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[97b] Deoxyribose 5-phosphate aldolase
[97b]
DEOXYRIBOSE 5-PHOSPHATE ALDOLASE
549
E]]ect o] pH. The purified enzyme shows a relatively sharp pH optimum with maximum activity at pH 6.3. Equilibrium Constants. The cleavage of deoxyribose 5-phosphate to yield acetaldehyde and glyceraldehyde 3-phosphate is freely reversible with a Keq of 2 X 10-4 M.
[97b] Deoxyribose 5-Phosphate Aldolase II. Liver B y D. P. GROTH
Deoxyribose5-phosphate ~ D-glyceraldehyde3-phosphate + acetaldehyde Deoxyribose aldolase has been purified from E. coli ~ and L. plantarum. ~ The procedures employed are not suitable for purification of the aldolase from rat liver, since the liver enzyme is very unstable. 3 With very mild conditions, however, the liver aldolase can be conveniently obtained in good yield.
Assay Methods Principle. The metabolism of deoxyribose 5-phosphate to acetaldehyde and glyceraldehyde 3-phosphate is measured either colorimetrically with diphenylamine ~ when the crude preparations are assayed or spectrophotometrieally with a modified procedure of Racker s when purified preparations are assayed. A very sensitive and specific assay can be employed with the highly purified preparations. In this assay the rate of incorporation of carbon-14 labeled acetaldehyde into deoxyribose 5-phosphate is measured. Reagents
Tris buffer, 0.05 M, pH 7.5, containing 0.01 M EDTA Sodium citrate, 0.1 M, pH 7.5 Deoxyribose 5-phosphate, 0.016 M pH 7.5 D P N H , 0.0O5 M Acetaldehyde-~4C, 0.02M, specific activity 5 X l0 s cpm per micromole 1E. Racker, J. Biol. Chem. 196, 347 (1952). 2W. E. Pricer, Jr. and B. L. tIorecker, J. Biol. Chem. 235, 1292 (1960). 3D. P. Groth, Federation Proc. 24, 666 (1965). 'G. Ashwell, Vol. III [12]. E. Racker, Vol. I [561.
550
ALDOLASES
[97b]
DL-Glyceraldehyde 3-phosphate, 0.02 M, pH 6.5 Crystalline yeast alcohol dehydrogenase, 5 mg/ml, (Sigma lyophilized), containing bovine serum albumin, 1 mg/ml Enzyme diluted with 0.005M Tris buffer, pH 7.5, containing 0.001 M EDTA and 0.005 M 2-mercaptoethanol to obtain 1001000 Racker 11 units per milliliter
Procedure. (a) WITH DIPHENYLAMINE.Add the following reagents to 8 X 70 mm test tubes: Tris buffer, 30 #l; sodium citrate, 10 ~l; deoxyribose 5-phosphate, 10 #l; distilled water, 200/xl; diluted enzyme, 50 ~l. Incubate at 22 ° for 30 minutes. Stop the reaction by the addition of 100 ~l of 3 M perchloric acid. After centrifugation for 5 minutes at 1500 rpm the residual deoxyribose 5-phosphate is estimated in the supernatant according to Disehe. 6 With crude enzyme preparations, an unincubated control is assayed at each enzyme concentration. (b) WITH ALCOHOLDEHYDROGENASE.Add the following reagents to a 1-cm silica cuvette of 0.5-ml capacity: Tris buffer, 30 #l; sodium citrate, 10 ~l; deoxyribose 5-phosphate, 10 ~l; alcohol dehydrogenase, 10 #l; D P N H , 10 /~l; distilled water, 180 /xl; and diluted enzyme, 50 ~l. A cuvette in which the deoxyribose 5-phosphate is omitted serves as control for each enzyme preparation. Absorbancy readings at 340 m~ are taken at 30-second intervals for 5 minutes at 22% (C) WITH 14C-LABELEDACETALDEHYDE.Add the following reagents to 8 X 70 mm test tubes: Tris buffer, 15 ~l; sodium citrate, 5 ~l; glyceraldehyde 3-phosphate, 10 ~l; acetaldehyde-14C, l0 ~l; distilled water, 60 ~l; and diluted enzyme 50 ~l. Incubate at 22 ° for 10 minutes. Stop the reaction by the addition of 50 ~l of 0.2 M formic acid. Dilute the mixture with 3 ml of distilled water and apply t e a 0.6 X 2 cm column of Dowex 1-formate. After addition of the sample, wash the column with 6 ml of distilled water. Enzymatically synthesized deoxyribose 5-phosphate-14C is then eluted from the column with 2 ml of 1 M sodium formate. The 14C is measured with a suitable liquid scintillation technique. Definition of Unit and Specific Activity. Protein concentration is determined by the biuret method up to the hydroxylapatite step in the purificatioa (below) and then spectrophotometrieally. ~ Units are expressed according to Racker2 Application o] Assay Methods to Crude Tissue Preparations. The diphenylamine method appears to be valid for crude mammalian tissue homogenates from which the large particulates have been removed by a 10-minute centrifugation at 10,000g. "Z. Dische, Mikrochcmie 8, 4 (1930). 7E. Lttync, Vol. I I I [73].
[97b] Purification
DEOXYRIBOSE 5-PHOSPHATE ALDOLASE
551
Procedure 3
All steps in the procedure were carried out at 2-4 °, and all solutions contained 0.005 M 2-mercaptoethanol and 0.001 M E D T A unless stated
otherwise. Step 1. Crude Extract. Seventy grams of rat liver (100-150 g animals are fasted overnight) is cut into small pieces and homogenized in 200 ml of 0.05 M Tris, p H 7.2, in a glass homogenizer with a tight-fitting Teflon pestle. Step ~. Separation o] Active Cell Fraction. Essentially all the activity of the homogenate is found to be localized in the soluble fraction not sedimented by centrifugation at 30,000 rpm for 90 minutes in a number 30 rotor in the Spinco Model L preparative ultracentrafuge. Step 3. Ammonium Sul]ate Fractionation. The soluble fraction is adjusted to 25% saturation by the slow addition of saturated ammonium sulfate, p H 7.4. The precipitate is collected by centrifugation and discarded. The ammonium sulfate concentration in the supernatant fraction is raised to 35% of saturation, and the resulting precipitate, after collection by centrifugation, is dissolved in 30 ml of 0.05 M Tris, p H 7.2. The solution is dialyzed twice for 45 minutes against 2-liter portions of 0.001 M phosphate buffer, p H 7.5, containing 0.005 M ammonium sulfate. The preparation at this stage can be stored frozen at --20 ° for at least 1 month with little loss in activity. Step ~. Chromatography on DEAE-CeUulose. s The ammonium sulfate fraction above is thawed, centrifuged to removed denatured protein, if necessary, and diluted with 500 ml of 0.001 M phosphate buffer, p H 7.5, containing 0.005 M ammonium sulfate. The diluted solution is applied to a 3 X 25 cm DEAE-cellulose column with a flow rate of 200 ml per hour. The column is eluted with 200 ml of 0.001 M phosphate buffer, p H 7.5, containing 0.05 M ammonium sulfate and then with 125 ml of 0.001 M phosphate buffer, pH 7.5, containing 0.1 M ammonium sulfate. Discard these fractions. The column is then eluted with an additional 150 ml of the buffered 0.1 M ammonium sulfate solution. The majority of the aldolase activity appears in this fraction. All elutions are carried out at a flow rate of 150-200 ml per hour. The aldolase activity is precipitated by the slow addition of 150 ml of saturated ammonium sulfate, p H 7.2, 8Whatman DE 50, after repeated cycling through 1 N Na0H and 1 N HC1, is sedimented several times by gravity in a large volume of water to remove fines. The very coarse cellulose particles are removed by homogenization in a Waring blendor for 1 minute. The column is packed under 2 psi pressure with the DEAE suspended in distilled water and is equilibrated with 9 liters of 0.001M phosphate buffer, pH 7.5, containing 0.005M ammonium sulfate.
552
ALDOLASES
[97b]
over a period of 30 minutes. The precipitate is collected by centrifugation and dissolved in 30 ml of 0.001 M phosphate buffer, pH 7.5, containing 0.005 M ammonium sulfate. This solution may be frozen and stored indefinitely at --20 ° , although freezing results in the loss of 40-50% of the activity. Step 5. Hydroxylapatite Chromatography2 Hydroxylapatite is prepared according to Tiselius et al. 1° sedimented repeatedly by gravity from a large volume of 10-~ M sodium phosphate buffer, pH 6.8, to remove fines, and stored in the cold. The column is prepared as follows: A suspension of hydroxylapatite is poured into a 2 X 80 cm glass column to give a final bed height of 70 cm. The column is equilibrated with 0.01 M potassium phosphate buffer, pH 8.0, at a flow rate of 20-30 ml per hour. Fifteen milliliters of the DEAE fraction above are concentrated to 1-2 ml by ultrafiltration against 500 ml of 0.001 M phosphate buffer, pH 7.5, containing 0.005 M ammonium sulfate and 0.0Ol M sodium ascorbate. The concentrated enzyme solution is applied to the hydroxylapatite column, and elution is carried out first with 70 ml of 0.01 M phosphate buffer, pH 8.0, and second with 300 ml of 0.03 M phosphate buffer, pH 8.0. Column flow rate is maintained at 15 ml per hour. The aldolase activity appears in the final 30-35 ml of the 0.03 M phosphate buffer eluate. The active fractions are concentrated by ultrafiltration as above. The concentrated enzyme solution is then rechromatographed on hydroxylapatite, which increases the specific activity approximately 3040% with little loss in enzyme activity. Freezing and storage at --20 ° for several months results in the loss of 50-70% of the activity, whereas the activity in the unfrozen solution has a half-life of approximately 1 week at 2 ° . Step 6. Rechromatography on DEAE-CeUulos.e. The aldolase from step 5 still contains small amounts of triosephosphate isomerase activity. This activity is removed as follows: a 0.8 X 10 cm column of DEAEcellulose is equilibrated with 0.001 M phosphate buffer, pH 7.5, containing 0.005 M ammonium sulfate and 0.001 M sodium citrate. Two-tenths milliliters of hydroxylapatite enzyme is diluted with 9 ml of the same buffer and 0.25 ml of 0.016 M deoxyribose 5-phosphate is added. After incubation at 22 ° for 30 minutes, the mixture is applied to the DEAEcellulose column. Elution is carried out at room temperature with successive 10-ml portions of the following solutions: (a) 0.001 M phosphate buffer, pH 7.5, 0.001 M citrate, 0.05 M ammonium sulfate; (b) 0.001 M phosphate buffer, pH 7.5, 0.001 M citrate, 0.1 M ammonium sulfate; (e) ' E D T A is omitted from the solutions used for chromatography of the enzyme on hydroxylapatite. '°A. Tiselius, S. Hjerten, and (J. Levin, Arch. Biochem. Biophys. 65, 132 (1956).
[97~
DEOXYRIBOSE 5-PHOSPHATE ALDOLASE
553
0.001 M phosphate buffer, p H 7.5, 0.001 M citrate, 0 . 2 M ammonium sulfate. One-milliliter fractions of each solution are collected every 2--3 minutes. Isomerase-free deoxyribose aldolase is eluted in milliliters 4-7 of the 0.2 M ammonium sulfate fraction. These active fractions are used as such. The activity has a half-life of 24 48 hours at 2 °. The purification procedure is summarized in the table. Step 5 is repeated twice and step 6 five times to completely purify the aldolase from 70 g of liver. PURIFICATION PROCEDURE FOR DEOXYRIBOSE ALDOLASE OF LIVER
Fraction 1. Crude extract 2. High speed supernatant 3. Ammonium sulfate fractionation 4. DEAE-cellulose chromatography 5. Hydroxylapatite chromatography 6. DEAE-cellulose
Aldolase Protein Volume activity concentration (ml) (units)~ (mg/ml)
Specific activity Recovery (units/rag) (%)
280 200
83,000 62,000
59 30
5 10
100 75
30
61,000
67.7
64
74
30
46,000
7.9
820
55
33,000
3.3
9,800
40
11,000
10
1.0 20
8,300
0.038
A unit of enzyme activity is defined as a change in absorbancy at 340 mu of 0.001 per minute at 22°. When the diphenylamine procedure is employed, this absorbancy change is calculated from the measured rate of metabolism of deoxyribose 5-phosphate. Properties
Contamination with Other Enzymes. The purified enzyme is free from triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, alcohol dehydrogenase, and glycerophosphate dehydrogenase. Specificity. The purified enzyme has no measurable activity with deoxyribose 1-phosphate, deoxyribose, purine deoxyribonucleotides, and purine deoxyribonucleosides. Optimal activity with deoxyribose 5-phosphate in either Tris or phosphate buffer is at p H 7.4-7.5. Activators. The purified enzyme is activated markedly by di- and triearboxylic acids. 11 Equilibrium. 2 The equilibrium constant for reaction (1) is approximately 2 X 10-4 at pH 7.5 and 22 °. Molecular Weight. The apparent minimal molecular weight of the 11N. Jiang and D. P. Groth, J. Biol. Chem. 237, 3339 (1962).
554
ALDOLASES
[97b]
aldolase is 253,000 as determined by the sucrose density gradient technique of Martin and Ames, TM Kinetic Properties2 The Km values for the substrates and activators are 1.7 X 10-4M, 2.67 X 10-4M, 2.0 X 10-4M, 1 X 10-4M, 2 X 10-4M, 5 X 10-4M for deoxyribose 5-phosphate, acetaldehyde, glyceraldehyde 3-phosphate, citrate, succinate, and glutarate, respectively.
1~R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372 (1961).
DEOXYRIBOSE 5-PHOSPHATE ALDOLASE
549
E]]ect o] pH. The purified enzyme shows a relatively sharp pH optimum with maximum activity at pH 6.3. Equilibrium Constants. The cleavage of deoxyribose 5-phosphate to yield acetaldehyde and glyceraldehyde 3-phosphate is freely reversible with a Keq of 2 X 10-4 M.
[97b] Deoxyribose 5-Phosphate Aldolase II. Liver B y D. P. GROTH
Deoxyribose5-phosphate ~ D-glyceraldehyde3-phosphate + acetaldehyde Deoxyribose aldolase has been purified from E. coli ~ and L. plantarum. ~ The procedures employed are not suitable for purification of the aldolase from rat liver, since the liver enzyme is very unstable. 3 With very mild conditions, however, the liver aldolase can be conveniently obtained in good yield.
Assay Methods Principle. The metabolism of deoxyribose 5-phosphate to acetaldehyde and glyceraldehyde 3-phosphate is measured either colorimetrically with diphenylamine ~ when the crude preparations are assayed or spectrophotometrieally with a modified procedure of Racker s when purified preparations are assayed. A very sensitive and specific assay can be employed with the highly purified preparations. In this assay the rate of incorporation of carbon-14 labeled acetaldehyde into deoxyribose 5-phosphate is measured. Reagents
Tris buffer, 0.05 M, pH 7.5, containing 0.01 M EDTA Sodium citrate, 0.1 M, pH 7.5 Deoxyribose 5-phosphate, 0.016 M pH 7.5 D P N H , 0.0O5 M Acetaldehyde-~4C, 0.02M, specific activity 5 X l0 s cpm per micromole 1E. Racker, J. Biol. Chem. 196, 347 (1952). 2W. E. Pricer, Jr. and B. L. tIorecker, J. Biol. Chem. 235, 1292 (1960). 3D. P. Groth, Federation Proc. 24, 666 (1965). 'G. Ashwell, Vol. III [12]. E. Racker, Vol. I [561.
550
ALDOLASES
[97b]
DL-Glyceraldehyde 3-phosphate, 0.02 M, pH 6.5 Crystalline yeast alcohol dehydrogenase, 5 mg/ml, (Sigma lyophilized), containing bovine serum albumin, 1 mg/ml Enzyme diluted with 0.005M Tris buffer, pH 7.5, containing 0.001 M EDTA and 0.005 M 2-mercaptoethanol to obtain 1001000 Racker 11 units per milliliter
Procedure. (a) WITH DIPHENYLAMINE.Add the following reagents to 8 X 70 mm test tubes: Tris buffer, 30 #l; sodium citrate, 10 ~l; deoxyribose 5-phosphate, 10 #l; distilled water, 200/xl; diluted enzyme, 50 ~l. Incubate at 22 ° for 30 minutes. Stop the reaction by the addition of 100 ~l of 3 M perchloric acid. After centrifugation for 5 minutes at 1500 rpm the residual deoxyribose 5-phosphate is estimated in the supernatant according to Disehe. 6 With crude enzyme preparations, an unincubated control is assayed at each enzyme concentration. (b) WITH ALCOHOLDEHYDROGENASE.Add the following reagents to a 1-cm silica cuvette of 0.5-ml capacity: Tris buffer, 30 #l; sodium citrate, 10 ~l; deoxyribose 5-phosphate, 10 ~l; alcohol dehydrogenase, 10 #l; D P N H , 10 /~l; distilled water, 180 /xl; and diluted enzyme, 50 ~l. A cuvette in which the deoxyribose 5-phosphate is omitted serves as control for each enzyme preparation. Absorbancy readings at 340 m~ are taken at 30-second intervals for 5 minutes at 22% (C) WITH 14C-LABELEDACETALDEHYDE.Add the following reagents to 8 X 70 mm test tubes: Tris buffer, 15 ~l; sodium citrate, 5 ~l; glyceraldehyde 3-phosphate, 10 ~l; acetaldehyde-14C, l0 ~l; distilled water, 60 ~l; and diluted enzyme 50 ~l. Incubate at 22 ° for 10 minutes. Stop the reaction by the addition of 50 ~l of 0.2 M formic acid. Dilute the mixture with 3 ml of distilled water and apply t e a 0.6 X 2 cm column of Dowex 1-formate. After addition of the sample, wash the column with 6 ml of distilled water. Enzymatically synthesized deoxyribose 5-phosphate-14C is then eluted from the column with 2 ml of 1 M sodium formate. The 14C is measured with a suitable liquid scintillation technique. Definition of Unit and Specific Activity. Protein concentration is determined by the biuret method up to the hydroxylapatite step in the purificatioa (below) and then spectrophotometrieally. ~ Units are expressed according to Racker2 Application o] Assay Methods to Crude Tissue Preparations. The diphenylamine method appears to be valid for crude mammalian tissue homogenates from which the large particulates have been removed by a 10-minute centrifugation at 10,000g. "Z. Dische, Mikrochcmie 8, 4 (1930). 7E. Lttync, Vol. I I I [73].
[97b] Purification
DEOXYRIBOSE 5-PHOSPHATE ALDOLASE
551
Procedure 3
All steps in the procedure were carried out at 2-4 °, and all solutions contained 0.005 M 2-mercaptoethanol and 0.001 M E D T A unless stated
otherwise. Step 1. Crude Extract. Seventy grams of rat liver (100-150 g animals are fasted overnight) is cut into small pieces and homogenized in 200 ml of 0.05 M Tris, p H 7.2, in a glass homogenizer with a tight-fitting Teflon pestle. Step ~. Separation o] Active Cell Fraction. Essentially all the activity of the homogenate is found to be localized in the soluble fraction not sedimented by centrifugation at 30,000 rpm for 90 minutes in a number 30 rotor in the Spinco Model L preparative ultracentrafuge. Step 3. Ammonium Sul]ate Fractionation. The soluble fraction is adjusted to 25% saturation by the slow addition of saturated ammonium sulfate, p H 7.4. The precipitate is collected by centrifugation and discarded. The ammonium sulfate concentration in the supernatant fraction is raised to 35% of saturation, and the resulting precipitate, after collection by centrifugation, is dissolved in 30 ml of 0.05 M Tris, p H 7.2. The solution is dialyzed twice for 45 minutes against 2-liter portions of 0.001 M phosphate buffer, p H 7.5, containing 0.005 M ammonium sulfate. The preparation at this stage can be stored frozen at --20 ° for at least 1 month with little loss in activity. Step ~. Chromatography on DEAE-CeUulose. s The ammonium sulfate fraction above is thawed, centrifuged to removed denatured protein, if necessary, and diluted with 500 ml of 0.001 M phosphate buffer, p H 7.5, containing 0.005 M ammonium sulfate. The diluted solution is applied to a 3 X 25 cm DEAE-cellulose column with a flow rate of 200 ml per hour. The column is eluted with 200 ml of 0.001 M phosphate buffer, p H 7.5, containing 0.05 M ammonium sulfate and then with 125 ml of 0.001 M phosphate buffer, pH 7.5, containing 0.1 M ammonium sulfate. Discard these fractions. The column is then eluted with an additional 150 ml of the buffered 0.1 M ammonium sulfate solution. The majority of the aldolase activity appears in this fraction. All elutions are carried out at a flow rate of 150-200 ml per hour. The aldolase activity is precipitated by the slow addition of 150 ml of saturated ammonium sulfate, p H 7.2, 8Whatman DE 50, after repeated cycling through 1 N Na0H and 1 N HC1, is sedimented several times by gravity in a large volume of water to remove fines. The very coarse cellulose particles are removed by homogenization in a Waring blendor for 1 minute. The column is packed under 2 psi pressure with the DEAE suspended in distilled water and is equilibrated with 9 liters of 0.001M phosphate buffer, pH 7.5, containing 0.005M ammonium sulfate.
552
ALDOLASES
[97b]
over a period of 30 minutes. The precipitate is collected by centrifugation and dissolved in 30 ml of 0.001 M phosphate buffer, pH 7.5, containing 0.005 M ammonium sulfate. This solution may be frozen and stored indefinitely at --20 ° , although freezing results in the loss of 40-50% of the activity. Step 5. Hydroxylapatite Chromatography2 Hydroxylapatite is prepared according to Tiselius et al. 1° sedimented repeatedly by gravity from a large volume of 10-~ M sodium phosphate buffer, pH 6.8, to remove fines, and stored in the cold. The column is prepared as follows: A suspension of hydroxylapatite is poured into a 2 X 80 cm glass column to give a final bed height of 70 cm. The column is equilibrated with 0.01 M potassium phosphate buffer, pH 8.0, at a flow rate of 20-30 ml per hour. Fifteen milliliters of the DEAE fraction above are concentrated to 1-2 ml by ultrafiltration against 500 ml of 0.001 M phosphate buffer, pH 7.5, containing 0.005 M ammonium sulfate and 0.0Ol M sodium ascorbate. The concentrated enzyme solution is applied to the hydroxylapatite column, and elution is carried out first with 70 ml of 0.01 M phosphate buffer, pH 8.0, and second with 300 ml of 0.03 M phosphate buffer, pH 8.0. Column flow rate is maintained at 15 ml per hour. The aldolase activity appears in the final 30-35 ml of the 0.03 M phosphate buffer eluate. The active fractions are concentrated by ultrafiltration as above. The concentrated enzyme solution is then rechromatographed on hydroxylapatite, which increases the specific activity approximately 3040% with little loss in enzyme activity. Freezing and storage at --20 ° for several months results in the loss of 50-70% of the activity, whereas the activity in the unfrozen solution has a half-life of approximately 1 week at 2 ° . Step 6. Rechromatography on DEAE-CeUulos.e. The aldolase from step 5 still contains small amounts of triosephosphate isomerase activity. This activity is removed as follows: a 0.8 X 10 cm column of DEAEcellulose is equilibrated with 0.001 M phosphate buffer, pH 7.5, containing 0.005 M ammonium sulfate and 0.001 M sodium citrate. Two-tenths milliliters of hydroxylapatite enzyme is diluted with 9 ml of the same buffer and 0.25 ml of 0.016 M deoxyribose 5-phosphate is added. After incubation at 22 ° for 30 minutes, the mixture is applied to the DEAEcellulose column. Elution is carried out at room temperature with successive 10-ml portions of the following solutions: (a) 0.001 M phosphate buffer, pH 7.5, 0.001 M citrate, 0.05 M ammonium sulfate; (b) 0.001 M phosphate buffer, pH 7.5, 0.001 M citrate, 0.1 M ammonium sulfate; (e) ' E D T A is omitted from the solutions used for chromatography of the enzyme on hydroxylapatite. '°A. Tiselius, S. Hjerten, and (J. Levin, Arch. Biochem. Biophys. 65, 132 (1956).
[97~
DEOXYRIBOSE 5-PHOSPHATE ALDOLASE
553
0.001 M phosphate buffer, p H 7.5, 0.001 M citrate, 0 . 2 M ammonium sulfate. One-milliliter fractions of each solution are collected every 2--3 minutes. Isomerase-free deoxyribose aldolase is eluted in milliliters 4-7 of the 0.2 M ammonium sulfate fraction. These active fractions are used as such. The activity has a half-life of 24 48 hours at 2 °. The purification procedure is summarized in the table. Step 5 is repeated twice and step 6 five times to completely purify the aldolase from 70 g of liver. PURIFICATION PROCEDURE FOR DEOXYRIBOSE ALDOLASE OF LIVER
Fraction 1. Crude extract 2. High speed supernatant 3. Ammonium sulfate fractionation 4. DEAE-cellulose chromatography 5. Hydroxylapatite chromatography 6. DEAE-cellulose
Aldolase Protein Volume activity concentration (ml) (units)~ (mg/ml)
Specific activity Recovery (units/rag) (%)
280 200
83,000 62,000
59 30
5 10
100 75
30
61,000
67.7
64
74
30
46,000
7.9
820
55
33,000
3.3
9,800
40
11,000
10
1.0 20
8,300
0.038
A unit of enzyme activity is defined as a change in absorbancy at 340 mu of 0.001 per minute at 22°. When the diphenylamine procedure is employed, this absorbancy change is calculated from the measured rate of metabolism of deoxyribose 5-phosphate. Properties
Contamination with Other Enzymes. The purified enzyme is free from triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, alcohol dehydrogenase, and glycerophosphate dehydrogenase. Specificity. The purified enzyme has no measurable activity with deoxyribose 1-phosphate, deoxyribose, purine deoxyribonucleotides, and purine deoxyribonucleosides. Optimal activity with deoxyribose 5-phosphate in either Tris or phosphate buffer is at p H 7.4-7.5. Activators. The purified enzyme is activated markedly by di- and triearboxylic acids. 11 Equilibrium. 2 The equilibrium constant for reaction (1) is approximately 2 X 10-4 at pH 7.5 and 22 °. Molecular Weight. The apparent minimal molecular weight of the 11N. Jiang and D. P. Groth, J. Biol. Chem. 237, 3339 (1962).
554
ALDOLASES
[97b]
aldolase is 253,000 as determined by the sucrose density gradient technique of Martin and Ames, TM Kinetic Properties2 The Km values for the substrates and activators are 1.7 X 10-4M, 2.67 X 10-4M, 2.0 X 10-4M, 1 X 10-4M, 2 X 10-4M, 5 X 10-4M for deoxyribose 5-phosphate, acetaldehyde, glyceraldehyde 3-phosphate, citrate, succinate, and glutarate, respectively.
1~R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372 (1961).