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Purification and Molecular Properties of a Cell-Wall β-Glucosyltransferase From Soybean Cells Cultured In Vitro
307
Plant.Science Letter., 28 (1982/83) 307-312 ElaeVler Scientific Publiahera Ireland Ltd.
PURIFICATION AND MOLECULAR PROPERTIES OF A CELL-WALL ~·GLUCOSYLTRANSFERASE FROM SOYBEAN CELLS CULTURED
IN VITRO
J. NARI, G. NOAT, J. RICARD, E. FRANCHINI and P. SAUVE ge ntre de Biochimie et de Biologie Moleculaire du CNRS, BP No. 71, 13277 Marseille, edex 9 (France) (ReceiVed June 11th 1982) (Accepted July 29th: 1982)
.
--------------------------------------------------------SUMMARY
fr A it-glUcosidase has been isolated and purified to apparent homo~eneity o~ soybean cell walls. The enzyme is a glycoprotein with a molecular weIght close to 60 000 and a sedimentation constant equal to 4.17. The protein is made up with one polypeptide chain only.
---------------------------------------------------------INTRODUCTION
It is CUrrently believed that loosening and extension of plant cell wall requires the functioning of glycosyltransferases [1]. Although both exo- and [ndo-glucanase activities have been shown to occur in some plant tissues b2-10], neither of them has been isolated from cell walls and purified to °flllogeneity, a necessary prerequisite to understanding the molecular basis o cell wall extension. Soybean (Glycine maximalis) cell culture in liquid medium provides a Useful material for studying the role of glycosyltransferases in cell wall ~tension for at least two reasons: firstly, it is extremely rich in cell wall g ucanases and secondly, as shown in the companion paper, the average cell \folUllle markedly varies during the culture. t The aim of this paper is therefore to purify to homogeneity a jj-glucosyll'Ilnsferase from cell walls of isolated soybean cells cultured in vitro in liquid IDedium and to determine some of its molecular properties. MATERIALS AND METHODS
d SOYbean cell clusters were cultured in vitro under sterile conditions as eacribed by Gamborgetal. (11]. GlUCosidase activity was followed by measuring the hydrolysis of p-nitro-
~a04.4211/83/0000-0000J$03.00 Pril983 Elaevier Scientific Publiaheralreland Ltd. nted and Published in Ireland
808
phenyl-I3-D-glucopyranoside. The standard incubation medium (3 ml) contained 0.05 M succinate buffer pH 5 an~ 1 • 10- 3 M p-nitrophenyli3-D-glucopyranoside. After 15 min of incu bation at 30°C, the reaction was stopped by addition of 1 ml of 0.2 M C03N~ and the absorbance of p-nitrophenol formed was read at 400 nm. One enzyme unit was defined as the amount of enzyme capable of hydrolyzing 1 ~M of substrate per minute under the above conditions. Protein estimation was carried out by the method of Bradford [12] or that of Bohlen et al. [13]. Carbohydrate concentration was estimated by the method of Dubois et al. [14]. Reducing sugars were titrated according to the Somogyi's technique [15]. Sephadex G-100 columns were equilibrated in 0.05 M succinate buffer (pH 5) containing NaCI at a final concentration of 1 M. Calibration of the columns for molecular weight estimation was performed according to the method of Whitaker [16] with the following standards: a-chymotrypsinogen (24700), pepsin (35 000), ovalbumin (45 000), serum albumin monomer (68 000) and serum albumin dimer (136 000). Hydroxyapatite (HA Ultrogel, Pharmindustrie, France) columns were equilibrated with 0.01 M phosphate buffer (pH 6.8). Columns were eluted first stepwise by 0.25 M phosphate buffer and then by a linear gradient of phosphate buffer of 0.25-0.4 M. Polyacrylamide gel electrophoresis under non-denaturing conditions was carried out by following the method of Gabriel [17]. Protein was stained on the gel by amidoblack. The glycosidase activity was detected on the gel after inCUbation at pH 6 with gentiobiose or cellobiose (25 mM) and visualization of glucose on the gel by tetrazolium nitroblue [18]. Glycoprotein was detected by the method of Zacharius et al. [19]. Electrophoresis under denaturing conditions was effected according to the method of Weber et al. [20] . Analytical centrifugation experiments were performed with a Spinco analytical model E ultracentrifuge equipped with an interferometric system for molecular weight determinations [21] and UV absorption optics for sedimentation constant estimation [22]. Chemicals were purchased from Sigma or Boehringer. RESULTS
Soybean cell clusters are taken during the exponential growth phase for enzyme isolation and purification. The clusters are filtered through miracloth (50 ~m) and rinsed. After grinding in a Waring blendor they are submitted to a pressure of 103 kg/cm 2 in a French press in presence of 0.4 M sucrose. Suspension of cell wall fragments is centrifuged 15 min at 1'100 X g. The pellet is suspended in 0.6 M sucrose solution and centrifuged again. The same operation is repeated several times with sucrose solutions of increasing molarity (up to 1 M). No lipid and lipoprotein can be detected on the cell wall fragments thus obtained, indicating that they are free from plasmalemma and cytoplasmic contaminations [23].
309
The pellet of cell wall fragments is then suspended in buffer (pH 5) con~ning 1 M NaCl. This treatment results in partial solubilization of ~-gluco sldase activity. The suspension is centrifuged again and the pellet is resuspended in the same succinate-NaCI solution. After a new centrifugation the two supernatants contain about 65% of total ~-glucosidase activity. Further NaCl treatments of cell wall fragments do not result in additional enzyme solubilization. Enzyme solubilization may also be obtained from intact unbroken cells, brsimply adding NaCl to the cell suspension. The amount of enzyme solu~Ulzed in that way is indeed smaller than the one obtained from a preparation of cell wall fragments. These results, however, show without ambiguity, that the ~-glucosidase studied in this work is exo-cellular and that location on the cell wall does not result from an artefact due to cell breakage. The collected supernatants containing ~-glucosidase activity are saturated ~o 30% by solid ammonium sulphate. After centrifugation, the precipitate ~s discarded and the supernatant is saturated again to 80% by solid ammonlUm SUlphate. After centrifugation the precipitate is collected with a small
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Fig. 1. ISOlation of p-glucosidase. A: molecular sieve chromatography on Sephadex G-lOO of "-glucosidase from cell walls. Four millilitres of a protein extract containing 30 mg of ~rotein and 8 mlf of polysaccharides are passed over a Sephadex G-IOO column (2.4 x 100 crn) equilibrated with pH 5,0.05 M luccinate buffer containing NaCI (final cone. M). The flow rate is 20 ml/h. The volume of the fractions collected is 3.6 ml. p-Nitro~henYI~-D-glucopyranOSide activity (0 - - - -0), protein concentration ( e _ ) and carboYdrate concentration (b.--A) are measured in the fractions (see Materials and ~ethodS). B: hydroxyapatite chromatograph), of cell wall P-gluc08idase. The active fracIOns frorn previous chromatography are collected under molecular sieving, concentrated ~nd Passed over the hydroxyapatite column (1.4 x 20 cm). Elution is effected with a near Kradient (---) of phoaphate (pH 6.8). The flow rate is 25 ml/h. Fractions of 6 ml ~e COllected. Protein concentration (e---e ) and p-glucosidase activity (0 - - -- - -0) are eterrnined in the fractions.
310
B
A ~0.2
+
E c
~ ~
0.1
v 0 u c a
.D
2
L
5l 02
.D
A
«
c
B
0.1
0 Migration
Fig. 2. Homogeneity of cell wall p-glucosidase. A: polyacrylamide gel electrophoresis of p-glucosidase under non-denaturing conditions. In each case, 25 J,l.g of protein is applied on the gel (see Materials and Methods). (A) Protein is stained with amidoblack. (B) P-glucosidase activity is detected on the gel by tetrazolium nitroblue. (C) Carbohydrates are detected on the gel. B: analytical centrifugation of p-glucosidase. Enzyme concentration is 1.2 mg/ml in a pH 5, 0.05 M succinate buffer containing 0.6 M NaCI. Migration is from left to right (rotation speed 60000 rev./min, temperature 25°C, UV optics). Sedimentation diagrams are obtained 20 min (1) or 40 min (2) after the start of ultracentrifugation. The value of the apparent sedimentation constant is 4.17.
S.4 L S.2
A
2 2 c v E v
a
u
(J)
a
-
B
2-
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ii v
b
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i
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'-
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e
4.2L-_ _ _ _-L._ _ _ _-"---1 1 1.S 2 VelVo
8'1
SO,S
51
51.S
(Rodius)2 (cm)2
Fig. 3. Molecular weight determination of p-glucosidase. A: molecular weight determination of p-glucosidase by molecular sieve chromatography. Exclusion volume of the column, VOl is determined with dextran blue. Points marked by the letters a, b, C, d, e, correspond to the elution ratios, Ve/Vo' of the various proteins used as standards (a-e, serum albumin dimer and monomer, ovalbumin, pepsin, a-chymotrypsinogen). The arrow shows the elution ratio of p-glucosidase and corresponds to a molecular weight of 62000 ± 1400 (mean of 6 determinations). The Sephadex G·100 column (1.2 x 100 cm) is equilibrated with 0.05 M succinate buffer containing NaCI (see Materials and Methods). B: molecular weight determination by eqUilibrium analytical centrifugation. The enzyme concentration is 1 mglml. Temperature is 25°C and rotation speed 30 000 rev./min. Time of centrifugation 24 h.
311
volume of 0.05 M succinate buffer (pH 5) containing NaCI (final molarity 1 M). About 80% of (3-glucosidase initially present in the soluble extract has been recovered after ammonium sulphate precipitation. This ammonium sulphate fractionation allows to discard most of the soluble polysaccharides present in the soluble extract of the cell walls. The soluble extract obtained after ammonium sulphate fractionation is submitted to molecular sieving on Sephadex G-100 equilibrated in the same succinate buffer containing NaCl. The resulting elution profile is shown in Fig. 1A. The fractions displaying fj-gll1cosidase activity are collected, concentrated by ultra-filtration on DIAFLO membranes (PM 30 Amicon), then diluted ten times with distilled water and submitted to hydroxyapatite chromatography. Elution is effected as described under Materials and Methods. The elution diagram is shown in Fig. lB. This (3-glucosidase appears homogenous in polyacrylamide gel electrophoresis and centrifugation (Fig. 2). The sedimentation constant is equal to 4.17 S. On electrophoresis, the protein band is clearly superimposable on enzyme activity. Moreover, results of Fig. 2 indicate that this enzyme is a glycoprotein. Both molecular sieving and analytical centrifugation give molecular weights of approx. 60 000 (Fig. 3). If polyacrylamide gel electrophoresis is performed under denaturing conditions the enzyme appears to Consist of a single polypeptide chain only (not shown). The enzyme hydrolyzes gentiobiose [(j(1-+ 6)glucoside], cellobiose [fj(1-+ 4)glucoside] and sophorose [(3(1-+ 2)glucoside]. DISCUSSION
~ fj-glucosidase has been solubilized from soybean cell walls and purified. ThIS enzyme is homogenous to analytical centrifugation and polyacrylamide gel electrophoresis. It appears to be a glycoprotein with a molecular weight of approx . 60000 and containing a single polypeptide. Detection of p-glucosidase activity on the cell wall does not result from an artefact consecutive to cell disruption and adsorption of a cytoplasmic enzyme to cell wall fragments, for the enzyme activity may be detected on unbrOken cells. This (3-glucosidase is thus typically exo-cellular. . .Only a few glycosidases have been highly purified from plant tissues and It IS therefore difficult to compare the enzyme from soybean cell walls with ~ther glucosidases obtained from different plant systems. To the best of our nowledge, the only (j-glycosidase purified so far to homogeneity from a plant tissue has been obtained from almond emulsion [24]. Although no definite information is available as to the intracellular location of this enzyme, it is very likely that it is not bound to cell walls. Moreover the fj-glycosidase from almond emulsin appears to be a dimer of 135000 moleCular Weight, in which one subunit reacts with (j-gIucosides, the other with fj-galactosides. This enzyme is in reality a bifunctional enzyme compl~ [24]. The (3-glucosidase purified from soybean cell walls is totally devoid of galactosidase activity and has a molecular weight close to that of a 'subunit' of the enzyme complex extracted from almond emulsin.
312
In order for a glycosidase to take part in cell wall extension, it must act as a true glycosyltransferase, that is, transfer the glycosyl moiety of a donor to a sugar acceptor and not solely to water [1]. It is therefore of importance to determine whether this enzyme, isolated from cell walls, is a true exo- or endo-glucosyltransferase. This matter is considered in the companion paper. REFERENCES 1 P. Albersheim, in: J. Bonner and J.E. Varner (Eds.), Plant Biochemistry, Academic Press, 1976, p. 225. 2 Y. Muuda, S. Oi and Y. Satomura, Plant Cell Physiol., 11 (1970) 631. 3 D.J. Nevins, Plant Cen Physiol.,16 (1975) 347. 4 D.J. Nevins, Plant Cell Physiol., 16 (1975) 495. 5 N. Sakurai and Y. Masuda, Plant Cell Physiol., 18 (1977) 587. 6 R. Yamamoto and D.J. Nevins, Plant Physiol., 51 (1981) 118. 7 A.N.J. Heyn, Arch. Biochem. Biophys., 132 (1969) 442. 8 D.J. Huber and D.J. Nevins, Plant Physiol., 65 (1980) 768. 9 D.J. Huber and D.J. Nevins, Planta, 151 (1981) 206. 10 R. Goldberg, Plant Sci. Lett., 8 (1977) 233. 11 O.L. Gamborg, R.A. Miller and K. Ojima, Exp. Cen Res., 50 (1968) 151. 12 M.M. Bradford, Anal. Biochem., 72 (1976) 248. 13 P. Bohlen, S. Stein, W. Dairman and S. Udenfriend, Arch. Biochem. Biophys., 155 (1973) 213. 14 M. Dubois, K.A. GUles, J.K. Hamilton, P.A. Rebers and F. Smith, Anal. Biochem., 28 (1956) 350. 15 M. Somogyi, J. BioI. Chem., 195 (1952) 19. 16 J.R. Whitaker, Anal. Biochem., 35 (1963) 1950. 17 O. Gabriel, Methods Enzymol., 22 (1971) 578. 18 H.M. Katzen and R.T. Schimke, Proc. Natl. Acad. Sci. U.S.A., 54 (1965) 1218. 19 R.M. ZachariuI, T.E. Zen, J.H. Morrison and J.J. Woodlock, Anal. Biochem., 30 (1969) 148. 20 K. Weber, J.R. Pringle and M. Osborn, Methods Enzymol., 26 (1972) 3. 21 D.A. Yphantis, Biochemistry, 3 (1964) 297. 22 J. Vinograd, R. Bruner, R. Kent and J. Weigle, Proc. Natl. Acad. Sci. U.S.A., 49 (1963) 902. 23 A.M. Cates80n, R. Goldberg and M.C. Winay, C.R. Acad. Sci., 272 (1971) 2078. 24 A.K. Grover, D.D. Macmurchie and R.J. CUlhley, Biochim. Biophys. Acta, 482 (1977) 98.
Plant.Science Letter., 28 (1982/83) 307-312 ElaeVler Scientific Publiahera Ireland Ltd.
PURIFICATION AND MOLECULAR PROPERTIES OF A CELL-WALL ~·GLUCOSYLTRANSFERASE FROM SOYBEAN CELLS CULTURED
IN VITRO
J. NARI, G. NOAT, J. RICARD, E. FRANCHINI and P. SAUVE ge ntre de Biochimie et de Biologie Moleculaire du CNRS, BP No. 71, 13277 Marseille, edex 9 (France) (ReceiVed June 11th 1982) (Accepted July 29th: 1982)
.
--------------------------------------------------------SUMMARY
fr A it-glUcosidase has been isolated and purified to apparent homo~eneity o~ soybean cell walls. The enzyme is a glycoprotein with a molecular weIght close to 60 000 and a sedimentation constant equal to 4.17. The protein is made up with one polypeptide chain only.
---------------------------------------------------------INTRODUCTION
It is CUrrently believed that loosening and extension of plant cell wall requires the functioning of glycosyltransferases [1]. Although both exo- and [ndo-glucanase activities have been shown to occur in some plant tissues b2-10], neither of them has been isolated from cell walls and purified to °flllogeneity, a necessary prerequisite to understanding the molecular basis o cell wall extension. Soybean (Glycine maximalis) cell culture in liquid medium provides a Useful material for studying the role of glycosyltransferases in cell wall ~tension for at least two reasons: firstly, it is extremely rich in cell wall g ucanases and secondly, as shown in the companion paper, the average cell \folUllle markedly varies during the culture. t The aim of this paper is therefore to purify to homogeneity a jj-glucosyll'Ilnsferase from cell walls of isolated soybean cells cultured in vitro in liquid IDedium and to determine some of its molecular properties. MATERIALS AND METHODS
d SOYbean cell clusters were cultured in vitro under sterile conditions as eacribed by Gamborgetal. (11]. GlUCosidase activity was followed by measuring the hydrolysis of p-nitro-
~a04.4211/83/0000-0000J$03.00 Pril983 Elaevier Scientific Publiaheralreland Ltd. nted and Published in Ireland
808
phenyl-I3-D-glucopyranoside. The standard incubation medium (3 ml) contained 0.05 M succinate buffer pH 5 an~ 1 • 10- 3 M p-nitrophenyli3-D-glucopyranoside. After 15 min of incu bation at 30°C, the reaction was stopped by addition of 1 ml of 0.2 M C03N~ and the absorbance of p-nitrophenol formed was read at 400 nm. One enzyme unit was defined as the amount of enzyme capable of hydrolyzing 1 ~M of substrate per minute under the above conditions. Protein estimation was carried out by the method of Bradford [12] or that of Bohlen et al. [13]. Carbohydrate concentration was estimated by the method of Dubois et al. [14]. Reducing sugars were titrated according to the Somogyi's technique [15]. Sephadex G-100 columns were equilibrated in 0.05 M succinate buffer (pH 5) containing NaCI at a final concentration of 1 M. Calibration of the columns for molecular weight estimation was performed according to the method of Whitaker [16] with the following standards: a-chymotrypsinogen (24700), pepsin (35 000), ovalbumin (45 000), serum albumin monomer (68 000) and serum albumin dimer (136 000). Hydroxyapatite (HA Ultrogel, Pharmindustrie, France) columns were equilibrated with 0.01 M phosphate buffer (pH 6.8). Columns were eluted first stepwise by 0.25 M phosphate buffer and then by a linear gradient of phosphate buffer of 0.25-0.4 M. Polyacrylamide gel electrophoresis under non-denaturing conditions was carried out by following the method of Gabriel [17]. Protein was stained on the gel by amidoblack. The glycosidase activity was detected on the gel after inCUbation at pH 6 with gentiobiose or cellobiose (25 mM) and visualization of glucose on the gel by tetrazolium nitroblue [18]. Glycoprotein was detected by the method of Zacharius et al. [19]. Electrophoresis under denaturing conditions was effected according to the method of Weber et al. [20] . Analytical centrifugation experiments were performed with a Spinco analytical model E ultracentrifuge equipped with an interferometric system for molecular weight determinations [21] and UV absorption optics for sedimentation constant estimation [22]. Chemicals were purchased from Sigma or Boehringer. RESULTS
Soybean cell clusters are taken during the exponential growth phase for enzyme isolation and purification. The clusters are filtered through miracloth (50 ~m) and rinsed. After grinding in a Waring blendor they are submitted to a pressure of 103 kg/cm 2 in a French press in presence of 0.4 M sucrose. Suspension of cell wall fragments is centrifuged 15 min at 1'100 X g. The pellet is suspended in 0.6 M sucrose solution and centrifuged again. The same operation is repeated several times with sucrose solutions of increasing molarity (up to 1 M). No lipid and lipoprotein can be detected on the cell wall fragments thus obtained, indicating that they are free from plasmalemma and cytoplasmic contaminations [23].
309
The pellet of cell wall fragments is then suspended in buffer (pH 5) con~ning 1 M NaCl. This treatment results in partial solubilization of ~-gluco sldase activity. The suspension is centrifuged again and the pellet is resuspended in the same succinate-NaCI solution. After a new centrifugation the two supernatants contain about 65% of total ~-glucosidase activity. Further NaCl treatments of cell wall fragments do not result in additional enzyme solubilization. Enzyme solubilization may also be obtained from intact unbroken cells, brsimply adding NaCl to the cell suspension. The amount of enzyme solu~Ulzed in that way is indeed smaller than the one obtained from a preparation of cell wall fragments. These results, however, show without ambiguity, that the ~-glucosidase studied in this work is exo-cellular and that location on the cell wall does not result from an artefact due to cell breakage. The collected supernatants containing ~-glucosidase activity are saturated ~o 30% by solid ammonium sulphate. After centrifugation, the precipitate ~s discarded and the supernatant is saturated again to 80% by solid ammonlUm SUlphate. After centrifugation the precipitate is collected with a small
E
0.4
B
A
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vi
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/
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0.10
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20
40
60 Fraction
80 numb~r
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-
.20.35 Q.
g
~
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E
,,
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/-;
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,
10
~
/
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0.05
:E
/
/~
50 Fraction
o
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0.25
100 numb~r
Fig. 1. ISOlation of p-glucosidase. A: molecular sieve chromatography on Sephadex G-lOO of "-glucosidase from cell walls. Four millilitres of a protein extract containing 30 mg of ~rotein and 8 mlf of polysaccharides are passed over a Sephadex G-IOO column (2.4 x 100 crn) equilibrated with pH 5,0.05 M luccinate buffer containing NaCI (final cone. M). The flow rate is 20 ml/h. The volume of the fractions collected is 3.6 ml. p-Nitro~henYI~-D-glucopyranOSide activity (0 - - - -0), protein concentration ( e _ ) and carboYdrate concentration (b.--A) are measured in the fractions (see Materials and ~ethodS). B: hydroxyapatite chromatograph), of cell wall P-gluc08idase. The active fracIOns frorn previous chromatography are collected under molecular sieving, concentrated ~nd Passed over the hydroxyapatite column (1.4 x 20 cm). Elution is effected with a near Kradient (---) of phoaphate (pH 6.8). The flow rate is 25 ml/h. Fractions of 6 ml ~e COllected. Protein concentration (e---e ) and p-glucosidase activity (0 - - -- - -0) are eterrnined in the fractions.
310
B
A ~0.2
+
E c
~ ~
0.1
v 0 u c a
.D
2
L
5l 02
.D
A
«
c
B
0.1
0 Migration
Fig. 2. Homogeneity of cell wall p-glucosidase. A: polyacrylamide gel electrophoresis of p-glucosidase under non-denaturing conditions. In each case, 25 J,l.g of protein is applied on the gel (see Materials and Methods). (A) Protein is stained with amidoblack. (B) P-glucosidase activity is detected on the gel by tetrazolium nitroblue. (C) Carbohydrates are detected on the gel. B: analytical centrifugation of p-glucosidase. Enzyme concentration is 1.2 mg/ml in a pH 5, 0.05 M succinate buffer containing 0.6 M NaCI. Migration is from left to right (rotation speed 60000 rev./min, temperature 25°C, UV optics). Sedimentation diagrams are obtained 20 min (1) or 40 min (2) after the start of ultracentrifugation. The value of the apparent sedimentation constant is 4.17.
S.4 L S.2
A
2 2 c v E v
a
u
(J)
a
-
B
2-
a
5
Q. I/)
ii v
b
4.8 4.6 4.4
g>
i
'i:
'-
d
e
4.2L-_ _ _ _-L._ _ _ _-"---1 1 1.S 2 VelVo
8'1
SO,S
51
51.S
(Rodius)2 (cm)2
Fig. 3. Molecular weight determination of p-glucosidase. A: molecular weight determination of p-glucosidase by molecular sieve chromatography. Exclusion volume of the column, VOl is determined with dextran blue. Points marked by the letters a, b, C, d, e, correspond to the elution ratios, Ve/Vo' of the various proteins used as standards (a-e, serum albumin dimer and monomer, ovalbumin, pepsin, a-chymotrypsinogen). The arrow shows the elution ratio of p-glucosidase and corresponds to a molecular weight of 62000 ± 1400 (mean of 6 determinations). The Sephadex G·100 column (1.2 x 100 cm) is equilibrated with 0.05 M succinate buffer containing NaCI (see Materials and Methods). B: molecular weight determination by eqUilibrium analytical centrifugation. The enzyme concentration is 1 mglml. Temperature is 25°C and rotation speed 30 000 rev./min. Time of centrifugation 24 h.
311
volume of 0.05 M succinate buffer (pH 5) containing NaCI (final molarity 1 M). About 80% of (3-glucosidase initially present in the soluble extract has been recovered after ammonium sulphate precipitation. This ammonium sulphate fractionation allows to discard most of the soluble polysaccharides present in the soluble extract of the cell walls. The soluble extract obtained after ammonium sulphate fractionation is submitted to molecular sieving on Sephadex G-100 equilibrated in the same succinate buffer containing NaCl. The resulting elution profile is shown in Fig. 1A. The fractions displaying fj-gll1cosidase activity are collected, concentrated by ultra-filtration on DIAFLO membranes (PM 30 Amicon), then diluted ten times with distilled water and submitted to hydroxyapatite chromatography. Elution is effected as described under Materials and Methods. The elution diagram is shown in Fig. lB. This (3-glucosidase appears homogenous in polyacrylamide gel electrophoresis and centrifugation (Fig. 2). The sedimentation constant is equal to 4.17 S. On electrophoresis, the protein band is clearly superimposable on enzyme activity. Moreover, results of Fig. 2 indicate that this enzyme is a glycoprotein. Both molecular sieving and analytical centrifugation give molecular weights of approx. 60 000 (Fig. 3). If polyacrylamide gel electrophoresis is performed under denaturing conditions the enzyme appears to Consist of a single polypeptide chain only (not shown). The enzyme hydrolyzes gentiobiose [(j(1-+ 6)glucoside], cellobiose [fj(1-+ 4)glucoside] and sophorose [(3(1-+ 2)glucoside]. DISCUSSION
~ fj-glucosidase has been solubilized from soybean cell walls and purified. ThIS enzyme is homogenous to analytical centrifugation and polyacrylamide gel electrophoresis. It appears to be a glycoprotein with a molecular weight of approx . 60000 and containing a single polypeptide. Detection of p-glucosidase activity on the cell wall does not result from an artefact consecutive to cell disruption and adsorption of a cytoplasmic enzyme to cell wall fragments, for the enzyme activity may be detected on unbrOken cells. This (3-glucosidase is thus typically exo-cellular. . .Only a few glycosidases have been highly purified from plant tissues and It IS therefore difficult to compare the enzyme from soybean cell walls with ~ther glucosidases obtained from different plant systems. To the best of our nowledge, the only (j-glycosidase purified so far to homogeneity from a plant tissue has been obtained from almond emulsion [24]. Although no definite information is available as to the intracellular location of this enzyme, it is very likely that it is not bound to cell walls. Moreover the fj-glycosidase from almond emulsin appears to be a dimer of 135000 moleCular Weight, in which one subunit reacts with (j-gIucosides, the other with fj-galactosides. This enzyme is in reality a bifunctional enzyme compl~ [24]. The (3-glucosidase purified from soybean cell walls is totally devoid of galactosidase activity and has a molecular weight close to that of a 'subunit' of the enzyme complex extracted from almond emulsin.
312
In order for a glycosidase to take part in cell wall extension, it must act as a true glycosyltransferase, that is, transfer the glycosyl moiety of a donor to a sugar acceptor and not solely to water [1]. It is therefore of importance to determine whether this enzyme, isolated from cell walls, is a true exo- or endo-glucosyltransferase. This matter is considered in the companion paper. REFERENCES 1 P. Albersheim, in: J. Bonner and J.E. Varner (Eds.), Plant Biochemistry, Academic Press, 1976, p. 225. 2 Y. Muuda, S. Oi and Y. Satomura, Plant Cell Physiol., 11 (1970) 631. 3 D.J. Nevins, Plant Cen Physiol.,16 (1975) 347. 4 D.J. Nevins, Plant Cell Physiol., 16 (1975) 495. 5 N. Sakurai and Y. Masuda, Plant Cell Physiol., 18 (1977) 587. 6 R. Yamamoto and D.J. Nevins, Plant Physiol., 51 (1981) 118. 7 A.N.J. Heyn, Arch. Biochem. Biophys., 132 (1969) 442. 8 D.J. Huber and D.J. Nevins, Plant Physiol., 65 (1980) 768. 9 D.J. Huber and D.J. Nevins, Planta, 151 (1981) 206. 10 R. Goldberg, Plant Sci. Lett., 8 (1977) 233. 11 O.L. Gamborg, R.A. Miller and K. Ojima, Exp. Cen Res., 50 (1968) 151. 12 M.M. Bradford, Anal. Biochem., 72 (1976) 248. 13 P. Bohlen, S. Stein, W. Dairman and S. Udenfriend, Arch. Biochem. Biophys., 155 (1973) 213. 14 M. Dubois, K.A. GUles, J.K. Hamilton, P.A. Rebers and F. Smith, Anal. Biochem., 28 (1956) 350. 15 M. Somogyi, J. BioI. Chem., 195 (1952) 19. 16 J.R. Whitaker, Anal. Biochem., 35 (1963) 1950. 17 O. Gabriel, Methods Enzymol., 22 (1971) 578. 18 H.M. Katzen and R.T. Schimke, Proc. Natl. Acad. Sci. U.S.A., 54 (1965) 1218. 19 R.M. ZachariuI, T.E. Zen, J.H. Morrison and J.J. Woodlock, Anal. Biochem., 30 (1969) 148. 20 K. Weber, J.R. Pringle and M. Osborn, Methods Enzymol., 26 (1972) 3. 21 D.A. Yphantis, Biochemistry, 3 (1964) 297. 22 J. Vinograd, R. Bruner, R. Kent and J. Weigle, Proc. Natl. Acad. Sci. U.S.A., 49 (1963) 902. 23 A.M. Cates80n, R. Goldberg and M.C. Winay, C.R. Acad. Sci., 272 (1971) 2078. 24 A.K. Grover, D.D. Macmurchie and R.J. CUlhley, Biochim. Biophys. Acta, 482 (1977) 98.