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[111] Branched-chain amino acid aminotransferase (pig heart mitochondria)
[ 111]
AMINOTRANSFERASE (PIG HEART, MITOCHONDRIA)
807
T h e r e is a separate enzyme with comparable specificity in mitochondria, t5
Escherichia coli) 4
14D. Rudman and A. Meister,J. Biol. Chem. 200, 591 (1953). tSK. Aki, K. Ogawa, A. Shirai and A. Ichihara, J. Biochem. (Tokyo) 62,610 (1967); see also this volume [ 111 ].
[111] B r a n c h e d - C h a i n
Amino Acid Aminotransferase
(Pig Heart Mitochondria) 1
By K. AKI and
A. ICHIHARA
Branched-chain L-amino acids (valine, leucine, and isoleucine) + tr-ketoglutarate branched-chain ,t-keto acids (a-ketoisovalerate, a-ketoisocaproate, and a-keto-/~-methylvalerate) + L-glutamate
Assay Method
Principle. T h e branched-chain a-keto acid formed was converted to the 2,4-dinitrophenylhydrazone, which was selectively extracted with toluene. T h e hydrazone was then transferred to sodium carbonate solution; the color was developed by addition of NaOH and measured at 440 mtt. This method TM is based on Friedemann and Haugen's method 2 with slight modifications, a Although cyclohexane is a more specific solvent for extraction of the hydrazone of a-ketoisocaproate, 4 toluene is routinely used because all hydrazones of these branched chain a-keto acids can be extracted equally well by toluene and contamination with the hydrazone of a-ketoglutarate is essentially negligible. Reagents Branched-chain L-amino acid, 0.1 M a-Ketoglutarate, 0.1 M. Adjust pH to 7.4 with sodium hydroxide solution and store in refrigerator. Pyridoxal phosphate, 0.002 M 1For the preparation of the branched-chain amino acid transferase from the soluble fraction of pig heart, see article [110]. The preparation from hog brain supernatant is described in article [112]. The leucine-specific aminotransferase from rat liver is described in article [113]. taA. Ichihara and E. Koyama, J. Biochem. 59, 160 (1966). 2T. E. Friedemann, Vol. III [66]. SH. Wada and E. E. Snell, J. Biol. Chem. 237, 127 (1962). 4R. T. Taylor and W. T. Jenkins, J. Biol. Chem. 241, 4391 (1966).
808
BRANCHED-CHAIN AMINO ACIDS
[111]
2-Mercaptoethanol, 0.25 M. Prepare daily. Potassium pyrophosphate buffer, 0.1 M (in pyrophosphate), pH 8.2 Trichloroacetic acid, 10% 2,4-Dinitrophenylhydrazine, 0.5% in 2 N HCI Toluene HC1, 0.5 N Sodium carbonate, 10% NaOH, 1.5 N Procedure. The reaction mixture contains in a total volume of 1.5 ml (in a small test tube): 0.3 ml of L-amino acid, 0.05 ml of ot-ketoglutarate, 0.05 ml of pyridoxal phosphate, 0.1 ml of 2-mercaptoethanol, 0.5 ml of pyrophosphate buffer, and an appropriate amount of enzyme. The reaction mixture is preincubated for 5 minutes at 37 °, and the reaction is started by addition of either substrate. The blank contains the reaction mixture without added amino acid. The incubation is usually carried out for 10 minutes, and the reaction is stopped by addition of 1.5 ml of trichloroacetic acid. The mixture is centrifuged, if necessary, and the supernatant is transferred to a test tube with a glass stopper and incubated at 25 ° for 5 minutes; 2 ml of 2,4-dinitrophenylhydrazine is added, and the mixture is incubated for another 5 minutes. Then 5 ml of toluene is added and the mixture is shaken vigorously for 2 minutes. The aqueous layer which separates after several minutes is carefully sucked off with a capillary pipette, and the toluene layer is washed in the same way with 5 ml of 0.5 N HCI. The mixture is centrifuged in a conical centrifuge tube, and 2 ml of toluene layer is transferred to a test tube and mixed with 2 ml of carbonate solution. The test tube is shaken vigorously to mix the two phases well; after several minutes, 1.5 ml of the carbonate layer is transferred to another test tube and 1.5 ml of N a O H is added. The color thus formed is measured at 440 m#. The hydrazones of the three branched-chain ot-keto acids had similar absorption maxima and extinction coefficients at 440 m#. Definition of Unit. One unit of activity was defined as the amount of enzyme which catalyzed conversion of 1 micromole of branched-chain amino acid to the corresponding keto acid per minute. Protein was measured by the method of Lowry et al. 5
Purification Procedure Considerable activity for transamination of branched-chain amino acids was found in both the supernatant and the mitochondrial fracsO. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see Vol. III [73].
[111]
AMINOTRANSFERASE (PIG HEART, MITOCHONDRIA)
809
tions, a'4'6'7 The method for purification of the mitochondrial enzyme from hog heart muscle has been described. 8 All procedures were carried out at 4 °. All buffers used for dialysis ("supplemented buffer") were supplemented with 2 × 10-3 M 2-mercaptoethanol, 1 × 10-3 M EDTA, and 1 × 10-s M pyridoxal phosphate. For column chromatography, the same supplements were added to the buffers, with the exception of pyridoxal phosphate, which was omitted. Step 1. Solubilization of Pig Heart Mitochondria. Pig heart mitochondria were isolated by the method of Hogeboom. 9 T h e mitochondrial fraction, containing 3.3 g of protein, was washed twice with 0.25 M sucrose solution and disrupted by homogenization in 100 ml of 0.05 M potassium pyrophosphate buffer (pH 8.6) in a Waring blendor for 5 minutes. The homogenate was centrifuged; the precipitate was washed with 50 ml of the same buffer. Solubilization can also be achieved by treatment with 0.5% deoxycholate.8 The combined supernatant was fractionated with solid ammonium sulfate. EDTA was added to the supernatant at a concentration of 1 × 10-3 M. The pH was adjusted to 7.2. The material precipitated between 30 and 50% saturation was dissolved in a small amount of 5 × 10-3 M potassium pyrophosphate buffer (pH 8.6) containing 2 × 10-3 M 2-mercaptoethanol, 1 × 10-3 M EDTA and 1 × 10-5 M pyridoxal phosphate. T h e ammonium sulfate precipitate was dialyzed overnight against the same buffer. Step 2. DEAE-Cellulose Column Chromatography. The dialyzed enzyme was then applied on a DEAE-cellulose column (1.2 × 30 cm) previously equilibrated with the same supplemented buffer (without pyridoxal phosphate). Enzyme was eluted with a linear concentration gradient of the same buffer from 5 × 10-3 M to 5 x 10-2 M (500 ml in each flask). Activity appeared, after 150 ml of eluent, at a concentration of 0.02 M buffer, and the active fractions (150 ml) were collected and concentrated by precipitation with 50% saturation of ammonium sulfate. Step 3. Hydroxylapatite Column Chromatography. The enzyme was dialyzed against supplemented 1 × 10 -3 M potassium phosphate buffer (pH 6.8) and applied on a hydroxylapatitea° column (1.2 x 30 cm) equilibrated with the same buffer. The column was washed with 200 ml of supplemented 4 × 10 -2 M potassium phosphate buffer (pH 7.8), and 6E. V. Rowsell, Biochem.J. 64, 235 (1956). ~K. H. B~/ssler and C. H. Hammar, Biochem. Z. 330, 555 (1958). SK. Aki, K. Ogawa, A. Shirai and A. Ichihara, J. Biochem. 62,610 (1967). 9G. H. Hogeboom, Vol. I [3]. t°A. Tiselius, S. Hjert~n, and O. Levin, Arch. Biochem. Biophys. 65, 132 (1956); see also Vol. V [2].
810
BRANCHED-CHAIN AMINO ACIDS
[111 ]
then the enzyme was eluted successively with 6 x 10 -~ M and 8 x 10 -z M buffers (200 ml each). Step 4. SephadexG-I O0 Column.Active fractions were present in eluates at both concentrations, and the enzyme was concentrated by precipitation with ammonium sulfate. It was then applied on a Sephadex G-100 column (2.3 x 100 cm) equilibrated with supplemented 5 x 10 -2 M potassium phosphate buffer (pH 7.8) and eluted with same buffer. T h e active fraction was eluted after 180 ml, in a total volume of 130 ml; this was collected, concentrated by precipitation with ammonium sulfate, and dialyzed against the same buffer. A summary of the purification procedure is given in the table. Properties s
Homogeneity. The purified enzyme was shown to be a single protein by electrophoresis on an acrylamide gel plate, by the immunodouble diffusion test, and by ultracentrifugation. The h0,w value was 5.0 S, which is very similar to that of the supernatant enzyme which has a molecular weight of 75,000.11 Properties in Common with Those of the Supernatant Enzyme. Enzyme activity was limited to valine, leucine, isoleucine, and a-ketoglutarate. Norvaline and norleucine were far less effective substrates. The optimal pH was 8.6 and activity was enhanced severalfold by addition of 20 mM 2-mercaptoethanol. These properties are very similar to those of the supernatant enzyme? a'n The purification procedure was carried out in the presence of pyridoxal phosphate. T h e requirement for pyridoxal phosphate and the Ks value for this cofactor may be determined after dialysis against buffers containing hydroxylamine?a Properties Distinct from Those of the Supernatant Enzyme. Attempts to separate the two enzymes by the usual methods of enzyme purification were unsuccessful, but significant separation was achieved on a Sephadex G-100 column. Electrophoresis on acrylamide gel revealed that the mitochondrial enzyme moved to the anode faster than the supernatant enzyme. The Ks values for substrates and cofactor were (mM): 1.3 for valine, 0.4 for leucine and isoleucine, 2.2 for a-ketoglutarate and 0.014 for pyridoxal phosphate. These values were very different from those of the supernatant enzyme?* Another difference in the properties of the two enzymes is seen in their stabilities. Thus, the supernatant enzyme retained almost full activity after treatment at 60 ° for 5 minutes, while the mitochondrial UR. T. Taylor and W. T. Jenkins, J. Biol. Chem. 241, 4396 (1966); see this volume [110].
[ 112]
AMINOTRANSFERASE(HOG BRAIN SUPERNATANT)
81 1
enzyme lost about 50% o f its activity. It should also be mentioned that the mitochondrial enzyme lost activity almost completely when stored at 4 ° for 48 hours without added 2-mercaptoethanol, while the supernatant enzyme was stable under these conditions. In the presence of 0.01 M 2-mercaptoethanol both enzymes were stable at this temperature, but they became very unstable when frozen. However, they were stable in the frozen state in the absence of 2-mercaptoethanol. PURIFICATION OF BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE FROM PIG HEART MITOCHONDRIA
Fraction
Total protein (mg)
Total activity for leucine (units)
Specific activity for leucine (units/mg protein)
3355 2103 1510 53 4 3
67.1 63.1 60.4 30.2 18.8 16.9
0.02 0.03 0.04 0.57 5.1 5.1
Mitochondria Crude extract A m m o n i u m sulfate DEAE-cellulose Hydroxylapatite
Sephadex G- 100
Relative activitya Valine Isoleucine 0.7 0.5 0.5 -
1.4 0.9 1.1 -
a Activity with leucine ---- 1.
[112] Branched-Chain
Amino
Acid Aminotransferase
(Hog Brain Supernatant)
By K .
AKI and
A. ICHIHARA
Branched-chain-L-amino acids (valine, leucine, and isoleucine) + a-ketoglutarate branched-chain a-keto acids (a-ketoisovalerate, ~,-ketoisocaproate, and a-keto-fl-methylvalerate) + I.-glutamate
A branched-chain amino acid transaminase was isolated from hog brain supernatant, and its properties were very much like those of the heart enzyme. 1 However, their chromatographic behavior on DEAEcellulose, their molecular weights, and immunochemical relationships were quite different, z 1K. Aki and A. Ichihara, see this volume [111]. 2K. Aki, A. Yokojima, and A. Ichihara,J. Biochem. 6 5 , 5 3 9 (1969).
AMINOTRANSFERASE (PIG HEART, MITOCHONDRIA)
807
T h e r e is a separate enzyme with comparable specificity in mitochondria, t5
Escherichia coli) 4
14D. Rudman and A. Meister,J. Biol. Chem. 200, 591 (1953). tSK. Aki, K. Ogawa, A. Shirai and A. Ichihara, J. Biochem. (Tokyo) 62,610 (1967); see also this volume [ 111 ].
[111] B r a n c h e d - C h a i n
Amino Acid Aminotransferase
(Pig Heart Mitochondria) 1
By K. AKI and
A. ICHIHARA
Branched-chain L-amino acids (valine, leucine, and isoleucine) + tr-ketoglutarate branched-chain ,t-keto acids (a-ketoisovalerate, a-ketoisocaproate, and a-keto-/~-methylvalerate) + L-glutamate
Assay Method
Principle. T h e branched-chain a-keto acid formed was converted to the 2,4-dinitrophenylhydrazone, which was selectively extracted with toluene. T h e hydrazone was then transferred to sodium carbonate solution; the color was developed by addition of NaOH and measured at 440 mtt. This method TM is based on Friedemann and Haugen's method 2 with slight modifications, a Although cyclohexane is a more specific solvent for extraction of the hydrazone of a-ketoisocaproate, 4 toluene is routinely used because all hydrazones of these branched chain a-keto acids can be extracted equally well by toluene and contamination with the hydrazone of a-ketoglutarate is essentially negligible. Reagents Branched-chain L-amino acid, 0.1 M a-Ketoglutarate, 0.1 M. Adjust pH to 7.4 with sodium hydroxide solution and store in refrigerator. Pyridoxal phosphate, 0.002 M 1For the preparation of the branched-chain amino acid transferase from the soluble fraction of pig heart, see article [110]. The preparation from hog brain supernatant is described in article [112]. The leucine-specific aminotransferase from rat liver is described in article [113]. taA. Ichihara and E. Koyama, J. Biochem. 59, 160 (1966). 2T. E. Friedemann, Vol. III [66]. SH. Wada and E. E. Snell, J. Biol. Chem. 237, 127 (1962). 4R. T. Taylor and W. T. Jenkins, J. Biol. Chem. 241, 4391 (1966).
808
BRANCHED-CHAIN AMINO ACIDS
[111]
2-Mercaptoethanol, 0.25 M. Prepare daily. Potassium pyrophosphate buffer, 0.1 M (in pyrophosphate), pH 8.2 Trichloroacetic acid, 10% 2,4-Dinitrophenylhydrazine, 0.5% in 2 N HCI Toluene HC1, 0.5 N Sodium carbonate, 10% NaOH, 1.5 N Procedure. The reaction mixture contains in a total volume of 1.5 ml (in a small test tube): 0.3 ml of L-amino acid, 0.05 ml of ot-ketoglutarate, 0.05 ml of pyridoxal phosphate, 0.1 ml of 2-mercaptoethanol, 0.5 ml of pyrophosphate buffer, and an appropriate amount of enzyme. The reaction mixture is preincubated for 5 minutes at 37 °, and the reaction is started by addition of either substrate. The blank contains the reaction mixture without added amino acid. The incubation is usually carried out for 10 minutes, and the reaction is stopped by addition of 1.5 ml of trichloroacetic acid. The mixture is centrifuged, if necessary, and the supernatant is transferred to a test tube with a glass stopper and incubated at 25 ° for 5 minutes; 2 ml of 2,4-dinitrophenylhydrazine is added, and the mixture is incubated for another 5 minutes. Then 5 ml of toluene is added and the mixture is shaken vigorously for 2 minutes. The aqueous layer which separates after several minutes is carefully sucked off with a capillary pipette, and the toluene layer is washed in the same way with 5 ml of 0.5 N HCI. The mixture is centrifuged in a conical centrifuge tube, and 2 ml of toluene layer is transferred to a test tube and mixed with 2 ml of carbonate solution. The test tube is shaken vigorously to mix the two phases well; after several minutes, 1.5 ml of the carbonate layer is transferred to another test tube and 1.5 ml of N a O H is added. The color thus formed is measured at 440 m#. The hydrazones of the three branched-chain ot-keto acids had similar absorption maxima and extinction coefficients at 440 m#. Definition of Unit. One unit of activity was defined as the amount of enzyme which catalyzed conversion of 1 micromole of branched-chain amino acid to the corresponding keto acid per minute. Protein was measured by the method of Lowry et al. 5
Purification Procedure Considerable activity for transamination of branched-chain amino acids was found in both the supernatant and the mitochondrial fracsO. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see Vol. III [73].
[111]
AMINOTRANSFERASE (PIG HEART, MITOCHONDRIA)
809
tions, a'4'6'7 The method for purification of the mitochondrial enzyme from hog heart muscle has been described. 8 All procedures were carried out at 4 °. All buffers used for dialysis ("supplemented buffer") were supplemented with 2 × 10-3 M 2-mercaptoethanol, 1 × 10-3 M EDTA, and 1 × 10-s M pyridoxal phosphate. For column chromatography, the same supplements were added to the buffers, with the exception of pyridoxal phosphate, which was omitted. Step 1. Solubilization of Pig Heart Mitochondria. Pig heart mitochondria were isolated by the method of Hogeboom. 9 T h e mitochondrial fraction, containing 3.3 g of protein, was washed twice with 0.25 M sucrose solution and disrupted by homogenization in 100 ml of 0.05 M potassium pyrophosphate buffer (pH 8.6) in a Waring blendor for 5 minutes. The homogenate was centrifuged; the precipitate was washed with 50 ml of the same buffer. Solubilization can also be achieved by treatment with 0.5% deoxycholate.8 The combined supernatant was fractionated with solid ammonium sulfate. EDTA was added to the supernatant at a concentration of 1 × 10-3 M. The pH was adjusted to 7.2. The material precipitated between 30 and 50% saturation was dissolved in a small amount of 5 × 10-3 M potassium pyrophosphate buffer (pH 8.6) containing 2 × 10-3 M 2-mercaptoethanol, 1 × 10-3 M EDTA and 1 × 10-5 M pyridoxal phosphate. T h e ammonium sulfate precipitate was dialyzed overnight against the same buffer. Step 2. DEAE-Cellulose Column Chromatography. The dialyzed enzyme was then applied on a DEAE-cellulose column (1.2 × 30 cm) previously equilibrated with the same supplemented buffer (without pyridoxal phosphate). Enzyme was eluted with a linear concentration gradient of the same buffer from 5 × 10-3 M to 5 x 10-2 M (500 ml in each flask). Activity appeared, after 150 ml of eluent, at a concentration of 0.02 M buffer, and the active fractions (150 ml) were collected and concentrated by precipitation with 50% saturation of ammonium sulfate. Step 3. Hydroxylapatite Column Chromatography. The enzyme was dialyzed against supplemented 1 × 10 -3 M potassium phosphate buffer (pH 6.8) and applied on a hydroxylapatitea° column (1.2 x 30 cm) equilibrated with the same buffer. The column was washed with 200 ml of supplemented 4 × 10 -2 M potassium phosphate buffer (pH 7.8), and 6E. V. Rowsell, Biochem.J. 64, 235 (1956). ~K. H. B~/ssler and C. H. Hammar, Biochem. Z. 330, 555 (1958). SK. Aki, K. Ogawa, A. Shirai and A. Ichihara, J. Biochem. 62,610 (1967). 9G. H. Hogeboom, Vol. I [3]. t°A. Tiselius, S. Hjert~n, and O. Levin, Arch. Biochem. Biophys. 65, 132 (1956); see also Vol. V [2].
810
BRANCHED-CHAIN AMINO ACIDS
[111 ]
then the enzyme was eluted successively with 6 x 10 -~ M and 8 x 10 -z M buffers (200 ml each). Step 4. SephadexG-I O0 Column.Active fractions were present in eluates at both concentrations, and the enzyme was concentrated by precipitation with ammonium sulfate. It was then applied on a Sephadex G-100 column (2.3 x 100 cm) equilibrated with supplemented 5 x 10 -2 M potassium phosphate buffer (pH 7.8) and eluted with same buffer. T h e active fraction was eluted after 180 ml, in a total volume of 130 ml; this was collected, concentrated by precipitation with ammonium sulfate, and dialyzed against the same buffer. A summary of the purification procedure is given in the table. Properties s
Homogeneity. The purified enzyme was shown to be a single protein by electrophoresis on an acrylamide gel plate, by the immunodouble diffusion test, and by ultracentrifugation. The h0,w value was 5.0 S, which is very similar to that of the supernatant enzyme which has a molecular weight of 75,000.11 Properties in Common with Those of the Supernatant Enzyme. Enzyme activity was limited to valine, leucine, isoleucine, and a-ketoglutarate. Norvaline and norleucine were far less effective substrates. The optimal pH was 8.6 and activity was enhanced severalfold by addition of 20 mM 2-mercaptoethanol. These properties are very similar to those of the supernatant enzyme? a'n The purification procedure was carried out in the presence of pyridoxal phosphate. T h e requirement for pyridoxal phosphate and the Ks value for this cofactor may be determined after dialysis against buffers containing hydroxylamine?a Properties Distinct from Those of the Supernatant Enzyme. Attempts to separate the two enzymes by the usual methods of enzyme purification were unsuccessful, but significant separation was achieved on a Sephadex G-100 column. Electrophoresis on acrylamide gel revealed that the mitochondrial enzyme moved to the anode faster than the supernatant enzyme. The Ks values for substrates and cofactor were (mM): 1.3 for valine, 0.4 for leucine and isoleucine, 2.2 for a-ketoglutarate and 0.014 for pyridoxal phosphate. These values were very different from those of the supernatant enzyme?* Another difference in the properties of the two enzymes is seen in their stabilities. Thus, the supernatant enzyme retained almost full activity after treatment at 60 ° for 5 minutes, while the mitochondrial UR. T. Taylor and W. T. Jenkins, J. Biol. Chem. 241, 4396 (1966); see this volume [110].
[ 112]
AMINOTRANSFERASE(HOG BRAIN SUPERNATANT)
81 1
enzyme lost about 50% o f its activity. It should also be mentioned that the mitochondrial enzyme lost activity almost completely when stored at 4 ° for 48 hours without added 2-mercaptoethanol, while the supernatant enzyme was stable under these conditions. In the presence of 0.01 M 2-mercaptoethanol both enzymes were stable at this temperature, but they became very unstable when frozen. However, they were stable in the frozen state in the absence of 2-mercaptoethanol. PURIFICATION OF BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE FROM PIG HEART MITOCHONDRIA
Fraction
Total protein (mg)
Total activity for leucine (units)
Specific activity for leucine (units/mg protein)
3355 2103 1510 53 4 3
67.1 63.1 60.4 30.2 18.8 16.9
0.02 0.03 0.04 0.57 5.1 5.1
Mitochondria Crude extract A m m o n i u m sulfate DEAE-cellulose Hydroxylapatite
Sephadex G- 100
Relative activitya Valine Isoleucine 0.7 0.5 0.5 -
1.4 0.9 1.1 -
a Activity with leucine ---- 1.
[112] Branched-Chain
Amino
Acid Aminotransferase
(Hog Brain Supernatant)
By K .
AKI and
A. ICHIHARA
Branched-chain-L-amino acids (valine, leucine, and isoleucine) + a-ketoglutarate branched-chain a-keto acids (a-ketoisovalerate, ~,-ketoisocaproate, and a-keto-fl-methylvalerate) + I.-glutamate
A branched-chain amino acid transaminase was isolated from hog brain supernatant, and its properties were very much like those of the heart enzyme. 1 However, their chromatographic behavior on DEAEcellulose, their molecular weights, and immunochemical relationships were quite different, z 1K. Aki and A. Ichihara, see this volume [111]. 2K. Aki, A. Yokojima, and A. Ichihara,J. Biochem. 6 5 , 5 3 9 (1969).