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[31] CMP-sialic acid synthetase (cytidine-5′-monophospho-sialic acid synthetase)
208
ENZYMES OF SUGAR ACTIVATION
[31]
hibited 91% and 12.6%, respectively, of the activity observed with N-acetylneuraminic acid 9-phosphate. Subsequent studies have shown that the majority of the phosphatase activity with D-ribose-5-P as substrate is found in the supernatant fluid of tile second 60% ammonium sulfate fraction in Step 2. This suggests that a separate enzyme is responsible for the hydrolysis of the pentose phosphate. 1 The following substrates were inactive G (i.e., less than 1% of the value obtained with N-acetylneuraminic acid 9-phosphate) ; UTP, UDP, UMP, UDP-glucose, a-glycerophosphate, glycerie acid-2-P, glyceric acid-3-P, P-enolpyruvate, dihydroxyacetone-P, p-nitrophenyl-P, fructose-l-P, galactese-l-P, fructose-6-P, erythrose-4-P, mannose-6-P, galactose-6-P, glucose-l-P, galactosamine-6-P, glucose-6-P, N-acetylglucosamine-6-P, N-acetylmannosamine-6-P, glucosamine-6-P, and PP~. Activators. Mg~÷ is required for enzyme activity; a sharp optimum is exhibited at a concentration of 1.92 micromoles per milliliter. A 5-fold change in Mg ÷÷ concentration results in a 33-50% loss in activity. The effect of other cations has not been tested. Effect of pH. The reaction proceeds at a maximum velocity in imidazole buffer at pH 7.0. Under standard assay conditions, activity with the purified enzyme is decreased by 85% in the presence of phosphate buffer, pH 7.0. Kinetic Properties. The approximate Km is 1.0 X 10-3M with Nacetylneuraminic acid 9-phosphate as substrate. Stability. While the crude extract is stable to storage in the frozen state, the purified preparation is not (in the presence and absence of albumin and 2-mercaptoethanol). The purified preparation is stable for at least 1 week at 4°; albumin increases the stability. All sugars were of the D configuration.
[31] CMP-Sialic
Acid Synthetase
(Cytidine-5'-monophospho-Sialic
By
Acid Synthetase)
EDWARD L. KEAN and SAUL ROSEMAN
Sialic acid d- CTP ~
• CMP-sialic acid d- PP~
Assay M e t h o d
Principle. The enzyme that catalyzes the synthesis of CMP-siaIic acid was first demonstrated in extracts obtained from hog submaxillary
[31]
CMP-SIALIC ACID S Y N T H E T A S E
209
glands1; it was also isolated from extracts of N e i s s e r i a m e n i n g i t i d i s Y The purification and properties of the hog submaxillary gland enzyme 3 are described below. Enzyme activity is determined by measuring the rate of formation of CMP-sialic acid. The determination of CMP-sialic acid in the presence of large quantities of the substrate, free sialic acid, is based on the principle that the substrate is quantitatively reduced by sodium borohydride under conditions where the product, a glycoside, is not reduced. Following destruction of the excess borohydride, the CMPsialic acid is hydrolyzed to free sialic acid and the latter is determined by the thiobarbituric acid assay, ~ or a radioactive method, 3 neither of which will detect reduced (presumably dihydro) sialic acid. Thiobarbituric Acid Assay Reagents
N-Aeetylneuraminic (NAN), 0.1 M, or N-glycolylneuraminic (NGN) acid 5 adjusted to pH 6 with K H C Q and stored at --18 ° CTP, 0.1 M, adjusted to pH 7 with KHC03 Tris-MgC12 buffer mixture: 9 parts of 1.0M Tris, pH 9.0, and 1 part of 1.0 M MgC12 NaBH4, 2.7 M, freshly prepared in cold H.~O Acetone Sodium metaperiodate, 0.2 M, in 9.0 M H3PO4 Sodium arsenite, 10%, in 0.5 M Na..,SO4 containing 0.1 N H..,SQ 2-Thiobarbiturie acid, 0.6%, in water (TBA) Saturated Na~SO4 Cyelohexanone Procedure. Each assay mixture contains the following components in a final volume of 0.5 ml: 0.025 ml of NAN or NGN; 0.025 ml of CTP; 0.1 ml of Tris-MgCl~ buffer mixture; and 0.01-0.2 unit of enzyme. Controls contain heat-inactivated in place of active enzyme, or lack one of the substrates (CTP or sialic acid), or are incubated for 0 minutes. After incubation for 30 minutes at 37 ° , the mixtures are treated with 0.075 ml of sodium borohydride solution. After frequent agitation at room temperature for 15 minutes, 0.075 ml of acetone is added, the mixture is allowed to stand for an additional 15 minutes and then ana-
~S. Roseman, Proc. Natl. Acad. Sci. U.S. 48, 437 (1962). -"L. Warren and R. S. Blacklow, J. Biol. Chem. 237, 3527 (1962). :~E. L. Kean and S. Roseman, manuscript submitted to J. Biol. Chem. ~L. Warren, J. Biol. Ct~em. 234, 1971 (1959). L. Svennerhohn, Vol. VI [65].
210
ENZYMES OF SUGAR ACTIVATION
[31]
lyzed by the T B A method. 4 Periodate, 0.25 ml, is added to each tube with mixing. After 20 minutes at room temperature, 1.0 ml of the arsenite solution is added with agitation to discharge the yellow-brown color. T B A solution (3.0 ml) is added, and the solution is placed in a boiling water bath for 15 minutes and then cooled in an ice bath. Prior to extracting the chromophore with 4.0 ml of cyclohexanone, 0.5 ml of saturated Na2S04 is added and the tubes are allowed to stand at room temperature for 10 minutes. The absorbancy of the organic phase is determined at 549 mt~. Standard curves are prepared by adding sialic acid, 0.005-0.075 micromole, to tubes containing 2.5 micromoles of reduced sialic acid, in addition to all the other components of the reaction mixture. (The sialic acid is added after the excess sodium borohydride is oxidized with acetone.)
Radioactive Assay Reagents (see thiobarbituric acid assay) NAN-l-14C, 0.1 M, or NGN-l-14C (1 X 10~ cpm/micromole) 6 C T P , 0.1 M Tris-MgC12 buffer mixture: 9 parts of 1.0 M Tris, p H 9.0, to 1 part of 1.0 M MgC12 NaBH~, 2.7 M Saturated glucose solution Cone. HC1 M e t h y l Cellosolve Ethanolamine "Labeled N-acetylneuraminic or N-glycolylneuraminic acids may be prepared by modifying the published procedure [P. Brunetti, G. W. Jourdian, and S. Roseman, J. Biol. Chem. 237, 2447 (1962)]. The modification consists of utilizing bacterial rather than hog kidney NAN-aldolase, and by increasing the concentration of the reactants. In a typical experiment, 0.17 mmole of sodium pyruvate-1240 (specific activity, 4.33 mC/millimole), 0.68 millimole unlabeled sodium pyruvate, 1.0 millimole of N-acetylmannosamine, 0.58 ml of 0.2 M potassium phosphate buffer, pH 7.5, and 0.t ml of purified bacterial NAN-aldolase (containing 52 units of enzyme; see Vol. VI [68]) were adjusted to pH 7.4 with KHC03 and incubated for 12 hours at 37°. Another 25 #l of enzyme was added, the incubation was continued for an additional 6 hours, adjusted to pH 3-4 with HC1, stored at --18 °, and purified by ion exchange chromatography on Dowex-1, formate resin, as described. The formic acid was removed by adsorption of the sialic acid on charcoal (Nuchar C-190), washed with water, and eluted with 1% Ntta in 97% MeOH. (The charcoal is activated with HC1, washed with water, extracted with NH3MeOH solution, washed with water, activated again with tiC1, and washed with water before use.) The solution is evaporated in a vacuum, and the labeled sialie acid is stored as the ammonium salt at --18 °. Yields of isolated material ranged from 60 to 70%.
[31]
CMP-SrALIC ACID SYNTHETASE
211
Procedure. In this assay, NAN-1-14C or NGN-l-14C is used as the substrate. The dihydrosialic acids are resistant under conditions where the sialic acids are decarboxylated7 in acid. Incubation mixtures prepared as described above, but containing ~4C-sialic acids, are placed in Thunberg tubes, the mixtures are treated with borohydride, and the latter is removed by adding 0.5 ml of glucose solution. After 15 minutes at room temperature, 0.5 ml of conc. HC1 is added. The caps of the Thunberg tubes contain 0.2 ml of a mixture of methyl CellosoIve and ethanolamine (1:1) as the C02 trapping agent. The tubes are assembled and evacuated to 200 mm Hg pressure for 15 seconds; the tubes are then closed and placed in a boiling water bath for 20 minutes. After gentle shaking at room temperature for 4 hours to aid diffusion of the C02, 2.5 ml of methyl Cellosolve is added to each cap, 2.0-ml aliquots are removed, and the ~4C02 is determined in a Packard Tri-carb Liquid Scintillation Spectrometer using a methyl Cellosolve counting system.8 Standard curves are prepared, as indicated previously, by adding sialic acid-l-14C (0.005-0.1 micromole; 1.0X 10~ cpm/micromole) after the excess borohydride is oxidized with glucose. The deearboxylation procedure is approximately 50% efficient, but it is directly proportional to the concentration of sialic acid. Both assay methods are applicable to estimation of the crude or purified enzyme. In crude enzyme preparations, a drop of n-octanol should be added to the assay tube, prior to the borohydride, to reduce foaming during reduction. In the case of the TBA assay, vigorous agitation is required to completely remove the yellow-brown color after the addition of arsenite. Enzyme purification was followed with both assays, whereas kinetic studies were performed using the TBA assay. Definition o] a Unit and Specific Activity. One unit of enzyme is defined as the quantity that converts 1 micromole of NAN to CMPNAN per hour, under the conditions described above. Specific activity is defined as units per milligram protein.
Purification Procedure Unless otherwise specified, the following operations are conducted between 0 ° and 4 °, centrifugations are at 35,000 g, and buffers contain 1% 2-mercaptoethanol. Step 1. Crude Extract. Hog submaxillary glands, obtained from a local slaughter house, are quickly separated from adipose tissue and stored in the frozen state. Thin slices of the frozen glands are shaken 7G. Blix, E. Lindberg, L. Odin and I. Werner, Acta Soc. Med. Upsalien. 61, 1 (1956). ~H. Jeffay and J. Alvarez, Anal. Chem. 33, 612 (1961).
212
ENZYMES OF SUGAR ACTIVATION
[31]
gently for 1 hour with 0.10 M potassium phosphate buffer, pH 7.6 (2 ml of buffer without 2-mercaptoethanol is used per gram wet weight of tissue). After centrifuging for 1 hour at 35,000 g, the supernatant fluid is filtered through glass wool and dialyzed for 4 hours against a 50-fold volume of 0.01 M Tris buffer, pH 7.6 (without 2-mercaptoethanol). The dialyzed solution, designated crude extract, is stable to storage in the frozen state. Step 2. DEAE-cellulose Step. The crude extract (12 ml) is transferred to a 2 X 3.5 cm column of DEAE-cellulose. [Diethylaminoethyl cellulose, type 20, purchased from Carl Schleicher and Schuell and Co., is prepared for use by treatment with large volumes of the following solutions in a sequential manner: 1 M KC1, 0.5M KOH, water, 0.1 N HC1 in 95% ethanol, 0.5 M KOH, 2 M KCI, and finally washed with water. The preparation is stored at 4°.] After the column has been washed with 150 ml of 0.01 M Tris buffer, pH 7.6, the enzyme is eluted with 400 ml of the buffer solution containing 0.075 M KC1. Step 3. Calcium Phosphate Gel Step. The DEAE-cellulose fraction is treated with calcium phosphate gel (prepared by the method of Singer and Kearneyg); 5 mg of gel per milligram of protein is optimal for adsorbing the enzyme. After gentle stirring for 20 minutes, the suspension is centrifuged at 10,000 g for 30 minutes, the precipitate is washed with 40 ml of 0.01 M Tris buffer, pH 7.6, and then three times with 25-ml portions of 0.01 M potassium phosphate buffer, pH 7.6 (in each case the gel is stirred for 5 minutes with the buffer and then centrifuged for 10 minutes). The enzyme is finally eluted from the gel by three such treatments, with 25 ml each, of 0.10 M potassium phosphate buffer, pH 7.6. Step 4. Hydroxylapatite Step. A suspension of Hypatite C (a form of hydroxylapatite, purchased from Clarkson Chemical Co. and equilibrated with phosphate buffer to pH 7.6) in 0.001 M potassium phosphate buffer, pH 7.6 (20 ml), is packed in a column under slight pressure so that the gel occupies a volume of 7.9 ml and the flow rate is 0.3 ml per minute. Pressure is required throughout the remainder of the procedure. The calcium phosphate gel eluate (70 ml) is placed on the column, followed by 60 ml of 0.10M potassium phosphate buffer, pH 7.6. The enzyme is then eluted with 75 ml of the phosphate buffer at a concentration of 0.20M. Step 5. Concentration Dialysis Step. The enzyme obtained from the Hypatite C is concentrated by a modified pressure dialysis technique. In this case, the sample is continuously added to a dialysis bag and maintained at atmospheric pressure while the bag is immersed in 3 1 of a 9T. P. Singer and E. B. Kearney, Arch. Biochem. 29, 190 (1950).
[31]
213
CMP-SIALIC ACID SYNTHETASE
buffer solution containing 0.05 M Tris, p H 7.6, the buffer solution being maintained at approximately 20 mm pressure. The dialysis medium is changed three times during the process, and the enzyme solution is concentrated about 10-fold at a rate of 2 ml per hour. The results of the purification procedure are summarized in the table. The purification data are presented at two pH values since the fractions PURIFICATION OF C M P - N A N SYNTHETASE FROM HOG SUBMAXILLARY GLANDS
Specific activity Step and fraction 1. 2. 3. 4. 5.
Crude extract DEAE-cellulose eluate Calcium phosphate gel Hydroxylapatite "Concentration dialysis"
Volume (ml)
Protein (mg/ml)
pH 7.6
pH 9.0
Yield (%)
12 400 75 75 5
12 0. 040 0.037 0. 0035 0.020-0.047
0.29 3.9 14 90 50
0.36 ---95-190
100 150 93 55 30-60
containing phosphate cannot be assayed at the optimum pH, 9.0, owing to precipitation of magnesium phosphate. Properties
Stability. The crude, dialyzed, extract is stable to storage in the frozen state. The purified enzyme is fully active for about 3 weeks when stored at 4 °, but is not stable to freezing and thawing, nor to lyophilization or to storage at room temperature for 12 hours. The addition of substrates partially stabilizes the purified enzyme to freezing and thawing. Effect o] Metals and SH Compounds. The purified enzyme shows an absolute requirement for a divalent cation. At a concentration where Mg ÷÷ shows maximum stimulation, 20 mM, Ca ++ and Mn ÷÷ are 20% as effective, Fe ÷÷ and Co ÷+ about 10%, while Cd ÷÷, Cu ÷÷, and Zn ÷÷ are inactive. All these ions, except Fe ÷÷, are inhibitory when incubated in the presence of Mg ÷÷ at the same concentration. An absolute requirement for SH compounds has not been demonstrated, although the purified enzyme retains its activity for longer periods of time when stored in a solution containing 1% 2-mercaptoethanol. The addition of 2-mercaptoethanol or glutathione over a range of concentrations between 3 and 30 m M stimulates the activity of the purified enzyme preparations 25-50% after the enzyme is dialyzed to remove 2-mercaptoethanol. Kinetic Properties. The following Km values were observed3: NAN, 0.8 X 10-3M; NGN, 2.3 X 10 3 M; CTP, 0.6 X 103 M.
214
ENZYMES OF SUGAR ACTIVATION
[31]
Specificity. Enzymes from a variety of mammalian tissues utilize NAN and N G N as substrates, while the following sugar derivatives are inactive (less than 3% compared with NAN) : NAN-9-P; NGN-9-P; Nacetyl-4-O-acetylneuraminic acid; N-acetyl-7-O-acetylneuraminic acid; N-acetyl-di-O-acetylneuraminic acid; 2-keto-3-deoxy-gluconate and heptonate. Questionable activity is detected with 2-keto-3-deoxy-octonate 1° (about 4% compared with NAN). None of the following nucleotides can substitute for CTP: ATP, GTP, ITP, UTP, TTP, ADP, GDP, IDP, UDP, and dCTP. A slight activity with CDP is ascribed to contamination with CTP. pH Optimum. A broad pH optimum is observed (between pH 8.5 and pH 9.3) with Tris buffer. With glycine buffers, a pH optimum of 9.3 is observed. Stoichiometry of the Reaction. Equimolar quantities of NAN and CTP react to give CMP-NAN and PPi. Reversibility o] the Reaction. The purified enzyme is capable of effecting the pyrophosphorolysis of CMP-NAN in the presence of PP~, thus reversing the reaction. The rate of the reverse reaction is about 1/1000 that of the forward reaction. Distribution o] the Enzyme. The enzyme is found in a variety of tissues obtained from the rat: liver, kidney, spleen, lung, epididymis, brain, and mammary gland. Activity was not detected in muscle and erythrocytes. In addition, the enzyme is found in submaxillary gland extracts obtained from hog, sheep, cow, and human. Enzymes obtained from the mammalian tissues can utilize either NAN or NGN as substrafe. Enzymatic activity is also detected in cell-free preparations obtained from Escherichia coli K-235, the organism from which CMPNAN was first isolated. 1~ In contrast to the enzymes obtained from mammalian sources, the E. coli enzyme cannot utilize N G N as substrate; similar results were reported with a preparation from Neisseria menir~gitidis. 2 Large-Scale Preparation o] Labeled CMP-Sialic Acid. In order to assay the sialyl-transferases (this volume [62] and [73]), labeled CMPsialic acid is used as substrate. For these purposes, it is unnecessary to prepare the highly purified synthetase. Instead, a 400-g sample of tissue is extracted as described above, and 650 ml of the crude extract is placed on a 6 )< 15 cm DEAE-cellulose column. The column is first washed with 1500 ml or more of 0.01 M Tris-HC1 buffer containing 1% 2-mercapto~°E. C. Heath and M. A. Ghalambor, Biochem. Biophys. Res. Commun. 10, 340, 346 (1963). 11:D. G. Comb, F. Shimizu, and S. Roseman, J. Am. Chem. Soc. 81, 5513 (1959).
[31]
CMP-SI&LIC ACID SYNTHETASE
215
ethanol; the end point is reached when the hemoglobin band is completely eluted. The enzyme is then eluted with 4500 ml of 0.01 M Tris buffer, containing 1% 2-mercaptoethanol and 0.075 M KC1, and precipitated by adjusting the solution to 90% of saturation with ammonium sulfate. The mixture is filtered through a Celite pad, and the enzyme is extracted from the pad first with 150 ml and then with 200 ml of 0.05 M Tris-HC1 buffer, pH 7.6, 0.01 M KC1, 1% 2-mercaptoethanol. After dialysis against the same buffer mixture, the enzyme is used for the synthesis of CMPsialic acid. The incubation mixture contains at least 200 units of enzyme, 360 micromoles of labeled sialic acid, 740 micromoles of CTP, and 20 ml of the Tris-Mg mixture used for the assay described above (final volume, 125 ml). After 1 hour at 37 °, an additional 160 units of enzyme and 370 micromoles of CTP are added. After an additional 2 hours at 37 °, the mixture is placed on a 450-ml column of Dowex-1, H C Q - , washed with 2 1 of water, and eluted with 4 1 of a linear gradient, from 0 to 1.0 M triethylamine bicarbonate, pH 7.4. The fractions containing CMP-sialie acid are pooled and evaporated to dryness in a vacuum; the product is converted to the ammonium salt and precipitated from ethanol. The usual yield of CMP-sialic acid ranges from 40 to 50%.
ENZYMES OF SUGAR ACTIVATION
[31]
hibited 91% and 12.6%, respectively, of the activity observed with N-acetylneuraminic acid 9-phosphate. Subsequent studies have shown that the majority of the phosphatase activity with D-ribose-5-P as substrate is found in the supernatant fluid of tile second 60% ammonium sulfate fraction in Step 2. This suggests that a separate enzyme is responsible for the hydrolysis of the pentose phosphate. 1 The following substrates were inactive G (i.e., less than 1% of the value obtained with N-acetylneuraminic acid 9-phosphate) ; UTP, UDP, UMP, UDP-glucose, a-glycerophosphate, glycerie acid-2-P, glyceric acid-3-P, P-enolpyruvate, dihydroxyacetone-P, p-nitrophenyl-P, fructose-l-P, galactese-l-P, fructose-6-P, erythrose-4-P, mannose-6-P, galactose-6-P, glucose-l-P, galactosamine-6-P, glucose-6-P, N-acetylglucosamine-6-P, N-acetylmannosamine-6-P, glucosamine-6-P, and PP~. Activators. Mg~÷ is required for enzyme activity; a sharp optimum is exhibited at a concentration of 1.92 micromoles per milliliter. A 5-fold change in Mg ÷÷ concentration results in a 33-50% loss in activity. The effect of other cations has not been tested. Effect of pH. The reaction proceeds at a maximum velocity in imidazole buffer at pH 7.0. Under standard assay conditions, activity with the purified enzyme is decreased by 85% in the presence of phosphate buffer, pH 7.0. Kinetic Properties. The approximate Km is 1.0 X 10-3M with Nacetylneuraminic acid 9-phosphate as substrate. Stability. While the crude extract is stable to storage in the frozen state, the purified preparation is not (in the presence and absence of albumin and 2-mercaptoethanol). The purified preparation is stable for at least 1 week at 4°; albumin increases the stability. All sugars were of the D configuration.
[31] CMP-Sialic
Acid Synthetase
(Cytidine-5'-monophospho-Sialic
By
Acid Synthetase)
EDWARD L. KEAN and SAUL ROSEMAN
Sialic acid d- CTP ~
• CMP-sialic acid d- PP~
Assay M e t h o d
Principle. The enzyme that catalyzes the synthesis of CMP-siaIic acid was first demonstrated in extracts obtained from hog submaxillary
[31]
CMP-SIALIC ACID S Y N T H E T A S E
209
glands1; it was also isolated from extracts of N e i s s e r i a m e n i n g i t i d i s Y The purification and properties of the hog submaxillary gland enzyme 3 are described below. Enzyme activity is determined by measuring the rate of formation of CMP-sialic acid. The determination of CMP-sialic acid in the presence of large quantities of the substrate, free sialic acid, is based on the principle that the substrate is quantitatively reduced by sodium borohydride under conditions where the product, a glycoside, is not reduced. Following destruction of the excess borohydride, the CMPsialic acid is hydrolyzed to free sialic acid and the latter is determined by the thiobarbituric acid assay, ~ or a radioactive method, 3 neither of which will detect reduced (presumably dihydro) sialic acid. Thiobarbituric Acid Assay Reagents
N-Aeetylneuraminic (NAN), 0.1 M, or N-glycolylneuraminic (NGN) acid 5 adjusted to pH 6 with K H C Q and stored at --18 ° CTP, 0.1 M, adjusted to pH 7 with KHC03 Tris-MgC12 buffer mixture: 9 parts of 1.0M Tris, pH 9.0, and 1 part of 1.0 M MgC12 NaBH4, 2.7 M, freshly prepared in cold H.~O Acetone Sodium metaperiodate, 0.2 M, in 9.0 M H3PO4 Sodium arsenite, 10%, in 0.5 M Na..,SO4 containing 0.1 N H..,SQ 2-Thiobarbiturie acid, 0.6%, in water (TBA) Saturated Na~SO4 Cyelohexanone Procedure. Each assay mixture contains the following components in a final volume of 0.5 ml: 0.025 ml of NAN or NGN; 0.025 ml of CTP; 0.1 ml of Tris-MgCl~ buffer mixture; and 0.01-0.2 unit of enzyme. Controls contain heat-inactivated in place of active enzyme, or lack one of the substrates (CTP or sialic acid), or are incubated for 0 minutes. After incubation for 30 minutes at 37 ° , the mixtures are treated with 0.075 ml of sodium borohydride solution. After frequent agitation at room temperature for 15 minutes, 0.075 ml of acetone is added, the mixture is allowed to stand for an additional 15 minutes and then ana-
~S. Roseman, Proc. Natl. Acad. Sci. U.S. 48, 437 (1962). -"L. Warren and R. S. Blacklow, J. Biol. Chem. 237, 3527 (1962). :~E. L. Kean and S. Roseman, manuscript submitted to J. Biol. Chem. ~L. Warren, J. Biol. Ct~em. 234, 1971 (1959). L. Svennerhohn, Vol. VI [65].
210
ENZYMES OF SUGAR ACTIVATION
[31]
lyzed by the T B A method. 4 Periodate, 0.25 ml, is added to each tube with mixing. After 20 minutes at room temperature, 1.0 ml of the arsenite solution is added with agitation to discharge the yellow-brown color. T B A solution (3.0 ml) is added, and the solution is placed in a boiling water bath for 15 minutes and then cooled in an ice bath. Prior to extracting the chromophore with 4.0 ml of cyclohexanone, 0.5 ml of saturated Na2S04 is added and the tubes are allowed to stand at room temperature for 10 minutes. The absorbancy of the organic phase is determined at 549 mt~. Standard curves are prepared by adding sialic acid, 0.005-0.075 micromole, to tubes containing 2.5 micromoles of reduced sialic acid, in addition to all the other components of the reaction mixture. (The sialic acid is added after the excess sodium borohydride is oxidized with acetone.)
Radioactive Assay Reagents (see thiobarbituric acid assay) NAN-l-14C, 0.1 M, or NGN-l-14C (1 X 10~ cpm/micromole) 6 C T P , 0.1 M Tris-MgC12 buffer mixture: 9 parts of 1.0 M Tris, p H 9.0, to 1 part of 1.0 M MgC12 NaBH~, 2.7 M Saturated glucose solution Cone. HC1 M e t h y l Cellosolve Ethanolamine "Labeled N-acetylneuraminic or N-glycolylneuraminic acids may be prepared by modifying the published procedure [P. Brunetti, G. W. Jourdian, and S. Roseman, J. Biol. Chem. 237, 2447 (1962)]. The modification consists of utilizing bacterial rather than hog kidney NAN-aldolase, and by increasing the concentration of the reactants. In a typical experiment, 0.17 mmole of sodium pyruvate-1240 (specific activity, 4.33 mC/millimole), 0.68 millimole unlabeled sodium pyruvate, 1.0 millimole of N-acetylmannosamine, 0.58 ml of 0.2 M potassium phosphate buffer, pH 7.5, and 0.t ml of purified bacterial NAN-aldolase (containing 52 units of enzyme; see Vol. VI [68]) were adjusted to pH 7.4 with KHC03 and incubated for 12 hours at 37°. Another 25 #l of enzyme was added, the incubation was continued for an additional 6 hours, adjusted to pH 3-4 with HC1, stored at --18 °, and purified by ion exchange chromatography on Dowex-1, formate resin, as described. The formic acid was removed by adsorption of the sialic acid on charcoal (Nuchar C-190), washed with water, and eluted with 1% Ntta in 97% MeOH. (The charcoal is activated with HC1, washed with water, extracted with NH3MeOH solution, washed with water, activated again with tiC1, and washed with water before use.) The solution is evaporated in a vacuum, and the labeled sialie acid is stored as the ammonium salt at --18 °. Yields of isolated material ranged from 60 to 70%.
[31]
CMP-SrALIC ACID SYNTHETASE
211
Procedure. In this assay, NAN-1-14C or NGN-l-14C is used as the substrate. The dihydrosialic acids are resistant under conditions where the sialic acids are decarboxylated7 in acid. Incubation mixtures prepared as described above, but containing ~4C-sialic acids, are placed in Thunberg tubes, the mixtures are treated with borohydride, and the latter is removed by adding 0.5 ml of glucose solution. After 15 minutes at room temperature, 0.5 ml of conc. HC1 is added. The caps of the Thunberg tubes contain 0.2 ml of a mixture of methyl CellosoIve and ethanolamine (1:1) as the C02 trapping agent. The tubes are assembled and evacuated to 200 mm Hg pressure for 15 seconds; the tubes are then closed and placed in a boiling water bath for 20 minutes. After gentle shaking at room temperature for 4 hours to aid diffusion of the C02, 2.5 ml of methyl Cellosolve is added to each cap, 2.0-ml aliquots are removed, and the ~4C02 is determined in a Packard Tri-carb Liquid Scintillation Spectrometer using a methyl Cellosolve counting system.8 Standard curves are prepared, as indicated previously, by adding sialic acid-l-14C (0.005-0.1 micromole; 1.0X 10~ cpm/micromole) after the excess borohydride is oxidized with glucose. The deearboxylation procedure is approximately 50% efficient, but it is directly proportional to the concentration of sialic acid. Both assay methods are applicable to estimation of the crude or purified enzyme. In crude enzyme preparations, a drop of n-octanol should be added to the assay tube, prior to the borohydride, to reduce foaming during reduction. In the case of the TBA assay, vigorous agitation is required to completely remove the yellow-brown color after the addition of arsenite. Enzyme purification was followed with both assays, whereas kinetic studies were performed using the TBA assay. Definition o] a Unit and Specific Activity. One unit of enzyme is defined as the quantity that converts 1 micromole of NAN to CMPNAN per hour, under the conditions described above. Specific activity is defined as units per milligram protein.
Purification Procedure Unless otherwise specified, the following operations are conducted between 0 ° and 4 °, centrifugations are at 35,000 g, and buffers contain 1% 2-mercaptoethanol. Step 1. Crude Extract. Hog submaxillary glands, obtained from a local slaughter house, are quickly separated from adipose tissue and stored in the frozen state. Thin slices of the frozen glands are shaken 7G. Blix, E. Lindberg, L. Odin and I. Werner, Acta Soc. Med. Upsalien. 61, 1 (1956). ~H. Jeffay and J. Alvarez, Anal. Chem. 33, 612 (1961).
212
ENZYMES OF SUGAR ACTIVATION
[31]
gently for 1 hour with 0.10 M potassium phosphate buffer, pH 7.6 (2 ml of buffer without 2-mercaptoethanol is used per gram wet weight of tissue). After centrifuging for 1 hour at 35,000 g, the supernatant fluid is filtered through glass wool and dialyzed for 4 hours against a 50-fold volume of 0.01 M Tris buffer, pH 7.6 (without 2-mercaptoethanol). The dialyzed solution, designated crude extract, is stable to storage in the frozen state. Step 2. DEAE-cellulose Step. The crude extract (12 ml) is transferred to a 2 X 3.5 cm column of DEAE-cellulose. [Diethylaminoethyl cellulose, type 20, purchased from Carl Schleicher and Schuell and Co., is prepared for use by treatment with large volumes of the following solutions in a sequential manner: 1 M KC1, 0.5M KOH, water, 0.1 N HC1 in 95% ethanol, 0.5 M KOH, 2 M KCI, and finally washed with water. The preparation is stored at 4°.] After the column has been washed with 150 ml of 0.01 M Tris buffer, pH 7.6, the enzyme is eluted with 400 ml of the buffer solution containing 0.075 M KC1. Step 3. Calcium Phosphate Gel Step. The DEAE-cellulose fraction is treated with calcium phosphate gel (prepared by the method of Singer and Kearneyg); 5 mg of gel per milligram of protein is optimal for adsorbing the enzyme. After gentle stirring for 20 minutes, the suspension is centrifuged at 10,000 g for 30 minutes, the precipitate is washed with 40 ml of 0.01 M Tris buffer, pH 7.6, and then three times with 25-ml portions of 0.01 M potassium phosphate buffer, pH 7.6 (in each case the gel is stirred for 5 minutes with the buffer and then centrifuged for 10 minutes). The enzyme is finally eluted from the gel by three such treatments, with 25 ml each, of 0.10 M potassium phosphate buffer, pH 7.6. Step 4. Hydroxylapatite Step. A suspension of Hypatite C (a form of hydroxylapatite, purchased from Clarkson Chemical Co. and equilibrated with phosphate buffer to pH 7.6) in 0.001 M potassium phosphate buffer, pH 7.6 (20 ml), is packed in a column under slight pressure so that the gel occupies a volume of 7.9 ml and the flow rate is 0.3 ml per minute. Pressure is required throughout the remainder of the procedure. The calcium phosphate gel eluate (70 ml) is placed on the column, followed by 60 ml of 0.10M potassium phosphate buffer, pH 7.6. The enzyme is then eluted with 75 ml of the phosphate buffer at a concentration of 0.20M. Step 5. Concentration Dialysis Step. The enzyme obtained from the Hypatite C is concentrated by a modified pressure dialysis technique. In this case, the sample is continuously added to a dialysis bag and maintained at atmospheric pressure while the bag is immersed in 3 1 of a 9T. P. Singer and E. B. Kearney, Arch. Biochem. 29, 190 (1950).
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213
CMP-SIALIC ACID SYNTHETASE
buffer solution containing 0.05 M Tris, p H 7.6, the buffer solution being maintained at approximately 20 mm pressure. The dialysis medium is changed three times during the process, and the enzyme solution is concentrated about 10-fold at a rate of 2 ml per hour. The results of the purification procedure are summarized in the table. The purification data are presented at two pH values since the fractions PURIFICATION OF C M P - N A N SYNTHETASE FROM HOG SUBMAXILLARY GLANDS
Specific activity Step and fraction 1. 2. 3. 4. 5.
Crude extract DEAE-cellulose eluate Calcium phosphate gel Hydroxylapatite "Concentration dialysis"
Volume (ml)
Protein (mg/ml)
pH 7.6
pH 9.0
Yield (%)
12 400 75 75 5
12 0. 040 0.037 0. 0035 0.020-0.047
0.29 3.9 14 90 50
0.36 ---95-190
100 150 93 55 30-60
containing phosphate cannot be assayed at the optimum pH, 9.0, owing to precipitation of magnesium phosphate. Properties
Stability. The crude, dialyzed, extract is stable to storage in the frozen state. The purified enzyme is fully active for about 3 weeks when stored at 4 °, but is not stable to freezing and thawing, nor to lyophilization or to storage at room temperature for 12 hours. The addition of substrates partially stabilizes the purified enzyme to freezing and thawing. Effect o] Metals and SH Compounds. The purified enzyme shows an absolute requirement for a divalent cation. At a concentration where Mg ÷÷ shows maximum stimulation, 20 mM, Ca ++ and Mn ÷÷ are 20% as effective, Fe ÷÷ and Co ÷+ about 10%, while Cd ÷÷, Cu ÷÷, and Zn ÷÷ are inactive. All these ions, except Fe ÷÷, are inhibitory when incubated in the presence of Mg ÷÷ at the same concentration. An absolute requirement for SH compounds has not been demonstrated, although the purified enzyme retains its activity for longer periods of time when stored in a solution containing 1% 2-mercaptoethanol. The addition of 2-mercaptoethanol or glutathione over a range of concentrations between 3 and 30 m M stimulates the activity of the purified enzyme preparations 25-50% after the enzyme is dialyzed to remove 2-mercaptoethanol. Kinetic Properties. The following Km values were observed3: NAN, 0.8 X 10-3M; NGN, 2.3 X 10 3 M; CTP, 0.6 X 103 M.
214
ENZYMES OF SUGAR ACTIVATION
[31]
Specificity. Enzymes from a variety of mammalian tissues utilize NAN and N G N as substrates, while the following sugar derivatives are inactive (less than 3% compared with NAN) : NAN-9-P; NGN-9-P; Nacetyl-4-O-acetylneuraminic acid; N-acetyl-7-O-acetylneuraminic acid; N-acetyl-di-O-acetylneuraminic acid; 2-keto-3-deoxy-gluconate and heptonate. Questionable activity is detected with 2-keto-3-deoxy-octonate 1° (about 4% compared with NAN). None of the following nucleotides can substitute for CTP: ATP, GTP, ITP, UTP, TTP, ADP, GDP, IDP, UDP, and dCTP. A slight activity with CDP is ascribed to contamination with CTP. pH Optimum. A broad pH optimum is observed (between pH 8.5 and pH 9.3) with Tris buffer. With glycine buffers, a pH optimum of 9.3 is observed. Stoichiometry of the Reaction. Equimolar quantities of NAN and CTP react to give CMP-NAN and PPi. Reversibility o] the Reaction. The purified enzyme is capable of effecting the pyrophosphorolysis of CMP-NAN in the presence of PP~, thus reversing the reaction. The rate of the reverse reaction is about 1/1000 that of the forward reaction. Distribution o] the Enzyme. The enzyme is found in a variety of tissues obtained from the rat: liver, kidney, spleen, lung, epididymis, brain, and mammary gland. Activity was not detected in muscle and erythrocytes. In addition, the enzyme is found in submaxillary gland extracts obtained from hog, sheep, cow, and human. Enzymes obtained from the mammalian tissues can utilize either NAN or NGN as substrafe. Enzymatic activity is also detected in cell-free preparations obtained from Escherichia coli K-235, the organism from which CMPNAN was first isolated. 1~ In contrast to the enzymes obtained from mammalian sources, the E. coli enzyme cannot utilize N G N as substrate; similar results were reported with a preparation from Neisseria menir~gitidis. 2 Large-Scale Preparation o] Labeled CMP-Sialic Acid. In order to assay the sialyl-transferases (this volume [62] and [73]), labeled CMPsialic acid is used as substrate. For these purposes, it is unnecessary to prepare the highly purified synthetase. Instead, a 400-g sample of tissue is extracted as described above, and 650 ml of the crude extract is placed on a 6 )< 15 cm DEAE-cellulose column. The column is first washed with 1500 ml or more of 0.01 M Tris-HC1 buffer containing 1% 2-mercapto~°E. C. Heath and M. A. Ghalambor, Biochem. Biophys. Res. Commun. 10, 340, 346 (1963). 11:D. G. Comb, F. Shimizu, and S. Roseman, J. Am. Chem. Soc. 81, 5513 (1959).
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CMP-SI&LIC ACID SYNTHETASE
215
ethanol; the end point is reached when the hemoglobin band is completely eluted. The enzyme is then eluted with 4500 ml of 0.01 M Tris buffer, containing 1% 2-mercaptoethanol and 0.075 M KC1, and precipitated by adjusting the solution to 90% of saturation with ammonium sulfate. The mixture is filtered through a Celite pad, and the enzyme is extracted from the pad first with 150 ml and then with 200 ml of 0.05 M Tris-HC1 buffer, pH 7.6, 0.01 M KC1, 1% 2-mercaptoethanol. After dialysis against the same buffer mixture, the enzyme is used for the synthesis of CMPsialic acid. The incubation mixture contains at least 200 units of enzyme, 360 micromoles of labeled sialic acid, 740 micromoles of CTP, and 20 ml of the Tris-Mg mixture used for the assay described above (final volume, 125 ml). After 1 hour at 37 °, an additional 160 units of enzyme and 370 micromoles of CTP are added. After an additional 2 hours at 37 °, the mixture is placed on a 450-ml column of Dowex-1, H C Q - , washed with 2 1 of water, and eluted with 4 1 of a linear gradient, from 0 to 1.0 M triethylamine bicarbonate, pH 7.4. The fractions containing CMP-sialie acid are pooled and evaporated to dryness in a vacuum; the product is converted to the ammonium salt and precipitated from ethanol. The usual yield of CMP-sialic acid ranges from 40 to 50%.