430
EPIMERASES AND ISOMERASES
[91]
[91] T r i o s e p h o s p h a t e I s o m e r a s e f r o m H u m a n a n d Horse Liver 1 By ROBERT SNYDER and EUN Woo LEE Glyceraldehyde 3-phosphate ~ dihydroxyacetone phosphate
Assay Method Principle. Triosephosphate isomerase converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate, a substrate for glycerol-Iphosphate dehydrogenase. NADH utilization during glycerol 1-phosphate production is stoichiometric with dihydroxyacetone phosphate production and is measured spectrophotometrically at 340 nm. Reagents DL-Glyceraldehyde 3-phosphate, 14.7 mM NADH, 4.2 mM Glycerol-l-phosphate dehydrogenase, 0.67 mg/ml (specific activity = 100-200 units/mg protein). Triethanolamine (2 m M ) - E D T A (5.4 mM) buffer, pH 7.9 Procedure. Place 0.1 ml of glyceraldehyde 3-phosphate, 0.1 ml of glycerol-l-phosphate dehydrogenase, 0.15 ml of NADH and 2.55 ml of triethanolamine-EDTA buffer in a 1-cm cuvette. Start the reaction by adding 0.1 ml of isomerase and follow the decrease in extinction at 340 nm. Definition of Unit and Specific Activity. One unit of triosephosphate isomerase is defined as the amount of enzyme that will isomerize 1 ~mole of D-glyceraldehyde 3-phosphate to dihydroxyacetone phosphate in 1 min. Specific activity is expressed as units per milligram of protein. Protein is measured by the method of Lowry et al. 2 Purification Procedure Either fresh or deep-frozen livers may be used. Step 1. Preparation of Water Extract. The liver is freed of connective 1E. W. Lee, J. A. Barriso, M. Pepe, and R. Snyder, Biochim. Biophys. Acta 242, 261 (1971). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, d. Biol. Chem. 193, 265 (1951).
[91]
T R I O S E P H O S P H A T E ISOMERASE FROM LIVER
431
tissue and blood vessels, diced, and put through a chilled meat grinder. The ground liver is then weighed and extracted with 2.5 parts of deionized water containing 0.05% EDTA for 4 hr with mechanical stirring at room temperature or overnight in the cold room at 3 ° . The water extract is obtained after centrifugation for 20 min at 8000 g. Step 2. Acetone Precipitation. Acetone is added in a fine stream to the mechanically stirred, ice cold, water extract over a 45-min period to a final concentration of 35% (v/v) (horse liver) or over a 60-min period to a final concentration of 50% (human liver). Stirring is continued for an additional 15-min period, and the resulting precipitate is removed by centrifugation for 20 min at 4000 g. Acetone is again added to the 35% (horse liver) or 50% (human liver) acetone extract over a 45-rain period to give a final concentration of 60%, and the resulting precipitate is collected after centrifugation as above. The residue is taken up in 8 volumes of deionized-water containing 0.05% EDTA and dialyzed against 0.05% EDTA overnight. Denatured protein formed during dialysis is discarded after centrifugation for 10 rain at 27,000 g. Step 3. Heat Treatment. The dialyzed solution is heated to 45 ° for 15 min, and the resulting precipitate of denatured protein is removed by centrifugation for 30 min at 16,000 g. The extract is again treated with heat at 50 ° for 30 min and centrifuged as above to remove denatured :protein. Step 4. Chromatography on QAE-Sephadex. The enzyme is next applied to a 2.3 X 28 cm QAE-Sephadex A-50 column equilibrated with 7 mM phosphate buffer, pH 7.8, at room temperature. Buffer is passed through the column until the extinction at 280 nm is no longer detected. Elution is performed with 0.1 M NaC1 in 7 mM phosphate buffer, pH 7.8. Most of the activity is found in the first (human liver) or second (horse liver) peak of the eluate. Step 5. Ammordum Sul]ate Precipitation. The enzyme eluted from the QAE-Sephadex column is successively dialyzed against 0.8 M, 1.3 M, and 2.6 M ammonium sulfate in 20 mM sodium acetate buffer, pH 5.3, for a total period of 2 days. The protein which is precipitated at 2.6 M ammonium sulfate is removed by centrifugation at 13,200 g for 1 hr. The supernatant, which contains most of the enzyme, is again dialyzed against 3.0 M and then 3.5 M ammonium sulfate in 20 mM sodium acetate buffer, pH 5.3 continuously with several changes of the ammonium sulfate solution in the cold room at 3 ° for 7 days. The resulting crystalline enzyrae (crude crystals) is collected after centrifugation as above. The "crude crystals" are dissolved by cautious, dropwise addition of a minimum amount of 0.05% EDTA solution at 3 °.
432
EPIMERASES
AND
[91]
ISOMERASES
v
8
if2
O
.2
d~ 8
O ~J
o
II
[91]
TRIOSEPHOSPHATE ISOMERASE FROM LIVER
433
TABLE II DISTRIBUTION OF TRIOSEPHOSPHATE ISOMERASE ACTIVITY AMONG ITS ISOZYMES
Isozymes 1 2 3
Rabbit muscle Horse liver Human liver (% of total (% of total (% of total activity) activity) activity) 22 44 24
4
6
50 38 12
----
--
--
4a
--
--
5 5a
4 --
---
6
--
--
62 -26 11
Step 6. Gel Filtration. The enzyme solution obtained from the preceding step is applied to a 2.5 X 40 cm column of Sephadex G-75 superfine equilibrated with 2 m M phosphate buffer, pH 7.8. The column is then washed with the same buffer until all the protein has passed through the column. Fractions of 8-10 ml are collected, and the enzyme is recovered after approximately 100-120 ml have passed through the column. A summary of the purification procedure is presented in Table I. Properties
Isozymes o] Horse and Human Liver Triosephosphate Isomerase. Upon polyacrylamide gel electrophoresis, horse and human liver triosephosphate isomerase can each be resolved into 3 isozymes. Table II shows the isozymes listed in order of decreasing electronegativity and the percentage of total activity for each. Rabbit muscle isomerase, which contains five isozymes, is shown for purpose of comparison. Orthogonal gel electrophoresis 3 of the horse liver isozymes results in equal migration ratios for the major isozymes suggesting that they are equal in molecular size and shape, but have different surface charges. Molecular Weight. Gel filtration and equilibrium sedimentation studies indicate a molecular weight of 46,000-49,000 for human liver triose-phosphate isomerase. Kinetic Constants. Maximum velocities, Michaelis constants, and equilibrium constants are shown in Table III. 3S. Raymond, Ann. N.Y. Acad. Sci. 121, 350 (1964).
434
[92]
EPIMERASES AND ISOMERASES
TABLE III MAXIMUM VELOCITIES~ MICHAELIS CONSTANTS, AND EQUILIBRIUM CONSTANTS OF TRIOSEPHOSPHATE ISOMERASE FROM HORSE LIVER AND HUMAN LIVER
Glyceraldehyde 3-phosphate to dihydroxyacetone phosphate
Dihydroacetone phosphate to glyceraldehyde 3-phosphate
gm
Source
V,~xa
Horseliver Humanliver
(raM)
Km
Vmax
(mM)
7935 _+ 456b 0.42 + 0.06 753 _+ 125 0.59 _+ 0.01 2302 + 119 0.40_+ 0.03 163 + 5 0.59 __ 0.01
Ko, 14.8 _+ 2.5 20.9 _+ 3.1
" V~x is expressed as IU/mg protein. b Variability is expressed as the standard deviation and was calculated when the experiment had been repeated in some cases three times but in most cases four times.
[92] T r i o s e p h o s p h a t e
Isomerase I from Yeast
By W. K. G. KRIETSCH D-Glyceraldehyde 3-phosphate ~ dihydroxyacetone phosphate Assay Method
Principle. The most convenient method of M e y e r - A r e n d t et al."- is used. The triosephosphate isomerase is assayed by measuring the rate of formation of dihydroxyacetone phosphate (DAP), which is converted to L-a-glycerophosphate (a-GP) by the indicator enzyme L-a-glycerophosphate dehydrogenase. The rate of D A P reduction to a - G P is equivalent to the rate of N A D H oxidation, which is measured by the absorbance decrease at 366 nm. Reagents Triethanolamine-HC1 buffer, 0.1 M, p H 7.5, 0.50 ml N A D H , 14 mM, 0.01 ml DL-Glyceraldehyde 3-phosphate, 90 mM, 0.02 ml L-a-Glycerophosphate dehydrogenase, 70 U / m l , 0.01ml
Procedure. Solutions in the indicated quantities were added to a glass 1D-Glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1. z E. Meyer-Arendt, G. Beisenherz, and T. Biicher, Naturwissenscha]ten 40, 59 (1953).
EPIMERASES AND ISOMERASES
[91]
[91] T r i o s e p h o s p h a t e I s o m e r a s e f r o m H u m a n a n d Horse Liver 1 By ROBERT SNYDER and EUN Woo LEE Glyceraldehyde 3-phosphate ~ dihydroxyacetone phosphate
Assay Method Principle. Triosephosphate isomerase converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate, a substrate for glycerol-Iphosphate dehydrogenase. NADH utilization during glycerol 1-phosphate production is stoichiometric with dihydroxyacetone phosphate production and is measured spectrophotometrically at 340 nm. Reagents DL-Glyceraldehyde 3-phosphate, 14.7 mM NADH, 4.2 mM Glycerol-l-phosphate dehydrogenase, 0.67 mg/ml (specific activity = 100-200 units/mg protein). Triethanolamine (2 m M ) - E D T A (5.4 mM) buffer, pH 7.9 Procedure. Place 0.1 ml of glyceraldehyde 3-phosphate, 0.1 ml of glycerol-l-phosphate dehydrogenase, 0.15 ml of NADH and 2.55 ml of triethanolamine-EDTA buffer in a 1-cm cuvette. Start the reaction by adding 0.1 ml of isomerase and follow the decrease in extinction at 340 nm. Definition of Unit and Specific Activity. One unit of triosephosphate isomerase is defined as the amount of enzyme that will isomerize 1 ~mole of D-glyceraldehyde 3-phosphate to dihydroxyacetone phosphate in 1 min. Specific activity is expressed as units per milligram of protein. Protein is measured by the method of Lowry et al. 2 Purification Procedure Either fresh or deep-frozen livers may be used. Step 1. Preparation of Water Extract. The liver is freed of connective 1E. W. Lee, J. A. Barriso, M. Pepe, and R. Snyder, Biochim. Biophys. Acta 242, 261 (1971). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, d. Biol. Chem. 193, 265 (1951).
[91]
T R I O S E P H O S P H A T E ISOMERASE FROM LIVER
431
tissue and blood vessels, diced, and put through a chilled meat grinder. The ground liver is then weighed and extracted with 2.5 parts of deionized water containing 0.05% EDTA for 4 hr with mechanical stirring at room temperature or overnight in the cold room at 3 ° . The water extract is obtained after centrifugation for 20 min at 8000 g. Step 2. Acetone Precipitation. Acetone is added in a fine stream to the mechanically stirred, ice cold, water extract over a 45-min period to a final concentration of 35% (v/v) (horse liver) or over a 60-min period to a final concentration of 50% (human liver). Stirring is continued for an additional 15-min period, and the resulting precipitate is removed by centrifugation for 20 min at 4000 g. Acetone is again added to the 35% (horse liver) or 50% (human liver) acetone extract over a 45-rain period to give a final concentration of 60%, and the resulting precipitate is collected after centrifugation as above. The residue is taken up in 8 volumes of deionized-water containing 0.05% EDTA and dialyzed against 0.05% EDTA overnight. Denatured protein formed during dialysis is discarded after centrifugation for 10 rain at 27,000 g. Step 3. Heat Treatment. The dialyzed solution is heated to 45 ° for 15 min, and the resulting precipitate of denatured protein is removed by centrifugation for 30 min at 16,000 g. The extract is again treated with heat at 50 ° for 30 min and centrifuged as above to remove denatured :protein. Step 4. Chromatography on QAE-Sephadex. The enzyme is next applied to a 2.3 X 28 cm QAE-Sephadex A-50 column equilibrated with 7 mM phosphate buffer, pH 7.8, at room temperature. Buffer is passed through the column until the extinction at 280 nm is no longer detected. Elution is performed with 0.1 M NaC1 in 7 mM phosphate buffer, pH 7.8. Most of the activity is found in the first (human liver) or second (horse liver) peak of the eluate. Step 5. Ammordum Sul]ate Precipitation. The enzyme eluted from the QAE-Sephadex column is successively dialyzed against 0.8 M, 1.3 M, and 2.6 M ammonium sulfate in 20 mM sodium acetate buffer, pH 5.3, for a total period of 2 days. The protein which is precipitated at 2.6 M ammonium sulfate is removed by centrifugation at 13,200 g for 1 hr. The supernatant, which contains most of the enzyme, is again dialyzed against 3.0 M and then 3.5 M ammonium sulfate in 20 mM sodium acetate buffer, pH 5.3 continuously with several changes of the ammonium sulfate solution in the cold room at 3 ° for 7 days. The resulting crystalline enzyrae (crude crystals) is collected after centrifugation as above. The "crude crystals" are dissolved by cautious, dropwise addition of a minimum amount of 0.05% EDTA solution at 3 °.
432
EPIMERASES
AND
[91]
ISOMERASES
v
8
if2
O
.2
d~ 8
O ~J
o
II
[91]
TRIOSEPHOSPHATE ISOMERASE FROM LIVER
433
TABLE II DISTRIBUTION OF TRIOSEPHOSPHATE ISOMERASE ACTIVITY AMONG ITS ISOZYMES
Isozymes 1 2 3
Rabbit muscle Horse liver Human liver (% of total (% of total (% of total activity) activity) activity) 22 44 24
4
6
50 38 12
----
--
--
4a
--
--
5 5a
4 --
---
6
--
--
62 -26 11
Step 6. Gel Filtration. The enzyme solution obtained from the preceding step is applied to a 2.5 X 40 cm column of Sephadex G-75 superfine equilibrated with 2 m M phosphate buffer, pH 7.8. The column is then washed with the same buffer until all the protein has passed through the column. Fractions of 8-10 ml are collected, and the enzyme is recovered after approximately 100-120 ml have passed through the column. A summary of the purification procedure is presented in Table I. Properties
Isozymes o] Horse and Human Liver Triosephosphate Isomerase. Upon polyacrylamide gel electrophoresis, horse and human liver triosephosphate isomerase can each be resolved into 3 isozymes. Table II shows the isozymes listed in order of decreasing electronegativity and the percentage of total activity for each. Rabbit muscle isomerase, which contains five isozymes, is shown for purpose of comparison. Orthogonal gel electrophoresis 3 of the horse liver isozymes results in equal migration ratios for the major isozymes suggesting that they are equal in molecular size and shape, but have different surface charges. Molecular Weight. Gel filtration and equilibrium sedimentation studies indicate a molecular weight of 46,000-49,000 for human liver triose-phosphate isomerase. Kinetic Constants. Maximum velocities, Michaelis constants, and equilibrium constants are shown in Table III. 3S. Raymond, Ann. N.Y. Acad. Sci. 121, 350 (1964).
434
[92]
EPIMERASES AND ISOMERASES
TABLE III MAXIMUM VELOCITIES~ MICHAELIS CONSTANTS, AND EQUILIBRIUM CONSTANTS OF TRIOSEPHOSPHATE ISOMERASE FROM HORSE LIVER AND HUMAN LIVER
Glyceraldehyde 3-phosphate to dihydroxyacetone phosphate
Dihydroacetone phosphate to glyceraldehyde 3-phosphate
gm
Source
V,~xa
Horseliver Humanliver
(raM)
Km
Vmax
(mM)
7935 _+ 456b 0.42 + 0.06 753 _+ 125 0.59 _+ 0.01 2302 + 119 0.40_+ 0.03 163 + 5 0.59 __ 0.01
Ko, 14.8 _+ 2.5 20.9 _+ 3.1
" V~x is expressed as IU/mg protein. b Variability is expressed as the standard deviation and was calculated when the experiment had been repeated in some cases three times but in most cases four times.
[92] T r i o s e p h o s p h a t e
Isomerase I from Yeast
By W. K. G. KRIETSCH D-Glyceraldehyde 3-phosphate ~ dihydroxyacetone phosphate Assay Method
Principle. The most convenient method of M e y e r - A r e n d t et al."- is used. The triosephosphate isomerase is assayed by measuring the rate of formation of dihydroxyacetone phosphate (DAP), which is converted to L-a-glycerophosphate (a-GP) by the indicator enzyme L-a-glycerophosphate dehydrogenase. The rate of D A P reduction to a - G P is equivalent to the rate of N A D H oxidation, which is measured by the absorbance decrease at 366 nm. Reagents Triethanolamine-HC1 buffer, 0.1 M, p H 7.5, 0.50 ml N A D H , 14 mM, 0.01 ml DL-Glyceraldehyde 3-phosphate, 90 mM, 0.02 ml L-a-Glycerophosphate dehydrogenase, 70 U / m l , 0.01ml
Procedure. Solutions in the indicated quantities were added to a glass 1D-Glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1. z E. Meyer-Arendt, G. Beisenherz, and T. Biicher, Naturwissenscha]ten 40, 59 (1953).
