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[113] Leucine aminotransferase (rat liver)
BRANCHED-CHAIN AMINO ACIDS
814
[ 113]
T h e s e two types o f e n z y m e e were f o u n d in the supernatants o f various hog tissues, such as h e a r t (I: 85%, III: 0), brain (I: 0, III: 96%), liver (I: 10%, III: 70%), and kidney (I: 30%, III: 60%). Studies on the subcellular distribution o f enzymes in brain also revealed that the supernatant contained only III, but the particulate fraction had I and III. Leucine-specific aminotransferase, r which was f o u n d in rat liver, and is described in article [113], was not f o u n d in hog organs. Various rat tissues (heart, kidney, adipose tissue, spleen, testis, a n d skeletal muscle) contained only type I enzyme. Liver c o n t a i n e d both I and the leucinespecific enzyme, while rat brain contained type III e n z y m e t o g e t h e r with a trace a m o u n t o f e n z y m e I. Recently, rat ascites h e p a t o m a cells (AH-130, a n d Yoshida sarcoma) were f o u n d to contain type III e n z y m e t o g e t h e r with a lesser a m o u n t o f e n z y m e I. s eFor convenience in comparative studies, the type of enzyme found in hog heart supernatant was named enzyme I, and the enzyme in hog brain supernatant was called enzyme III. The values given are based on the differential elution on DEAE-chromatography. rK. Aki, K. Ogawa, and A. Ichihara, Biochim. Biophys. ,4cta 159, 276 (1968). 8K. Ogawa and A. Ichihara, Proc. Syrup. Chem. Physiol. Pathol. 7, 167 (1967).
[113] Leucine Aminotransferase 1 (Rat Liver) By K. AKI and A. ICHIHARA
L-Leucine + a-ketoglutarate ~ a-ketoisocaproate + L-glutamate Leucine-specific a m i n o t r a n s f e r a s e was isolated f r o m rat liver? a This e n z y m e was eluted by 0.18 M p h o s p h a t e buffer f r o m a DEAE-cellulose column. T h e various properties o f the e n z y m e were quite different f r o m those o f the b r a n c h e d - c h a i n amino acid a m i n o t r a n s f e r a s e f o u n d in o t h e r tissues. T h e n a m e "leucine transaminase" o r "leucine aminotransferase" has been used often for the branched-chain amino acid aminotransferases described in the previous articles. However, use o f this n a m e should be limited to the e n z y m e described in this article.
Assay Method T h e assay m e t h o d o f e n z y m e activity is very similar to those described in article [111], 2 e x c e p t that 2 - m e r c a p t o e t h a n o l was omitted 1EC 2.6.1.6; L-ieucine: 2-oxoglutarate aminotransferase. I"K. Aki, K. Ogawa and A. Ichihara, Biachim. Biophys. Acta 159, 276 (1968). ~K. Aki and A. Ichihara, see this volume [111].
[ 113]
LEUCINE AMINOTRANSFERASE
815
from the reaction mixture. Owing to the low enzyme activity in the crude homogenate, the mixture should be incubated for at least 1 hour. Calculation of Enzyme Activity. Rat liver contains two aminotransferase activities for leucine; one is active for all branched-chain amino acids, and the other is specific for leucine. Therefore, the activities in the crude homogenate are calculated by assuming that the activity for valine (or isoleucine) represents that of the branched-chain amino acid aminotransferase and the difference between the activity for leucine and that for valine (or isoleucine) is the activity of the leucine aminotransferase. This difference, however, is not the activity at maximal velocity of the leucine aminotransferase, because the Km of the enzyme for leucine is very high and solubility of leucine is low, so that it is difficult to add a sufficient amount to saturate the enzyme. Therefore, the Vmax must be calculated from the activities at various concentrations of the substrate; routinely the observed activity was multiplied by a factor of 5.5 to obtain Vmax.
Definition of Unit and Specific Activity. One unit of enzyme activity was defined as the amount of enzyme which catalyzed transamination of 1 micromole of substrate per minute. Specific activity was expressed as units per milligram of protein. Protein was measured by the method of Lowry et al.a
Purification Procedure All procedures were carried out at 4 °. All buffers used in the purification ("supplemented" buffers) were supplemented with 2 × 10 -a M 2-mercaptoethanol, 1 x 10 -3 M EDTA, and 1 × l0 -5 M pyridoxal phosphate. For column chromatography, the same supplements were added to the buffers, with the exception of pyridoxal phosphate, which was omitted. In each chromatography, the column was equilibrated with the buffer used for previous dialysis of the enzyme (except for pyridoxal phosphate). Step 1. Crude Extract of Rat Liver. About 30 livers were homogenized in 0.25 M sucrose solution, and the supernatant was obtained by the method of Hogeboom. 4 The supernatant was fractionated with solid ammonium sulfate at pH 7.4 in the presence of 1 × 10 -s M EDTA. The material precipitated between 30 and 75% saturation was dissolved in a small amount of supplemented 5 × 10 -3 M potassium phosphate aO. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see also Vol. III [73]. *G. H. Hogeboom, Vol. I [3].
816
[113]
BRANCHED-CHAIN AMINO ACIDS
buffer (pH 7.8). T h e enzyme preparation was dialyzed against the same buffer. Step 2. DEAE-Cellulose Column Chromatography. The dialyzed enzyme was applied on a DEAE-cellulose column (1.2 x 30 cm). T h e enzyme was eluted with a linear concentration gradient of supplemented potassium phosphate buffer (pH 7.8) from 5 X 10 -3 M to 3 x 10 -1 M (500 ml in each flask). The activity for leucine appeared in the nonadsorbed fraction and in the 0.02 M and 0.18 M buffer fractions. The activity in the nonadsorbed fraction was very small, and the activity in the fraction with 0.02 M buffer was identified as that of the enzyme found in hog heart described in article [ 110]. The active fraction, eluted after 380 ml in a total volume of 220 ml, (0.18 M buffer) was collected and concentrated by ammonium sulfate fractionation with 75% saturation. Step 3. Hydroxylapatite Column Chromatography. Enzyme was dialyzed against supplemented 1 x 10 -2 M potassium phosphate buffer (pH 6.8) and then applied on a hydroxylapatite s column (1.2 x 30 cm). The enzyme was eluted with a linear concentration gradient of supplemented potassium phosphate buffer (pH 7.8) from 2 x 10 -t M to 1.0 M (500 ml in each flask). The active fraction appearing after 150 ml at a concentration of about 0.4 M was collected (total volume 160 ml), concentrated by ammonium sulfate precipitation, and dissolved in supplemented 5 x 10 -2 M potassium pyrophosphate buffer (pH 8.2). Step 4. Sephadex G-200 Column. This enzyme preparation was applied to a Sephadex G-200 column (2.3 X 100 cm). Active fractions were collected after 180 ml, in a total volume of 120 ml. These were concentrated by ammonium sulfate precipitation and dialyzed against supPURIFICATIONOF LEUCINEAMINOTRANSFERASE FROM RAT LIVER
Fraction
Total protein (mg)
Total activity (u nits)
Specific activity (units/mg protein)
Yield (% )
Crude extract Ammonium sulfate DEAE-celluiose Hydroxylapatite Sephadex G-200
18,250 4,700 380 27 3
31.0 23.5 17.6 9.8 3.1
0.002 0.005 0.046 0.36 1.03
100 76 57 32 10
5A. Tiselius, S. Hjert~n, and O. Levin, Arch. Biochem. Biophys. 65, 132 (1956); see also Vol. V [2].
[ 113]
LEUCINE AMINOTRANSFERASE
817
plemented 5 x 10 -~ M potassium pyrophosphate buffer (pH 8.2). The final preparation showed a 600-fold purification (see the table).
Properties The enzyme was active only with leucine and a-ketoglutarate. Valine, isoleucine, and other amino acids did not serve as substrates. The Km for leucine was very high (2.5 × 10 -2 M); and for ot-ketoglutarate, 6.5 × 10 -5 M. The Km for pyridoxal phosphate was 4 × 10 -e M. 5a 2-Mercaptoethanol did not affect activity, but PCMB at a concentration of 1 × 10 -3 M inhibited activity completely. The optimum pH was 8.7. One-quarter of the activity in the crude homogenate was present in the supernatant and the rest in the mitochondria. Administration of glucocorticoid or a high protein diet to rats induced a severalfold increase in activity in the supernatant, but not in the mitochondrial fraction, e'¢ This enzyme was not found in rat fetal liver, but it appeared after birth and increased to the adult level within 1 week. s Distribution of aminotransferases for branched-chain amino acids in various rat tissues showed that major tissues (heart, kidney, adipose, spleen, testis, and skeletal muscle) contained only the enzyme found in pig heart, which was described in article [110]. Liver contained leucinespecific enzyme as well, and brain had the third type of enzyme, described in article [ 112]. 9 It is interesting that rat ascites hepatoma cells (AH-130 and Yoshida sarcoma) contained this brain-type enzyme with a lesser amount of heart-type enzyme and that there was no leucine aminotransferase. 10
5aSince the purification of the enzyme was carried out in the presence of pyridoxal phosphate, the final preparation was dialyzed against buffers containing hydroxylamine prior to determination of the Ks value for pyridoxal phosphate (see reference la). 8A. Ichihara, H. Takahashi, K. Aki, and A. Shirai, Biochem. Biophys. Res. Commun. 26, 674 (1967). ~A. lchihara, K. Aki, K. Ogawa, A. Shirai, and H. Takahashi, in "International Symposium on Pyridoxal Enzymes" (K. Yamada, N. Katunuma, and H. Wada, eds.), p. 165. Maruzen, Tokyo, 1968. 8A. Ichihara and H. Takahashi, Biochim. Biophys. Acta 167, 274 (1968). 9K. Aki and A. Ichihara, see this volume [112]. l°K. Ogawa and A. Ichihara, Proc. Syrup. Chem. Physiol. Pathol. 7, 167 (1967).
814
[ 113]
T h e s e two types o f e n z y m e e were f o u n d in the supernatants o f various hog tissues, such as h e a r t (I: 85%, III: 0), brain (I: 0, III: 96%), liver (I: 10%, III: 70%), and kidney (I: 30%, III: 60%). Studies on the subcellular distribution o f enzymes in brain also revealed that the supernatant contained only III, but the particulate fraction had I and III. Leucine-specific aminotransferase, r which was f o u n d in rat liver, and is described in article [113], was not f o u n d in hog organs. Various rat tissues (heart, kidney, adipose tissue, spleen, testis, a n d skeletal muscle) contained only type I enzyme. Liver c o n t a i n e d both I and the leucinespecific enzyme, while rat brain contained type III e n z y m e t o g e t h e r with a trace a m o u n t o f e n z y m e I. Recently, rat ascites h e p a t o m a cells (AH-130, a n d Yoshida sarcoma) were f o u n d to contain type III e n z y m e t o g e t h e r with a lesser a m o u n t o f e n z y m e I. s eFor convenience in comparative studies, the type of enzyme found in hog heart supernatant was named enzyme I, and the enzyme in hog brain supernatant was called enzyme III. The values given are based on the differential elution on DEAE-chromatography. rK. Aki, K. Ogawa, and A. Ichihara, Biochim. Biophys. ,4cta 159, 276 (1968). 8K. Ogawa and A. Ichihara, Proc. Syrup. Chem. Physiol. Pathol. 7, 167 (1967).
[113] Leucine Aminotransferase 1 (Rat Liver) By K. AKI and A. ICHIHARA
L-Leucine + a-ketoglutarate ~ a-ketoisocaproate + L-glutamate Leucine-specific a m i n o t r a n s f e r a s e was isolated f r o m rat liver? a This e n z y m e was eluted by 0.18 M p h o s p h a t e buffer f r o m a DEAE-cellulose column. T h e various properties o f the e n z y m e were quite different f r o m those o f the b r a n c h e d - c h a i n amino acid a m i n o t r a n s f e r a s e f o u n d in o t h e r tissues. T h e n a m e "leucine transaminase" o r "leucine aminotransferase" has been used often for the branched-chain amino acid aminotransferases described in the previous articles. However, use o f this n a m e should be limited to the e n z y m e described in this article.
Assay Method T h e assay m e t h o d o f e n z y m e activity is very similar to those described in article [111], 2 e x c e p t that 2 - m e r c a p t o e t h a n o l was omitted 1EC 2.6.1.6; L-ieucine: 2-oxoglutarate aminotransferase. I"K. Aki, K. Ogawa and A. Ichihara, Biachim. Biophys. Acta 159, 276 (1968). ~K. Aki and A. Ichihara, see this volume [111].
[ 113]
LEUCINE AMINOTRANSFERASE
815
from the reaction mixture. Owing to the low enzyme activity in the crude homogenate, the mixture should be incubated for at least 1 hour. Calculation of Enzyme Activity. Rat liver contains two aminotransferase activities for leucine; one is active for all branched-chain amino acids, and the other is specific for leucine. Therefore, the activities in the crude homogenate are calculated by assuming that the activity for valine (or isoleucine) represents that of the branched-chain amino acid aminotransferase and the difference between the activity for leucine and that for valine (or isoleucine) is the activity of the leucine aminotransferase. This difference, however, is not the activity at maximal velocity of the leucine aminotransferase, because the Km of the enzyme for leucine is very high and solubility of leucine is low, so that it is difficult to add a sufficient amount to saturate the enzyme. Therefore, the Vmax must be calculated from the activities at various concentrations of the substrate; routinely the observed activity was multiplied by a factor of 5.5 to obtain Vmax.
Definition of Unit and Specific Activity. One unit of enzyme activity was defined as the amount of enzyme which catalyzed transamination of 1 micromole of substrate per minute. Specific activity was expressed as units per milligram of protein. Protein was measured by the method of Lowry et al.a
Purification Procedure All procedures were carried out at 4 °. All buffers used in the purification ("supplemented" buffers) were supplemented with 2 × 10 -a M 2-mercaptoethanol, 1 x 10 -3 M EDTA, and 1 × l0 -5 M pyridoxal phosphate. For column chromatography, the same supplements were added to the buffers, with the exception of pyridoxal phosphate, which was omitted. In each chromatography, the column was equilibrated with the buffer used for previous dialysis of the enzyme (except for pyridoxal phosphate). Step 1. Crude Extract of Rat Liver. About 30 livers were homogenized in 0.25 M sucrose solution, and the supernatant was obtained by the method of Hogeboom. 4 The supernatant was fractionated with solid ammonium sulfate at pH 7.4 in the presence of 1 × 10 -s M EDTA. The material precipitated between 30 and 75% saturation was dissolved in a small amount of supplemented 5 × 10 -3 M potassium phosphate aO. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see also Vol. III [73]. *G. H. Hogeboom, Vol. I [3].
816
[113]
BRANCHED-CHAIN AMINO ACIDS
buffer (pH 7.8). T h e enzyme preparation was dialyzed against the same buffer. Step 2. DEAE-Cellulose Column Chromatography. The dialyzed enzyme was applied on a DEAE-cellulose column (1.2 x 30 cm). T h e enzyme was eluted with a linear concentration gradient of supplemented potassium phosphate buffer (pH 7.8) from 5 X 10 -3 M to 3 x 10 -1 M (500 ml in each flask). The activity for leucine appeared in the nonadsorbed fraction and in the 0.02 M and 0.18 M buffer fractions. The activity in the nonadsorbed fraction was very small, and the activity in the fraction with 0.02 M buffer was identified as that of the enzyme found in hog heart described in article [ 110]. The active fraction, eluted after 380 ml in a total volume of 220 ml, (0.18 M buffer) was collected and concentrated by ammonium sulfate fractionation with 75% saturation. Step 3. Hydroxylapatite Column Chromatography. Enzyme was dialyzed against supplemented 1 x 10 -2 M potassium phosphate buffer (pH 6.8) and then applied on a hydroxylapatite s column (1.2 x 30 cm). The enzyme was eluted with a linear concentration gradient of supplemented potassium phosphate buffer (pH 7.8) from 2 x 10 -t M to 1.0 M (500 ml in each flask). The active fraction appearing after 150 ml at a concentration of about 0.4 M was collected (total volume 160 ml), concentrated by ammonium sulfate precipitation, and dissolved in supplemented 5 x 10 -2 M potassium pyrophosphate buffer (pH 8.2). Step 4. Sephadex G-200 Column. This enzyme preparation was applied to a Sephadex G-200 column (2.3 X 100 cm). Active fractions were collected after 180 ml, in a total volume of 120 ml. These were concentrated by ammonium sulfate precipitation and dialyzed against supPURIFICATIONOF LEUCINEAMINOTRANSFERASE FROM RAT LIVER
Fraction
Total protein (mg)
Total activity (u nits)
Specific activity (units/mg protein)
Yield (% )
Crude extract Ammonium sulfate DEAE-celluiose Hydroxylapatite Sephadex G-200
18,250 4,700 380 27 3
31.0 23.5 17.6 9.8 3.1
0.002 0.005 0.046 0.36 1.03
100 76 57 32 10
5A. Tiselius, S. Hjert~n, and O. Levin, Arch. Biochem. Biophys. 65, 132 (1956); see also Vol. V [2].
[ 113]
LEUCINE AMINOTRANSFERASE
817
plemented 5 x 10 -~ M potassium pyrophosphate buffer (pH 8.2). The final preparation showed a 600-fold purification (see the table).
Properties The enzyme was active only with leucine and a-ketoglutarate. Valine, isoleucine, and other amino acids did not serve as substrates. The Km for leucine was very high (2.5 × 10 -2 M); and for ot-ketoglutarate, 6.5 × 10 -5 M. The Km for pyridoxal phosphate was 4 × 10 -e M. 5a 2-Mercaptoethanol did not affect activity, but PCMB at a concentration of 1 × 10 -3 M inhibited activity completely. The optimum pH was 8.7. One-quarter of the activity in the crude homogenate was present in the supernatant and the rest in the mitochondria. Administration of glucocorticoid or a high protein diet to rats induced a severalfold increase in activity in the supernatant, but not in the mitochondrial fraction, e'¢ This enzyme was not found in rat fetal liver, but it appeared after birth and increased to the adult level within 1 week. s Distribution of aminotransferases for branched-chain amino acids in various rat tissues showed that major tissues (heart, kidney, adipose, spleen, testis, and skeletal muscle) contained only the enzyme found in pig heart, which was described in article [110]. Liver contained leucinespecific enzyme as well, and brain had the third type of enzyme, described in article [ 112]. 9 It is interesting that rat ascites hepatoma cells (AH-130 and Yoshida sarcoma) contained this brain-type enzyme with a lesser amount of heart-type enzyme and that there was no leucine aminotransferase. 10
5aSince the purification of the enzyme was carried out in the presence of pyridoxal phosphate, the final preparation was dialyzed against buffers containing hydroxylamine prior to determination of the Ks value for pyridoxal phosphate (see reference la). 8A. Ichihara, H. Takahashi, K. Aki, and A. Shirai, Biochem. Biophys. Res. Commun. 26, 674 (1967). ~A. lchihara, K. Aki, K. Ogawa, A. Shirai, and H. Takahashi, in "International Symposium on Pyridoxal Enzymes" (K. Yamada, N. Katunuma, and H. Wada, eds.), p. 165. Maruzen, Tokyo, 1968. 8A. Ichihara and H. Takahashi, Biochim. Biophys. Acta 167, 274 (1968). 9K. Aki and A. Ichihara, see this volume [112]. l°K. Ogawa and A. Ichihara, Proc. Syrup. Chem. Physiol. Pathol. 7, 167 (1967).