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[100] Cerebroside glycosidases
[100]
CEREBROSIDE GLYCOSIDASES
591
For maltose, isomaltose, trehalose, cellobiose, and gentiobiose n----2. For sucrose, lactose, turanose, and palatinose n = 1. one unit of disaccharidase is the activity hydrolyzing 1 micromole of substrate per minute.
units:
Disaccharidase units/ml sample
(a - b) • d. 2 n. 180- 0.1 • 60
(a - b) - d n • 540
Variations in Procedure. When preparations with low disaccharidase activity are measured, the amount of protein may cause turbidity in the solution for spectrophotometry. Sometimes the turbidity can be removed by centrifugation just before reading. Otherwise the 0.8 ml of water added to the enzyme-disaccharide reaction mixture before boiling can be replaced with 0.4 ml each of Somogyi's 21 Zn-Ba protein precipitation reagents. In this case the boiling is omitted. 0ne-half milliliter of the clear supernatant after centrifugation is used for incubation with the TGO reagent. With highly purified glucose oxidase preparations the reaction can be performed in a single step, so that the disaccharide hydrolysis and the coupled glucose oxidase reaction occur in the same reaction mixture. 22 Phosphate buffer then has to be added to speed up the mutarotation of glucose (the purified glucose oxidase preparations do not seem to contain enough mutarotase). This modified reaction is very suitable for use on the ultramicro scale.
51M. Somogyi, J. Biol. Chem. 160, 69 (1945). 2~M. Messer and A. Dahlqvist, Anal. Biochem. in press (1966).
[ 100]
Cerebroside Glycosidases
B y Roscoe O. BRADY and JULIAN N. KANFER
I. Glucoeerebroside Glycosidase from H u m a n Spleen Glueoeerebroside W H20 ---,glucose -5 eeramide Assay Method Principle. The activity of the enzyme can be determined by the liberation of glucose from glucocerebroside. This process may be conveniently followed by measuring the amount of water-soluble radioactivity arising from glucose-1-14C-cerebroside. 1 Alternatively, the
I R. O. Brady, J. Kanfer, and D. Shapiro, J. Biol. Chem. 240, 39 (1965).
592
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[100]
liberated hexose may be determined by conventional procedures for free glucose. Reagents
Glucose-l-14C-cerebrosidel Potassium phosphate buffer, pH 6.0, 1.0M Cutscum solution, 50 mg per milliliter of H20 (Fisher Chemical Company) Chloroform-methanol solution, 2:1 (v/v) Perchloric acid solution, 7% Potassium hydroxide solution, 14% "Theoretical upper phase water"2: chloroform-methanol-water, 3:48:47 (v/v/v) Scintillation counting solution3:62.5 g naphthalene, 150 mg POPOP and 6.0 g DPO, q.s. to 500 ml with dioxane (Packard Instrument Company, Inc.) Procedure. The assay mixture contains 25 micromoles of potassium phosphate buffer, pH 6.0, 75 millimicromoles of glucose-l-14C-eerebro side, 0.4 mg of Cutscum, and the enzyme in a final volume of 0.25 ml. The labeled substrate (2 mg/ml) is suspended in a solution of Cutseum (16 mg/ml), warmed, and allowed to cool until a clear solution is obtained before adding a 0.025-mi aliquot to the incubation mixture. After incubation for 1 hour at 37 °, the reaction is stopped by the addition of 0.17 ml of 7% HCI04. The precipitate is removed by centrifugation and the supernatant solution is transferred to another centrifuge tube followed by neutralization with 0.09 ml of 14% KOH. KCI04 is removed by centrifugation and the supernatant solution is transferred to a glassstoppered centrifuge tube to which 6 ml of chloroform-methanol, 2:1 is added. The solutions are mixed and 0.5 ml of H20 is added to the homogenate solution. The tubes are agitated and are clarified by centrifugation; the aqueous phase is carefully removed with a capillary pipette. Of "theoretical upper phase water," 1.25 ml is added to the chloroform phase. The mixture is agitated and again clarified by centrifugation. The upper phase is combined with the preceding aqueous fraction. To a 1-ml aliquot of the combined aqueous phase is added 9 ml of the scintillation counting solution and the radioactivity determined by liquid scintillation spectrometry. Definition o] Unit and Specific Activity. A unit of activity is defined
~J. Folch, M. Lees, and G. H. Sloane Stanley, J. Biol. Chem. 226, 497 (1957). s H. Werbin, I. C. Chaikoff, and B. N. Imada, Proc. Soc. Exptl. Biol. Med. I~2~ 8 (1959).
[100]
CEREBROSIDE GLYCOSIDASES
593
as the amount of enzyme catalyzing the hydrolysis of 1 millimicromole of eerebroside per hour. Specific activity is expressed as units per milligrams of protein. Protein is estimated by the procedure of Itzhaki and Gill. 4
Purification Procedure Human spleen tissue obtained at operation is frozen in dry ice. After several hours it is thawed, minced and homogenized in 2 volumes of 0.1 M potassium phosphate buffer, pH 7.0 in an all-glass Ten Broeck homogenizer having 0.02 inch clearance between pestle and sleeve. The suspension is centrifuged at 600 g for 12 minutes. The precipitate is discarded, and the supernatant fraction is centrifuged at 100,000 g for 1 hour. Ammonium Sulfate Fractionation. Solid ammonium sulfate is added to the supernatant solution and stirred for 5 minutes at 2°; the precipitate is collected by centrifugation. Protein precipitating at 0-25% and 25-35% saturation with ammonium sulfate (70.5 g/100 ml ~ 100% saturation at 0 °) is dissolved in 0.01 M potassium phosphate buffer, pH 7.0. The enzyme is found primarily in the 25-35% ammonium sulfate fraction. Acid Precipitation. To the preceding 25-35% ammonium sulfate fraction is added 0.1 volume of 1.0M potassium citrate buffer, pH 5.0. The mixture is kept at 0 ° for 10 minutes and then centrifuged. The supernatant solution is incubated at 37 ° for 5 minutes and then centrifuged at 2 °. The supernatant solution is dialyzed overnight against 500 volumes of 0.01 M potassium phosphate buffer, pH 7.0. The sediment which appears is removed by centrifugation. Ethanol Fractionation. Ethanol is added to the enzyme solution immersed in an ice-salt bath at --12 ° . The protein precipitating between 39 and 56% by volume with ethanol is collected by centrifugation and dissolved in 0.01 M potassium phosphate buffer, pH 7.0. Acetone Fractionation. Acetone chilled to --12 ° is added dropwise to the preceding alcohol fraction. The protein precipitating between 50 and 62% by volume with acetone is collected by centrifugation and dissolved in 0.01 M potassium phosphate buffer, pH 7.0. The final enrichment is 82-fold and is obtained in 35% yield. The purification procedure is summarized in Table I. Properties Stability. The most highly purified fraction is completely stable over a period of 1 month when kept frozen at --20 °. 4R. F. Itzhaki and D. M. Gill, Anal. Biochem. 9, 401 (1964).
594
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[100]
TABLE I PURIFICATION OF GLUCOCEREBROSIDE-CLEAVING ENZYME FROM HUMAN SPLEEN TISSUE
Fraction I. Supernatant solution (100,000 g) II. Ammoniumsulfate, 0.25-0.35 III. Dialyzed pH 5.0 supernatant IV. Alcohol,0.39-0.56 V. Acetone, 0.50-0.62
Specific activity (units/mg protein)
Volume (ml)
Total activity units
100
630
4200
0.15
20
473
840
0.57
20 5 5
489 244 219
278 39 18
Protein (mg)
1.8 6.2 12.3
Activators and Inhibitors. The activity is enhanced by the presence of Cutseum in the incubation mixture. The optimal amount is 1.3 mg/ml. The enzyme is strongly inhibited by Tris buffer and moderately (23%) inhibited by 1 X 10-4 M p-hydroxymercuribenzoate. Ef]ect o] pH. The optimum pH is 6.0. Specificity. The most highly purified enzyme fraction catalyzes the hydrolysis of glucocerebroside. Galactocerebroside is inactive as a subtrate for this enzyme, p-nitrophenyl-fl-D-glucopyranoside is hydrolyzed at 45% the rate of glucocerebroside, o-nitrophenyl-fl-D-galactopyranoside is not hydrolyzed, p-nitrophenyl-~-D-2-acetamido-2-deoxy-glucopyranoside is hydrolyzed at 13 times the rate of glucocerebroside. The hydrolysis of glucocerebroside is not diminished in the presence of a 10-fold excess of p-nitrophenyl-fl-D-2-acetamido-2-deoxy-glueopyranoside. This observation suggests that the hydrolysis of these materials is catalyzed by different enzymes present in Fraction ¥. Substrate Affinity. The Km value for glucocerebroside is 4.2 X 10-~ M. Nature of the Reaction. The enzyme catalyzes the hydrolytic cleavage of the glycosidic bond of glucocerebroside. Ceramide (N-acylsphingosine) and free glucose are the products of the reaction. The amount of enzyme in spleens obtained from patients with Gaucher's disease appears to be considerably diminished from the level observed in non-Gaucher's human spleen preparations2 It is considered that a deficiency of this enzyme may be responsible for the accumulation of glucocerebroside in various tissues of patients with Gaucher's disease. R. O. Brady, J. N. Kanfer, and D. Shapiro, Biochem. Biophys. Res. Commun. 18, 221 (1965).
[100]
CEREBROSIDE GLYCOSIDASES
595
II. Glucosyl and Galactosyl Ceramide Glycosidase from Rat Intestine Cerebroside + H~O--* hexose ~- ceramide Assay Method P~/nc/ple. The activity of the enzyme can be determined as in Section I using glucose-l-14C-cerebrosidel or galaetose-l-14C-cerebroside, 6 or by conventional procedures for free glucose or galactose.
Reagents. Same as listed in Section I plus: Galactose-1 J4C-cerebroside 6 Sodium eholate solution, 50 mg/ml, (Matheson, Coleman and Bell)
Procedure. The assay mixture contains 12.5 mieromoles of potassium phosphate buffer, pH 6.0, 540 millimicromoles of glueose-l-~C-cerebroside or galaetose-l-14C-cerebroside, 1.5 mg of sodium cholate, 0.2 mg of Cutseum, and the enzyme in a final volume of 0.15 ml. The labeled substrate (10 mg/ml) is suspended in a solution of sodium cholate (50 mg/ml) and warmed until a clear solution is obtained before adding an aliquot of 0.0375 ml to the incubation mixture. After incubation for 1 hour at 37 °, the extent of the reaction is determined as described in Section I. Definition o] Unit and Specific Activity. A unit of activity is defined as the amount of enzyme catalyzing the hydrolysis of 1 m~mole of eerebroside per minute. Specific activity is expressed as units per milligram of protein. Purification Procec~ure Small intestinal tissue is obtained from 14-day-old Sprague-Dawley rats. The portion 1 em distal to the pylorus and 2 cm proximal to the cecum is employed. It is slit longitudinally and washed copiously with isotonic saline to remove the contents of the intestinal lumen. The tissue is chilled, minced, and homogenized in 9 volumes of 0.25 M sucrose in an all glass Ten Broeck homogenizer having 0.02-inch clearance between pestle and sleeve. The suspension is centrifuged at 600 g for 12 minutes. The precipitate is discarded and the supernatant fraction centrifuged at 8400 g for 12 minutes. SoIubilization of Enzyme. The particles sedimenting between 600 and 8400 g are suspended in 0.25 M sucrose and recentrifuged at 8400 g for 12 minutes. The washed particles are suspended in a solution containing 6R. O. Brady, A. E. Gal, J. N. Kanfer, and R. M. Bradley, J. Biol. Chem. 240, 3766 (1966).
596
[100]
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
per milliliter 5 mg of sodium cholate, equal to one-half of the original volume of the homogenate. The unfrozen suspension is kept at 0 ° overnight and then centrifuged at 100,000 g. for 1 hour. The enzyme in the supernatant solution is dialzed overnight against 2000 volumes of 0.01 M potassium phosphate buffer, pH 6.0. Ammonium Sul]ate Fractionation. Solid ammonium sulfate is added to the dialyzed enzyme, stirred for 5 minutes at 2 °, and the precipitate is collected by centrifugation. Protein precipitating at 0-25%, 25-35%, and 35-50% saturation with ammonium sulfate is dissolved in 0.01 M potassium phosphate buffer, pH 6.0. The enzyme is found primarily in the 35-50% ammonium sulfate fraction. Chromatography on TEAE-cellulose. A 5 X 18 mm column of TEAEcellulose is equilibrated with 0.01 M potassium phosphate buffer, pH 6.0. To this column 568 ~g of protein from the preceding 35-50% ammonium sulfate fraction is applied in 1.9 ml of 0.01 M potassium phosphate buffer, pH 6.0. Fractions are collected consisting of column pass, and 2-ml portions of 0.01, 0.035, 0.065 and 0.1 M potassium phosphate buffer solutions, pH 6.0. The cerebroside-cleaving enzyme is found principally in the 0.065 M fraction. The final enrichment is approximately 2300-fold with respect to the original 600-8400 g particulate preparation and is obtained in 38% yield. The purification procedure is summarized in Table II. TABLE II PURIFICATION OF RAT INTESTINAL GLUCOSYL ANn GALACTOSYL CERAMIDE GLYCOSIDASE
Fraction I. 8400 g resuspended particles II. 100,000 g cholate-treatedsupernatant solution IIL Ammoniumsulfate, 0.35-0.50 IV. TEAE eluate, 0.065 M potassium phosphate, pH 6.0
Total Volume activity (ml) units 117 58
89.4 78.8
6 6
52.8 33.8
Specific activity Protein (units/mg (mg) protein) 470 5.9 2.1 0.078
0.19 13.2 25 433
Properties
Stability. The most highly purified fraction is completely stable over a period of 1 month when kept unfrozen at 0 °. Activators and Inhibitors. The activity is enhanced by the presence of sodium eholate and Cutscum in the incubation mixture. The optimal amounts are 10. and 1.3 mg/ml, respectively. The enzyme is strongly
3-ASPARTYL-N-ACETYLGLUCOSAMINADASE
[101]
597
inhibited by Tris buffer, and is noncompetitively inhibited by cellobiose whose Ki is 3.3 X 10-3 M. Effect of pH. The optimum pH is 6.0. Specificity. The enzyme catalyzes the hydrolysis of both gluco- and galactocerebroside. Higher ceramide oligosaccharides (ceramide lactose and globoside) are hydrolyzed at somewhat slower rates. Psychosines are hydrolyzed at 5% of the rate of the respective cerebrosides, o-nitrophenyl-fl-D-galactopyranoside, p-nitrophenyl-fl-D-glucopyranoside, and p-nitrophenyl-fl-D-2-acetamido-2-deoxyglucopyranoside are hydrolyzed at 58, 80, and 25%, respectively, compared with the rate of hydrolysis of galactocerebroside. Substrate Affinity. The Km value for both gluco- and galactocerebroside is 4.9 X 10-4 M. Nature of the Reaction. The enzyme catalyzes the hydrolytic cleavage of the glycosidic bond of gluco- and galactocerebrosides. Ceramide (N-acylsphingosine) and free hexose are the products of the reaction.
[ 101] f l - A s p a r t y l - N - a c e t y l g l u c o s a m i n a d a s e f r o m E p i d i d y m i s B y EDWIN"H. EYLAR and MAKOTOMURAKAMI
1-B-Aspartyl-2-acetamido-l,2-dideoxy-D-glucosylamine--~ N-acetylglucosamine+ asparagine Assay Method Principle. The structure 1-5 of the carbohydrate-protein linkage in ovalbumin has been established as 1-fl-aspartyl-2-acetamido-l,2-dideoxy-D-glucosylamine by a comparison of the synthetic compound with that isolated from an ovalbumin glycopeptide. The fl-aspartylglycosylamine linkage in this compound is cleaved by the N-glycosidase6 to Nacetylglucosamine and asparagine. The N-acetylglucosamine liberated by the enzyme is determined colorimetrically by a modification of the Morgan-Elson reaction. 7
1G. S. Marks, R. D. Marshall, and A. Neuberger, Biochem. J. 87, 274 (1963). ~I. Yamashina, K. Ban-i, and M. Makino, Biochim. Biophys. Acta 78, 382 (1963). 3H. Tsukamoto, A. Yamamoto, and C. Miyashita, Biochem. Biophys. Res. Commun. 1.5, 151 (1064). V. P. Bogdanov, E. D. Kaverzneva, and A. P. Andreyeva, Biochim. Biophys. Acta 83, 69 (1964). "R. D. Marshall and A. Neuberger, Biochemistry 3, 1596 (1964). 6M. Murakami and E. H. Eylar, J. Biol. Chem. 240, PC556 (1965). ~J. L. Reissig, J. L. Strominger, and L. F. Leloir, J. Biol. Chem. 217, 959 (1955).
CEREBROSIDE GLYCOSIDASES
591
For maltose, isomaltose, trehalose, cellobiose, and gentiobiose n----2. For sucrose, lactose, turanose, and palatinose n = 1. one unit of disaccharidase is the activity hydrolyzing 1 micromole of substrate per minute.
units:
Disaccharidase units/ml sample
(a - b) • d. 2 n. 180- 0.1 • 60
(a - b) - d n • 540
Variations in Procedure. When preparations with low disaccharidase activity are measured, the amount of protein may cause turbidity in the solution for spectrophotometry. Sometimes the turbidity can be removed by centrifugation just before reading. Otherwise the 0.8 ml of water added to the enzyme-disaccharide reaction mixture before boiling can be replaced with 0.4 ml each of Somogyi's 21 Zn-Ba protein precipitation reagents. In this case the boiling is omitted. 0ne-half milliliter of the clear supernatant after centrifugation is used for incubation with the TGO reagent. With highly purified glucose oxidase preparations the reaction can be performed in a single step, so that the disaccharide hydrolysis and the coupled glucose oxidase reaction occur in the same reaction mixture. 22 Phosphate buffer then has to be added to speed up the mutarotation of glucose (the purified glucose oxidase preparations do not seem to contain enough mutarotase). This modified reaction is very suitable for use on the ultramicro scale.
51M. Somogyi, J. Biol. Chem. 160, 69 (1945). 2~M. Messer and A. Dahlqvist, Anal. Biochem. in press (1966).
[ 100]
Cerebroside Glycosidases
B y Roscoe O. BRADY and JULIAN N. KANFER
I. Glucoeerebroside Glycosidase from H u m a n Spleen Glueoeerebroside W H20 ---,glucose -5 eeramide Assay Method Principle. The activity of the enzyme can be determined by the liberation of glucose from glucocerebroside. This process may be conveniently followed by measuring the amount of water-soluble radioactivity arising from glucose-1-14C-cerebroside. 1 Alternatively, the
I R. O. Brady, J. Kanfer, and D. Shapiro, J. Biol. Chem. 240, 39 (1965).
592
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[100]
liberated hexose may be determined by conventional procedures for free glucose. Reagents
Glucose-l-14C-cerebrosidel Potassium phosphate buffer, pH 6.0, 1.0M Cutscum solution, 50 mg per milliliter of H20 (Fisher Chemical Company) Chloroform-methanol solution, 2:1 (v/v) Perchloric acid solution, 7% Potassium hydroxide solution, 14% "Theoretical upper phase water"2: chloroform-methanol-water, 3:48:47 (v/v/v) Scintillation counting solution3:62.5 g naphthalene, 150 mg POPOP and 6.0 g DPO, q.s. to 500 ml with dioxane (Packard Instrument Company, Inc.) Procedure. The assay mixture contains 25 micromoles of potassium phosphate buffer, pH 6.0, 75 millimicromoles of glucose-l-14C-eerebro side, 0.4 mg of Cutscum, and the enzyme in a final volume of 0.25 ml. The labeled substrate (2 mg/ml) is suspended in a solution of Cutseum (16 mg/ml), warmed, and allowed to cool until a clear solution is obtained before adding a 0.025-mi aliquot to the incubation mixture. After incubation for 1 hour at 37 °, the reaction is stopped by the addition of 0.17 ml of 7% HCI04. The precipitate is removed by centrifugation and the supernatant solution is transferred to another centrifuge tube followed by neutralization with 0.09 ml of 14% KOH. KCI04 is removed by centrifugation and the supernatant solution is transferred to a glassstoppered centrifuge tube to which 6 ml of chloroform-methanol, 2:1 is added. The solutions are mixed and 0.5 ml of H20 is added to the homogenate solution. The tubes are agitated and are clarified by centrifugation; the aqueous phase is carefully removed with a capillary pipette. Of "theoretical upper phase water," 1.25 ml is added to the chloroform phase. The mixture is agitated and again clarified by centrifugation. The upper phase is combined with the preceding aqueous fraction. To a 1-ml aliquot of the combined aqueous phase is added 9 ml of the scintillation counting solution and the radioactivity determined by liquid scintillation spectrometry. Definition o] Unit and Specific Activity. A unit of activity is defined
~J. Folch, M. Lees, and G. H. Sloane Stanley, J. Biol. Chem. 226, 497 (1957). s H. Werbin, I. C. Chaikoff, and B. N. Imada, Proc. Soc. Exptl. Biol. Med. I~2~ 8 (1959).
[100]
CEREBROSIDE GLYCOSIDASES
593
as the amount of enzyme catalyzing the hydrolysis of 1 millimicromole of eerebroside per hour. Specific activity is expressed as units per milligrams of protein. Protein is estimated by the procedure of Itzhaki and Gill. 4
Purification Procedure Human spleen tissue obtained at operation is frozen in dry ice. After several hours it is thawed, minced and homogenized in 2 volumes of 0.1 M potassium phosphate buffer, pH 7.0 in an all-glass Ten Broeck homogenizer having 0.02 inch clearance between pestle and sleeve. The suspension is centrifuged at 600 g for 12 minutes. The precipitate is discarded, and the supernatant fraction is centrifuged at 100,000 g for 1 hour. Ammonium Sulfate Fractionation. Solid ammonium sulfate is added to the supernatant solution and stirred for 5 minutes at 2°; the precipitate is collected by centrifugation. Protein precipitating at 0-25% and 25-35% saturation with ammonium sulfate (70.5 g/100 ml ~ 100% saturation at 0 °) is dissolved in 0.01 M potassium phosphate buffer, pH 7.0. The enzyme is found primarily in the 25-35% ammonium sulfate fraction. Acid Precipitation. To the preceding 25-35% ammonium sulfate fraction is added 0.1 volume of 1.0M potassium citrate buffer, pH 5.0. The mixture is kept at 0 ° for 10 minutes and then centrifuged. The supernatant solution is incubated at 37 ° for 5 minutes and then centrifuged at 2 °. The supernatant solution is dialyzed overnight against 500 volumes of 0.01 M potassium phosphate buffer, pH 7.0. The sediment which appears is removed by centrifugation. Ethanol Fractionation. Ethanol is added to the enzyme solution immersed in an ice-salt bath at --12 ° . The protein precipitating between 39 and 56% by volume with ethanol is collected by centrifugation and dissolved in 0.01 M potassium phosphate buffer, pH 7.0. Acetone Fractionation. Acetone chilled to --12 ° is added dropwise to the preceding alcohol fraction. The protein precipitating between 50 and 62% by volume with acetone is collected by centrifugation and dissolved in 0.01 M potassium phosphate buffer, pH 7.0. The final enrichment is 82-fold and is obtained in 35% yield. The purification procedure is summarized in Table I. Properties Stability. The most highly purified fraction is completely stable over a period of 1 month when kept frozen at --20 °. 4R. F. Itzhaki and D. M. Gill, Anal. Biochem. 9, 401 (1964).
594
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[100]
TABLE I PURIFICATION OF GLUCOCEREBROSIDE-CLEAVING ENZYME FROM HUMAN SPLEEN TISSUE
Fraction I. Supernatant solution (100,000 g) II. Ammoniumsulfate, 0.25-0.35 III. Dialyzed pH 5.0 supernatant IV. Alcohol,0.39-0.56 V. Acetone, 0.50-0.62
Specific activity (units/mg protein)
Volume (ml)
Total activity units
100
630
4200
0.15
20
473
840
0.57
20 5 5
489 244 219
278 39 18
Protein (mg)
1.8 6.2 12.3
Activators and Inhibitors. The activity is enhanced by the presence of Cutseum in the incubation mixture. The optimal amount is 1.3 mg/ml. The enzyme is strongly inhibited by Tris buffer and moderately (23%) inhibited by 1 X 10-4 M p-hydroxymercuribenzoate. Ef]ect o] pH. The optimum pH is 6.0. Specificity. The most highly purified enzyme fraction catalyzes the hydrolysis of glucocerebroside. Galactocerebroside is inactive as a subtrate for this enzyme, p-nitrophenyl-fl-D-glucopyranoside is hydrolyzed at 45% the rate of glucocerebroside, o-nitrophenyl-fl-D-galactopyranoside is not hydrolyzed, p-nitrophenyl-~-D-2-acetamido-2-deoxy-glucopyranoside is hydrolyzed at 13 times the rate of glucocerebroside. The hydrolysis of glucocerebroside is not diminished in the presence of a 10-fold excess of p-nitrophenyl-fl-D-2-acetamido-2-deoxy-glueopyranoside. This observation suggests that the hydrolysis of these materials is catalyzed by different enzymes present in Fraction ¥. Substrate Affinity. The Km value for glucocerebroside is 4.2 X 10-~ M. Nature of the Reaction. The enzyme catalyzes the hydrolytic cleavage of the glycosidic bond of glucocerebroside. Ceramide (N-acylsphingosine) and free glucose are the products of the reaction. The amount of enzyme in spleens obtained from patients with Gaucher's disease appears to be considerably diminished from the level observed in non-Gaucher's human spleen preparations2 It is considered that a deficiency of this enzyme may be responsible for the accumulation of glucocerebroside in various tissues of patients with Gaucher's disease. R. O. Brady, J. N. Kanfer, and D. Shapiro, Biochem. Biophys. Res. Commun. 18, 221 (1965).
[100]
CEREBROSIDE GLYCOSIDASES
595
II. Glucosyl and Galactosyl Ceramide Glycosidase from Rat Intestine Cerebroside + H~O--* hexose ~- ceramide Assay Method P~/nc/ple. The activity of the enzyme can be determined as in Section I using glucose-l-14C-cerebrosidel or galaetose-l-14C-cerebroside, 6 or by conventional procedures for free glucose or galactose.
Reagents. Same as listed in Section I plus: Galactose-1 J4C-cerebroside 6 Sodium eholate solution, 50 mg/ml, (Matheson, Coleman and Bell)
Procedure. The assay mixture contains 12.5 mieromoles of potassium phosphate buffer, pH 6.0, 540 millimicromoles of glueose-l-~C-cerebroside or galaetose-l-14C-cerebroside, 1.5 mg of sodium cholate, 0.2 mg of Cutseum, and the enzyme in a final volume of 0.15 ml. The labeled substrate (10 mg/ml) is suspended in a solution of sodium cholate (50 mg/ml) and warmed until a clear solution is obtained before adding an aliquot of 0.0375 ml to the incubation mixture. After incubation for 1 hour at 37 °, the extent of the reaction is determined as described in Section I. Definition o] Unit and Specific Activity. A unit of activity is defined as the amount of enzyme catalyzing the hydrolysis of 1 m~mole of eerebroside per minute. Specific activity is expressed as units per milligram of protein. Purification Procec~ure Small intestinal tissue is obtained from 14-day-old Sprague-Dawley rats. The portion 1 em distal to the pylorus and 2 cm proximal to the cecum is employed. It is slit longitudinally and washed copiously with isotonic saline to remove the contents of the intestinal lumen. The tissue is chilled, minced, and homogenized in 9 volumes of 0.25 M sucrose in an all glass Ten Broeck homogenizer having 0.02-inch clearance between pestle and sleeve. The suspension is centrifuged at 600 g for 12 minutes. The precipitate is discarded and the supernatant fraction centrifuged at 8400 g for 12 minutes. SoIubilization of Enzyme. The particles sedimenting between 600 and 8400 g are suspended in 0.25 M sucrose and recentrifuged at 8400 g for 12 minutes. The washed particles are suspended in a solution containing 6R. O. Brady, A. E. Gal, J. N. Kanfer, and R. M. Bradley, J. Biol. Chem. 240, 3766 (1966).
596
[100]
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
per milliliter 5 mg of sodium cholate, equal to one-half of the original volume of the homogenate. The unfrozen suspension is kept at 0 ° overnight and then centrifuged at 100,000 g. for 1 hour. The enzyme in the supernatant solution is dialzed overnight against 2000 volumes of 0.01 M potassium phosphate buffer, pH 6.0. Ammonium Sul]ate Fractionation. Solid ammonium sulfate is added to the dialyzed enzyme, stirred for 5 minutes at 2 °, and the precipitate is collected by centrifugation. Protein precipitating at 0-25%, 25-35%, and 35-50% saturation with ammonium sulfate is dissolved in 0.01 M potassium phosphate buffer, pH 6.0. The enzyme is found primarily in the 35-50% ammonium sulfate fraction. Chromatography on TEAE-cellulose. A 5 X 18 mm column of TEAEcellulose is equilibrated with 0.01 M potassium phosphate buffer, pH 6.0. To this column 568 ~g of protein from the preceding 35-50% ammonium sulfate fraction is applied in 1.9 ml of 0.01 M potassium phosphate buffer, pH 6.0. Fractions are collected consisting of column pass, and 2-ml portions of 0.01, 0.035, 0.065 and 0.1 M potassium phosphate buffer solutions, pH 6.0. The cerebroside-cleaving enzyme is found principally in the 0.065 M fraction. The final enrichment is approximately 2300-fold with respect to the original 600-8400 g particulate preparation and is obtained in 38% yield. The purification procedure is summarized in Table II. TABLE II PURIFICATION OF RAT INTESTINAL GLUCOSYL ANn GALACTOSYL CERAMIDE GLYCOSIDASE
Fraction I. 8400 g resuspended particles II. 100,000 g cholate-treatedsupernatant solution IIL Ammoniumsulfate, 0.35-0.50 IV. TEAE eluate, 0.065 M potassium phosphate, pH 6.0
Total Volume activity (ml) units 117 58
89.4 78.8
6 6
52.8 33.8
Specific activity Protein (units/mg (mg) protein) 470 5.9 2.1 0.078
0.19 13.2 25 433
Properties
Stability. The most highly purified fraction is completely stable over a period of 1 month when kept unfrozen at 0 °. Activators and Inhibitors. The activity is enhanced by the presence of sodium eholate and Cutscum in the incubation mixture. The optimal amounts are 10. and 1.3 mg/ml, respectively. The enzyme is strongly
3-ASPARTYL-N-ACETYLGLUCOSAMINADASE
[101]
597
inhibited by Tris buffer, and is noncompetitively inhibited by cellobiose whose Ki is 3.3 X 10-3 M. Effect of pH. The optimum pH is 6.0. Specificity. The enzyme catalyzes the hydrolysis of both gluco- and galactocerebroside. Higher ceramide oligosaccharides (ceramide lactose and globoside) are hydrolyzed at somewhat slower rates. Psychosines are hydrolyzed at 5% of the rate of the respective cerebrosides, o-nitrophenyl-fl-D-galactopyranoside, p-nitrophenyl-fl-D-glucopyranoside, and p-nitrophenyl-fl-D-2-acetamido-2-deoxyglucopyranoside are hydrolyzed at 58, 80, and 25%, respectively, compared with the rate of hydrolysis of galactocerebroside. Substrate Affinity. The Km value for both gluco- and galactocerebroside is 4.9 X 10-4 M. Nature of the Reaction. The enzyme catalyzes the hydrolytic cleavage of the glycosidic bond of gluco- and galactocerebrosides. Ceramide (N-acylsphingosine) and free hexose are the products of the reaction.
[ 101] f l - A s p a r t y l - N - a c e t y l g l u c o s a m i n a d a s e f r o m E p i d i d y m i s B y EDWIN"H. EYLAR and MAKOTOMURAKAMI
1-B-Aspartyl-2-acetamido-l,2-dideoxy-D-glucosylamine--~ N-acetylglucosamine+ asparagine Assay Method Principle. The structure 1-5 of the carbohydrate-protein linkage in ovalbumin has been established as 1-fl-aspartyl-2-acetamido-l,2-dideoxy-D-glucosylamine by a comparison of the synthetic compound with that isolated from an ovalbumin glycopeptide. The fl-aspartylglycosylamine linkage in this compound is cleaved by the N-glycosidase6 to Nacetylglucosamine and asparagine. The N-acetylglucosamine liberated by the enzyme is determined colorimetrically by a modification of the Morgan-Elson reaction. 7
1G. S. Marks, R. D. Marshall, and A. Neuberger, Biochem. J. 87, 274 (1963). ~I. Yamashina, K. Ban-i, and M. Makino, Biochim. Biophys. Acta 78, 382 (1963). 3H. Tsukamoto, A. Yamamoto, and C. Miyashita, Biochem. Biophys. Res. Commun. 1.5, 151 (1064). V. P. Bogdanov, E. D. Kaverzneva, and A. P. Andreyeva, Biochim. Biophys. Acta 83, 69 (1964). "R. D. Marshall and A. Neuberger, Biochemistry 3, 1596 (1964). 6M. Murakami and E. H. Eylar, J. Biol. Chem. 240, PC556 (1965). ~J. L. Reissig, J. L. Strominger, and L. F. Leloir, J. Biol. Chem. 217, 959 (1955).