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The interaction of concanavalin A with methyl α-D -glucopyranoside
BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY
BBA
303
NOTES
21226
The interaction of concanavalin A with methyl ~-D-glucopyranoside C o n c a n a v a l i n A, a s a c c h a r i d e - b i n d i n g p r o t e i n f r o m J a c k b e a n was i s o l a t e d b y c r y s t a l l i z a t i o n m a n y y e a r s ago 1. C o n c a n a v a l i n A is n o t a h o m o g e n e o u s m o l e c u l a r species in t h e p H r a n g e w h e r e it b i n d s s a c c h a r i d e s 2, 3. I t s e e m e d possible t h a t b i n d i n g p a r a m e t e r s for a s m a l l s a c c h a r i d e like m e t h y l ~-I~-glucopyranoside c o u l d f u r n i s h a p h y s i c o c h e m i c a l c h a r a c t e r i z a t i o n of this p r o t e i n . I n d e e d a single b i n d i n g c o n s t a n t a n d an e q u i v a l e n t w e i g h t d e s c r i b e t h e b i n d i n g of t h e g l u c o s i d e b y c o n c a n a v a l i n A. B i n d i n g of t h e s a c c h a r i d e , we find, d e p e n d s on d i v a l e n t m e t a l c o n t e n t of t h e p r o t e i n . R e m o v a l of m e t a l d e s t r o y s t h e b i n d i n g sites for t h e s a c c h a r i d e . T h e p r o t e i n was i s o l a t e d f r o m J a c k b e a n m e a l ( S i g m a C h e m i c a l Co., St. Louis, Mo.) b y t w o c r y s t a l l i z a t i o n s as d e s c r i b e d 4. T h e satd. NaC1 filtrate, w h i c h c o n t a i n e d 24 m g / m l p r o t e i n a n d will be r e f e r r e d to as s t o c k s o l u t i o n , was k e p t in t h e r o o m for a few w e e k s w i t h o u t d e t e r i o r a t i o n . N a t i v e p r o t e i n is s t o c k s o l u t i o n a p p r o p r i a t e l y d i l u t e d a n d d i a l y z e d a g a i n s t a few c h a n g e s of a buffer s o l u t i o n in t h e cold. P a r t i a l l y d e - m e t a l l i z e d p r o t e i n was p r e p a r e d as d e s c r i b e d 5 b y m a k i n g t h e p r o t e i n o . I M in HC1 a n d d i a l y z i n g first a g a i n s t m e t a l - f r e e w a t e r a n d t h e n a g a i n s t m e t a l - f r e e buffer. D e - m e t a l l i z a t i o n of p r o t e i n was, h o w e v e r , n o t e x a c t l y r e p r o d u c i b l e a n d d i f f e r e n t b a t c h e s v a r i e d in t h e i r m e t a l c o n t e n t (Table I). TABLE I EFFECT
O F M E T A L ON T H E B I N D I N G O F M E T H Y L ~ - D - G L U C O P Y R A N O S I D E
BY CONCANAVALIN
A
Protein concn., 6-8 mg/ml. The compositions of acetate buffer (pH 5.2) and of phosphate buffer (pH 6.8) are given in the text. /t'ass and methyl ~-D-glucopyranoside bound were calculated from plots, such as in Fig. i. Metal content is that of the isolated protein prior to its use in the binding experiment. Metal was added as an equimolar solution of the chlorides of calcium, magnesium and manganese. Protein
Ka,8 × IO -~ Glucoside (l/mole) bound (moles/ 320o0g)
Native 3.92 Native -Native 4.28 De-metallized (I) 3.97 De-metallized (I) 4.18 De-metallized (2) - De-metallized (2) - Native 3.7 ° De-metallized (3) - -
I.OO 0.98* 0.68 0.57 0.94, 0.20 0.98* o.95 o.04*
Buffer
Acetate Acetate Acetate Acetate Acetate Acetate Acetate Phosphate Phosphate
Metal content of protein (moles/32 ooo g) Ca ~+
M g ~+ M n ~+ S u m
0.64 0.64 o.64 0.34 0.34 0.20 0.20 0.8o o.22
0.35 0.09 0.35 0.09 0.35 o.09 0.34 0.04 0.34 0.04 0.06 o.oi 0.06 o.oi I.OO - 0.07 o.oo
1.o8 1.o8 1.o8 0.72 0.72 0.27 0.27 -0.29
C o n c n . × ~ 0 '~ of each added metal (M)
0.25 2. 5 --0.25 -0.25 ---
* Calculated from a single experiment with Kas84.0 × IO~ 1/mole assumed. Bio chim. Biophys. Acta, I65 (1968) 303-305
304
PRELIMINARY
NOTES
For a binding experiment, radioactive methyl e-D@4C]glucopyranoside (3.2 mC/mmole, The Radiochemical Centre, Amersham) diluted with unlabelled methyl ~-I~-glucopyranoside (Pfanstiehl Laboratories, Waukegan, Ill.) was used. The specific counting rate of labelled methyl ~-I~-glucopyranoside was based on its sugar content in the phenol-sulphuric acid test 6 with methyl e-~-glucopyranoside as standard. Binding experiments were performed in dialysis cells (Technilab Instruments, Los Angeles, Calif.) with membranes of Visking 2o/32 inch tubing t h a t were boiled in three changes of IO -~ M disodium EDTA. One c o m p a r t m e n t was filled with the protein solution (6-8 mg/ml), the other with buffer. The labelled methyl ~-~-glucopyranoside was added to protein or buffer. After equilibration (16 h, 4 °) samples were removed for determination of counting rate and absorbance at 28o mff. Concentration of protein was calculated from absorbance at 28o raft: E ~~ %m at 28o m~ in o.o5 M ~% sodium phosphate (pH 6.8) with 0.2 M NaC1 is 13.7; E,C m at 280 mff in 0.05 M sodium acetate (pH 5.2) with 0.2 M NaC1 is 12.4. The specific absorbances were determined on the basis of weight concentrations calculated from refractive index increments measured with the Rayleigh optical system of an ultracentrifuge and a capillary-type b o u n d a r y forming cell, and an assumed value of dn/dc of o.oo185 dl/g. The calcium, magnesium and manganese were determined b y atomic absorption s p e c t r o p h o t o m e t r y (Model 303, P e r k i n - E l m e r Corp., Norwalk, Conn.). From the binding experiments the following terms were calculated: r, moles of methyl ~-D-glucopyranoside bound per g of protein and f, concentration of the free glucoside. In Fig. I, results of binding experiments are plotted according to the following formula ~: r/f
=
- K r + NK
{_ex5' C o
, , r ~
I ~ ~ ~\, I lo 20 t ' ( m o l e s / g ) x 1Q 6
~
, , , . 30
Fig. I. Plot of methyl ~-D-glucopyranoside binding by native and partially de-metallized concanavalin A. O, native protein in phosphate (pH 6.8) ; ~, native protein in acetate (pH 5.2) ; &, demetallized protein in acetate (pH 5.2). It can be seen t h a t the binding of methyl e-1)-glucopyranoside both b y native protein and b y de-metallized protein at p H 5.2 and 6.8, irrespective of whether phosphate or acetate were used as buffers, can be described b y straight lines of similar slopes but differing in their intercept on the abscissa. The maximal value of N was obtained in experiments where metal was added (Table I). In this case, the equivalent binding weight is calculated to be 32 ooo. Considering t h a t the molecular weight of concanavalin A was found to be 55o00, this would suggest t h a t a molecule Biochim. Biophys. Acta, 165 (1968) 3o3-3o5
PRELIMINARY NOTES
305
of concanavalin A has two binding sites for methyl a-D-glucopyranoside 8. Recently it has been suggested that concanavalin A may be composed of a 165o0 molecular weight subunit9. Results of binding experiments are summarized in Table I. They show that the binding constant for methyl a-D-glucopyranoside is not affected by the concentration of metal. However, the amount of methyl ~-I)-glucopyranoside bound increases with rising concentration of metal to a maximum, and is independent of pH and buffer. Investigation of metal binding by concanavalin A and its role in binding of methyl ~-o-glucopyranoside, is now in progress in this laboratory.
Department of Biophysics, The Weizmann Institute of Science, Rehovoth (Israel)
J. YARIV A. JOSEPH KALB A. LEVlTZKI
J. B. SUMNER, J. Biol. Chem., 37 (1919) 137. J. B. SUMNER, N. GRALI~N AND I. n . ERIKSSON-QUENSEL,J. Biol. Chem., 125 (1938) 45. B. B. L. AGRAWAL AND I. J. GOLDSTEIN, Biochim. Biophys. Acta, 133 (1967) 376. J. n . SUMNER AND S. V. HOWELL, J. Bacteriol., 32 (1936) 227. J. B. SUMNER AND S. F. HOWELL, J. Biol. Chem., 115 (1936) 583 • M. DUBOlS, K. A. GILLES, J. t~. HAMILTON, P. A. REBERS AND F. SMITH, Anal. Chem., 28 (1956) 350. 7 G. SCATCHARD,Ann. N . Y . Acad. Sci., 51 (1949) 660. 8 A. J. I~ALB AND A. LUSTIG, Biochim. Biophys. Acta, in the press. 9 M. O. J. OLSON AND I. E. LIENER, Biochemistry, 6 (1967) 38Ol. I 2 3 4 5 6
Received May i3th, 1968 Biochim. Biophys. Ac~a, 165 (1968) 3o3-3o5
PRELIMINARY
BBA
303
NOTES
21226
The interaction of concanavalin A with methyl ~-D-glucopyranoside C o n c a n a v a l i n A, a s a c c h a r i d e - b i n d i n g p r o t e i n f r o m J a c k b e a n was i s o l a t e d b y c r y s t a l l i z a t i o n m a n y y e a r s ago 1. C o n c a n a v a l i n A is n o t a h o m o g e n e o u s m o l e c u l a r species in t h e p H r a n g e w h e r e it b i n d s s a c c h a r i d e s 2, 3. I t s e e m e d possible t h a t b i n d i n g p a r a m e t e r s for a s m a l l s a c c h a r i d e like m e t h y l ~-I~-glucopyranoside c o u l d f u r n i s h a p h y s i c o c h e m i c a l c h a r a c t e r i z a t i o n of this p r o t e i n . I n d e e d a single b i n d i n g c o n s t a n t a n d an e q u i v a l e n t w e i g h t d e s c r i b e t h e b i n d i n g of t h e g l u c o s i d e b y c o n c a n a v a l i n A. B i n d i n g of t h e s a c c h a r i d e , we find, d e p e n d s on d i v a l e n t m e t a l c o n t e n t of t h e p r o t e i n . R e m o v a l of m e t a l d e s t r o y s t h e b i n d i n g sites for t h e s a c c h a r i d e . T h e p r o t e i n was i s o l a t e d f r o m J a c k b e a n m e a l ( S i g m a C h e m i c a l Co., St. Louis, Mo.) b y t w o c r y s t a l l i z a t i o n s as d e s c r i b e d 4. T h e satd. NaC1 filtrate, w h i c h c o n t a i n e d 24 m g / m l p r o t e i n a n d will be r e f e r r e d to as s t o c k s o l u t i o n , was k e p t in t h e r o o m for a few w e e k s w i t h o u t d e t e r i o r a t i o n . N a t i v e p r o t e i n is s t o c k s o l u t i o n a p p r o p r i a t e l y d i l u t e d a n d d i a l y z e d a g a i n s t a few c h a n g e s of a buffer s o l u t i o n in t h e cold. P a r t i a l l y d e - m e t a l l i z e d p r o t e i n was p r e p a r e d as d e s c r i b e d 5 b y m a k i n g t h e p r o t e i n o . I M in HC1 a n d d i a l y z i n g first a g a i n s t m e t a l - f r e e w a t e r a n d t h e n a g a i n s t m e t a l - f r e e buffer. D e - m e t a l l i z a t i o n of p r o t e i n was, h o w e v e r , n o t e x a c t l y r e p r o d u c i b l e a n d d i f f e r e n t b a t c h e s v a r i e d in t h e i r m e t a l c o n t e n t (Table I). TABLE I EFFECT
O F M E T A L ON T H E B I N D I N G O F M E T H Y L ~ - D - G L U C O P Y R A N O S I D E
BY CONCANAVALIN
A
Protein concn., 6-8 mg/ml. The compositions of acetate buffer (pH 5.2) and of phosphate buffer (pH 6.8) are given in the text. /t'ass and methyl ~-D-glucopyranoside bound were calculated from plots, such as in Fig. i. Metal content is that of the isolated protein prior to its use in the binding experiment. Metal was added as an equimolar solution of the chlorides of calcium, magnesium and manganese. Protein
Ka,8 × IO -~ Glucoside (l/mole) bound (moles/ 320o0g)
Native 3.92 Native -Native 4.28 De-metallized (I) 3.97 De-metallized (I) 4.18 De-metallized (2) - De-metallized (2) - Native 3.7 ° De-metallized (3) - -
I.OO 0.98* 0.68 0.57 0.94, 0.20 0.98* o.95 o.04*
Buffer
Acetate Acetate Acetate Acetate Acetate Acetate Acetate Phosphate Phosphate
Metal content of protein (moles/32 ooo g) Ca ~+
M g ~+ M n ~+ S u m
0.64 0.64 o.64 0.34 0.34 0.20 0.20 0.8o o.22
0.35 0.09 0.35 0.09 0.35 o.09 0.34 0.04 0.34 0.04 0.06 o.oi 0.06 o.oi I.OO - 0.07 o.oo
1.o8 1.o8 1.o8 0.72 0.72 0.27 0.27 -0.29
C o n c n . × ~ 0 '~ of each added metal (M)
0.25 2. 5 --0.25 -0.25 ---
* Calculated from a single experiment with Kas84.0 × IO~ 1/mole assumed. Bio chim. Biophys. Acta, I65 (1968) 303-305
304
PRELIMINARY
NOTES
For a binding experiment, radioactive methyl e-D@4C]glucopyranoside (3.2 mC/mmole, The Radiochemical Centre, Amersham) diluted with unlabelled methyl ~-I~-glucopyranoside (Pfanstiehl Laboratories, Waukegan, Ill.) was used. The specific counting rate of labelled methyl ~-I~-glucopyranoside was based on its sugar content in the phenol-sulphuric acid test 6 with methyl e-~-glucopyranoside as standard. Binding experiments were performed in dialysis cells (Technilab Instruments, Los Angeles, Calif.) with membranes of Visking 2o/32 inch tubing t h a t were boiled in three changes of IO -~ M disodium EDTA. One c o m p a r t m e n t was filled with the protein solution (6-8 mg/ml), the other with buffer. The labelled methyl ~-~-glucopyranoside was added to protein or buffer. After equilibration (16 h, 4 °) samples were removed for determination of counting rate and absorbance at 28o mff. Concentration of protein was calculated from absorbance at 28o raft: E ~~ %m at 28o m~ in o.o5 M ~% sodium phosphate (pH 6.8) with 0.2 M NaC1 is 13.7; E,C m at 280 mff in 0.05 M sodium acetate (pH 5.2) with 0.2 M NaC1 is 12.4. The specific absorbances were determined on the basis of weight concentrations calculated from refractive index increments measured with the Rayleigh optical system of an ultracentrifuge and a capillary-type b o u n d a r y forming cell, and an assumed value of dn/dc of o.oo185 dl/g. The calcium, magnesium and manganese were determined b y atomic absorption s p e c t r o p h o t o m e t r y (Model 303, P e r k i n - E l m e r Corp., Norwalk, Conn.). From the binding experiments the following terms were calculated: r, moles of methyl ~-D-glucopyranoside bound per g of protein and f, concentration of the free glucoside. In Fig. I, results of binding experiments are plotted according to the following formula ~: r/f
=
- K r + NK
{_ex5' C o
, , r ~
I ~ ~ ~\, I lo 20 t ' ( m o l e s / g ) x 1Q 6
~
, , , . 30
Fig. I. Plot of methyl ~-D-glucopyranoside binding by native and partially de-metallized concanavalin A. O, native protein in phosphate (pH 6.8) ; ~, native protein in acetate (pH 5.2) ; &, demetallized protein in acetate (pH 5.2). It can be seen t h a t the binding of methyl e-1)-glucopyranoside both b y native protein and b y de-metallized protein at p H 5.2 and 6.8, irrespective of whether phosphate or acetate were used as buffers, can be described b y straight lines of similar slopes but differing in their intercept on the abscissa. The maximal value of N was obtained in experiments where metal was added (Table I). In this case, the equivalent binding weight is calculated to be 32 ooo. Considering t h a t the molecular weight of concanavalin A was found to be 55o00, this would suggest t h a t a molecule Biochim. Biophys. Acta, 165 (1968) 3o3-3o5
PRELIMINARY NOTES
305
of concanavalin A has two binding sites for methyl a-D-glucopyranoside 8. Recently it has been suggested that concanavalin A may be composed of a 165o0 molecular weight subunit9. Results of binding experiments are summarized in Table I. They show that the binding constant for methyl a-D-glucopyranoside is not affected by the concentration of metal. However, the amount of methyl ~-I)-glucopyranoside bound increases with rising concentration of metal to a maximum, and is independent of pH and buffer. Investigation of metal binding by concanavalin A and its role in binding of methyl ~-o-glucopyranoside, is now in progress in this laboratory.
Department of Biophysics, The Weizmann Institute of Science, Rehovoth (Israel)
J. YARIV A. JOSEPH KALB A. LEVlTZKI
J. B. SUMNER, J. Biol. Chem., 37 (1919) 137. J. B. SUMNER, N. GRALI~N AND I. n . ERIKSSON-QUENSEL,J. Biol. Chem., 125 (1938) 45. B. B. L. AGRAWAL AND I. J. GOLDSTEIN, Biochim. Biophys. Acta, 133 (1967) 376. J. n . SUMNER AND S. V. HOWELL, J. Bacteriol., 32 (1936) 227. J. B. SUMNER AND S. F. HOWELL, J. Biol. Chem., 115 (1936) 583 • M. DUBOlS, K. A. GILLES, J. t~. HAMILTON, P. A. REBERS AND F. SMITH, Anal. Chem., 28 (1956) 350. 7 G. SCATCHARD,Ann. N . Y . Acad. Sci., 51 (1949) 660. 8 A. J. I~ALB AND A. LUSTIG, Biochim. Biophys. Acta, in the press. 9 M. O. J. OLSON AND I. E. LIENER, Biochemistry, 6 (1967) 38Ol. I 2 3 4 5 6
Received May i3th, 1968 Biochim. Biophys. Ac~a, 165 (1968) 3o3-3o5