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[196] Cystathionine synthase (rat liver)
454
SULFUR AMINO ACIDS
[ 196]
[196] Cystathionine Synthase I (Rat Liver) By M. SUDA, H. NAKAGAWA, and H. KIMURA HOOC-- CHNH~-- CI-I2OH
+
H8-- CH2-- CH~-- CHNH2--COOH
Homocysteine
Serine
HOOC -- C HNI-I.-- C I-I~- - S -- CH2- - CIS~- C HNI-I~-- COOH
Cystathionine
Assay Method
Principle. The radioactive cystathionine formed from homocysteine and serine-14C is determined after it is separated from radioactive serine and pyruvate on a column of a cationic ion exchanger, Dowex 50W-X8(H+). The method described here is a modification of that of Mudd et al. TM Reagents L-Serine-U-~4C, 0.1 M. Radioactive serine-U-14C is diluted with carrier L-serine to give a final specific radioactivity of approximately 10,000 cpm per micromole in a 0.1 M solution of the amino acid. L-Homocysteine, 0.2 M. L-Homocysteine thiolactone hydrochloride, 30.7 mg, is dissolved in 0.2 ml of 5 N N a O H , incubated for 5 minutes at 37 °, and then neutralized with 2 N HCI (approximately 0.4 ml). The final volume is adjusted with 0.05 M Tris-HCl buffer, pH 8.6, to 1 ml. z Pyridoxal phosphate, 10 -3 M DL-Allocystathionine, 0.05 M Tris-HCl buffer, 0.5 M, p H 8.6, containing 5 × 10 -3 M ethylenediamine tetraacetate (EDTA)
1See article [195] for the p r e p a r a t i o n o f cystathionine synthase f r o m o t h e r sources. See
also Vol. IV [127a] for an earlier rat liver preparation. la S. H. Mudd, J. A. Finkeistein, F. I r r e v e r r e , a n d L. Laster,J. Biol. Chem. 240, 4382 (1965). 2j. A. D u e r r e and C. H. Miller, Anal. Biochem. 17, 310 (1966).
[ 196]
CVSTATmONINE SYNTHASE(RAT LIVER)
455
Trichloroacetic acid (TCA), 8% HC1, 0.4 N Pyridine, 1 N Dowex 50W-X8(H+), 200-400 mesh
Procedure. One-tenth milliliter of homocysteine, 0.05 ml of pyridoxal phosphate, 0.1 ml of Tris-HC1 buffer containing EDTA, and enzyme are put into a thick-walled conical centrifuge tube. The volume is adjusted with water to 0.45 ml. The tube is warmed in a 37 ° water bath for 5 minutes. The reaction is started with 0.05 ml of serine-~4C and is linear with time for at least 30 minutes. In our procedure, the reaction is terminated with 0.5 ml of 8% TCA 15 minutes after incubation at 37 °. Any precipitate formed after the preparation has stood for 10 minutes standing in an ice bath is removed by centrifugation. A 0.5 ml aliquot of the supernatant is mixed with 0.01 ml of cystathionine as a carrier and then diluted with 12 ml of water. The solution is applied on a Dowex 50 column (0.9 x 3 cm). The column is washed with 5 ml of" water followed by 30 ml of 0.4 N HC1 with which serine is eluted. To the column is then added a 4 ml portion, followed by a 5-ml portion, of 1 N pyridine. Cystathionine is eluted in the 5-ml fraction. An aliquot of the eluate is applied to a planchet and dried; then the radioactivity was determined with a gas flow counter. For a control tube, heat-inactivated enzyme is added in place of the active enzyme. Definition of Unit and Specific Activity. One unit is defined as the amount of enzyme which catalyzes the formation of 1 micromole ofcystathionine per minute. Specific activity is expressed as units per milligram of protein. Application of Assay Method to Crude Preparation. When rat liver homogenate is prepared in phosphate buffer as described in the purification procedure, cystathionase as well as cystathionine synthase is tound in 105,000 g supernatant. At pH 8.6, however, cystathionase does not interfere with cystathionine synthase assay, since there is no difference between cystathionine synthase activities at this pH in the presence and absence of a neutralization antibody against cystathionase obtained from a rabbit immunized with crystalline rat liver cystathionase. 3'4 Crude liver homogenates also have serine dehydratase activity, but since this enzyme activity is inhibited by homocysteine 5 under the assay conditions, this should not usually interfere with the assay for liver cystathionine synthase. It is recommended, however, that a neutralization antibody against liver serine dehydratase, prepared from rabbits 3y. Matsuo and D. M. Greenbevg, J. Biol. Chem. 230, 561 (1958). 4A. Kato, M. Ogura, H. Kimura, T. Kawai and M. Suda,J. Biochem. (Tokyo) 59, 34 (1966). "SA. S, S. M. Selim and D. M. Greenberg, Biochim. Biophys. Acta 42, 211 (1960).
456
SULFUR AMINO ACIDS
[ 196]
immunized with crystalline serine dehydratase (this volume [182], be used for the liver enzyme assay when serine dehydratase is elevated by feeding a high protein diet, administration of glucocorticoid, or starvation. Purification Procedure All preparations are carried out at 2° , unless otherwise specified. Step 1. Preparation of Crude Extract. One h u n d r e d male Wistar rats, weighing 150-250 g each, are sacrificed by decapitation, and the livers are homogenized with 3 volumes of buffer A (0.05 M potassium phosphate buffer, pH 7.2, 0.15 M KCI, and 10-3 M EDTA). The homogenate is centrifuged for 20 minutes at 10,000 g. Step 2. Acid Treatment. The supernatant fraction is adjusted to pH 5.5 by dropwise addition of 50% acetic acid and then allowed to stand for 30 minutes with mechanical stirring. The precipitate formed is removed by centrifugation for 20 minutes, and the supernatant is ad.justed to pH 7.2 with 2 N NaOH. This procedure is not effective for the purification of the synthase, but is very effective in removing serine dehydratase, which is unstable at an acidic pH. Step 3. Ammonium Sulfate Fractionation. Solid ammonium sulfate is added with mechanical stirring to the above fraction to 25% saturation, and the pH is adjusted to 7.2 with 10% ammonium hydroxide. One hour after addition of the salt, the precipitate is removed by centrifugation for 20 minutes at 10,000 g and the supernatant is brought to 37.5% saturation with ammonium sulfate. The precipitate is collected by centrifugation for 20 minutes at 10,000 g and dissolved in one-tenth volume of buffer B (0.01 M potassium phosphate buffer, pH 7.8, and 10 -3 M EDTA). The fraction is dialyzed against 2 changes of 10 volumes of buffer B. Most cystathionase activity is removed by this procedure. Step 4. First DEAE-Cellulose Column Chromatography. The dialyzed fraction is applied on a DEAE-cellulose column (5 × 20 cm) which has been equilibrated with buffer B. The column is washed with 1000 ml of buffer B and then eluted with 1000 ml of 0.05 M potassium phosphate buffer, pH 7.8, containing 10 -3 M EDTA followed by 1000 ml of 0.1 M potassium phosphate buffer, pH 7.8, containing 10 -3 M EDTA. The last eluates containing cystathionine synthase are pooled and brought to 40% saturation with solid ammonium sulfate. The pH is adjusted to 7.2 with 10% ammonium hydroxide solution. After 30 minutes with mechanical stirring, the precipitate is collected by centrifugation and dissolved in a small volume of buffer A. Step 5. Treatment with P-cellulose. The fraction is applied on a column (4.5 × 44 cm) of Sephadex G-25 which has been equilibrated with 0.01 M maleate buffer (potassium salt), pH 5.5 containing 10 -3 M EDTA to
[196]
CYSTATHIONINE SYNTHASE (RAT LIVER)
457
remove ammonium sulfate. The column is eluted with the same buffer. The protein is followed spectrophotometrically, and the fractions are pooled (volume is approximately 250 ml) and mixed with a 100-ml portion (10 g dry weight) of P-cellulose which has been equilibrated with 0.01 M maleate buffer, pH 5.5, containing 10 -3 M EDTA. The mixture is allowed to stand for 30 minutes with mechanical stirring and then centrifuged for 20 minutes at 10,000 g. The precipitate is washed once with 200 ml of the same buffer by centrifugation. The supernatant and washing are combined and brought to 40% saturation with solid ammonium sulfate. The precipitate is collected as described above and dissolved in a small volume of buffer A. Step 6. Sephadex G-IO0 Column Chromatography. The above fraction is applied on a Sephadex G-100 column (3 × 90 cm) which has been equilibrated with buffer B. The column is eluted with the same buffer. Fractions of 5 ml are collected in a fraction collector. The enzyme activity is usually fbund in tubes 25 to 36. Step 7. Second DEAE-Cellulose Column Chromatography. The pooled active fraction is applied to a DEAE-cellulose column (1.2 X 5 cm) which has been equilibrated with buffer B. The column is eluted by linear gradient elution technique; 35 ml of buffer B is contained in the mixing chamber, and 35 ml of 0.2 M potassium phosphate buffer, pH 7.8, containing 10 -3 M EDTA, is added slowly with stirring during elution. Fractions of 1.2 ml are collected in a fraction collector. The active fractions (tubes 23 to 34) are combined. The enzyme at this step can be stored without loss of activity at - 20 °. A representative purification is given in the table. PURIVlCATION OF (]YSTATHIONINE SYNTHASE fROM RAT LIVER
Step Crude extract Acid treatment (N H4)2SO4 fraction (25-37.5% saturation) First DEAE-cellulose column chromatography, (NH4)~SO4 fraction P-cellulose eluate, (NH4)2SO4 fraction Sephadex G-100 column chromatography Second DEAE-cellulose column chromatography
Total volume (ml)
Total units
Total protein (rag)
2960 2890 265
354 286 190
69,900 53,200 6,35(I
24
76
500
Specific activity (units/rag protein x 10 -:~) 5.1 5.4 30.0
Yield (%) 100 80.8 53.7
152
21.4
7.0
15.9
33.5
473
4.5
56.0
7.7
10.0
704
2.2
30.0
4.0
3.1
1212
I. 1
458
SULFUR AMINO ACIDS
[ 196]
Properties of Liver Cystathionine Synthase
Specificity. As discussed in articles [182] and [195i], cystathionine synthase has been considered to be the same enzyme as serine dehydratase; however, the purified preparation of cystathionine synthase has no serine dehydratase activity. T h e purified preparation also has no cystathionase activity. Biological Significance. Cystathionine synthase is found in various tissues of various animals. High activity is found in the liver and pancreas of the rat, rabbit, and monkey.' H u m a n liver also has high activity. Higher activity is found in kidney, brain, and adipose tissue of various animals. It has been reported that cystathionine synthase, a key enzyme of biosynthesis of cysteine from methionine in mammalian liver, is repressed by the end product, dietary cystine. 6a It has also been reported that the liver of a patient with homocystinuria has no cystathionine synthase, 8 suggesting that, in the absence of the synthase, homocystine accumulates. Coenzyme. Crude liver preparations are not activated by pyridoxal phosphate; however, the purified preparation is completely inhibited by 10 -3 M NH2OH and the inhibition is reversed by pyridoxal phosphate. Other Properties. Km values for serine and homocysteine are 1.6 × 10 -a M and 1.1 x 10 -z M, respectively. T h e enzyme exhibits the highest activity at pH 8.6 with a sharp decrease of the activity toward an alkaline pH.
aM. Suda, Advan. Enzyme Regulation 5, 181 (1967). ~J. D. Finkelstein and S. H. Mudd, J. Biol. Chem. 242, 873 (1967). aS. H. Mudd, J. D. Finkelstein, F. Irreverre, and L. Laster, Biochem. Biophys. Res. Commun. 19, 665 (1965).
SULFUR AMINO ACIDS
[ 196]
[196] Cystathionine Synthase I (Rat Liver) By M. SUDA, H. NAKAGAWA, and H. KIMURA HOOC-- CHNH~-- CI-I2OH
+
H8-- CH2-- CH~-- CHNH2--COOH
Homocysteine
Serine
HOOC -- C HNI-I.-- C I-I~- - S -- CH2- - CIS~- C HNI-I~-- COOH
Cystathionine
Assay Method
Principle. The radioactive cystathionine formed from homocysteine and serine-14C is determined after it is separated from radioactive serine and pyruvate on a column of a cationic ion exchanger, Dowex 50W-X8(H+). The method described here is a modification of that of Mudd et al. TM Reagents L-Serine-U-~4C, 0.1 M. Radioactive serine-U-14C is diluted with carrier L-serine to give a final specific radioactivity of approximately 10,000 cpm per micromole in a 0.1 M solution of the amino acid. L-Homocysteine, 0.2 M. L-Homocysteine thiolactone hydrochloride, 30.7 mg, is dissolved in 0.2 ml of 5 N N a O H , incubated for 5 minutes at 37 °, and then neutralized with 2 N HCI (approximately 0.4 ml). The final volume is adjusted with 0.05 M Tris-HCl buffer, pH 8.6, to 1 ml. z Pyridoxal phosphate, 10 -3 M DL-Allocystathionine, 0.05 M Tris-HCl buffer, 0.5 M, p H 8.6, containing 5 × 10 -3 M ethylenediamine tetraacetate (EDTA)
1See article [195] for the p r e p a r a t i o n o f cystathionine synthase f r o m o t h e r sources. See
also Vol. IV [127a] for an earlier rat liver preparation. la S. H. Mudd, J. A. Finkeistein, F. I r r e v e r r e , a n d L. Laster,J. Biol. Chem. 240, 4382 (1965). 2j. A. D u e r r e and C. H. Miller, Anal. Biochem. 17, 310 (1966).
[ 196]
CVSTATmONINE SYNTHASE(RAT LIVER)
455
Trichloroacetic acid (TCA), 8% HC1, 0.4 N Pyridine, 1 N Dowex 50W-X8(H+), 200-400 mesh
Procedure. One-tenth milliliter of homocysteine, 0.05 ml of pyridoxal phosphate, 0.1 ml of Tris-HC1 buffer containing EDTA, and enzyme are put into a thick-walled conical centrifuge tube. The volume is adjusted with water to 0.45 ml. The tube is warmed in a 37 ° water bath for 5 minutes. The reaction is started with 0.05 ml of serine-~4C and is linear with time for at least 30 minutes. In our procedure, the reaction is terminated with 0.5 ml of 8% TCA 15 minutes after incubation at 37 °. Any precipitate formed after the preparation has stood for 10 minutes standing in an ice bath is removed by centrifugation. A 0.5 ml aliquot of the supernatant is mixed with 0.01 ml of cystathionine as a carrier and then diluted with 12 ml of water. The solution is applied on a Dowex 50 column (0.9 x 3 cm). The column is washed with 5 ml of" water followed by 30 ml of 0.4 N HC1 with which serine is eluted. To the column is then added a 4 ml portion, followed by a 5-ml portion, of 1 N pyridine. Cystathionine is eluted in the 5-ml fraction. An aliquot of the eluate is applied to a planchet and dried; then the radioactivity was determined with a gas flow counter. For a control tube, heat-inactivated enzyme is added in place of the active enzyme. Definition of Unit and Specific Activity. One unit is defined as the amount of enzyme which catalyzes the formation of 1 micromole ofcystathionine per minute. Specific activity is expressed as units per milligram of protein. Application of Assay Method to Crude Preparation. When rat liver homogenate is prepared in phosphate buffer as described in the purification procedure, cystathionase as well as cystathionine synthase is tound in 105,000 g supernatant. At pH 8.6, however, cystathionase does not interfere with cystathionine synthase assay, since there is no difference between cystathionine synthase activities at this pH in the presence and absence of a neutralization antibody against cystathionase obtained from a rabbit immunized with crystalline rat liver cystathionase. 3'4 Crude liver homogenates also have serine dehydratase activity, but since this enzyme activity is inhibited by homocysteine 5 under the assay conditions, this should not usually interfere with the assay for liver cystathionine synthase. It is recommended, however, that a neutralization antibody against liver serine dehydratase, prepared from rabbits 3y. Matsuo and D. M. Greenbevg, J. Biol. Chem. 230, 561 (1958). 4A. Kato, M. Ogura, H. Kimura, T. Kawai and M. Suda,J. Biochem. (Tokyo) 59, 34 (1966). "SA. S, S. M. Selim and D. M. Greenberg, Biochim. Biophys. Acta 42, 211 (1960).
456
SULFUR AMINO ACIDS
[ 196]
immunized with crystalline serine dehydratase (this volume [182], be used for the liver enzyme assay when serine dehydratase is elevated by feeding a high protein diet, administration of glucocorticoid, or starvation. Purification Procedure All preparations are carried out at 2° , unless otherwise specified. Step 1. Preparation of Crude Extract. One h u n d r e d male Wistar rats, weighing 150-250 g each, are sacrificed by decapitation, and the livers are homogenized with 3 volumes of buffer A (0.05 M potassium phosphate buffer, pH 7.2, 0.15 M KCI, and 10-3 M EDTA). The homogenate is centrifuged for 20 minutes at 10,000 g. Step 2. Acid Treatment. The supernatant fraction is adjusted to pH 5.5 by dropwise addition of 50% acetic acid and then allowed to stand for 30 minutes with mechanical stirring. The precipitate formed is removed by centrifugation for 20 minutes, and the supernatant is ad.justed to pH 7.2 with 2 N NaOH. This procedure is not effective for the purification of the synthase, but is very effective in removing serine dehydratase, which is unstable at an acidic pH. Step 3. Ammonium Sulfate Fractionation. Solid ammonium sulfate is added with mechanical stirring to the above fraction to 25% saturation, and the pH is adjusted to 7.2 with 10% ammonium hydroxide. One hour after addition of the salt, the precipitate is removed by centrifugation for 20 minutes at 10,000 g and the supernatant is brought to 37.5% saturation with ammonium sulfate. The precipitate is collected by centrifugation for 20 minutes at 10,000 g and dissolved in one-tenth volume of buffer B (0.01 M potassium phosphate buffer, pH 7.8, and 10 -3 M EDTA). The fraction is dialyzed against 2 changes of 10 volumes of buffer B. Most cystathionase activity is removed by this procedure. Step 4. First DEAE-Cellulose Column Chromatography. The dialyzed fraction is applied on a DEAE-cellulose column (5 × 20 cm) which has been equilibrated with buffer B. The column is washed with 1000 ml of buffer B and then eluted with 1000 ml of 0.05 M potassium phosphate buffer, pH 7.8, containing 10 -3 M EDTA followed by 1000 ml of 0.1 M potassium phosphate buffer, pH 7.8, containing 10 -3 M EDTA. The last eluates containing cystathionine synthase are pooled and brought to 40% saturation with solid ammonium sulfate. The pH is adjusted to 7.2 with 10% ammonium hydroxide solution. After 30 minutes with mechanical stirring, the precipitate is collected by centrifugation and dissolved in a small volume of buffer A. Step 5. Treatment with P-cellulose. The fraction is applied on a column (4.5 × 44 cm) of Sephadex G-25 which has been equilibrated with 0.01 M maleate buffer (potassium salt), pH 5.5 containing 10 -3 M EDTA to
[196]
CYSTATHIONINE SYNTHASE (RAT LIVER)
457
remove ammonium sulfate. The column is eluted with the same buffer. The protein is followed spectrophotometrically, and the fractions are pooled (volume is approximately 250 ml) and mixed with a 100-ml portion (10 g dry weight) of P-cellulose which has been equilibrated with 0.01 M maleate buffer, pH 5.5, containing 10 -3 M EDTA. The mixture is allowed to stand for 30 minutes with mechanical stirring and then centrifuged for 20 minutes at 10,000 g. The precipitate is washed once with 200 ml of the same buffer by centrifugation. The supernatant and washing are combined and brought to 40% saturation with solid ammonium sulfate. The precipitate is collected as described above and dissolved in a small volume of buffer A. Step 6. Sephadex G-IO0 Column Chromatography. The above fraction is applied on a Sephadex G-100 column (3 × 90 cm) which has been equilibrated with buffer B. The column is eluted with the same buffer. Fractions of 5 ml are collected in a fraction collector. The enzyme activity is usually fbund in tubes 25 to 36. Step 7. Second DEAE-Cellulose Column Chromatography. The pooled active fraction is applied to a DEAE-cellulose column (1.2 X 5 cm) which has been equilibrated with buffer B. The column is eluted by linear gradient elution technique; 35 ml of buffer B is contained in the mixing chamber, and 35 ml of 0.2 M potassium phosphate buffer, pH 7.8, containing 10 -3 M EDTA, is added slowly with stirring during elution. Fractions of 1.2 ml are collected in a fraction collector. The active fractions (tubes 23 to 34) are combined. The enzyme at this step can be stored without loss of activity at - 20 °. A representative purification is given in the table. PURIVlCATION OF (]YSTATHIONINE SYNTHASE fROM RAT LIVER
Step Crude extract Acid treatment (N H4)2SO4 fraction (25-37.5% saturation) First DEAE-cellulose column chromatography, (NH4)~SO4 fraction P-cellulose eluate, (NH4)2SO4 fraction Sephadex G-100 column chromatography Second DEAE-cellulose column chromatography
Total volume (ml)
Total units
Total protein (rag)
2960 2890 265
354 286 190
69,900 53,200 6,35(I
24
76
500
Specific activity (units/rag protein x 10 -:~) 5.1 5.4 30.0
Yield (%) 100 80.8 53.7
152
21.4
7.0
15.9
33.5
473
4.5
56.0
7.7
10.0
704
2.2
30.0
4.0
3.1
1212
I. 1
458
SULFUR AMINO ACIDS
[ 196]
Properties of Liver Cystathionine Synthase
Specificity. As discussed in articles [182] and [195i], cystathionine synthase has been considered to be the same enzyme as serine dehydratase; however, the purified preparation of cystathionine synthase has no serine dehydratase activity. T h e purified preparation also has no cystathionase activity. Biological Significance. Cystathionine synthase is found in various tissues of various animals. High activity is found in the liver and pancreas of the rat, rabbit, and monkey.' H u m a n liver also has high activity. Higher activity is found in kidney, brain, and adipose tissue of various animals. It has been reported that cystathionine synthase, a key enzyme of biosynthesis of cysteine from methionine in mammalian liver, is repressed by the end product, dietary cystine. 6a It has also been reported that the liver of a patient with homocystinuria has no cystathionine synthase, 8 suggesting that, in the absence of the synthase, homocystine accumulates. Coenzyme. Crude liver preparations are not activated by pyridoxal phosphate; however, the purified preparation is completely inhibited by 10 -3 M NH2OH and the inhibition is reversed by pyridoxal phosphate. Other Properties. Km values for serine and homocysteine are 1.6 × 10 -a M and 1.1 x 10 -z M, respectively. T h e enzyme exhibits the highest activity at pH 8.6 with a sharp decrease of the activity toward an alkaline pH.
aM. Suda, Advan. Enzyme Regulation 5, 181 (1967). ~J. D. Finkelstein and S. H. Mudd, J. Biol. Chem. 242, 873 (1967). aS. H. Mudd, J. D. Finkelstein, F. Irreverre, and L. Laster, Biochem. Biophys. Res. Commun. 19, 665 (1965).