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[101] Acetylation of amines with pigeon liver enzyme
608
ENZYMES OF LIPID METABOLISM
[101]
[101] A c e t y l a t i o n of A m i n e s w i t h P i g e o n L i v e r E n z y m e 1 RNH2 + Ac~-~SCoA --~ RNHCOCH3 + CoA B y HERBERT TABOR
Assay Method Principle. Acetyl coenzyme A (Ac~--SCoA) acetylates various amines in the presence of an enzyme prepared from pigeon liver. Although the Ac~SCoA can be added directly, in the method described below it is generated in the incubation mixture from acetyl phosphate and CoA in the presence of phosphotransacetylase. The method originally used by Kaplan and Lipmann ~depended on the acetylation of sulfanilamide. Free sulfanilamide was measured in aliquots of the incubation mixture by the Bratton-Marshall diazotization procedure. The modification described below is based on the spectral shift obtained when p-nitroaniline is acetylated to p-nitroacetanilide. At 420 m~ the molar extinction coefficient of p-nitroaniline is 6000, whereas that of p-nitroacetanilide is negligible at this wavelength. This permits direct observation of the course of the acetylation reaction in the spectrophotometer. Because of the sulfhydryl nature of the acetylating enzyme, a reducing agent (sodium thioglycolate) and a chelating agent (disodium ethylenediaminetetraacetate) are also added to the assay. Reagents
p-Nitroaniline solution (0.001 M). Dissolve 138 mg. of commercial p-nitroaniline in 10 ml. of ethanol. Add water with stirring to a final volume of 1 1. 0.1 M potassium phosphate buffer, pH 6.8. Enzyme. Dilute the enzyme with water to a concentration of approximately 100 to 600 units of enzyme per milliliter. (See definition below.) 0.25 M dilithium acetyl phosphate. Dissolve 330 mg. in 10 ml. of water. (See Vol. III [39] for synthesis of dilithium acetylphosphate.) Transacetylase. Dilute with water to obtain 80 units/ml. (See Vol. I [98] for preparation of this enzyme.) 1The method described here has been reported by H. Tabor, A. H. Mehler, and E. R. Stadtman, J. Biol. Chem. 204, 127 (1953). 2N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174p 37 (1948).
[101]
ACETYLATION OF &MINES WITH PIGEON LIVER ENZYME
609
Coenzyme A solution containing 2.4 micromoles/ml. Commercial samples are available and are satisfactory. For methods of isolation and purification of CoA, see Vol. I I I [131]. 0.1 M sodium thioglycolate. Dissolve 114 mg. of sodium thioglycolate in 10 ml. of water. Other reducing agents, such as cysteine, glutathione, and H2S, are also satisfactory. 0.1 M disodium ethylenediaminetetraacetate (EDTA). 337 mg. are dissolved in 10 ml. of water.
Procedure) Both the blank and the experimental cells (1-cm. light path) contain 0.1 ml. of the phosphate buffer, 0.05 ml. of sodium thioglycolate, 0.05 ml. of EDTA, 0.05 ml. of dilithium acetyl phosphate, 0.05 ml. of transacetylase, 0.05 ml. of CoA, pigeon liver enzyme (usually 0.05 ml.), and water (added to a total volume of 0.9 ml.). An additional 0.1 ml. of water is included in the blank cell. 0.1 ml. of the p-nitroaniline solution is added to the experimental cell; after thorough mixing, readings are taken at 420 m~ at 15-second intervals. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which causes an initial rate of decrease in optical density at 420 m~ of 0.001 per minute at 25° under the above conditions. Specific activity is expressed as units per milligram of protein. Protein concentrations can be determined from the optical density at 280 m~, using the absorption at 260 m~ to correct for nucleic acid. (See Vol. I I I [73].) Application of Assay Method to Crude Tissue Preparations. The assay method described is suitable for use with crude as well as with purified preparations. Purification Procedure
The use of pigeon liver acetone powders for the preparation of active extracts for the acetylation of aromatic amines was first reported by Kaplan and Lipmann 2 and has been repeated by many laboratories. Chou and Lipmann 4 have used acetone fractionations of pigeon liver extracts in their studies on the acetylation enzyme. Step 1. Preparation of Pigeon Liver Acetone Powder. Freshly removed pigeon livers are blended with 10 vol. of cold ( - 10°) acetone for 1 minute in a Waring blendor. After filtration on a Biichner funnel with suction, the semidry filter cake is blended with 10 vol. of cold acetone and again 3 Crude extracts of Cl. kluyveri (0.05 ml.) can be s u b s t i t u t e d for the transacetylase preparation. The acetyl phosphate, CoA, and transacetylase additions can be replaced b y 0.1 micromole of A c ~ S C o A . (See Vol. I I I [137] for preparation of Ac ~ S C o A . ) 4 T. C. Chou a n d F. Lipmann, J. Biol. Chem. 19{}, 89 (1952).
610
ENZYM~.S OF LIPID MET&BOLISM
[101]
filtered. The filter cake is pulverized b y hand, spread over a large surface, and allowed to dry at room temperature. I t is then stored at 0% Under these conditions the powder is stable for several weeks, b u t then it gradually deteriorates. Step ~. Preparation of Crude Extract. 12 g. of the pigeon liver powder is ground in a m o r t a r with 120 ml. of distilled water for 10 to 15 minutes at room temperature. T h e suspension is then centrifuged at 22,000 X g for 10 minutes. Step 3. The supernatant (96 ml.) is cooled to 0 ° and treated with 76 ml. of cold (0 °) acetone. T h e precipitate is collected b y centrifugation and dissolved in 15 ml. of cold water. Step ~. The enzyme solution is then treated with 90 ml. of aged alumina gel C~ (11 mg./ml.) (see Vol. I [11] for preparation of this gel). After 5 minutes the mixture is centrifuged, and the precipitate collected. The precipitate is washed with 100 ml. of water and then eluted with 100 ml. of 0.01 M potassium phosphate buffer (pH 7.8) in three portions (eluate I). F u r t h e r elution with 50 ml. of the buffer yields additional activity of slightly lower specific activity (eluate II). Properties
Stability. The enzyme solutions can be stored at - 1 5 ° ; the activity is essentially unchanged for at least three months. More concentrated enzyme solutions can be obtained b y lyophilization after the addition of 100 micromoles of E D T A per 100 ml. Specificity. Numerous amines, 5 including p-nitroaniline, m-nitroaniline, p-aminobenzoic acid, o-phenylenediamine, o-toluidine, m-toluidine, o-bromoaniline, p-bromoaniline, o-anisidine, p-anisidine, histamine, phenethylamine, and glucosamine are acetylated, e No evidence for 6Pigeon liver extracts have been used in other laboratories for the acetylation of sulfanilamide and p-aminobenzoic acid, 2 4-aminoazobenzene JR. E. Handschumacher, G. C. Mueller, and F. M. Strong, J. Biol. Chem. 189, 335 (1951)], 4-aminoazobenzene-4'-sulfonic acid [S. P. Bessman and F. Lipmann, Arch. Biochem. and Biophys. 46, 252 (1953)], histamine [R. C. Millican, S. M. Rosenthal, and H. Tabor, J. Pharmacol. Exptl. Therap. 97, 4 (1949)], and glucosamine [T. C. Chou and M. Soodak, J. Biol. Chem. 196, 105 (1952), and Vol. I [102].
e Acetylation of the aromatic amines can be studied with spectrophotometric techniques; the acetylation of aliphatic amines is followed by the disappearance of ninhydrin-reacting material, using the quantitative ninhydrin method of Moore and Stein (see Vol. III [76]). With histamine, the reaction may also be followed by measuring the formation of acetylhistamine (Vol. III [90]). Glucosamine acetylation can be studied with the method of Morgan and Elson (see' Vol. I [102] and Vol. III [121). The incubation mixture used to study the acetylation of histamine contains 2
[101]
ACETYLATION
OF AMINES
WITH
PIGEON
LIVER
ENZYME
611
acetylation is found with o-aminobenzoic acid, o-nitroaniline, orthanilic acid, sulfanilic acid, or p-nitromethylaniline. Acylation of p-nitroaniline also takes place when butyryl CoA is used as the acetylating agent instead of A c ~ S C o A ; however, the rate is only 4 % of t h a t observed with A c ~ S C o A . No acylation is observed with palmityl CoA. Effect of pH. With p-nitroaniline as the substrate, the activity is essentially constant from p H 6 to over 9.5. With histamine as the substrate, the reaction falls off sharply below pH 8.5. Activators and Inhibitors. The acetylating enzyme is a sulfhydryl enzyme and is completely inhibited b y 10-5 M p-chloromercuribenzoate. The enzyme is also spontaneously inactivated in the usual incubation mixtures (particularly at pH 8 and higher), presumably owing to the effect of heavy metal impurities on the sulfhydryl groups. This inactivation can be completely reversed by reducing agents, such as sodium thioglycolate, cysteine, or hydrogen sulfide. Since the enzyme shows no inactivation in the presence of E D T A , this is routinely added to all incubation mixtures. The acetylating enzyme is inhibited b y free CoA. 0.09 micromole of free CoA causes a 50% inhibition in an incubation mixture containing 0.05 micromole of A c ~ S C o A . Marked inhibition is produced b y palmityl CoA. Stoichiometry. With limiting amounts of p-nitroaniline and excess A c ~ S C o A (or with the phosphotransacetylase-acetyl-P system), the reaction proceeds until the amine is completely acetylated. With limiting amounts of Ac--~SCoA and excess p-nitroaniline, the quantity of p-nitroaniline acetylated is exactly equivalent to the a m o u n t of A c ~ S C o A prese n t / This affords a convenient assay for AC--~SCoA (see Vol. I I I [131]). A~nity of the Enzyme for CoA and for p-Nitroaniline. With varying amounts of CoA, approximately 50% of the maximal rate is observed with a CoA concentration of 0.015 micromole/ml.; CoA concentrations of
micromoles of histamine, 300 micromoles of potassium pyrophosphate buffer (pH 9.3), 10 mieromoles of lithium acetyl phosphate, 4 units of transacetylase, 0.1 micromole of CoA, 10 micromoles of sodium thioglycolate, 5 mieromoles of EDTA, and 160 units of acetylating enzyme in 1 mh final volume. Approximately 1 micromole of histamine is aeetylated in 30 minutes. Under similar conditions with phenethylamine as the substrate 0.2 micromole is aeetylated. 7 In experiments with stoiehiometri¢ quantities of Ac~SCoA, it is advisable to omit the sodium thioglycolate from the incubation mixture to avoid any loss due to the nonenzymatic acetylation of the thioglycolate [E. R. Stadtman, J. Biol. Chem. 196, 535 (1952)].
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.
ENZYMES OF LIPID METABOLISM
[101]
[101] A c e t y l a t i o n of A m i n e s w i t h P i g e o n L i v e r E n z y m e 1 RNH2 + Ac~-~SCoA --~ RNHCOCH3 + CoA B y HERBERT TABOR
Assay Method Principle. Acetyl coenzyme A (Ac~--SCoA) acetylates various amines in the presence of an enzyme prepared from pigeon liver. Although the Ac~SCoA can be added directly, in the method described below it is generated in the incubation mixture from acetyl phosphate and CoA in the presence of phosphotransacetylase. The method originally used by Kaplan and Lipmann ~depended on the acetylation of sulfanilamide. Free sulfanilamide was measured in aliquots of the incubation mixture by the Bratton-Marshall diazotization procedure. The modification described below is based on the spectral shift obtained when p-nitroaniline is acetylated to p-nitroacetanilide. At 420 m~ the molar extinction coefficient of p-nitroaniline is 6000, whereas that of p-nitroacetanilide is negligible at this wavelength. This permits direct observation of the course of the acetylation reaction in the spectrophotometer. Because of the sulfhydryl nature of the acetylating enzyme, a reducing agent (sodium thioglycolate) and a chelating agent (disodium ethylenediaminetetraacetate) are also added to the assay. Reagents
p-Nitroaniline solution (0.001 M). Dissolve 138 mg. of commercial p-nitroaniline in 10 ml. of ethanol. Add water with stirring to a final volume of 1 1. 0.1 M potassium phosphate buffer, pH 6.8. Enzyme. Dilute the enzyme with water to a concentration of approximately 100 to 600 units of enzyme per milliliter. (See definition below.) 0.25 M dilithium acetyl phosphate. Dissolve 330 mg. in 10 ml. of water. (See Vol. III [39] for synthesis of dilithium acetylphosphate.) Transacetylase. Dilute with water to obtain 80 units/ml. (See Vol. I [98] for preparation of this enzyme.) 1The method described here has been reported by H. Tabor, A. H. Mehler, and E. R. Stadtman, J. Biol. Chem. 204, 127 (1953). 2N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174p 37 (1948).
[101]
ACETYLATION OF &MINES WITH PIGEON LIVER ENZYME
609
Coenzyme A solution containing 2.4 micromoles/ml. Commercial samples are available and are satisfactory. For methods of isolation and purification of CoA, see Vol. I I I [131]. 0.1 M sodium thioglycolate. Dissolve 114 mg. of sodium thioglycolate in 10 ml. of water. Other reducing agents, such as cysteine, glutathione, and H2S, are also satisfactory. 0.1 M disodium ethylenediaminetetraacetate (EDTA). 337 mg. are dissolved in 10 ml. of water.
Procedure) Both the blank and the experimental cells (1-cm. light path) contain 0.1 ml. of the phosphate buffer, 0.05 ml. of sodium thioglycolate, 0.05 ml. of EDTA, 0.05 ml. of dilithium acetyl phosphate, 0.05 ml. of transacetylase, 0.05 ml. of CoA, pigeon liver enzyme (usually 0.05 ml.), and water (added to a total volume of 0.9 ml.). An additional 0.1 ml. of water is included in the blank cell. 0.1 ml. of the p-nitroaniline solution is added to the experimental cell; after thorough mixing, readings are taken at 420 m~ at 15-second intervals. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which causes an initial rate of decrease in optical density at 420 m~ of 0.001 per minute at 25° under the above conditions. Specific activity is expressed as units per milligram of protein. Protein concentrations can be determined from the optical density at 280 m~, using the absorption at 260 m~ to correct for nucleic acid. (See Vol. I I I [73].) Application of Assay Method to Crude Tissue Preparations. The assay method described is suitable for use with crude as well as with purified preparations. Purification Procedure
The use of pigeon liver acetone powders for the preparation of active extracts for the acetylation of aromatic amines was first reported by Kaplan and Lipmann 2 and has been repeated by many laboratories. Chou and Lipmann 4 have used acetone fractionations of pigeon liver extracts in their studies on the acetylation enzyme. Step 1. Preparation of Pigeon Liver Acetone Powder. Freshly removed pigeon livers are blended with 10 vol. of cold ( - 10°) acetone for 1 minute in a Waring blendor. After filtration on a Biichner funnel with suction, the semidry filter cake is blended with 10 vol. of cold acetone and again 3 Crude extracts of Cl. kluyveri (0.05 ml.) can be s u b s t i t u t e d for the transacetylase preparation. The acetyl phosphate, CoA, and transacetylase additions can be replaced b y 0.1 micromole of A c ~ S C o A . (See Vol. I I I [137] for preparation of Ac ~ S C o A . ) 4 T. C. Chou a n d F. Lipmann, J. Biol. Chem. 19{}, 89 (1952).
610
ENZYM~.S OF LIPID MET&BOLISM
[101]
filtered. The filter cake is pulverized b y hand, spread over a large surface, and allowed to dry at room temperature. I t is then stored at 0% Under these conditions the powder is stable for several weeks, b u t then it gradually deteriorates. Step ~. Preparation of Crude Extract. 12 g. of the pigeon liver powder is ground in a m o r t a r with 120 ml. of distilled water for 10 to 15 minutes at room temperature. T h e suspension is then centrifuged at 22,000 X g for 10 minutes. Step 3. The supernatant (96 ml.) is cooled to 0 ° and treated with 76 ml. of cold (0 °) acetone. T h e precipitate is collected b y centrifugation and dissolved in 15 ml. of cold water. Step ~. The enzyme solution is then treated with 90 ml. of aged alumina gel C~ (11 mg./ml.) (see Vol. I [11] for preparation of this gel). After 5 minutes the mixture is centrifuged, and the precipitate collected. The precipitate is washed with 100 ml. of water and then eluted with 100 ml. of 0.01 M potassium phosphate buffer (pH 7.8) in three portions (eluate I). F u r t h e r elution with 50 ml. of the buffer yields additional activity of slightly lower specific activity (eluate II). Properties
Stability. The enzyme solutions can be stored at - 1 5 ° ; the activity is essentially unchanged for at least three months. More concentrated enzyme solutions can be obtained b y lyophilization after the addition of 100 micromoles of E D T A per 100 ml. Specificity. Numerous amines, 5 including p-nitroaniline, m-nitroaniline, p-aminobenzoic acid, o-phenylenediamine, o-toluidine, m-toluidine, o-bromoaniline, p-bromoaniline, o-anisidine, p-anisidine, histamine, phenethylamine, and glucosamine are acetylated, e No evidence for 6Pigeon liver extracts have been used in other laboratories for the acetylation of sulfanilamide and p-aminobenzoic acid, 2 4-aminoazobenzene JR. E. Handschumacher, G. C. Mueller, and F. M. Strong, J. Biol. Chem. 189, 335 (1951)], 4-aminoazobenzene-4'-sulfonic acid [S. P. Bessman and F. Lipmann, Arch. Biochem. and Biophys. 46, 252 (1953)], histamine [R. C. Millican, S. M. Rosenthal, and H. Tabor, J. Pharmacol. Exptl. Therap. 97, 4 (1949)], and glucosamine [T. C. Chou and M. Soodak, J. Biol. Chem. 196, 105 (1952), and Vol. I [102].
e Acetylation of the aromatic amines can be studied with spectrophotometric techniques; the acetylation of aliphatic amines is followed by the disappearance of ninhydrin-reacting material, using the quantitative ninhydrin method of Moore and Stein (see Vol. III [76]). With histamine, the reaction may also be followed by measuring the formation of acetylhistamine (Vol. III [90]). Glucosamine acetylation can be studied with the method of Morgan and Elson (see' Vol. I [102] and Vol. III [121). The incubation mixture used to study the acetylation of histamine contains 2
[101]
ACETYLATION
OF AMINES
WITH
PIGEON
LIVER
ENZYME
611
acetylation is found with o-aminobenzoic acid, o-nitroaniline, orthanilic acid, sulfanilic acid, or p-nitromethylaniline. Acylation of p-nitroaniline also takes place when butyryl CoA is used as the acetylating agent instead of A c ~ S C o A ; however, the rate is only 4 % of t h a t observed with A c ~ S C o A . No acylation is observed with palmityl CoA. Effect of pH. With p-nitroaniline as the substrate, the activity is essentially constant from p H 6 to over 9.5. With histamine as the substrate, the reaction falls off sharply below pH 8.5. Activators and Inhibitors. The acetylating enzyme is a sulfhydryl enzyme and is completely inhibited b y 10-5 M p-chloromercuribenzoate. The enzyme is also spontaneously inactivated in the usual incubation mixtures (particularly at pH 8 and higher), presumably owing to the effect of heavy metal impurities on the sulfhydryl groups. This inactivation can be completely reversed by reducing agents, such as sodium thioglycolate, cysteine, or hydrogen sulfide. Since the enzyme shows no inactivation in the presence of E D T A , this is routinely added to all incubation mixtures. The acetylating enzyme is inhibited b y free CoA. 0.09 micromole of free CoA causes a 50% inhibition in an incubation mixture containing 0.05 micromole of A c ~ S C o A . Marked inhibition is produced b y palmityl CoA. Stoichiometry. With limiting amounts of p-nitroaniline and excess A c ~ S C o A (or with the phosphotransacetylase-acetyl-P system), the reaction proceeds until the amine is completely acetylated. With limiting amounts of Ac--~SCoA and excess p-nitroaniline, the quantity of p-nitroaniline acetylated is exactly equivalent to the a m o u n t of A c ~ S C o A prese n t / This affords a convenient assay for AC--~SCoA (see Vol. I I I [131]). A~nity of the Enzyme for CoA and for p-Nitroaniline. With varying amounts of CoA, approximately 50% of the maximal rate is observed with a CoA concentration of 0.015 micromole/ml.; CoA concentrations of
micromoles of histamine, 300 micromoles of potassium pyrophosphate buffer (pH 9.3), 10 mieromoles of lithium acetyl phosphate, 4 units of transacetylase, 0.1 micromole of CoA, 10 micromoles of sodium thioglycolate, 5 mieromoles of EDTA, and 160 units of acetylating enzyme in 1 mh final volume. Approximately 1 micromole of histamine is aeetylated in 30 minutes. Under similar conditions with phenethylamine as the substrate 0.2 micromole is aeetylated. 7 In experiments with stoiehiometri¢ quantities of Ac~SCoA, it is advisable to omit the sodium thioglycolate from the incubation mixture to avoid any loss due to the nonenzymatic acetylation of the thioglycolate [E. R. Stadtman, J. Biol. Chem. 196, 535 (1952)].
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.