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[102] Acetylation of d-glucosamine by pigeon liver extracts
612
[102]
ENZYMES OF LIPID METABOLISM
over 0.05 micromole/ml, give maximal rates. With p-nitroaniline 50% saturation of the enzyme is attained at approximately 0.1 micromole/ml. SUMMARY OF PURIFICATION PROCEDURE
Fraction 1 and 2. Crude extract 3. Acetone fraetionation
Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 96 16
406 1620
100 50
150 70
39,000 38 26,000
10.6
-67
4. C~
Eluate I Eluate II
15,000 0.55 3,500 0.38
275 184
38 9
[102] A c e t y l a t i o n of D - G l u c o s a m i n e by P i g e o n Liver E x t r a c t s A c~ SCoA + v-Glucosamine--. N-Acetyl-D-glucosamine + CoA
By MORRIS SOODAK Assay Method
Principle. Pigeon liver extracts contain an acetokinase which transfers the acetyl group of A c ~ S C o A to glucosamine and galactosamine. 1 The appearance of the acetylated amino sugar is followed by a modification of the Morgan and Elson method, 2 which is specific for N-acetylamino sugars. The formation of N-acetyl-D-glucosamine is confirmed chromatographically on paper. Reagents O.1M N-acetylglucosamine.3 0 . 1 M D-glucosamine.HCl. 1.0 M KAc. 0 . 1 M cysteine.HC1. 0 . 1 M MgC12. O.1M ATP (Na + or K+). 1.0 M KF. CoA solution, 200 units/ml. 2.0 M Tris, pH 8.2. 0.25 M lithium acetyl phosphate. t T. C. Chou a n d M. Soodak, J. Biol. Chem. 196, 105 (1952). t W. T. J. Morgan and L. A. Elson, Biochem. J. 26, 988 (1934).
N-Acetylglucosamine was obtained from Krishell Laboratories, Portland, Oregon.
[102] ACETYLATION OF I)-GLUCOSAMINE BY PIGEON LIVER EXTRACTS 613
1.0 M NH~C1. Enzymes. (1) Crude and aged extract of pigeon liver acetone powder. 4 (2a) Partially purified glucosamine acceptor enzyme from pigeon liver ~ (see below). (2b) Partially purified transacetylase from Clostridium kluyveri2 5% TCA. 1.0 M Na~CO3. 95 % ethanol. Ehrlich's reagent. Dissolve 1.6 g. of p-dimethylaminobenzaldehyde 7 in a cooled mixture of 30 ml. of conc. HC1 and 30 ml. of 95% ethanol.
Procedure 1. Acetylation with Aged Pigeon Liver Extract. The reaction mixture, final volume 1 ml., which is incubated for 2 hours at 37 °, in 16 × 100-mm. test tubes, is as follows: 10 micromoles glucosamine.HC1 (0.1 ml.), 50 micromoles acetate (0.05 ml.), 10 micromoles cysteine.HCl (0.1 ml.), 10 micromoles MgCI~ (0.1 ml.), 10 micromoles ATP (0.i ml.), 50 micromoles KF (0.05 ml.), 20 units CoA (0.1 ml.), 200 micromoles Tris (0.1 ml.), and 0.3 ml. aged pigeon liver extract (corresponding to 30 mg. acetone powder). Procedure 2. Acetylation with Partially Purified Glucosamine Acceptor System from Pigeon Liver (Fraction A-60). ~ This preparation no longer contains the acetate-ATP reaction found in crude pigeon liver extracts. A c ~ S C o A is here supplied by the transacetylase system. The reaction mixture, which is incubated for 2 hours at 30 °, is as follows: 10 micromoles glucosamine.HC1 (0.1 ml.), 25 micromoles acetyl phosphate (0.1 ml.), 10 micromoles cysteine.HC1 (0.1 ml.), 5 micromoles MgCl~ (0.05 ml.), 100 micromoles NH4C1 (0.1 ml.), 20 units CoA (0.1 ml.), 10 units transacetylase (0.05 ml.), 0.1 ml. water, and 0.3 ml. liver Fraction A-60. Control tubes are run which contain no ATP or acetyl phosphate. To another set of control tubes is added 0.5 to 2 micromoles of N-acetylglucosamine. These latter tubes serve as internal standards. The enzymatic reaction is terminated by the addition of 2 ml. of TCA. The protein precipitate is spun down. A 1-ml. aliquot is transferred to a 16 × 150-mm. test tube, followed by 0.5 ml. of water and 0.5 ml. of M Na2CO3. The tubes are heated in a boiling water bath for 10 minutes 4 N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174~ 37 (1948). The preparation of pigeon Iiver acetone powder and of the extracts therefrom is described in Vol. I [101]; and also see Vol. I I I [132]. 6 T. C. Chou and F. Lipmann, J. Biol. Chem. 196, 89 (1952). 6 See section on transacetylase (Vol. I [98]). 7 A preparation of p-dimethylaminobenzaldehyde (highest purity) from Phanstiehl Chemical Co., Waukegan, Illinois, may be used without recrystallization.
614
ENZYMES OF LIPID METABOLISM
[102]
and cooled in cold water. 2.5 ml. of 95 % ethanol is added to each tube with shaking, followed by 0.5 ml. of Ehrlich's reagent. After shaking, the tubes are kept at 37 ° for 30 minutes for color development and then read on a Klett colorimeter with a 540-m~ filter. In the experiments with transacetylase plus the partially purified glucosamine acceptor enzyme, sufficient N-acetylglucosamine is formed to confirm its appearance by paper chromatography. In this case, after the enzymatic incubation, a 0.5-ml. aliquot is removed and treated with 1 ml. of TCA for the colorimetric determination as above. The remainder of the incubation mixture (0.5 ml.) is treated in the following manner. 3 ml. of 95% ethanol is added, and the tubes are allowed to stand haft an hour for protein precipitation. After spinning, the supernatants are evaporated to dryness in a bath at 40 ° or below by means of a stream of air. The residue is extracted with 2 ml. of 95 % ethanol, and the extracts are brought to dryness again. The final residue is dissolved in 0.1 to 0.2 ml. of water, and appropriate aliquots are applied to Whatman No. 1 sheets. Ascending chromatograms are run for 16 hours with 80% propanol containing 0.8% ammonium acetate as the solvent. The spots are developed by the methods described by Partridge. 8 The RI for glucosamine and galactosamine is 0.32. The R / f o r acetylglucosamine is 0.46.
Preparation of the Partially Purified Glucosamine Acceptor Enzyme from Pigeon Liver Extracts. s The fraction precipitating between 40 and 60% acetone concentration contains the glucosamine acetokinase. All operations are carried out in the cold. For centrifugation a refrigerated centrifuge at - 1 5 ° is used throughout. A 10 % suspension of pigeon liver acetone powder in a 25% acetone solution containing 0.1 M Tris at pH 8.2 is stirred gently for 5 to 10 minutes at about - 5 °. The insoluble material is centrifuged at 4000 r.p.m, for 10 minutes and discarded. The supernatant is brought to 40% acetone concentration by the cautious addition of cold acetone (about - 5 ° ) . The precipitate is centrifuged as above. The supernatant is further treated with acetone to attain a 60 % concentration. This 40 to 60% fraction is spun down. The 25 to 40% (Fraction A-40) and the 40 to 60% (Fraction A-60) precipitates are separately dissolved in 1 ml./g, of original powder of M Tris at pH 8.2 and dialyzed for 12 hours with one change of fluid against 4.5 1. of a solution containing 0.001 M cysteine.HC1, 0.02 M KHCO~, and 0.5% KC1 (pH 8 adjusted with K~C03). Fraction A-40 contains the acetate-ATP reaction, and the enzymes for the synthesis of citrate, acetoacetate, and glutamine. Fraction A-60 contains the arylamine acetylation acceptor enzyme as well as that for the amino sugars (see below). All the enzymes used are stored at - 1 5 to - 3 0 ° and are stable for many months. 8 S. M. Partridge, Biochem. J. 42, 238 (1948).
[102]
ACETYLATIOI~ OF D-GLUCOSAMINE BY PIGEON LIVER EXTRACTS 6 1 5
Results. Crude aged pigeon liver extracts (0.3 ml.) under the conditions stated above, or when combined with the transacetylase system as acetyl~SCoA source, synthesize from 0.1 to 0.25 micromole of acetylglucosamine. With the transacetylase system plus Fraction A-60, from 1 to 2 micromoles is formed. This corresponds to about a seven-fold concentration of the enzyme in the A-60 fraction. In the latter case, the acetylglucosamine formation is confirmed by paper chromatography. The synthesis of acetylgalactosamine via the Fraction A-60 system when followed by the Morgan and Elson colorimetric method using acetylglucosamine as a standard is about 30% of that of acetylglucosamine. Aminoff et al., 9 however, have recently shown that the color yield of acetylgalactosamine is only 20 % of that of acetylglucosamine. Taking this into account, similar amounts of acetylglucosamine and acetylgalactosamine are formed. C o m m e n t s . Any source of Ac~SCoA, Ac~SCoA itself, the transacetylase system, or an acetate-ATP-CoA system may be used in conjunction with Fraction A-60. Since the first two sources may not be available, a convenient source is the acetate-ATP-CoA reaction of yeast of Jones et al. 1° The identity of the amino sugar acetokinase has not been definitely established. Tabor et al. H have purified the enzyme from pigeon liver which acetylates aromatic amines some 25-fold, by acetone fractionation and alumina C~ adsorption. This preparation acetylates glucosamine at 4 % of the rate of p-nitroaniline acetylation and also acetylates histamine and ~-phenylethylamine. The enzyme contains an essential sulfhydryl group. Bessman and Lipmann ~ have found that their arylamine acetokinase preparation from pigeon liver, purified 38-fold by acetone fractionation, can also carry out a low-energy acetyl transfer between various aromatic amines. Some of their unpublished observations indicate that low-energy acetyl is transferred to glucosamine at a slow rate. Further work is necessary to prove the existence of a special acetokinase for the amino sugars. Katz et al. 13 have shown that dried-ceU preparations of C l o s t r i d i u m k l u y v e r i in the presence of acetyl phosphate catalyze a cyanide-induced acetylation of glucosamine.
9 D. Aminoff,W. T. J. Morgan, and W. M. Watkins, Biochem. J. 51~ 379 (1952). 1oM. E. Jones, S. Black, R. M. Flynn, and F. Lipmann, Biochim. et Biophys. Acta 12~ 141 (1953); see also Vol. I [96]. 11H. Tabor, A. H. Mehler, and E. R. Stadtman, J. Biol. Chem. 204~ 127 (1953). is S. P. Bessman and F. Lipmann, Arch. Biochem. and Biophys. 46, 252 (1953). 13j. Katz, I. Lieberman, and H. A. Barker, J. Biol. Chem. 200t 417 (1953).
[102]
ENZYMES OF LIPID METABOLISM
over 0.05 micromole/ml, give maximal rates. With p-nitroaniline 50% saturation of the enzyme is attained at approximately 0.1 micromole/ml. SUMMARY OF PURIFICATION PROCEDURE
Fraction 1 and 2. Crude extract 3. Acetone fraetionation
Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 96 16
406 1620
100 50
150 70
39,000 38 26,000
10.6
-67
4. C~
Eluate I Eluate II
15,000 0.55 3,500 0.38
275 184
38 9
[102] A c e t y l a t i o n of D - G l u c o s a m i n e by P i g e o n Liver E x t r a c t s A c~ SCoA + v-Glucosamine--. N-Acetyl-D-glucosamine + CoA
By MORRIS SOODAK Assay Method
Principle. Pigeon liver extracts contain an acetokinase which transfers the acetyl group of A c ~ S C o A to glucosamine and galactosamine. 1 The appearance of the acetylated amino sugar is followed by a modification of the Morgan and Elson method, 2 which is specific for N-acetylamino sugars. The formation of N-acetyl-D-glucosamine is confirmed chromatographically on paper. Reagents O.1M N-acetylglucosamine.3 0 . 1 M D-glucosamine.HCl. 1.0 M KAc. 0 . 1 M cysteine.HC1. 0 . 1 M MgC12. O.1M ATP (Na + or K+). 1.0 M KF. CoA solution, 200 units/ml. 2.0 M Tris, pH 8.2. 0.25 M lithium acetyl phosphate. t T. C. Chou a n d M. Soodak, J. Biol. Chem. 196, 105 (1952). t W. T. J. Morgan and L. A. Elson, Biochem. J. 26, 988 (1934).
N-Acetylglucosamine was obtained from Krishell Laboratories, Portland, Oregon.
[102] ACETYLATION OF I)-GLUCOSAMINE BY PIGEON LIVER EXTRACTS 613
1.0 M NH~C1. Enzymes. (1) Crude and aged extract of pigeon liver acetone powder. 4 (2a) Partially purified glucosamine acceptor enzyme from pigeon liver ~ (see below). (2b) Partially purified transacetylase from Clostridium kluyveri2 5% TCA. 1.0 M Na~CO3. 95 % ethanol. Ehrlich's reagent. Dissolve 1.6 g. of p-dimethylaminobenzaldehyde 7 in a cooled mixture of 30 ml. of conc. HC1 and 30 ml. of 95% ethanol.
Procedure 1. Acetylation with Aged Pigeon Liver Extract. The reaction mixture, final volume 1 ml., which is incubated for 2 hours at 37 °, in 16 × 100-mm. test tubes, is as follows: 10 micromoles glucosamine.HC1 (0.1 ml.), 50 micromoles acetate (0.05 ml.), 10 micromoles cysteine.HCl (0.1 ml.), 10 micromoles MgCI~ (0.1 ml.), 10 micromoles ATP (0.i ml.), 50 micromoles KF (0.05 ml.), 20 units CoA (0.1 ml.), 200 micromoles Tris (0.1 ml.), and 0.3 ml. aged pigeon liver extract (corresponding to 30 mg. acetone powder). Procedure 2. Acetylation with Partially Purified Glucosamine Acceptor System from Pigeon Liver (Fraction A-60). ~ This preparation no longer contains the acetate-ATP reaction found in crude pigeon liver extracts. A c ~ S C o A is here supplied by the transacetylase system. The reaction mixture, which is incubated for 2 hours at 30 °, is as follows: 10 micromoles glucosamine.HC1 (0.1 ml.), 25 micromoles acetyl phosphate (0.1 ml.), 10 micromoles cysteine.HC1 (0.1 ml.), 5 micromoles MgCl~ (0.05 ml.), 100 micromoles NH4C1 (0.1 ml.), 20 units CoA (0.1 ml.), 10 units transacetylase (0.05 ml.), 0.1 ml. water, and 0.3 ml. liver Fraction A-60. Control tubes are run which contain no ATP or acetyl phosphate. To another set of control tubes is added 0.5 to 2 micromoles of N-acetylglucosamine. These latter tubes serve as internal standards. The enzymatic reaction is terminated by the addition of 2 ml. of TCA. The protein precipitate is spun down. A 1-ml. aliquot is transferred to a 16 × 150-mm. test tube, followed by 0.5 ml. of water and 0.5 ml. of M Na2CO3. The tubes are heated in a boiling water bath for 10 minutes 4 N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174~ 37 (1948). The preparation of pigeon Iiver acetone powder and of the extracts therefrom is described in Vol. I [101]; and also see Vol. I I I [132]. 6 T. C. Chou and F. Lipmann, J. Biol. Chem. 196, 89 (1952). 6 See section on transacetylase (Vol. I [98]). 7 A preparation of p-dimethylaminobenzaldehyde (highest purity) from Phanstiehl Chemical Co., Waukegan, Illinois, may be used without recrystallization.
614
ENZYMES OF LIPID METABOLISM
[102]
and cooled in cold water. 2.5 ml. of 95 % ethanol is added to each tube with shaking, followed by 0.5 ml. of Ehrlich's reagent. After shaking, the tubes are kept at 37 ° for 30 minutes for color development and then read on a Klett colorimeter with a 540-m~ filter. In the experiments with transacetylase plus the partially purified glucosamine acceptor enzyme, sufficient N-acetylglucosamine is formed to confirm its appearance by paper chromatography. In this case, after the enzymatic incubation, a 0.5-ml. aliquot is removed and treated with 1 ml. of TCA for the colorimetric determination as above. The remainder of the incubation mixture (0.5 ml.) is treated in the following manner. 3 ml. of 95% ethanol is added, and the tubes are allowed to stand haft an hour for protein precipitation. After spinning, the supernatants are evaporated to dryness in a bath at 40 ° or below by means of a stream of air. The residue is extracted with 2 ml. of 95 % ethanol, and the extracts are brought to dryness again. The final residue is dissolved in 0.1 to 0.2 ml. of water, and appropriate aliquots are applied to Whatman No. 1 sheets. Ascending chromatograms are run for 16 hours with 80% propanol containing 0.8% ammonium acetate as the solvent. The spots are developed by the methods described by Partridge. 8 The RI for glucosamine and galactosamine is 0.32. The R / f o r acetylglucosamine is 0.46.
Preparation of the Partially Purified Glucosamine Acceptor Enzyme from Pigeon Liver Extracts. s The fraction precipitating between 40 and 60% acetone concentration contains the glucosamine acetokinase. All operations are carried out in the cold. For centrifugation a refrigerated centrifuge at - 1 5 ° is used throughout. A 10 % suspension of pigeon liver acetone powder in a 25% acetone solution containing 0.1 M Tris at pH 8.2 is stirred gently for 5 to 10 minutes at about - 5 °. The insoluble material is centrifuged at 4000 r.p.m, for 10 minutes and discarded. The supernatant is brought to 40% acetone concentration by the cautious addition of cold acetone (about - 5 ° ) . The precipitate is centrifuged as above. The supernatant is further treated with acetone to attain a 60 % concentration. This 40 to 60% fraction is spun down. The 25 to 40% (Fraction A-40) and the 40 to 60% (Fraction A-60) precipitates are separately dissolved in 1 ml./g, of original powder of M Tris at pH 8.2 and dialyzed for 12 hours with one change of fluid against 4.5 1. of a solution containing 0.001 M cysteine.HC1, 0.02 M KHCO~, and 0.5% KC1 (pH 8 adjusted with K~C03). Fraction A-40 contains the acetate-ATP reaction, and the enzymes for the synthesis of citrate, acetoacetate, and glutamine. Fraction A-60 contains the arylamine acetylation acceptor enzyme as well as that for the amino sugars (see below). All the enzymes used are stored at - 1 5 to - 3 0 ° and are stable for many months. 8 S. M. Partridge, Biochem. J. 42, 238 (1948).
[102]
ACETYLATIOI~ OF D-GLUCOSAMINE BY PIGEON LIVER EXTRACTS 6 1 5
Results. Crude aged pigeon liver extracts (0.3 ml.) under the conditions stated above, or when combined with the transacetylase system as acetyl~SCoA source, synthesize from 0.1 to 0.25 micromole of acetylglucosamine. With the transacetylase system plus Fraction A-60, from 1 to 2 micromoles is formed. This corresponds to about a seven-fold concentration of the enzyme in the A-60 fraction. In the latter case, the acetylglucosamine formation is confirmed by paper chromatography. The synthesis of acetylgalactosamine via the Fraction A-60 system when followed by the Morgan and Elson colorimetric method using acetylglucosamine as a standard is about 30% of that of acetylglucosamine. Aminoff et al., 9 however, have recently shown that the color yield of acetylgalactosamine is only 20 % of that of acetylglucosamine. Taking this into account, similar amounts of acetylglucosamine and acetylgalactosamine are formed. C o m m e n t s . Any source of Ac~SCoA, Ac~SCoA itself, the transacetylase system, or an acetate-ATP-CoA system may be used in conjunction with Fraction A-60. Since the first two sources may not be available, a convenient source is the acetate-ATP-CoA reaction of yeast of Jones et al. 1° The identity of the amino sugar acetokinase has not been definitely established. Tabor et al. H have purified the enzyme from pigeon liver which acetylates aromatic amines some 25-fold, by acetone fractionation and alumina C~ adsorption. This preparation acetylates glucosamine at 4 % of the rate of p-nitroaniline acetylation and also acetylates histamine and ~-phenylethylamine. The enzyme contains an essential sulfhydryl group. Bessman and Lipmann ~ have found that their arylamine acetokinase preparation from pigeon liver, purified 38-fold by acetone fractionation, can also carry out a low-energy acetyl transfer between various aromatic amines. Some of their unpublished observations indicate that low-energy acetyl is transferred to glucosamine at a slow rate. Further work is necessary to prove the existence of a special acetokinase for the amino sugars. Katz et al. 13 have shown that dried-ceU preparations of C l o s t r i d i u m k l u y v e r i in the presence of acetyl phosphate catalyze a cyanide-induced acetylation of glucosamine.
9 D. Aminoff,W. T. J. Morgan, and W. M. Watkins, Biochem. J. 51~ 379 (1952). 1oM. E. Jones, S. Black, R. M. Flynn, and F. Lipmann, Biochim. et Biophys. Acta 12~ 141 (1953); see also Vol. I [96]. 11H. Tabor, A. H. Mehler, and E. R. Stadtman, J. Biol. Chem. 204~ 127 (1953). is S. P. Bessman and F. Lipmann, Arch. Biochem. and Biophys. 46, 252 (1953). 13j. Katz, I. Lieberman, and H. A. Barker, J. Biol. Chem. 200t 417 (1953).