Planta
Planta 144, 367-371 (1979)
9 by Springer-Verlag 1979
Affinity Chromatography of a Lectin from Robinia pseudoacacia L. and Demonstration of Lectins in Sieve-Tube Sap from Other Tree Species C h r i s t i n e Gietl 1, H e i n r i c h K a u s s 2, a n d H u b e r t Ziegler 1 1 Institut ffir Botanik und Mikrobiologie der Technischen Universit/it, Arcisstral3e 21, D-8000 Mfinchen 2, and 2 Fachbereich Biologie der Universitfit, Postfach 3049, D-6750 Kaiserslautern, Federal Republic of Germany
Abstract. A c a r b o h y d r a t e - b i n d i n g p r o t e i n in the sievet u b e sap o f Robinia pseudoacacia L. was i s o l a t e d by affinity c h r o m a t o g r a p h y on D - g a l a c t o s a m i n e c o u p l e d to C H - S e p h a r o s e 4B. T h e yield o f the purified p r o t e i n was 41,3 g g / m l o f sieve-tube sap or 5,7% o f the t o t a l protein. In disk e l e c t r o p h o r e s i s at an a l k a l i n e a n d acidic p H the lectin a p p e a r e d to be h o m o g e n o u s . It has a m o l e c u l a r weight o f a b o u t 100,000 a n d consists o f f o u r types o f subunits (tool wt. 70,000, 60,000, 32,500, 25,000). T h e c o m p l e x f o r m a t i o n b e t w e e n the Robinia lectin a n d e r y t h r o c y t e s is i n h i b i t e d b y simple sugars, especially N - a c e t y l - D - g a l a c t o s a m i n e , but n o t by g l y c o p r o t e i n s c o n t a i n i n g N - a c e t y l - g l u c o s a m i n e o r by a p a r t i a l h y d r o l y s a t e o f chitin. Sieve-tube sap o f 63 different species out o f 21 families were e x a m i n e d for their lectin-activity. W i t h the e x c e p t i o n o f the species o f the families A c e r a c e a e a n d Oleaceae, all a n a l y z e d species a g g l u t i n a t e d t r y p s i n i z e d r a b b i t erythrocytes. 15 species were e x a m i n e d for their sugar specifity. T h e i r lectin-activity was n o t i n h i b i t e d by simple sugars, b u t by g l y c o p r o t e i n s c o n t a i n i n g N - a c e t y l - g l u c o s a m i n e a n d b y a p a r t i a l h y d r o l y s a t e o f chitin. T h e s a m e result was f o u n d for the lectin f r o m the p h l o e m e x u d a t e o f Cucurbita pepo. Key words" A f f i n i t y c h r o m a t o g r a p h y - C a r b o h y d r a t e binding protein- Lectin- N-Acetyl-D-galactosamine - N - A c e t y l - g l u c o s a m i n e o l i g o m e r s - Robinia Sieve t u b e sap.
Introduction R e c e n t l y a c a r b o h y d r a t e b i n d i n g p r o t e i n (lectin) was d e m o n s t r a t e d in the sieve-tube sap f r o m Robinia pseudoacacia ( K a u s s a n d Ziegler, 1974). The a g g l u t i n a t i o n 9 o f e r y t h r o c y t e s by this lectin was strongly i n h i b i t e d b y N - a c e t y l - D - g a l a c t o s a m i n e a n d to a lesser degree
by D-fucose, lactose a n d 7 s t r u c t u r a l l y r e l a t e d sugars. F o r s o y b e a n seed lectin, which exhibits a similar sugar specifity, a p u r i f i c a t i o n p r o c e d u r e by affinity c h r o m a t o g r a p h y on D - g a l a c t o s a m i n e c o u p l e d to C H - S e p h a rose has been d e s c r i b e d by A l l e n a n d N e u b e r g e r (1975). In the p r e s e n t study we describe the i s o l a t i o n a n d c h a r a c t e r i s a t i o n o f the c a r b o h y d r a t e b i n d i n g p r o t e i n in the sieve-tube sap o f Robinia pseudoacacia with the use o f the same m e t h o d . F u r t h e r m o r e lectin activity a n d sugar specifity was s t u d i e d in the sievet u b e sap o f several other tree species a n d in the p h l o e m e x u d a t e o f Cucurbita pepo, for which Sabnis a n d H a r t (1978) r e p o r t e d high lectin activity b u t no sugar specifity.
Materials and Methods Sieve tube sap was collected according to the method of Hartig (1860) in September 1977, phloem exudate from Cucurbita pepo was produced according to Sabnis and Hart (1978). Sap of Robinia pseudoacacia came from trees of different age in the city of Munich; sap of the other tree species came from the Botanical Gardens in Munich and Darmstadt and also from the forest Garden in Grafrath. Cucurbita pepo was grown in an open culture chamber on normal garden soil. The sap was frozen in an ice-NaCl-mixture and stored at -18 ~ C up to the time of the experiments. The agglutination test and the evaluation of the sugar specifity was performed according to Kauss and Glaser (1974) and Kauss and Ziegler (1974). For the affinity chromatography of Robinia lectin sieve-tube sap (22.5 ml) was dialyzed over night against 0.05 M K/Na phosphate buffer (pH 7.4)+0.9% NaC1 ("PBS") diluted 1 : 2, and then centrifuged (50,000 g, 20 min). The clear supernatant was applied to a 1.00 x 10 cm column of Sepharose-N-caproyl galactosamine conjugate, prepared according to Allen and Neuberger (1975) and equilibrated with PBS. Fractions of 2.4 ml were collected. After elution of unbound protein with PBS (absorbance at 280 nm < 0,01), the lectin was eluted completely by 0.2 M lactose in PBS: A subsequent elution with 0.01 M N-acetyl-galactosamine in PBS, and with 0.1 N acetic acid did not reveal further fractions with agglutinating activity. The fractions eluted with lactose were dialyzed against PBS to remove lactose. The agglutination titers,
0032-0935/79/0144/0367/$01.00
368
Chr. Gietl et al. : Lectins in Sieve-Tube Sap
as well as the protein content were determined in every fraction. The fractions eluted with PBS, and with lactose respectively were combined. The titer and the protein content was redetermined in the pooled fractions. Protein was determined according to Lowry et al. (1951) with bovine serum albumin as a standard. The partial chitin hydrolysate containing a mixture of fl l ~ 4 linked oligomers of N-acetyl-D-glucosamine was produced according to Rupley (1964). The hydrolysate contained reducing sugars equivalent to 0.1 mg glucose/ml (demonstrated by the anthronesulfuric acid reaction; Whistler and Wolfrom, 1962), showed on paper-chromatography a continous line of spots from the monomer N-acetyl-glucosamine up to the unhydrolysated chitin. The following methods were used for the characterization of sieve-tube sap lectin from Robinia pseudoacacia: Disc electrophoresis in 7.5% polyacrylamide gel at pH 8.9 and pH 4.3 (Maurer, 1968), and polyacrylamide gel electrophoresis in the presence of sodium-dodecyl-sulfate (SDS; Weber and Osborn, 1969). Estimation of the molecular weight was performed either by column gel filtration on Sephadex G200 (Andrews, 1965) using a 1.50 x 80 cm column at 2~ C, 8 ml/h, 1.3 ml fractions. In addition, ultracentrifugation in a sucrose-gradient (5-25%; Martin and Ames, 1961) was used. The glycoproteins fetuine, ovomucoid and thyroglobuline were obtained from Sigma, Munich, W. Germany. The sieve-tube sap of the other tree species was treated like that from Robinia: dialyzed, diluted and centrifuged.
0./+00
400
e.o.,.,o....o,..,,'~176 o
0.300
o-e
o
Planta 144, 367-371 (1979)
9 by Springer-Verlag 1979
Affinity Chromatography of a Lectin from Robinia pseudoacacia L. and Demonstration of Lectins in Sieve-Tube Sap from Other Tree Species C h r i s t i n e Gietl 1, H e i n r i c h K a u s s 2, a n d H u b e r t Ziegler 1 1 Institut ffir Botanik und Mikrobiologie der Technischen Universit/it, Arcisstral3e 21, D-8000 Mfinchen 2, and 2 Fachbereich Biologie der Universitfit, Postfach 3049, D-6750 Kaiserslautern, Federal Republic of Germany
Abstract. A c a r b o h y d r a t e - b i n d i n g p r o t e i n in the sievet u b e sap o f Robinia pseudoacacia L. was i s o l a t e d by affinity c h r o m a t o g r a p h y on D - g a l a c t o s a m i n e c o u p l e d to C H - S e p h a r o s e 4B. T h e yield o f the purified p r o t e i n was 41,3 g g / m l o f sieve-tube sap or 5,7% o f the t o t a l protein. In disk e l e c t r o p h o r e s i s at an a l k a l i n e a n d acidic p H the lectin a p p e a r e d to be h o m o g e n o u s . It has a m o l e c u l a r weight o f a b o u t 100,000 a n d consists o f f o u r types o f subunits (tool wt. 70,000, 60,000, 32,500, 25,000). T h e c o m p l e x f o r m a t i o n b e t w e e n the Robinia lectin a n d e r y t h r o c y t e s is i n h i b i t e d b y simple sugars, especially N - a c e t y l - D - g a l a c t o s a m i n e , but n o t by g l y c o p r o t e i n s c o n t a i n i n g N - a c e t y l - g l u c o s a m i n e o r by a p a r t i a l h y d r o l y s a t e o f chitin. Sieve-tube sap o f 63 different species out o f 21 families were e x a m i n e d for their lectin-activity. W i t h the e x c e p t i o n o f the species o f the families A c e r a c e a e a n d Oleaceae, all a n a l y z e d species a g g l u t i n a t e d t r y p s i n i z e d r a b b i t erythrocytes. 15 species were e x a m i n e d for their sugar specifity. T h e i r lectin-activity was n o t i n h i b i t e d by simple sugars, b u t by g l y c o p r o t e i n s c o n t a i n i n g N - a c e t y l - g l u c o s a m i n e a n d b y a p a r t i a l h y d r o l y s a t e o f chitin. T h e s a m e result was f o u n d for the lectin f r o m the p h l o e m e x u d a t e o f Cucurbita pepo. Key words" A f f i n i t y c h r o m a t o g r a p h y - C a r b o h y d r a t e binding protein- Lectin- N-Acetyl-D-galactosamine - N - A c e t y l - g l u c o s a m i n e o l i g o m e r s - Robinia Sieve t u b e sap.
Introduction R e c e n t l y a c a r b o h y d r a t e b i n d i n g p r o t e i n (lectin) was d e m o n s t r a t e d in the sieve-tube sap f r o m Robinia pseudoacacia ( K a u s s a n d Ziegler, 1974). The a g g l u t i n a t i o n 9 o f e r y t h r o c y t e s by this lectin was strongly i n h i b i t e d b y N - a c e t y l - D - g a l a c t o s a m i n e a n d to a lesser degree
by D-fucose, lactose a n d 7 s t r u c t u r a l l y r e l a t e d sugars. F o r s o y b e a n seed lectin, which exhibits a similar sugar specifity, a p u r i f i c a t i o n p r o c e d u r e by affinity c h r o m a t o g r a p h y on D - g a l a c t o s a m i n e c o u p l e d to C H - S e p h a rose has been d e s c r i b e d by A l l e n a n d N e u b e r g e r (1975). In the p r e s e n t study we describe the i s o l a t i o n a n d c h a r a c t e r i s a t i o n o f the c a r b o h y d r a t e b i n d i n g p r o t e i n in the sieve-tube sap o f Robinia pseudoacacia with the use o f the same m e t h o d . F u r t h e r m o r e lectin activity a n d sugar specifity was s t u d i e d in the sievet u b e sap o f several other tree species a n d in the p h l o e m e x u d a t e o f Cucurbita pepo, for which Sabnis a n d H a r t (1978) r e p o r t e d high lectin activity b u t no sugar specifity.
Materials and Methods Sieve tube sap was collected according to the method of Hartig (1860) in September 1977, phloem exudate from Cucurbita pepo was produced according to Sabnis and Hart (1978). Sap of Robinia pseudoacacia came from trees of different age in the city of Munich; sap of the other tree species came from the Botanical Gardens in Munich and Darmstadt and also from the forest Garden in Grafrath. Cucurbita pepo was grown in an open culture chamber on normal garden soil. The sap was frozen in an ice-NaCl-mixture and stored at -18 ~ C up to the time of the experiments. The agglutination test and the evaluation of the sugar specifity was performed according to Kauss and Glaser (1974) and Kauss and Ziegler (1974). For the affinity chromatography of Robinia lectin sieve-tube sap (22.5 ml) was dialyzed over night against 0.05 M K/Na phosphate buffer (pH 7.4)+0.9% NaC1 ("PBS") diluted 1 : 2, and then centrifuged (50,000 g, 20 min). The clear supernatant was applied to a 1.00 x 10 cm column of Sepharose-N-caproyl galactosamine conjugate, prepared according to Allen and Neuberger (1975) and equilibrated with PBS. Fractions of 2.4 ml were collected. After elution of unbound protein with PBS (absorbance at 280 nm < 0,01), the lectin was eluted completely by 0.2 M lactose in PBS: A subsequent elution with 0.01 M N-acetyl-galactosamine in PBS, and with 0.1 N acetic acid did not reveal further fractions with agglutinating activity. The fractions eluted with lactose were dialyzed against PBS to remove lactose. The agglutination titers,
0032-0935/79/0144/0367/$01.00
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Chr. Gietl et al. : Lectins in Sieve-Tube Sap
as well as the protein content were determined in every fraction. The fractions eluted with PBS, and with lactose respectively were combined. The titer and the protein content was redetermined in the pooled fractions. Protein was determined according to Lowry et al. (1951) with bovine serum albumin as a standard. The partial chitin hydrolysate containing a mixture of fl l ~ 4 linked oligomers of N-acetyl-D-glucosamine was produced according to Rupley (1964). The hydrolysate contained reducing sugars equivalent to 0.1 mg glucose/ml (demonstrated by the anthronesulfuric acid reaction; Whistler and Wolfrom, 1962), showed on paper-chromatography a continous line of spots from the monomer N-acetyl-glucosamine up to the unhydrolysated chitin. The following methods were used for the characterization of sieve-tube sap lectin from Robinia pseudoacacia: Disc electrophoresis in 7.5% polyacrylamide gel at pH 8.9 and pH 4.3 (Maurer, 1968), and polyacrylamide gel electrophoresis in the presence of sodium-dodecyl-sulfate (SDS; Weber and Osborn, 1969). Estimation of the molecular weight was performed either by column gel filtration on Sephadex G200 (Andrews, 1965) using a 1.50 x 80 cm column at 2~ C, 8 ml/h, 1.3 ml fractions. In addition, ultracentrifugation in a sucrose-gradient (5-25%; Martin and Ames, 1961) was used. The glycoproteins fetuine, ovomucoid and thyroglobuline were obtained from Sigma, Munich, W. Germany. The sieve-tube sap of the other tree species was treated like that from Robinia: dialyzed, diluted and centrifuged.
0./+00
400
e.o.,.,o....o,..,,'~176 o
0.300
o-e
o
