THE JOURNAL OF BIOLOGICAL CHE~STRY Vol. 243, No. 1, Issue of January 10, PP. 103-111, 1968

Printed in U.S.A.

Glycosidases of Phaseolus vulgaris

II. ISOLATION AND GENERAL PROPERTIES*

(Received for publication, August 17,1X17)

K. M. L. AGRAWAL AND OM P. BAHL

From the Department of Biochemistry, State University of New Yodc at Buffalo, Bufalo, New York 1421.4

SUMMARY

The enzymes, cr-galactosidase, P-galactosidase, a-man- nosidase, P-glucosidase, and p-acetylglucosaminidase, have been purified from the germinating seeds of Phaseolus vulgaris. All five enzymes have been simultaneously iso- lated in a highly active form. The pH optimum, K,,,, and energy of activation of each glycosidase for the reaction of hydrolysis of the appropriate P-nitrophenyl glycoside have been determined. The specificity of the enzymes has been studied by using synthetic and natural substrates such as melibiose, r&ose, stachyose, lactose, mannobiose, methyl cx-D-mannopyranoside, cellobiose, gentiobiose, sophorose, and methyl P-D-glucopyranoside. All these enzymes appear to be highly specific for the glycopyranosyl group and the anomeric configuration of the glycosidic linkage. Their action on macromolecules, such as galactomannans from guar and locust bean gums, desialyzed fetuin and its tryptic glycopeptides, a glycopeptide from orosomucoid, and ri- bonuclease B, has been studied.

The carbohydrate moieties of glycoproteins in general, in- cluding hemagglutinins, cell wall polysaccharides, and blood group substances, are complex heteropolysaccharides (1). The classical chemical procedures, such as methylation (Z), periodate oxidation (3), and partial acid hydrolysis (4), although they yield valuable information about the fine structure of polysaccharides, are inadequate for the determination of the sequence of the mono- saccharides and of the anomeric configuration of the glycosidic bonds. In addition, these methods necessitate the use of large amounts of the starting materials. Thus, they have a limited application in the case of glycoproteins only available in smaller quantities, such as human chorionic gonadotropin and follicle- stimulating hormones. These problems of sequence determina- tion, the anomeric configuration of the glycosidic bonds in the polysaccharide moiety, and greatly increased analytical sensi-

* This work was supported by Research Grant AM 10273-01 from the United States Public Health Service.

tivity might be solved through the use of highly specific glycosi- dases.

Glycosidases are widely distributed in animals (5-9), micro- organisms (l&13), and plants (14-17). Although their isolation from certain mammalian tissues and microorganisms (5-17) has been the subject of many earlier communications, the present studies were undertaken with a view to finding active and more readily available sources for the preparation of the enzymes. The seeds of Phaseolus vulgaris were chosen because seeds in general are rich in hemagglutinins (S-20) and in galactomannans, glucomannans, and other complex polysaccharides (211, and these polymers are degraded during the germination process to provide energy and intermediates for early embryonic growth, which suggests that the seeds would be rich in glycosidases. An examination of an aqueous extract of the seeds of P. vulgaris showed an exceptionally high activity of the following enzymes: P-acetylglucosaminidase (P-2-acetamido-2-deoxy-n-glucoside ac- etamidoglucohydrolase (E.C. 3.2.1.30)), cY-galactosidase (CY-D- galactoside galactohydrolase (E.C. 3.2.1.22))) /3-galactosidase (b-n-galactoside galactohydrolase (E.C. 3.2.1.23)), cY-manno- sidase (a-n-mannoside mannohydrolase (E.C. 3.2.1.24))) and /3-glucosidase (P-D-glucoside glucohydrolase (E.C. 3.2.1.21)), and the specific activity of each of these enzymes increased up to 30-fold during the course of germination. In this report, the purification of the above five enzymes, as well as their general kinetic properties, is described. The specificity of each enzyme, studied extensively by using glycosides, oligosaccharides, poly- saccharides, and glycoproteins, is also discussed.

MATERIALS AND METHODS

Pinto beans were obtained from Charter Seed Company, Twin Falls, Idaho, or locally. DEAE- and CM-Sephadexes were obtained from Pharmacia Fine Chemicals, Inc., and alu- minum oxide from Merck and Company.

Enzyme Substrates-p-Nitrophenyl CY- and /3-n-glucopyrano- sides, cellobiose, melibiose, raffinose, and gentiobiose were pur- chased from California Corporation for Biochemical Research. Methyl fi-n-glucopyranoside, methyl a-D-mannopyranoside, lactose, and stachyose were purchased from Mann Research Laboratories. p-Nitrophenyl ok- and @-n-galactopyranoside, p- nitrophenyl a- and ,8-L-fucopyranosides, p-nitrophenyl p-D-xylo-

103

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104 Glycosidases of Phaseolus vulgaris. II Vol. 243, No. 1

pyranoside, p-nitrophenyl 2-acetamido-2-deoxy-@n-glucopy- ranoside, and bovine pancreatic ribonuclease B (Batch No. 28375) were purchased from Pierce Chemical Company, Rock- ford, Illinois. p-Nitrophenyl 2-acetamido-2-deoxy-p-n-galacto- pyranoside was purchased from Cycle Chemical Company, Los Angeles.

Fetuin was prepared according to the procedure described by Spiro (22). Galactomannans from guar and locust bean meals were purified by forming their copper complexes (21). Phenyl 2-acetamido-2-deoxy-a-n-glucopyranoside was prepared by the method of Weissmann (23). The glycopeptide from desialyzed orosomucoid, which had been previously treated with P-galacto- sidase, was a gift from Dr. R. J. Winzler. Sophorose and 4-O-/3- n-mannopyranosyl-n-mannose were gifts from Dr. I. J. Gold- stein.

Enzyme Assays-p-Nitrophenyl n-glycopyranosides were used as substrates for enzyme assays. Each enzyme was assayed at its pH optimum in 0.05 M sodium citrate buffer. The liberation of p-nitrophenol was followed by a slightly modified procedure of Findlay, Levvy, and Marsh. The details of the method are described elsewhere (24).

A unit of enzyme was defined as the amount which would liberate 1 pmole of p-nitrophenol per min at 30”. The specific activity was expressed as enzyme units per mg of protein. The protein determinations were made by the method of Lowry et al., with the use of crystalline bovine serum albumin as a standard (2.9.

Identijication and Estimation of Sugars-The mono- and oligosaccharides released by the enzymatic digestion were iden- tified by paper, as well as by thin layer, chromatography. Paper chromatography was carried out in two solvent systems, (a) l- butanol-pyridine-water (5:3:2, v/v (26)) and (b) l-butanol- ethanol-water (4: 1:5, v/v, upper phase (27)). The spots were visualized by silver nitrate spray (28). Thin layer chromatog- raphy was carried out on cellulose with ethyl acetate-pyridine- water (2: 1: 2, v/v) as a developing solvent (29). The sugars were detected by spraying the plates with silver nitrate (28) or diphenylamine reagent (30).

The neutral monosaccharides formed during enzymatic hy- drolysis were estimated by gas-liquid chromatography and by the method of Somogyi and Nelson (31). For gas-liquid chro- matography, the sugars were converted into their trimethylsilyl ethers (32), which were then analyzed with an Aerograph 1520 gas chromatograph, equipped with hydrogen flame detector and matrix temperature programmer. The release of N-acetyl- glucosamine’ during enzymatic hydrolysis was followed by the method of Reissig et al. (33).

Isolation. of Mmo- and Oligosaccharides from Enzymatic Di- gests-In order to isoIate the monosaccharides from the enzymatic digest, it was placed on a column (1 x 5 cm) of charcoal-Celite mixture (1: 1) and Amberlite MB-l. The charcoal-Celite mix- ture was placed in the bottom half of the column, and Amberlite MB-l was layered over it. The column was washed with 50 ml of water, and the eluate was concentrated to dryness by a rotary evaporator at 3540”. Where both mono- and oligosaccharides were sought, the digest was simply passed through a column (1 X 2.5 cm) of Amberlite MB-l. The column was washed, and the washings were dried as above.

1 The abbreviations used are: N-acetylglucosamine, L-acetam- ido-2-deoxy-n-glucose; N-acetylgalactosamine, 2-acetamidod- deoxy-n-galactose.

High Voltage Paper Electrophmesis-High voltage paper electrophoresis was carried out in a pyridine acetate buffer, pH 4.7 (34). The peptides were detected by ninhydrin spray (34).

Germination of Pinto Beans-The pinto beans were germinated at 26-28”, in the dark, in sand flats. The beans were sprinkled on the sand, sprayed with Orthocide garden fungicide (507, captan), and covered with a 0.25.inch layer of sand. Finally, the flats were covered with black cloth and watered four times a day. The cotyledons were removed at different stages of germi- nation and extracted with buffer as described below. The specific activities were measured by the standard assays.

Purijkation of Enzymes

All operations of extraction and purification were carried out at 4” unless otherwise specified.

Step 1: Extraction-The cotyledons (1,000 g), obtained by germinating the seeds for 6 days, were washed with water, sus- pended in 1,600 ml of 0.2 M sodium citrate buffer, pH 6.0, and homogenized in a Waring Blendor for three closely spaced 30-set intervals. After the homogenate was allowed to stand for 1 hour, it was centrifuged for 1 hour in a Servall RC-2 refrigerated centrifuge at 14,900 x g. The clear supernatant was collected, and the sediment was discarded.

Step 6: Ammonium Sulfate Fractionafion-To the above super- natant, 43 g of solid ammonium sulfate per 100 ml of the super- natant were added gradually over a period of 30 to 45 min, with continuous stirring, to bring the mixture to 65% saturation. The resulting mixture was centrifuged for 1 hour at 37,000 X g. The brownish precipitate thus formed was dissolved in 400 ml of 0.2 M citrate buffer, pH 6.0, and the ammonium sulfate precipi- tation was repeated once more in the same manner. Finally, the precipitate was dissolved in 400 ml of 0.05 M citrate buffer, pH 4.6, and allowed to stand in the cold for 3 hours. The turbid solution was again centrifuged at 37,000 x g for 1 hour, and the crude mixture of the enzymes was precipitated from the clear supernatant by bringing it to 100% saturation with solid am- monium sulfate. The precipitate was redissolved in 400 ml of 0.05 M citrate buffer, pH 4.6, and the resulting solution was assayed for various glycosidases.

Step S: Chromatography on DEAE-Sephadex-A column (5 x 110 cm) of DEAE-Sephadex A-50 was equilibrated with 0.04 M

sodium phosphate buffer, pH 6.5, until the pH of the effluent was 6.5. A 175-ml portion of the enzyme solution (2.79 g of protein), previously dialyzed against phosphate buffer, pH 6.5, was applied to the column. The column was eluted with 4 liters of a linear gradient between 0.04 M sodium phosphate buffer, pH 7.5, and 1 M sodium chloride in the same buffer. The eluate was collected in 1%ml fractions which were both assayed for enzyme activities and read at 280 rnp for protein content. Tubes 20 through 70 (Fraction Dl) contained a- and ,&galactosidases, cr-mannosidase, and ,&glucosidase, and tubes 79 through 100 (Fraction D2) contained only P-acetylglucosaminidase activity. The enzymes were recovered from these two fractions either by 100% saturation of the solution with solid ammonium sulfate or, alternatively, by lyophilization of the solution in the presence of ammonium sulfate (5 mg per ml). In either procedure, no apparent loss of activity was observed. The chromatogram is presented in Fig. 1.

Step 4: Chromatography of Fraction Dl on CM-Sephadex-A column (2.5 X 70 cm) of CM-Sephadex C-50 in 0.25 M sodium

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Issue of January 10, 1968 K. llf. L. Agrawal and 0. P, Bahl 105

acetate buffer, pH 5.8, was equilibrated with the same buffer 0.5 i > 0.5 until the pH of the effluent was 5.8. A 5-ml aliquot of Fraction pH 4.4 pH 5.0 pH6.0

Dl, previously dialyzed against the same buffer (60 mg of protein, obtained from DEAE-Sephadex chromatography), was applied t 0.4. -0.4 1

to the column, which subsequently was eluted with a linear pH 2 2 gradient set up between 400 ml each of 0.25 M sodium acetate g 0.3.

1

0.3 - buffer, pH 5.8 and pH 4.0. The eluate was collected in 7-ml s: fractions, and each fraction was assayed for enzymatic activities z

: 0.2- 0.2 z and protein content as described previously. Tubes were pooled into three major fractions, CM1 (tubes 20 through 25), E

/

t CM2 (tubes 35 through 44), and CM3 (tubes 45 through 58) g 0.1 - 0.1 a (Fig. 2). The enzymes were recovered either by 100% satura-

cv

tion with ammonium sulfate or by lyophilization in the presence &/d of ammonium sulfate. 0 IO 20 60 90

FRACTION NO. 0.7 FIG. 3. Column chromatography of Fraction Dl on alumina.

A l-ml aliquot (3 mg of protein) of Fraction D1 was applied to 3 0.6 the column (1 X 10 cm) previously equilibrated with 0.2 M sodium

phosphate-citric acid buffer, pH 4.4. The column was eluted % 0.5 3 successively with 40, 80, and 60 ml of 0.2 M sodium phosphate- 5 zz $2

citric acid buffers of pH 4.4, 5.0, and 6.0, respectively; 2.0-ml 04 > fractions were collected. --, 280 rnp absorbance and A---&

s E fi-galactosidase activity. U, units. 2 0.3 -

E' 1 Chromatography of Fraction Dl on Alumina Column (Id)-

01 02 cl L it

Chromatographic grade aluminum oxide was washed several

0. I times with water, followed by a thorough washing with 0.2 M sodium phosphate-citric acid buffer, pH 4.4, A column (1 x 10 cm) of alumina was equilibrated with the above buffer. A l-ml

FRACTION NO portion of Fraction Dl from Step 3 was applied to the column,

FIN. 1. Column chromatography of partially purified enzyme which was eluted successively with 0.25 M sodium citrate buffers

mixture from P. vulgaris on DEAE-Sephadex A-50. A 175-ml of pH 4.4, (40 ml), 5.0 (80 ml), and 6.0 (60 ml). The elution solution of the crude enzyme (2.79 g of protein) was applied to a profile is shown in Fig. 3. column of DEAE-Sephadex A-50 (5 X 110 cm) previously equi- Further Puri$cation of /3-Acetylglucosaminidase (Fra&m Of?) librated with 0.04 M sodium phosphate buffer, pH 6.5. The column cm DEAE-Xephudez-Fraction D2 obtained from DEAE-Sepha- was eluted with a linear gradient of 4000 ml of 0.04 M phosphate buffer, pH 7.5, and 1 M NaCl in the same buffer. Fractions of dex was further purified on a smaller column (2.5 X 60 cm) of

18 ml were collected. -, absorbance at 280 rn,u. Enzyme ac- DEAE-Sephadex A-50, which was prepared under the same con- tivity: A-A, or-galactosidase; A----+, B-galactosidase; ditions as before. After a 5-ml sample (19 units) was applied O-----U, @-glucosidase; D-----W, a-mannosrdase; O-0, fl- to the column, the gradient elution was carried out with 200 ml acetylglucosaminidase; and -, fractions pooled. U, units. of 0.04 M phosphate buffer, pH 7.5, and 200 ml of 1 M NaCl in

the same buffer. The eluate was collected in 4-ml fractions. The enzyme activity eluted in a single peak in tubes 40 through 44, which were pooled (Fraction D4 in Fig. 4).

Chromatography of Fraction D.4 on Sephadex G-100-A 7-ml sample of Fraction D4 (50.0 mg) from the previous step was placed on a column of Sephadex G-100 (2 x 180 cm), previously equilibrated with 0.04 M sodium phosphate buffer, pH 7.5. The column was eluted with 500 ml of the same buffer, and 4.6-ml fractions were collected. Tubes 38 through 44, which were found to contain all the P-acetylglucosaminidaxe activity, were pooled (Fraction 81 (Fig. 5)).

Action of Glycosidases on Low Molecular Weight Matd&-A IO 20 30 40 50 60 70 80 90 100 0.25- to 2.0-mg sample of a low molecular weight substrate

FRACTION NO (Table III, below) in 0.1 M triethylammonium acetate buffer of FIG. 2. Column chromatography of Fraction Dl on CM-Sepha-

dex C-50. A 5-ml aliquot of Fraction Dl (60 mg of protein) was optimum pH was incubated for 1 to 4 hours with 0.1 to 0.5 unit of

applied to a column (2.5 X 70 cm) previously equilibrated with the enzyme solution. The monosaccharides were isolated as 0.25 M sodium acetate buffer, pH 5.8. The column was eluted above and estimated by gas-liquid chromatography. with a linear pH gradient set up between 400 ml each of 0.25 M Action of ar-Galactosidase on Galactomannam from Guar and sodium acetate buffer, pH 5.8 and pH 4.0; 7-ml fractions were Locust BeansTo a 3.5~ml sample of a 0.38% solution of a collected. O-0, absorbance at 280 m~c. Enzyme activity: A-A, a-galactosidase; A-+, p-galactosidase.; t+---0, galactomannan from guar or locust bean meal in 0.05 M sodium B;gluuT;dase; W----B, a-mannosrdase; and -, fractions pooled. citrate buffer, pH 6.0, 500 ,ul of the enzyme solution (1.16 units)

9 were added. The mixture was incubated at 40” for 24 hours.

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106 Glycosidases of Phaseolus vulgaris. II Vol. 243, No. 1

To avoid any bacterial contamination, 50 ~1 of toluene were added to the digest. Appropriate enzyme and substrate con- trols were run concurrently. The galactose was isolated from the hydrolysate as above and estimated by gas-liquid chro- matography.

Action of &Galactosidase on Desialyzed Fetuin and its Tryptic Glycopeptides-Fetuin (5 mg) was desialyzed by mild acid hy- drolysis (22). The resulting desialyzed protein was dissolved in 400 ~1 of 0.05 M sodium citrate buffer, pH 3.9, and treated with 1 ml of the enzyme solution (6 units) in the same buffer. After 100 ~1 of toluene were added the digest was incubated at 37”. Aliquots of 100 ~1 were withdrawn at regular intervals; a

D5

il

20 40 60 00 100

FRACTION NO

FIG. 4. Purification of fi-acetylglucosaminidase (Fraction D2) on DEAE-Sephadex A-50. A 5-ml aliquot of Fraction D2 (50 mg of protein) was applied to the column (2.5 X 60 cm) prepared as before. The column was eluted with a linear gradient of 400 ml of 0.64 M sodium phosphate buffer, pH 7.5, and 1 M sodium chlo- ride in the same buffer; 4-ml fractions were collected. -, absorbance at 280 mp; O-0, p-acetylglucosaminidase activity; and -, fractions pooled.

i 4

0. I

20 40 60 80 FRACTION NO

FIG. 5. Column chromatography of Fraction D4 (p-acetylglu- cosaminidase) on Sephadex G-100. A 7-ml aliquot of Fraction D4 (50 mg of protein) was applied to the column (2 X 180 cm), equilibrated with 0.04 M sodium phosphate buffer, pH 7.5. The column was eluted with the same buffer and 4.6~ml fractions were collected. -, absorbance at 280 rnp; O-0, P-acetylglucos- aminidase activity; and -, fractions pooled.

Q IOO-

ii

2 80.

20 40 60

TIME (HR.1

FIG. 6. Rate curves for the hydrolysis of the desialyzed fetuin (0-O) and its tryptic glycopeptides (O-0) with p-galac- tosidase. A solution of 5 mg of desialyzed fetuin in 400 ~1 of 0.05 M sodium citrate buffer, pH 3.9, was incubated with a l-ml solution of fl-galactosidase (6 units) at 37’. Portions of 100 ~1 were removed at regular time intervals and assayed for galactose by the method of Somogyi and Nelson; a zero time aliquot was used as a blank. The tryptic glycopeptides obtained from 10 mg of desialyzed fetuin were dissolved in 600 ~1 of 0.05 M citrate buffer, pH 3.9, and the mixture was incubated with 200 ~1 of @-ga- lactosidase (0.76 unit) at 37”. Aliquots of 200 ~1 were withdrawn at different time intervals and analyzed for galactose as its tri- methylsilyl ether derivative by gas-liquid chromatography (see text for details).

zero time sample was used as a blank. The galactose in each aliquot was determined by the Somogyi and Nelson method (31) after its isolation as above. The rate of release of galactose with time is shown in Fig. 6.

To an aqueous solution of 10 mg of desialyzed fetuin in 200 ~1 of water, an equal volume of ethanol was added. The resulting mixture was kept at 30” for 10 hours to denature the protein. After the sample was evaporated to dryness, the residue was dissolved in 600 ~1 of 0.02 M ammonium bicarbonate solution, pH 8.5, and the resulting solution was incubated with 10 ~1 of trypsin solution in 0.001 N HCl (1 mg per ml) at 30” for 6 hours. An additional 10 ~1 of trypsin solution were added, and the reaction mixture was further incubated for 6 hours. High voltage electrophoresis at pH 4.7 (34) showed that extensive degradation of the protein had occurred as a result of digestion with trypsin.

The solution was boiled for 3 min and then evaporated to dryness. After the dissolution of the residue in 600 ~1 of 0.05 M

citrate buffer, pH 3.9, 200 ~1 of ,&galactosidase (0.76 unit) were added. After the addition of 10 ~1 of toluene, the reaction mix- ture was incubated at 37”. Aliquots were withdrawn after 0, 15,. 30,45, and 60 hours. An equal volume of ethanol was added to each aliquot, and the resulting precipitate of glycopeptides was removed by centrifugation in a clinical centrifuge. The clear supernatant was evaporated to dryness, and the residue was dissolved in 1 ml of water. The galactose was isolated from the resulting solution as above and estimated by gas-liquid chromatography. The rate of release of galactose with time is shown in Fig. 6.

Action of /I-Acetylglucosaminidase on Galactose;free Tryptic Glycopeptides of Desialyzed Fetuin, Glycopeptide from Or- osomucoid, and Ribmuclease B-The experimental details of the action of /3-acetylglucosaminidase on the above glycoproteins and glycopeptides are described elsewhere (24).

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Issue of January 10, 1968 K. M. L. Agrawal and 0. P. Bahl

RESULTS

Germination Studies-The cotyledons were picked at different stages of germination, washed with water, and extracted with 0.2 M citrate buffer, pH 6.0. The extracts exhibited the presence of w and /3-galactosidases, cY-mannosidase, /3-glucosidase, and ,&acetylglucosaminidase. The enzymes, (Y- and P-L-fucosidases, /3-xylosidase, and oc-glucosidase, were not detected. During the process of germination, the specific activities of the enzymes increased gradually until they reached a maximum after 6 days, after which they gradually declined (Fig. 7).

Purijication of Glycos-idasesBriefly, the purification involved four major steps: (a) extraction, (b) fractionation by ammonium sulfate, (c) chromatography on DEAE-Sephadex A-50, and (d) chromatography on CM-Sephadex C-50. Ammonium sulfate precipitation was carried out twice. The resulting partially purified precipitate of the enzymes thus obtained was fractionated on a DEAE-Sephadex column, which resulted in three major protein fractions, Dl to D3 (Fig. 1). Fraction Dl was a mix- ture of CX- and ,&galactosidases, cr-mannosidase, and P-glucosidase. Fraction D2 contained /I-acetylglucosaminidase activity ex- clusively, whereas D3 did not carry any activity. When Dl was further fractionated on CM-Sephadex, it yielded three ma- jor fractions, CM1 to CM3 (Fig. 2). Fraction CMl, which came off in the void volume of the column, showed P-galactosid- ase activity only (specific activity, 5.2). Fraction CM2 had cu-mannosidase activity only (specific activity, 3.0). Fraction CM3 was an unresolved mixture of /3-glucosidase (specific ac- tivity, 2.1) and a-galactosidase (specific activity, 5.0). The recoveries from DEAE-Sephadex ranged from 70 to 80 To, except cr-mannosidase, where the recovery was 50%. The recoveries of all the enzymes from the CM-Sephadex column varied be- tween 60 and 8Oa/,.

Fraction D2 from DEAE-Sephadex A-50 was further purified by repeating the DEAE-Sephadex step once more. Two frac- tions, designated D4 and D5, were obtained (Fig. 3). Frac- tion D4, which contained all the P-acetylglucosaminidase ac- tivity, was subjected to chromatography on Sephadex G-100 (Fig. 5), which gave a preparation with a specific activity of 8.

Chromatography of Dl on an alumina column gave only P-galactosidase (specific activity, 4.2), in 50% yield. Other glycosidases could not be eluted from the column (Fig. 3). The results of purification are summarized in Table I.

General Properties of Enzymes-All the enzyme preparations were unstable to freezing and thawing. However, they could be stored at 4” without any loss of activity. The cr-mannosidase

2 4 6 8 IO 12 14 16

PERIOD OF GERMINATION (DAYS)

FIG. 7. Effect of the time of germination on the specific activi- ties of P. vulgaris glycosidases. P. vulgaris seeds were germinated on sand flats in the dark for 3,6,10, and 15 days. The cotyledons were homogenized in 0.2 M citrate buffer, pH 6. After centrifuga- tion at 14,900 X g, the enzymes in the supernatant were assayed by the standard assay. Protein determinations were made by the method of Lowry et al. The results are plotted as change in specific activity of an enzyme with time. a----a, a-galactosi- dase; A--A, p-galactosidase; U-0, p-glucosidase; ~4, cy-mannosidase; and 0-0, fi-acetylglucosaminidase.

was an exception; it lost activity gradually, even at 4”. The enzyme preparations maintained stability over a period of 50 mm at 30” (Fig. 8). All the enzyme preparations were free of chymotryptic or tryptic activity (35).

E$ect of pH, Substrate Concentration, and Temperature on Rates of Enzyme Catalysis-The effect of pH on the activity of all the glycosidases was studied by using p-nitrophenyl glycosides as substrates and phosphate-citrate buffers of pH 2.4 to 8 at 30” for 10 min. The pH-activity curves are shown in Fig. 9. The enzymes were found to be stable around their pH optima (Table II). The experiments on the pH stability of the enzymes were carried out by incubating the enzymes at 30” at pH values ranging from 2.4 to 8 for 1 hour before the assay at their pH optima. An untreated sample of each enzyme was used as a control and was assumed to have 100% activity. The results are plotted as percentage of the control activity, as shown in Fig. 10.

The effect of substrate concentration on the velocity of en- zyme reaction was investigated with the use of varying con- centrations of the appropriate p-nitrophenyl glycoside (0.1 mM

TABLE I

PurQication of glycosidases of P. vulgaris from 1000 g of cotyledons

Fraction a-Galacto- (5Galacto- WMXlllO- @-GlUCO- @-A&y!@

sidase spec- Recovery sldase spec- Recovery sidase spec- Recovery sidase spec- Recovery cosamm’- ific activity Xc activity ific activity ific activity dase spec-

ific actwit]

~~ ~____ x 101 units x 101 unils x 103 units x IO’ w&s x 10’

Crude extract (before germination). 2 2 1 1 2

Crude extract (after germination). 63 401 45 286 35 223 22 140 72

Ammonium sulfate precipitation, 409 337 270 223 227 198 136 119 475

DEAE-Sephadex A-50.. _. 2100 260 1700 157 1200 96 690 95 980

DEAE-Sephadex A-50.. 3900

Sephadex G-100.. 8000 CM-Sephadex C-50 5022 188 5152 134 3024 58 2150 71 Alumina........................... 4160 86 I -

i _

- I ReCOVery

units

458 430 343 274 238

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108 Glycosidmes of Phaseolus vulgaris. II VoI. 243, No. 1

TIME (MINS)

FIG. 8. Effect of the time of incubation on the hydrolysis of p-nitrophenyl glycosides by the glycosidases of P. vu2garis. The enzymes were incubated at 30” with 5 mM p-nitrophenyl gluco- pyranosides in citrate buffer at their pH optima for 5 to 50 min. I (A), a-galactosidase.; B (A), 8-galactosidase; 3 (O), &glucosi- dase; 4’ (w), a-mannosldase; and 6 (o), p-acetylglucosaminidase.

PH

FIG. 9. pH dependence of P. vulgaris glycosidases. The en- zymes were incubated with 5 mM p-nitrophenyl glycosides for 10 min in the phosphate-citrate buffers of pH 2.4 to 8 at 30”. pH curves: A-A, cu-galactosidase; A-A, p-galactosidase; O---O, p-glucosidase; FM, cY-mannosidase; and O-O, B-acetylglucosaminidase.

TABLE II Properties of glycosidases of P. vulgaris

______ PH PH M x 10-4

a-Galactosidase.. . 6.5-6.7 4.6-8 6.57 fl-Galactosidase.. 3.84.0 4.6-8 9.18 or-Mannosidase.. 3.8-4.0 6-7 11.7 @-Glucosidase. . 4.8-5.0 4.6-6.8 0.83 P-Acetylglucosa-

minidase. 4.6-4.8 4.6-8 4.68

a E,, activation energy.

i

E.”

hi/mole

13.6 12.4 10.1 11.6

2.5 1.9 2.7 1.6 2.2 1.8 1.7 1.8

9.8 1.9 1.7

-

j 1: -

j-25” 5-35”

90

80 = 70 5 60 F 2 50

40

g 30 20 IO

2 3 4 5 6 7 8 9

PH

FIG. 10. Stability of P. vulgaris glycosidases at different pH values. The enzymes were incubated with 5 mM p-nitrophenyl glycosides for 1 hour at 30” in phosphate-citrate buffers of pH 2.4 to 8.0. The enzymes were assayed at their pH optima. An untreated sample of enzyme was used in each case as a control; the results are plotted as percentage of the control activity. A-A, oc-galactosidase; A-A, p-galactosidase; D----O, fl-glucosidase ; D---m a-mannosidase ; and l - l , @-acetyl- glucosaminidase.

I/S (tnM)-’

FIG. 11. Lineweaver-Burk plots of P. vulgaris glycosidases. The enzymes were incubated for 10 min with varying concentra- tions (0.1 to 5 mM) of p-nitrophenyl glycosides in sodium citrate buffers at their pH optima at 30”. A-A, or-galactosidase; A-A, fi-galactosidase; o-0, o-glucosidase; +m, a-man- nosidase; and O-O, B-acetylglucosaminidase.

to 5 InM) in 0.05 M citrate buffers under optimum pH conditions. The Lineweaver-Burk plots showed straight line relationships

(Fig. 11).

The effect of temperature on the rate of hydrolysis was studied in the range of 15-45’. The Arrhenius plots (Fig. 12), which showed linear relationships, were used to calculate the activation energies of various glycosidases.

The pH optima, pH stability, K,, activation energies, and & 10 values of the glycosidases are summarized in Table II.

Substrate Specificity Studies-The enzyme ol-galactosidase hydrolyzed p-nitrophenyl a-n-galactopyranoside but was unable to hydrolyze the p-nitrophenyl P-glycoside. It cleaved meli-

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hue of January 10, 1968 K. M. L. Agrawal and 0. P. Bahl 109

3.0 3.1 3.2 3.3 3.4 35 3.6 I T

FIG. 12. Arrhenius plots for P. vulgaris glvcosidases. The enzymes were incubated for 10 min with 5 GM p-nitrophenyl glycosides in sodium citrate buffers at their DH ontima at tem-

A 1

peratures from 15 to 45”. A---& a-galactosidase; A-A, &galactosidase; O--O, fi-glucosidase; +B, a-mannosidase; and 0-0, P-acetylglucosaminidase.

biose (6-O-cr-D-galactopyranosyl-D-glucose) into galactose and glucose; raffinose (O-cr-D-galactopyranosyl-(1 + 6)-O-a-D-glu- copyranosyl-(1 + 2)-P-D-fructofuranoside) into galactose and sucrose; and stachyose (0-a-D-galactopyranosyL(1 -+ 6)-0-a-~- galactopyranosyl-( 1 --f 6) -@a-D-glucopyranosyl-( 1 + 2) -@-D-

fructofuranoside) into galactose, r&nose, and sucrose. The identification of the mono- and oligosaccharides was carried out by paper chromatography (26). The enzyme was also able to remove, under the conditions of hydrolysis used, 27% of the total 1 -+ 6-a-linked D-galactose residues from the galacto- mannans from guar gum and 30% of these residues from locust bean gum.

The /3-galactosidase hydrolyzed the p-nitrophenyl/3-D-galacto- pyranoside, but failed to cause the hydrolysis of the correspond- ing p-nitrophenyl a-glycoside. The enzyme cleaved lactose (4-0-P-D-galactopyranosyl-D-glucose) i&o galactose and glucose. It released 40% of 1 ---f 4-B-linked D-galactopyranose residues, as followed by the Somogyi-Nelson method (31), from the non- reducing termini of the carbohydrate chains of desialyzed fetuin. The hydrolysis was apparently complete in 60 hours, as shown in Fig. 6. When the tryptic glycopeptides of desialyzed fetuin were digested with P-galactosidase, 76% of the total galactose was released in 60 hours.

The P-acetylglucosaminidase was capable of hydrolyzing p- nitrophenyl 2-acetamido-2-deoxy-P-D-glucopyranoside, but had no action on the corresponding phenyl cr-glycoside. It also hydrolyzed p-nitrophenyl 2-acetamido-2-deoxy-p-D-galacto- pyranoside, an indication of the presence of P-acetylgalactos- aminidase activity. The enzyme was able to liberate 14% of the terminal N-acetylglucosamine residues from the tryptic glycopeptides of desialyzed fetuin that had been previously treated with ,&galactosidase. Also, the enzyme removed 50% of N-acetylglucosamine residues from an orosomucoid glycopep- tide that had been digested previously with &galactosidase. Finally, bovine pancreatic ribonuclease B, on treatment with the P-acetylglucosaminidase, yielded 1 mole of N-acetylglucosa- mine per mole of the protein.

The P-glucosidase did not exhibit any a-glucosidase activity. It was able to hydrolyze sophorose (0-/3-D-glucopyranosyl-(1 + 2)-D-glucose), cellobiose (O-O-D-glucopyranosyl-(1 -+ 4)-D-glu-

case), gentiobiose (O-/i?-D-ghCOpyranOSy1-(I + 6)-D-glucose), and methyl P-D-glucopyranoside; glucose was yielded in each case.

The a-mannosidase was able to hydrolyze p-nitrophenyl (Y-D-

mannopyranoside and methyl a-D-mannopyranoside. It failed to hydrolyze 0-/3-D-mannopyranosyl-(1 -+ 4)-D-mannose.

DISCUSSION

The present studies were initiated with a view to finding suita- ble sources of glycosidases and developing procedures for their isolation. The results reported in this communication indicate that P. vu2guris is indeed an active source of several glycosidases, which can be isolated by the simple procedure detailed above. The enzymes, a-galactosidase, /Lgalactosidase, Lu-mannosidase, P-glucosidase, and fi-acetylglucosaminidase, have been obtained in a highly active form. One of the reasons for the effectiveness of this procedure may be the initial step, germination of the seeds. The process of germination not only causes a degradation of the polysaccharide gums and proteins but also results in the elevation of the specific activity of the enzymes in the seeds. The gums in the seeds of P. vulgaris, like those of any other leguminous plant (21), form a viscous solution in water. I f this polymeric material is not removed in the initial stages of purification, the re- sulting viscous solution of the crude enzymes causes some diffi- culty in the subsequent chromatographic steps. In the present scheme of isolation, the removal of the polysaccharides is effected partly by germination and partly by repeated precipitations with ammonium sulfate. In view of the above considerations, fractionation by acetone or alcohol in the initial stages would not be desirable, as both the polysaccharides and the proteins would be precipitated together.

By the above procedure, P-galactosidase activity was separated from fl-glucosidase activity. In rat intestinal mucosa (36) and almond emulsin (15) enzymes, both activities seem to reside in the same molecule. On the other hand, &galactosidases from calf liver (37) and rat brain (37) do not show any /%glucosidase activity, and thus behave like the P. vulgaris enzyme. How- ever, the @-acetylglucosaminidase exhibited ,&acetylgalac- tosaminidase activity also, as it catalyzed the hydrolysis of p-nitrophenyl 2-acetamido-2-deoxy$-D-galactopyranoside. N- Acetylglucosamine or N-acetylgalactosamine was found to in- hibit both the activities, which suggests that the two activities might be present in the same molecule. However, further work would be necessary to clarify this point. The analogous enzymes isolated from ram testis (6) and pig epididymis (5) also show both the activities, presumably associated with the same en- zyme site.

The specificity studies indicate that these glycosidases are highly specific for glycosyl residues and the anomeric configura- tion of the glycosidic bonds. For instance, cr-galactosidase and a-mannosidase hydrolyze only the ol-glycosidic bonds, and do not show any P-glycosidase activity. Similarly, P-galacto- sidase, P-glucosidase, and fi-acetylglucosaminidase do not appear to have any ar-glycosidase activity. Apparently, the suscepti- bility of a glycosidic bond to hydrolysis by a glycosidase does not depend on either the nature of the aglycon (38) or its linkage to the glycosyl group (Table III). For instance, P-glucosidase catalyzes the hydrolysis of 1 -+ 2-p-, 1 --f 4-p-, and 1 + S-p- glycosidic bonds in sophorose, cellobiose, and gentiobiose, re- spectively. The rate of hydrolysis, however, does depend upon the nature of the aglycon (38), as well as the type of linkage.

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110

TABLE III with oc-galactosidase, galactose was released; this observation Substrate specijkity of glycosidases of P. vulgaris indicates that these residues must be involved in a-glycosidic

Enzyme or substrate

cl-Galactosidase Meiibiose...................... Raffinose...................... Stachyose Guar gum. Locust bean gum.

p-Galactosidase Lactose. Desialyzed fetuin.. Glycopeptides of desialyzed

fetuin....................... au-Mannosidase

4.O-p-o-Mannopyranosyl-D- mannose..........

Methyl-a-a-mannopyranoside &Glucosidase

Cellobiose.................... Gentiobiose.................... Sophorose Methyl-p-n-glucopyranoside..

p-Acetylglucosaminidase Galactose-free glycopeptides of

desialyzed fetuin %alactose-free glycopeptide of

desialyzed orosomucoid. Ribonuclease B..

%E.yme 2x? ‘eriod ot linkages in the structure of galactomannans, a result which is

incuba- Sugar consistent with the above structures. strate tion I &ased Since P. vulgaris glycosidases degrade glycoproteins, glycopep-

-

vnits mg % tides, and polysaccharides so readily and specifically, and are easy to isolate, they are expected to find an extensive application

0.58 1.0 1 16.9 in the study of the complex macromolecules.

0.58 1.0 2 27.0 0.58 2.0 4 9.4 Acknozobdgments-We wish to thank Dr. R. J. Winder for

1.16 3.5 24 27.1 his continued interest and advice during the course of this work.

1.16 3.5 24 30.0 We would also like to thank Drs. David Straus and Jonathan Brodie for their comments and helpful suggestions in the prep-

0.43 0.9 1 28.2 aration of the manuscript. 6.00 5.0 30 40.0

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(1964). As previously observed by other investigators (31), the size of the substrate also affects the rate of hydrolysis. This is quite obvious from the rate curves of the hydrolysis of desialyzed fetuin and its tryptic glycopeptides with /3-galactosidase (Fig. S), and those of orosomucoid glycopeptide and bovine pancreatic ribonuclease B with /?-acetylglucosaminidase (24).

Glycoproteins and glycopeptides are effective substrates of P. vulgaris enzymes. When desialyzed fetuin, its tryptic glycopeptides, and the glycopeptide from desialyzed orosomucoid were successively treated with ,&galactosidase and /3-acetylglu- cosaminidase, galactose and N-acetylglucosamine were released. Therefore, the sequence of the monosaccharides at the nonre- ducing ends of the carbohydrate chains of fetuin and orosomu- coid is N-acetylneuraminic acid-n-galactose-N-acetylglucosamine. Furthermore, the glycosidic linkages involving galactose and N- acetylglucosamine residues are of /3 configuration. These results are consistent with earlier findings (39, 40). Since &acetyl- glucosaminidase liberates 1 of the 2 residues of N-acetylglucos- amine per mole of ribonuclease B, the carbohydrate unit of ribonuclease B appears to be terminated by a ,&N-acetylglucos- amine residue. If the 2nd N-acetylglucosamine residue is in- volved in the attachment of the carbohydrate to the polypeptide chain, then the rest of the oligosaccharide chain must be com- posed of mannose residues (41).

The galactomannans from guar and locust bean gums consist of a main chain of mannose residues, to which n-galactopyranose residues are attached by 1 + 6-a linkages on every other mannose residue in guar gum and on every 4th mannose residue in locust bean rrum (21). When these galactomannans were digested u .I

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K. M. L. Agrawal and Om P. BahlPROPERTIES

: II. ISOLATION AND GENERALPhaseolus vulgarisGlycosidases of

1968, 243:103-111.J. Biol. Chem. 

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