suggested (4,5) .
It appears then, that once CS has been
activated by IAA in vivo or in vitro the activity is irreversible . It would seem then, that the allosteric property of citrate synthase with regard to IAA is extremely labile .
The sensitivity of the enzyme to the
hormone thus appears to be regulated by the hormone itself .
It is suggested
that investigations of detailed mechanism of the citrate synthase : IAA interactions be carried out with an enzyme isolated from tissues where it is not desensitized by heat or activated by the hormone itself or possible by other metabolites . Acknowledgement This work was supported by National Science Foundation Grant GB - 7618 . References 1.
J . MONOD, J . P . CHANCEUX and F . JACOB, J . Mol . Biol ., 6, 306 (1963) .
2.
I .V . SARKISSLAN, Physiol . Plantaru~ 19, 328 (1966) .
3.
I . V . SARKISSIAN, Biochemistry of Plant Growth Hormones , Runge Press, Ottawa, in press .
4.
I . V . SARKISSIAN and F .C . SCHI9ALSTIEG, Federation Proc ., 28, 414 (1969) .
5.
I .V . SARKISSIAN and F .C . SCHMALSTIEG, Naturwissenschaften , 56, 1969, in press .
6.
0 . WARBURG and W . CHRISTIAN, Biochem Z . , 310, 384 (1941-2) .