- Email: [email protected]
[45] Purification of putrescine aminopropyltransferase (spermidine synthase) from eukaryotic tissues
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2.2 x 10 - 6 M. The enzyme is also active with spermidine and cadaverine as the acceptor, but the rates are much slower than with putrescine as the acceptor. No activity is found with 1,3-diaminopropane or monoacetylputrescine. The enzyme is inhibited by p-hydroxymercuribenzoate (0.05 mM) and N-ethylmaleimide (1 mM in 0.1 M Tris-HCl buffer, pH 8.2). No inhibition occurs with carbonyl-binding reagents. The end products of the reaction, 5'-methylthioadenosine and spermidine, both inhibit the reaction.
[45] P u r i f i c a t i o n o f P u t r e s c i n e A m i n o p r o p y l t r a n s f e r a s e (Spermidine Synthase) from Eukaryotic Tissues 1 B y KEIJIRO S A M E J I M A , A A R N E R A I N A , BANRI Y A M A N O H A , and TERHO ELORANTA
Spermidine synthase has been partially purified from several eukaryotic tissues, including rat brain, 2 liver, 3 and ventral prostate. 4 It appears, however, that in no case was the final enzyme preparation more than 10% pure. The recent design of a new affinity chromatographic adsorbent, 5 S-adenosyl(5')-3-thiopropylamine linked to Sepharose (ATPASepharose), has proved to be an important advance in the purification of spermidine synthase. In this chapter we describe purification of spermidine synthase to homogeneity from bovine brain and rat ventral prostate. As the purification procedures for these two tissues are somewhat different, they are treated separately. Preparation of ATPA-Sepharose s S-Adenosyl(5')-3-thiopropylamine (ATPA) hydrogen sulfate is prepared by the method of Jamieson. 6 Bromoacetylation of AH-Sepharose (Pharmacia) is carried out by a slightly modified method of Cuatrecasas. 7 To 6 ml of AH-Sepharose 4B is added 0.24 ml of o-bromoacetyl-N-hySee also this volume [47]. 2 A. Raina and P. Hannonen, FEBS Lett. 16, 1 (1971). 3 p. Hannonen, J. J/inne, and A. Raina, Biochem. Biophys. Res. Commun. 46, 341 (1972). 4 H. Hibasami, R. T. Borchardt, S, Y. Chen, J. K. Coward, and A. E. Pegg, Biochem. J. 187, 419 (1980). 5 K. Samejima and B. Yamanoha, Arch. Biochem. Biophys. 216, 213 (1982). 6 G. A. Jamieson, J. Org. Chem. 28, 2397 (1963). 7 p. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970).
METHODS IN ENZYMOLOGY, VOL. 94
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9
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droxysuccinimide solution prepared by mixing 1.2 mmol of N-hydroxysuccinimide, 1.0 mmol of bromoacetic acid, and i. 1 mmol of N,N'-dicyclohexylcarbodiimide in 8 ml of dioxane. The suspension is gently stirred for 30 min at 4°. Thereafter the Sepharose is washed with 800 ml of 0.1 M NaCI and 400 ml of water, and equilibrated with 5% acetic acid. To 5.5 ml of bromoacetamidohexyl-Sepharose 5 ml of ATPA hydrogen sulfate solution (790 mg dissolved in 5% acetic acid) is added, and the mixture is stirred gently for 20 hr at 37°. In the following masking procedures of unreacted bromoacetyl groups, a careful removal of air bubbles is indispensable for obtaining ATPA-Sepharose that gives reproducible results and a high yield in the purification of spermidine synthase. After removal of excess ATPA, 2-mercaptoethanol prepared in 5% acetic acid is added to the suspension to a final concentration of 2 M. The suspension in a round-bottom flask is deaerated in v a c u o for 1 hr at room temperature in a rotary evaporator connected to an aspirator (25 mm Hg), and then kept at room temperature for 3 days. The suspension is washed with 5% acetic acid and water and is equilibrated at 4 ° with 25 mM sodium phosphate buffer, pH 7.2, containing 0.3 mM ethylenediaminetetraacetic acid (EDTA) and 0.5 mM dithiothreitol (buffer A). After adding 2-ethanolamine hydrochloride to a final concentration of 1 M, the suspension is placed in v a c u o at 4° for 3 hr. The suspension is then allowed to stand for 2 days at 4 ° and is washed with buffer A before use. ATPA-Sepharose thus obtained contains 1 ttmol of ATPA/ml. Assay Methods S p e r m i d i n e S y n t h a s e . The assay of spermidine synthase activity is carried out as described in this volume [42], using [propylamine-l-14C]de carboxy-AdoMet as the labeled substrate. The activity of the purified enzyme is assayed in the presence of albumin (0.2 mg/ml) in the reaction mixture. As an alternative procedure, the method of Janne et al. 8 with some modifications 9 can be used. The reaction mixture contains, in a total volume of 0.5 ml, 0.1 M sodium phosphate buffer, pH 7.2, 1 mM putrescine, 50/zM decarboxy-AdoMet, 5 mM dithiothreitol, and enzyme protein. After incubation for 30 min at 37°, spermidine formed is determined by high-performance liquid chromatography.I° 8 j. J~inne, A. Schenone, and H. G. Williams-Ashman, Biochem. Biophys. Res. Commun. 42, 758 (1971). 9 K. Samejima and Y. Nakazawa, Arch. Biochem. Biophys. 201, 241 (1980). 10 K. Samejima, M. Kawase, S. Sakamoto, M. Okada, and Y. Endo, Anal. Biochem. 76, 392 (1976).
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Definition o f Unit and Specific Activity. One unit of enzyme activity represents the formation of I nmol of spermidine in 1 min under standard assay conditions. Specific activity is expressed as units per milligram of protein. Protein is measured by the method of Lowry et al. Jl either directly or after precipitation with trichloroacetic acid, with crystalline bovine serum albumin as a standard or by ultraviolet absorption.12
Purification of Spermidine Synthase from Bovine Brain Step 1. Extraction o f Tissues. Fresh bovine brains (4.2 kg) obtained from a local slaughterhouse and immediately cooled in ice are washed in 0.25 M sucrose and homogenized in 125-g portions with an Ultra-Turrax (Janke and Kunkel) homogenizer in two volumes of ice-cold 0.25 M sucrose containing 1 mM EDTA, 5 mM 2-mercaptoethanol, and 0.1 mM dithiothreitol. All subsequent operations are performed at 0-4 °. The homogenate is centrifuged at 25,000 g for 30 rain. The supernatant fraction is filtered through glass wool. Step 2. A m m o n i u m Sulfate Fractionation. The crude supernatant (6270 ml) is fractionated with solid ammonium sulfate (Mann, special enzyme grade). The proteins precipitated between 40 and 60% saturation of ammonium sulfate are collected by centrifugation at 14,000 g for 30 min, dissolved in 500 ml of 10 mM Tris-HC1, pH 7.5 (measured at room temperature), containing 0.08 M NaC1, 1 mM 2-mercaptoethanol, and 0.1 mM dithiothreitol (buffer B), and dialyzed for 24 hr against 25 volumes of buffer B, changing the buffer twice during dialysis. The dialyzed enzyme preparation is freed of insoluble material by centrifugation at 14,000 g for 15 min. Step 3. DEAE-Cellulose Chromatography. The dialyzed ammonium sulfate fraction (635 ml) is applied to a DEAE-cellulose column (Whatman DE-52, 8.5 × 30 cm) equilibrated with buffer B. The column is washed with 900 ml of buffer B containing 1 mM dithiothreitol and connected to a linear gradient of 0.08 to 0.5 M sodium chloride in buffer B (total gradient volume is 4000 ml). Spermidine synthase is eluted between 0.11-0.16 M sodium chloride. The active fractions (540 ml) are pooled and brought to 0.70 saturation with ammonium sulfate to concentrate the enzyme. The precipitate is divided into two equal portions that are purified separately in the following steps. Step 4. Chromatography on Hydroxyapatite. This step is included to
u O. H. Lowry,N. J. Rosebrough, A. L. Farr, and R. J. Randall,J. Biol. Chem. 193, 265 (1951). 12H. M. Kalckar,J. Biol. Chem. 167, 461 (1947).
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remove contaminating S-adenosylmethionine decarboxylase and spermine synthase activities, which also may have a high affinity for ATPASepharose. These two enzymes are more strongly adsorbed to hydroxyapatite as compared to spermidine synthase. 2,3 Half of the enzyme preparation obtained in step 3 is dissolved in 17 ml of 0.02 M potassium phosphate buffer, pH 7.2, containing 1 mM 2-mercaptoethanol and 0.1 mM dithiothreitol (buffer C) and dialyzed against 5 liters of buffer C, changing the buffer once during dialysis. After centrifugation at 14,000 g for 10 min, the supernatant (27 ml) is applied to a hydroxyapatite column (Hypatite C, Clarkson Chemicals Co.; 2.6 x 17 cm) equilibrated with buffer C. The column is first eluted with buffer C (40 ml) containing 1 mM dithiothreitol, followed by a linear gradient of 0.02 to 0.4 M potassium phosphate buffer, pH 7.2, containing 1 mM 2-mercaptoethanol and 1 mM dithiothreitol (total gradient volume is 500 ml). Spermidine synthase is eluted practically unadsorbed. The active fractions (140 ml) are pooled and concentrated by the addition of ammonium sulfate (80% saturation). Step 5. Affinity Chromatography on ATPA-Sepharose. The precipitate obtained in step 4 is dissolved in 0.02 M potassium phosphate buffer, pH 7.2, containing 0.3 M NaC1, 0.1 mM EDTA, 1 mM 2-mercaptoethanol, and 1 mM dithiothreitol (buffer D), and passed through a Sephadex G-25 column (1.5 x 17 cm). The desalted enzyme preparation (12.8 ml) is applied at a flow rate of 0.1 ml/min to an ATPA-Sepharose column (1.0 x 2.6 cm) equilibrated with buffer D. The column is washed with 15 ml of buffer D, followed by buffer D containing 0.6 M NaC1 (15 ml) until no protein is being eluted (monitored by UV absorption). Spermidine synthase is then eluted with 6 ml of buffer D containing 0.25 mM decarboxy-AdoMet.13 The enzyme solution is concentrated by ultrafiltration to 1.4 ml. At this stage the enzyme preparation is nearly homogeneous, but polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis show a faint band of contaminating protein in the high-molecular-weight region. Step 6. Gel Filtration. A portion of the concentrated enzyme preparation (0.8 ml) is applied to a Sephadex G-150 column (Pharmacia, 1.5 x 45 cm) equilibrated with buffer D and eluted with the same buffer, collecting 2-ml fractions. The active fractions are pooled (9.5 ml), concentrated to 1.1 ml by ultrafiltration, and stored at 0-4 °. The enzyme is homogeneous as judged by polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. A typical purification is summarized in the table. 13 Synthesis of d e c a r b o x y - A d o M e t is described in K. Samejima, Y. N a k a z a w a , and I. Matsunaga, Chem. Pharm. Bull. 26, 1480 (1978).
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PURIFICATION OF SPERMIDINE SYNTHASE FROM BOVINE BRAIN
Step 1. 2. 3. 4. 5. 6.
Crude extract Ammonium sulfate DEAE-cellulose Hydroxyapatite ATPA-Sepharose Sephadex G-150
Total protein (mg) 50,400 10,200 1480 460 0.92 0.32
Specific Total activity activity (units/mg) (units) 0.10 0.26 1.58 3.60 521 712
Yield" (%)
5050 2670 2330 1650 480 230
100 53 46 33 9.5 4.5
Purification (fold) 1 3 16 36 5200 7100
a The yield at the affinity chromatography step is somewhat variable and can be considerably higher than in this particular experiment depending on the batch of ATPASepharose used.
Purification of Spermidine Synthase from R a t Prostate s
Step 1. Extraction of Tissues. Rat ventral prostates are collected and stored frozen at - 2 0 °. F r o z e n prostates (68 g) of Wistar rats are homogenized in 2 volumes of 25 m M Tris-HCl buffer, p H 7.2, containing 0.3 m M E D T A and l0 m M 2-mercaptoethanol. The 10,000 g supernatant is passed through glass wool. Step 2. Ammonium Sulfate Fractionation. 3 The crude supernatant is fractionated with solid a m m o n i u m sulfate at 0 °. The proteins precipitated between 30 and 60% saturation of a m m o n i u m sulfate are collected. The precipitate is dissolved in a small volume of 25 m M sodium phosphate, p H 7.2, containing 0.3 m M E D T A and 0.5 m M dithiothreitol (buffer A) and passed through a Sephadex G-25 column (equilibrated with buffer A) to remove salt. Step 3. DEAE-Cellulose Chromatography. 3 The desalted protein fraction (1.7 g, 120 ml) is applied to a DEAE-cellulose column (Whatman DE-52, 2.6 x 33 cm) equilibrated with buffer A. The column is eluted with a linear gradient of 0 to 0.4 M NaCI prepared in buffer A (total gradient volume 1600 ml). Fractions containing the synthase activity (110 ml) eluted between 0.08 and 0.1 M sodium chloride are concentrated and equilibrated with buffer A containing 0.3 M NaCI by ultrafiltration in a Centriflo cone (CF 25, Ultrafiltration m e m b r a n e cones, Amicon) to a final volume of 10 ml. Step 4. Affinity Chromatography on ATPA-Sepharose. Affinity chromatography is carried out at 4 ° in a column p a c k e d with A T P A - S e p h a r o s e (0.5 ml) and equilibrated with buffer A containing 0.3 M NaCI. The concentrated e n z y m e solution (77.4 mg of protein, 10 ml) is applied to the
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column at a flow rate of 0.1-0.2 ml/min. The column is washed with 10 ml of equilibration buffer. More than 99% of the protein is removed by the washing. The synthase is then eluted with 4 ml of equilibration buffer containing 2.5 mM decarboxy-AdoMet. 13 Most of the activity is eluted in the first 2 ml. The effluent is pooled and concentrated by ultrafiltration in a Centriflo cone to a final volume of 0.5 ml (containing 0.4 mg of protein). The synthase is nearly homogeneous in this fraction. Step 5. Gel Filtration. The concentrated synthase solution is applied to a Sephacryl S-300 column (Superfine from Pharmacia, 1.2 × 100 cm) equilibrated with buffer A containing 0.3 M NaCI, and eluted with the same buffer at a flow rate of 3.7 ml/hr. The synthase is eluted in 60-70 ml. The active fraction is stored on ice after concentration to 0.5 ml by ultrafiltration. The final preparation has a specific activity of 1300 units/mg, representing 4480-fold purification over the crude supernatant fraction. The yield of the enzyme is 39%. Properties
Stability. Both brain and prostatic spermidine synthases purified through the affinity chromatography step are stable for several months when stored on ice in a buffer containing 0.3 M NaCl, 1 mM dithiothreitol, and 0.05-0.25 mM decarboxy-AdoMet. Purity. The enzyme preparations after the final step show no evidence of contamination as judged by polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. No S-adenosylmethionine decarboxylase or spermine synthase activities can be detected in the final preparations. Subunit Molecular Weight and Molecular Size. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gives a subunit molecular weight of 35,800 and 37,000 for brain and prostatic enzyme, respectively. Gel filtration on a calibrated column of Sephacryl S-300 reveals a molecular weight of approximately 73,000 for the prostatic enzyme. These data suggest that the native enzyme is composed of two subunits of equal size. Kinetics. An apparent Km for decarboxy-AdoMet is about 1 /zM for both brain and prostatic spermidine synthases. A Km of about 0.03 mM for putrescine is obtained for the brain enzyme, whereas a somewhat higher value (0.1 raM) is obtained for the prostatic spermidine synthase. Both enzymes show a strong substrate inhibition with decarboxy-AdoMet that complicates the study of enzyme kinetics. The enzyme is also weakly inhibited by 5'-methylthioadenosine, one of the reaction products.
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Substrate Specificity. Cadaverine, in addition to putrescine, can serve as the propylamine acceptor, although cadaverine gives a reaction rate of only 5-7% of that obtained with putrescine under standard assay conditions (1 mM amine). 1,6-Diaminohexane shows some activity (1.5%), whereas 1,3-diaminopropane is totally inactive as a substrate.
[46] P u r i f i c a t i o n o f S p e r m i d i n e A m i n o p r o p y l t r a n s f e r a s e (Spermine Synthase) from Bovine Brain
By
AARNE RAINA, RAIJA-LEENA PAJULA,
and TERHO ELORANTA
Spermine synthase has been partially purified from rat brain I and ventral prostate, 2 tissues that contain the highest activity of this enzyme in t h e r a t . 3 In this chapter we describe the purification of spermine synthase from bovine brain, which also is rich in spermine synthase and is easily obtained in amounts sufficient for large-scale purification. The use of affinity chromatography on spermine-Sepharose considerably facilitates the purification procedure. 4,5 Assay Methods
Spermine Synthase. The assay of spermine synthase activity is carried out as described elsewhere in this volume [42], using [propylamine1-14C]decarboxy-AdoMet as the labeled substrate. Definition of Unit and Specific Activity. One unit of enzyme activity represents the formation of 1 nmol of spermine in 1 min under standard assay conditions. Specific activity is expressed as units per milligram of protein. Protein is measured by the method of Lowry et al. 6 either directly or after precipitation with trichloroacetic acid, with crystalline bovine serum albumin as a standard, or by ultraviolet absorption. 7 P. Hannonen, J. J~inne, and A. Raina, Biochim. Biophys. Acta 289, 225 (1972). 2 H. Hibasami, R. T. Borchardt, S. Y. Chen, J. K. Coward, and A. E. Pegg, Biochern. J. 187, 419 (1980). 3 A. Raina, R.-L. Pajula, and T. O. Eloranta, FEBS Lett. 67, 252 (1976). 4 R.-L. Pajula, A. Raina, and J. Kekoni, FEBS Lett. 90, 153 (1978). 5 R.-L. Pajula, A Raina, and T. Eloranta, Eur. J. Biochern. 101, 619 (1979). 6 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 7 H. M. Kalckar, J. Biol. Chem. 167, 461 (1947).
METHODS IN ENZYMOLOGY, VOL. 94
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2.2 x 10 - 6 M. The enzyme is also active with spermidine and cadaverine as the acceptor, but the rates are much slower than with putrescine as the acceptor. No activity is found with 1,3-diaminopropane or monoacetylputrescine. The enzyme is inhibited by p-hydroxymercuribenzoate (0.05 mM) and N-ethylmaleimide (1 mM in 0.1 M Tris-HCl buffer, pH 8.2). No inhibition occurs with carbonyl-binding reagents. The end products of the reaction, 5'-methylthioadenosine and spermidine, both inhibit the reaction.
[45] P u r i f i c a t i o n o f P u t r e s c i n e A m i n o p r o p y l t r a n s f e r a s e (Spermidine Synthase) from Eukaryotic Tissues 1 B y KEIJIRO S A M E J I M A , A A R N E R A I N A , BANRI Y A M A N O H A , and TERHO ELORANTA
Spermidine synthase has been partially purified from several eukaryotic tissues, including rat brain, 2 liver, 3 and ventral prostate. 4 It appears, however, that in no case was the final enzyme preparation more than 10% pure. The recent design of a new affinity chromatographic adsorbent, 5 S-adenosyl(5')-3-thiopropylamine linked to Sepharose (ATPASepharose), has proved to be an important advance in the purification of spermidine synthase. In this chapter we describe purification of spermidine synthase to homogeneity from bovine brain and rat ventral prostate. As the purification procedures for these two tissues are somewhat different, they are treated separately. Preparation of ATPA-Sepharose s S-Adenosyl(5')-3-thiopropylamine (ATPA) hydrogen sulfate is prepared by the method of Jamieson. 6 Bromoacetylation of AH-Sepharose (Pharmacia) is carried out by a slightly modified method of Cuatrecasas. 7 To 6 ml of AH-Sepharose 4B is added 0.24 ml of o-bromoacetyl-N-hySee also this volume [47]. 2 A. Raina and P. Hannonen, FEBS Lett. 16, 1 (1971). 3 p. Hannonen, J. J/inne, and A. Raina, Biochem. Biophys. Res. Commun. 46, 341 (1972). 4 H. Hibasami, R. T. Borchardt, S, Y. Chen, J. K. Coward, and A. E. Pegg, Biochem. J. 187, 419 (1980). 5 K. Samejima and B. Yamanoha, Arch. Biochem. Biophys. 216, 213 (1982). 6 G. A. Jamieson, J. Org. Chem. 28, 2397 (1963). 7 p. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970).
METHODS IN ENZYMOLOGY, VOL. 94
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9
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droxysuccinimide solution prepared by mixing 1.2 mmol of N-hydroxysuccinimide, 1.0 mmol of bromoacetic acid, and i. 1 mmol of N,N'-dicyclohexylcarbodiimide in 8 ml of dioxane. The suspension is gently stirred for 30 min at 4°. Thereafter the Sepharose is washed with 800 ml of 0.1 M NaCI and 400 ml of water, and equilibrated with 5% acetic acid. To 5.5 ml of bromoacetamidohexyl-Sepharose 5 ml of ATPA hydrogen sulfate solution (790 mg dissolved in 5% acetic acid) is added, and the mixture is stirred gently for 20 hr at 37°. In the following masking procedures of unreacted bromoacetyl groups, a careful removal of air bubbles is indispensable for obtaining ATPA-Sepharose that gives reproducible results and a high yield in the purification of spermidine synthase. After removal of excess ATPA, 2-mercaptoethanol prepared in 5% acetic acid is added to the suspension to a final concentration of 2 M. The suspension in a round-bottom flask is deaerated in v a c u o for 1 hr at room temperature in a rotary evaporator connected to an aspirator (25 mm Hg), and then kept at room temperature for 3 days. The suspension is washed with 5% acetic acid and water and is equilibrated at 4 ° with 25 mM sodium phosphate buffer, pH 7.2, containing 0.3 mM ethylenediaminetetraacetic acid (EDTA) and 0.5 mM dithiothreitol (buffer A). After adding 2-ethanolamine hydrochloride to a final concentration of 1 M, the suspension is placed in v a c u o at 4° for 3 hr. The suspension is then allowed to stand for 2 days at 4 ° and is washed with buffer A before use. ATPA-Sepharose thus obtained contains 1 ttmol of ATPA/ml. Assay Methods S p e r m i d i n e S y n t h a s e . The assay of spermidine synthase activity is carried out as described in this volume [42], using [propylamine-l-14C]de carboxy-AdoMet as the labeled substrate. The activity of the purified enzyme is assayed in the presence of albumin (0.2 mg/ml) in the reaction mixture. As an alternative procedure, the method of Janne et al. 8 with some modifications 9 can be used. The reaction mixture contains, in a total volume of 0.5 ml, 0.1 M sodium phosphate buffer, pH 7.2, 1 mM putrescine, 50/zM decarboxy-AdoMet, 5 mM dithiothreitol, and enzyme protein. After incubation for 30 min at 37°, spermidine formed is determined by high-performance liquid chromatography.I° 8 j. J~inne, A. Schenone, and H. G. Williams-Ashman, Biochem. Biophys. Res. Commun. 42, 758 (1971). 9 K. Samejima and Y. Nakazawa, Arch. Biochem. Biophys. 201, 241 (1980). 10 K. Samejima, M. Kawase, S. Sakamoto, M. Okada, and Y. Endo, Anal. Biochem. 76, 392 (1976).
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Definition o f Unit and Specific Activity. One unit of enzyme activity represents the formation of I nmol of spermidine in 1 min under standard assay conditions. Specific activity is expressed as units per milligram of protein. Protein is measured by the method of Lowry et al. Jl either directly or after precipitation with trichloroacetic acid, with crystalline bovine serum albumin as a standard or by ultraviolet absorption.12
Purification of Spermidine Synthase from Bovine Brain Step 1. Extraction o f Tissues. Fresh bovine brains (4.2 kg) obtained from a local slaughterhouse and immediately cooled in ice are washed in 0.25 M sucrose and homogenized in 125-g portions with an Ultra-Turrax (Janke and Kunkel) homogenizer in two volumes of ice-cold 0.25 M sucrose containing 1 mM EDTA, 5 mM 2-mercaptoethanol, and 0.1 mM dithiothreitol. All subsequent operations are performed at 0-4 °. The homogenate is centrifuged at 25,000 g for 30 rain. The supernatant fraction is filtered through glass wool. Step 2. A m m o n i u m Sulfate Fractionation. The crude supernatant (6270 ml) is fractionated with solid ammonium sulfate (Mann, special enzyme grade). The proteins precipitated between 40 and 60% saturation of ammonium sulfate are collected by centrifugation at 14,000 g for 30 min, dissolved in 500 ml of 10 mM Tris-HC1, pH 7.5 (measured at room temperature), containing 0.08 M NaC1, 1 mM 2-mercaptoethanol, and 0.1 mM dithiothreitol (buffer B), and dialyzed for 24 hr against 25 volumes of buffer B, changing the buffer twice during dialysis. The dialyzed enzyme preparation is freed of insoluble material by centrifugation at 14,000 g for 15 min. Step 3. DEAE-Cellulose Chromatography. The dialyzed ammonium sulfate fraction (635 ml) is applied to a DEAE-cellulose column (Whatman DE-52, 8.5 × 30 cm) equilibrated with buffer B. The column is washed with 900 ml of buffer B containing 1 mM dithiothreitol and connected to a linear gradient of 0.08 to 0.5 M sodium chloride in buffer B (total gradient volume is 4000 ml). Spermidine synthase is eluted between 0.11-0.16 M sodium chloride. The active fractions (540 ml) are pooled and brought to 0.70 saturation with ammonium sulfate to concentrate the enzyme. The precipitate is divided into two equal portions that are purified separately in the following steps. Step 4. Chromatography on Hydroxyapatite. This step is included to
u O. H. Lowry,N. J. Rosebrough, A. L. Farr, and R. J. Randall,J. Biol. Chem. 193, 265 (1951). 12H. M. Kalckar,J. Biol. Chem. 167, 461 (1947).
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remove contaminating S-adenosylmethionine decarboxylase and spermine synthase activities, which also may have a high affinity for ATPASepharose. These two enzymes are more strongly adsorbed to hydroxyapatite as compared to spermidine synthase. 2,3 Half of the enzyme preparation obtained in step 3 is dissolved in 17 ml of 0.02 M potassium phosphate buffer, pH 7.2, containing 1 mM 2-mercaptoethanol and 0.1 mM dithiothreitol (buffer C) and dialyzed against 5 liters of buffer C, changing the buffer once during dialysis. After centrifugation at 14,000 g for 10 min, the supernatant (27 ml) is applied to a hydroxyapatite column (Hypatite C, Clarkson Chemicals Co.; 2.6 x 17 cm) equilibrated with buffer C. The column is first eluted with buffer C (40 ml) containing 1 mM dithiothreitol, followed by a linear gradient of 0.02 to 0.4 M potassium phosphate buffer, pH 7.2, containing 1 mM 2-mercaptoethanol and 1 mM dithiothreitol (total gradient volume is 500 ml). Spermidine synthase is eluted practically unadsorbed. The active fractions (140 ml) are pooled and concentrated by the addition of ammonium sulfate (80% saturation). Step 5. Affinity Chromatography on ATPA-Sepharose. The precipitate obtained in step 4 is dissolved in 0.02 M potassium phosphate buffer, pH 7.2, containing 0.3 M NaC1, 0.1 mM EDTA, 1 mM 2-mercaptoethanol, and 1 mM dithiothreitol (buffer D), and passed through a Sephadex G-25 column (1.5 x 17 cm). The desalted enzyme preparation (12.8 ml) is applied at a flow rate of 0.1 ml/min to an ATPA-Sepharose column (1.0 x 2.6 cm) equilibrated with buffer D. The column is washed with 15 ml of buffer D, followed by buffer D containing 0.6 M NaC1 (15 ml) until no protein is being eluted (monitored by UV absorption). Spermidine synthase is then eluted with 6 ml of buffer D containing 0.25 mM decarboxy-AdoMet.13 The enzyme solution is concentrated by ultrafiltration to 1.4 ml. At this stage the enzyme preparation is nearly homogeneous, but polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis show a faint band of contaminating protein in the high-molecular-weight region. Step 6. Gel Filtration. A portion of the concentrated enzyme preparation (0.8 ml) is applied to a Sephadex G-150 column (Pharmacia, 1.5 x 45 cm) equilibrated with buffer D and eluted with the same buffer, collecting 2-ml fractions. The active fractions are pooled (9.5 ml), concentrated to 1.1 ml by ultrafiltration, and stored at 0-4 °. The enzyme is homogeneous as judged by polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. A typical purification is summarized in the table. 13 Synthesis of d e c a r b o x y - A d o M e t is described in K. Samejima, Y. N a k a z a w a , and I. Matsunaga, Chem. Pharm. Bull. 26, 1480 (1978).
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PURIFICATION OF SPERMIDINE SYNTHASE FROM BOVINE BRAIN
Step 1. 2. 3. 4. 5. 6.
Crude extract Ammonium sulfate DEAE-cellulose Hydroxyapatite ATPA-Sepharose Sephadex G-150
Total protein (mg) 50,400 10,200 1480 460 0.92 0.32
Specific Total activity activity (units/mg) (units) 0.10 0.26 1.58 3.60 521 712
Yield" (%)
5050 2670 2330 1650 480 230
100 53 46 33 9.5 4.5
Purification (fold) 1 3 16 36 5200 7100
a The yield at the affinity chromatography step is somewhat variable and can be considerably higher than in this particular experiment depending on the batch of ATPASepharose used.
Purification of Spermidine Synthase from R a t Prostate s
Step 1. Extraction of Tissues. Rat ventral prostates are collected and stored frozen at - 2 0 °. F r o z e n prostates (68 g) of Wistar rats are homogenized in 2 volumes of 25 m M Tris-HCl buffer, p H 7.2, containing 0.3 m M E D T A and l0 m M 2-mercaptoethanol. The 10,000 g supernatant is passed through glass wool. Step 2. Ammonium Sulfate Fractionation. 3 The crude supernatant is fractionated with solid a m m o n i u m sulfate at 0 °. The proteins precipitated between 30 and 60% saturation of a m m o n i u m sulfate are collected. The precipitate is dissolved in a small volume of 25 m M sodium phosphate, p H 7.2, containing 0.3 m M E D T A and 0.5 m M dithiothreitol (buffer A) and passed through a Sephadex G-25 column (equilibrated with buffer A) to remove salt. Step 3. DEAE-Cellulose Chromatography. 3 The desalted protein fraction (1.7 g, 120 ml) is applied to a DEAE-cellulose column (Whatman DE-52, 2.6 x 33 cm) equilibrated with buffer A. The column is eluted with a linear gradient of 0 to 0.4 M NaCI prepared in buffer A (total gradient volume 1600 ml). Fractions containing the synthase activity (110 ml) eluted between 0.08 and 0.1 M sodium chloride are concentrated and equilibrated with buffer A containing 0.3 M NaCI by ultrafiltration in a Centriflo cone (CF 25, Ultrafiltration m e m b r a n e cones, Amicon) to a final volume of 10 ml. Step 4. Affinity Chromatography on ATPA-Sepharose. Affinity chromatography is carried out at 4 ° in a column p a c k e d with A T P A - S e p h a r o s e (0.5 ml) and equilibrated with buffer A containing 0.3 M NaCI. The concentrated e n z y m e solution (77.4 mg of protein, 10 ml) is applied to the
[45]
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column at a flow rate of 0.1-0.2 ml/min. The column is washed with 10 ml of equilibration buffer. More than 99% of the protein is removed by the washing. The synthase is then eluted with 4 ml of equilibration buffer containing 2.5 mM decarboxy-AdoMet. 13 Most of the activity is eluted in the first 2 ml. The effluent is pooled and concentrated by ultrafiltration in a Centriflo cone to a final volume of 0.5 ml (containing 0.4 mg of protein). The synthase is nearly homogeneous in this fraction. Step 5. Gel Filtration. The concentrated synthase solution is applied to a Sephacryl S-300 column (Superfine from Pharmacia, 1.2 × 100 cm) equilibrated with buffer A containing 0.3 M NaCI, and eluted with the same buffer at a flow rate of 3.7 ml/hr. The synthase is eluted in 60-70 ml. The active fraction is stored on ice after concentration to 0.5 ml by ultrafiltration. The final preparation has a specific activity of 1300 units/mg, representing 4480-fold purification over the crude supernatant fraction. The yield of the enzyme is 39%. Properties
Stability. Both brain and prostatic spermidine synthases purified through the affinity chromatography step are stable for several months when stored on ice in a buffer containing 0.3 M NaCl, 1 mM dithiothreitol, and 0.05-0.25 mM decarboxy-AdoMet. Purity. The enzyme preparations after the final step show no evidence of contamination as judged by polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. No S-adenosylmethionine decarboxylase or spermine synthase activities can be detected in the final preparations. Subunit Molecular Weight and Molecular Size. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gives a subunit molecular weight of 35,800 and 37,000 for brain and prostatic enzyme, respectively. Gel filtration on a calibrated column of Sephacryl S-300 reveals a molecular weight of approximately 73,000 for the prostatic enzyme. These data suggest that the native enzyme is composed of two subunits of equal size. Kinetics. An apparent Km for decarboxy-AdoMet is about 1 /zM for both brain and prostatic spermidine synthases. A Km of about 0.03 mM for putrescine is obtained for the brain enzyme, whereas a somewhat higher value (0.1 raM) is obtained for the prostatic spermidine synthase. Both enzymes show a strong substrate inhibition with decarboxy-AdoMet that complicates the study of enzyme kinetics. The enzyme is also weakly inhibited by 5'-methylthioadenosine, one of the reaction products.
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PUTRESCINE AND SPERMIDINE AMINOPROPYLTRANSFERASES
[46]
Substrate Specificity. Cadaverine, in addition to putrescine, can serve as the propylamine acceptor, although cadaverine gives a reaction rate of only 5-7% of that obtained with putrescine under standard assay conditions (1 mM amine). 1,6-Diaminohexane shows some activity (1.5%), whereas 1,3-diaminopropane is totally inactive as a substrate.
[46] P u r i f i c a t i o n o f S p e r m i d i n e A m i n o p r o p y l t r a n s f e r a s e (Spermine Synthase) from Bovine Brain
By
AARNE RAINA, RAIJA-LEENA PAJULA,
and TERHO ELORANTA
Spermine synthase has been partially purified from rat brain I and ventral prostate, 2 tissues that contain the highest activity of this enzyme in t h e r a t . 3 In this chapter we describe the purification of spermine synthase from bovine brain, which also is rich in spermine synthase and is easily obtained in amounts sufficient for large-scale purification. The use of affinity chromatography on spermine-Sepharose considerably facilitates the purification procedure. 4,5 Assay Methods
Spermine Synthase. The assay of spermine synthase activity is carried out as described elsewhere in this volume [42], using [propylamine1-14C]decarboxy-AdoMet as the labeled substrate. Definition of Unit and Specific Activity. One unit of enzyme activity represents the formation of 1 nmol of spermine in 1 min under standard assay conditions. Specific activity is expressed as units per milligram of protein. Protein is measured by the method of Lowry et al. 6 either directly or after precipitation with trichloroacetic acid, with crystalline bovine serum albumin as a standard, or by ultraviolet absorption. 7 P. Hannonen, J. J~inne, and A. Raina, Biochim. Biophys. Acta 289, 225 (1972). 2 H. Hibasami, R. T. Borchardt, S. Y. Chen, J. K. Coward, and A. E. Pegg, Biochern. J. 187, 419 (1980). 3 A. Raina, R.-L. Pajula, and T. O. Eloranta, FEBS Lett. 67, 252 (1976). 4 R.-L. Pajula, A. Raina, and J. Kekoni, FEBS Lett. 90, 153 (1978). 5 R.-L. Pajula, A Raina, and T. Eloranta, Eur. J. Biochern. 101, 619 (1979). 6 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 7 H. M. Kalckar, J. Biol. Chem. 167, 461 (1947).
METHODS IN ENZYMOLOGY, VOL. 94
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9