Planta

Planta (1981)152:578-586

9 Springer-Verlag 1981

Immunohistoehemical localization of -glucosidases in lignin and isoflavone metabolism in Cicer arietinum L. seedlings* Gerd Burmeister** and Wolfgang H6sel Lehrstuhl fiir Biochemieder Pflanzender WestffilischenWilhelms-Universit~it,Hindenburgplatz55, D-4400 Mfinster, Federal Republic of Germany

Abstract. Coniferin specific- and isoflavone 7-gluco-

side specific fi-glucosidases have been localized in stem and root sections of chick pea (Cicer arietinum L.) seedlings by the indirect immunofluorometrical method. The coniferin specific/~-glucosidase has been found in the cell walls of the tracheary elements and of the endo-, epi-, and exodermis. All these tissues are known to contain either lignin or polymers, like suberin and cutin, which consist partially of phenylpropanoid elements. The localization of this fl-glucosidase is therefore in agreement with its postulated relationship to the phenylpropanoid metabolism. The isoflavone 7-glucoside specific /?-glucosidase, on the other hand, is predominantly located in the parenchymatic cortex cells, and obviously in the cytoplasm. These cells are known to contain the isoflavone formononetin, which has been shown to undergo turnover in chick pea seedlings. We therefore have good reason to assume that this fl-glucosidase is involved in the metabolism of the 7-glucoside of this isoflavone. Key words" Cicer /%Glucosidases (localization) Immunofluorescence Isoflavones Lignin.

Introduction

The occurrence of isoflavones and cinnamyl alcohols in the form of glycosylated products in higher plants is well known (e.g. Wong 1975; Freudenberg and Abbreviations: SDS=sodium dodecylsulfate;PBS=physiological

phosphate saline * The results are part of the thesis of Gerd Burmeister, 1980, University of Mtinster ** Present address." Universitiits-Hautklinik,Abt. ffir Experimentelle Dermatologie, Von-Esmarch-Str. 56, 4400 Mfinster

0032-0935/81/0152/0578/$01.80

Harkin 1963; Sarkanen and Ludwig 1971). Some of these compounds have been shown to undergo turnover in vivo, e.g., the isoflavone formononetin in chick pea seedlings (Barz 1969; Barz and H6sel 1971 ; Amrhein and Diederich 1980) and the cinnamyl alcohol glucoside coniferin in spruce seedlings (Marcinowski and Grisebach 1977). Moreover, several /?glycosidases have been characterized in these two plants which display specificity for these compounds. Thus, Cicer arietinurn L. contains different/~-glycosidases with specificity for isoflavone 7-glycosides (H6sel and Barz 1975; H6sel 1976) and coniferin (H6sel et al. 1978; Surholt and H6sel 1981). Picea abies hypocotyls have been shown to contain a coniferin specific /%glucosidase (Marcinowski and Grisebach 1978), which is similar to the one described for Cicer arietinum L. Recently a /%glucosidase has been described in Glycine max. which displays a very high degree of specificity for coniferin and syringin (H6sel and Todenhagen 1980). Concomitant induction of coniferin hydrolyzing/?-glucosidase activity with lignin biosynthetic enzymes and lignification in several plant cell cultures corroborated further the relationship between this/%glucosidase and lignification (H6sel and Borgmann 1978; H6sel et al., in preparation). However, the exact role of this/%glucosidase in lignification is still not known. There are several reports on the localization of /~-glucosidases in plant tissues. /~-Glucosidase activity has been demonstrated in lignifying tissues of various plants using indican as a substrate (Freudenberg et al. 1955; Reznik 1955; Koblitz 1961). Gierse and Barz (1976) investigated the distribution of /%glucosidase activity in stem and root sections of chick pea seedlings using the artificial substrate 4-methylumbelliferyl-p-glucoside. They detected /%glucosidase activity predominantly in the cortex cells, but also in several other tissues, among them xylem and epidermis. Most of these studies suffer from the use of artificial sub-

G. Burmeister and W. H6sel: Immunohistochemical localization of/?-glucosidases in Cicer strates for v a r i o u s r e a s o n s . T h u s , it is n e a r l y i m p o s s i ble to d i f f e r e n t i a t e a m o n g t h e s e v e r a l / ~ - g l u c o s i d a s e s w h i c h m i g h t be p r e s e n t in the s a m e p l a n t a n d e v e n in t h e s a m e tissue. F u r t h e r m o r e , it is n o t p o s s i b l e to use d e n a t u r a t i n g f i x a t i o n m e t h o d s since o n e h a s to rely o n e n z y m a t i c a l a c t i v i t y f o r the s t a i n i n g . T h e low molecular weight aglycones diffuse very rapidly a n d h e n c e p r e v e n t e x a c t l o c a l i z a t i o n studies, e s p e c i a l ly o n t h e i n t r a c e l l u l a r level. F i n a l l y , s t r o n g p l a n t b a c k g r o u n d f l u o r e s c e n c e d u e to e n d o g e n o u s p h e n o l i c s u b s t a n c e s at t i m e s s e v e r e l y h a m p e r s t h e use o f fluorogenic substrates. I n o r d e r to l o c a l i z e t h e i s o f l a v o n e 7 - g l u c o s i d e spec i f i c / ~ - g l u c o s i d a s e (" i s o f l a v o n e g l u c o s i d a s e " ) a n d t h e c o n i f e r i n specific /?-glucosidase ( " c o n i f e r i n g l u c o s i d a s e " ) in c h i c k p e a seedlings, we c h o s e an i m m u n o h i s t o c h e m i c a l a p p r o a c h to a v o i d t h e d i s a d v a n t a g e s m e n t i o n e d a b o v e , in p a r t i c u l a r the l a c k o f s p e c i f i c i t y o f the s y n t h e t i c s u b s t r a t e s . I n c o n t r a s t to a n i m a l s and microorganisms, immunofluorescence has been u s e d o n l y r a r e l y to l o c a l i z e p r o t e i n s in plants. T h i s is p r o b a b l y d u e to s o m e i n h e r e n t d i s a d v a n t a g e s o f p l a n t tissues f o r this t y p e o f study, m a i n l y t h e prese n c e o f t h e r i g i d cell walls a n d the v a c u o l e s . A m o n g the proteins localized were: pollen wall proteins ( K n o x 1971); a c i d r i b o n u c l e a s e ( B a u m g a r t n e r a n d M a t i l e 1976); u r e a s e ( M u r r a y a n d K n o x 1977); lectins ( C l a r k e et al. 1975; H a p n e r a n d H a p n e r 1978); a n d ribulose-l,5-bisphosphate carboxylase (Hattersley e t a l . 1977). I n a r e c e n t s t u d y M a r c i n o w s k i et al. (1979) d e m o n s t r a t e d t h e l o c a l i z a t i o n o f t h e c o n i f e r i n s p e c i f i c / ? - g l u c o s i d a s e in h y p o c o t y l s e c t i o n s o f s p r u c e s e e d l i n g s w i t h this t e c h n i q u e . W e d e s c r i b e h e r e the i m m u n o f l u o r e s c e n t l o c a l i z a t i o n o f t h e c o n i f e r i n gluc o s i d a s e a n d t h e i s o f l a v o n e g l u c o s i d a s e in s t e m a n d r o o t s o f c h i c k p e a seedlings. T h e use o f t w o a n t i b o d y p r e p a r a t i o n s w i t h specificity f o r t w o d i f f e r e n t / % g l u c o sidases in t h e s a m e p l a n t s h o u l d p r o v i d e a g o o d i n t e r n a l c o n t r o l f o r t h e i m m u n o f l u o r o m e t r i c a l results, since t h e t w o / ? - g l u c o s i d a s e s w e r e likely to b e p r e s e n t at d i f f e r e n t sites in t h e tissues a n d t h e cells.

Materials and methods Material. Commercially available chick pea seeds were soaked for two days in tap water and grown in vermiculite at 24 ~ C under a photoregime of 15 h light and 9 h dark. The plants were grown in water instead of vermiculite when the roots were harvested. For enzyme isolations 10-12 day-old plants were used. Chick pea cell suspension cultures were cultivated as described earlier (Burmeister and H6sel 1980). Six-day-old cultures, after inoculation, were used for enzyme isolations. The glycosidic substrates and the material for protein purifications were obtained as described previously (H6sel et al. 1978). Complete Freund's adjuvant (Behring, Marburg/Lahn, FRG), fluorescein isothiocyanate conjugated goat anti-rabbit IgG (Miles, Frankfurt a.M., FRG), tissue tek OCT (WKF Forschungsgerfite GmbH, Brandau/Darmstadt, FRG), agarose and salicin (Serva, Heidelberg, FRG), conca-

579

navalin A-Sepharose 4 B (Sigma, Taufkirchen, FRG), CNBr- and epoxy-activated Sepharose (Pharmacia, Freiburg, FRG) were obtained as noted. All other chemicals were purchased from Merck, Darmstadt, FRG.

Purification of [3-gIucosidases. Isoflavone glucosidase was purified from chick pea roots as described earlier (H6sel and Barz 1975). In addition, isoelectric focusing in a column was performed and the main isoflavone glucosidase isoenzyme peak (p~= 7.6) isolated as previously described (Burmeister and H6sel 1980). The preparation showed only one protein band in SDS disc gel electrophoresis on staining with Coomassie blue. It was used at this stage of purification for the immunization of the rabbit. Before coupling to CNBr-Sepharose, the isoflavone glucosidase was further purified by chromatography on a concanavalin A Sepharose column (1,8.10 cm). The column was equilibrated with acetate buffer 10 mM, pH 5.9, which contained 200 mM NaC1, 50 mM CaC12, 50 mM MnCI2, and 0,02% NAN3. After the protein (ca. 2 mg) was loaded, the column was washed with the same buffer and eluted with 100 ml of a linear ~-methylmannoside 0-200 mM gradient (see Fig. 3 b). The coniferin glucosidase was purified from cell suspension cultures as previously described, except that the isoelectric focusing step was omitted (H6sel et al. 1978). In addition, a chromatography on 6-gluconolactone Sepharose 6B was performed according to the procedure described by Kanfer et al. (1973, 1974). For the coupling of 6-gluconolactone to epoxyactivated Sepharose 6B the procedure outlined by Pharmacia was followed. The actual coupling mixtm-e consisted of 5 g (dry weight) epoxyactivated Sepharose 6B suspended in 25 ml borate buffer 0.1 M, pH 11.8, and 2 g 6-gluconolactone. The mixture was kept for 50 h at 42 ~ C under gentle shaking. The gel was then washed several times according to the Pharmacia procedure. A column (1.6.6 cm) of this gel was prepared, equilibrated with citrate phosphate buffer, pH 5, 0.025 M, and the protein loaded under these conditions. The bound protein was eluted with a linear NaC1 gradient from 0-0.5 M. The coniferin glucosidase eluted between 0.02 M 0.15 M (cf. Surholt 1979). This protein was used for the immunization of the rabbit. The coniferin glucosidase was also chromatographed on concanavalin A Sepharose before coupling to the Sepharose as described above. The glucosidase peak indicated in Fig. 3a was used for the coupling to Sepharose. Coupling of the glucosidases to CNBr-Sepharose 4 B. One milligram of coniferin- and isoflavone glucosidase, respectively, were bound to 0,5 g CNBr activated Sepharose 4B, according to the method described by Axen et al. (1967). Measurement of glucosidase activity. Incubations and measurements of the /~-glucosidases were performed as described earlier (H6sel and Barz 1975; H6sel et al. 1978). The following substrates were used: 4-nitrophenyl-/3-glucoside, coniferin, syringin, and formononetin 7-/~-glucoside. Low glucosidase activities, such as in the antibody-antigen precipitation assays, were measured fluorometrically with 4-methylumbelliferyl-fl-glucoside. The excition wavelength was 357 nm; the emission was measured at 450 nm in NazCO3 I M (ISA spectrofluo JY 3 D fluoremeter, Jobin, Yvon). For quantitative calculations a standard curve of 4-methylumbelliferone obtained under the same conditions was used. Glucosidase activity in the gels was detected by incubating the gels with 2 mM solution of this substrate in a suitable buffer and observing the fluorescence in UV light of 354 nm. Preparation ofantisera. 700 ~tg of the isoflavone glucosidase, purified as described above was erflulsified in 1.5 ml physiological NaCI solution and the same volume of Freund's complete adjuvant.

580

G. Burmeister and W. H6seh Immunohistochemical locaIization of fl-glucosidases in Cicer

One-half of the emulsion was injected into a 6-month-oId rabbit subcutaneously at different places; the remainder was injected 8 weeks later, intramuscularly. A third injection of 100 ~tg enzyme in physiological NaC1 solution was given another 14 weeks later without any adjuvant into the veins. Two weeks later 40 ml blood were taken from the ear of the rabbit. The serum was obtained by keeping the blood for 1 h at room temperature, another 12 h at 4 ~ C, followed by centrifugation. It was stored at - 2 0 ~ in small portions. This serum will be referred to as "Is-serum". Antibodies against the coniferin specific ]Lglucosidase were obtained in the same way except that the intervals between the injections were only 5 weeks. Blood was taken 8 days after the last injection and the serum obtained as described above. This serum will be referred to as "C-serum'. Control sera were taken from each rabbit before immunization. The ?-globulin fractions of the sera were prepared as described by Harboe and Ingild (1973).

Purification of the ?-globulins by immunoadsorption. Purification of the ?-globulins was carried out using a modified method of Hudson and Hay (1976). Three milligrams y-globulins obtained from C-serum were applied to the coniferin glucosidase linked Sepharose column, which had been equilibrated with physiological phosphate saline buffer, pH 7.2 (PBS buffer). The non-binding antibodies were eluted with this buffer. Bound antibodies were eluted subsequentiy in three steps using: 1. sodium-acetate buffer 0.2 M, pH 4, 2. glycine-HC1 buffer 0.1 M, pH 2.5, 3. glycine-HCI buffer 0.1 M, pH 2.5, containing 10% dioxane (v/v). The fractions eluted at pH 2.5 were immediately neutralized by adding suitable amounts of solid Tris. The column was thoroughly washed afterwards with PBS buffer before a new run was started. Six milligrams y-globulins obtained from Is-serum were similarly chromatographed on the isoflavone glucoside linked Sepharose column with the exception that bound antibodies were only eluted with glycine-HC1 buffer 0.i M, pH 2.5, containing 10% dioxane. Antibody-antigen precipitation tests. Suitable amounts of/~-glucosidases dissolved in 100 ~tl PBS buffer were incubated with a series of different antibody concentrations or control sera for 1 h at room temperature and then for 15 h at 4 ~ C. After centrifugation for 1 rain at 15,000g, /Lglucosidase activity was determined in 100 g1 of the supernatant with 4-nitrophenyl-/~-glucoside as the substrate. Since the control serum enhanced the glucosidase activity to some extent these values were set to 100% and used for the calculation of the precipitated glucosidase activities. The percentage of glucosidase activities in the supernatants was plotted on a linear scale versus the antibody dilutions on a logarithmic scale. The antibody diIution which precipitated 50% of the glucosidase activity was taken as the titre or cross reactivity value. In a second type of assay, 50 ~1 glucosidase and 50 pl antibody dilution were incubated and centrifuged in the same way as described above. The glucosidase activity was determined in an aliquot of the supernatant and used for the calculation of the total activity in the supernatant. The activity of the resuspended pellet in the remaining supernatant was also measured as a control and used for the calculation of the precipitated activity. This was possible because precipitation of the glucosidase by the antibodies did not influence the enzymatic activity at all The sum of the activities in the supernatant and pellet were set to 100% and the titre values calculated as described above. This type of assay was usually done fluorometrically with 4-methylumbelliferyl-/~-glucoside as the substrate when low amounts of glucosidase activities had to be measured. It avoided the necessity of using the control serum.

Immunodiffusion and-electrophoresis. Ouchterlony type double immunodiffusions were performed according to the prescription of Zwilling (1977). The layers contained 0.7% agarose, 0.425% sodium chloride, and 0.1% sodium azide (w/v respectively). Diffusion was allowed to proceed for 24 h in a moist chamber. Immunoelectrophoresis was performed as described by Zwilling (1977) in Michaelis buffer, pH 8.2, using 5 mA/slide for about two hours. A iongitudinal depot was then cut into the agarose layer in the middle of the slide where the sera were placed. Diffusion was allowed to proceed for 24 h.

Immunofluorometrical localization of glucosidases. Three-millimeter-long pieces of stems and roots of C. arietinum seedlings were frozen in OCT compound tissue tek using liquid carbon dioxide. They were kept 1 h at - 18~ C. Then, 14 ~m thick sections were cut at the same temperature with a microtome. The slices were washed two times with PBS buffer, pH 7.2, at room temperature and incubated for 5 min in a 2.5% salicin solution in PBS buffer to block the lectins, as suggested by Clarke et al. (1975). The slices were then incubated for 30 rain with either C-y-globulins, Is-y-globulins, or control ?-globulins at a concentration of 0.3 mg m1-1. The sections were washed for 5 rain with PBS buffer to remove unbound antibodies. This procedure was followed by an incubation with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin G. After another 10-rain washing step with PBS buffer the sections were mounted in glycerol/PBS 8/2 (v/v). Examination was done by fluorescence microscopy with a Zeiss microscope equipped with a mercury arc source and a FITC specific filter system. Lignin was stained in fresh stem and root sections using the Wiesner and M/iule tests (Braune et al. 1967).

Protein determinations. Protein was measured by the methods of Lowry et al. (t951) and Bradford (1976). Protein staining in gels was either done with Coomassie blue (SDS gels) or amidoblack (agarose gels) according to Zwilling (1977).

Results Characteristics o f the antisera. V a r i o u s / % g l u c o s i d a s e s f r o m chick pea as well as f r o m o t h e r s o u r c e s were t e s t e d w i t h t h e Is- a n d t h e C - a n t i s e r u m , r e s p e c t i v e l y , by measuring the precipitated amounts of glucosidase activities by different antisera dilutions. The results a r e g i v e n in Fig. 1 a l o n g w i t h t h e p r e c i p i t a t i o n s o b t a i n e d by the t w o i m m u n o g e n s . It can be seen t h a t f o r e i g n / L g l u c o s i d a s e s , like e m u l s i n f r o m a l m o n d s o r t h e c o n i f e r i n s p e c i f i c / % g l u c o s i d a s e f r o m Glycine max. ( H 6 s e l a n d T o d e n h a g e n 1980), a r e n o t p r e c i p i t a t e d a t all b y e i t h e r a n t i s e r u m . T h e / L g l u c o s i d a s e s i s o l a t e d from chick pea display some kind of cross reactivity with the two sera, p r i m a r i l y with the Is-serum. The c r o s s r e a c t i o n s d i s p l a y e d w i t h t h e C - a n t i s e r u m are n e a r l y u n d e t e c t a b l e in t h i s k i n d o f t e s t in w h i c h s i m i lar a m o u n t s o f g l u c o s i d a s e a c t i v i t i e s w e r e e m p l o y e d . W h e n t h e a m o u n t s o f / % g l u c o s i d a s e s , e.g., o f t h e i s o flavone glucosidase, are varied, an appreciable degree of precipitation then becomes detectable, indicating the presence of some cross-reacting antibodies. There is o n l y o n e e x c e p t i o n , n a m e l y t h e s o - c a l l e d c o n i f e r i n

G. Burmeister and W. H6scl: Immunohistochemical localization of fl-glucosidases in o

o

o

@

100'

z

;

581

o

.,o

i

Cicer

o

/ _.--.

/ / ~ A -

50. . . . . . . . . . .

,

,-------z---"~~//

.

.

.

.

2

4

8

1'6

1oo. ~

a

a ~ ,

i

B

10"

~2

~

128

G o

yJ 2

|

*.

,~_--.x 1'6

DILUT~ON

32

6/.

1~)8

OF A N T I S E R U M

Fig. 1a, b. Precipitation of various ~-glycosidases by different dilutions of C-antiserum (a) and Is-antiserum (b). The experiments were performed as described under methods. 4-Nitrophenyl-/~-glucoside was the substrate used with exception of the /3-glucosidase from soybean, where coniferin was used. 9 9 coniferin specific /~-glucosidase from C. arietinum ; x - - x isoflavone 7-glucoside specific/Lglucosidase from C. arietinum; x - - - isoflavone 7-diglycoside specific fl-glycosidase from C. arietinum leaves; v - - v flglucosidase II front C. arietinum cell suspension cultures; n - - ~ fl-glucosidases (specificity unknown) from C. arietinum cell suspension cultures (cf. Burmeister and H6sel 1980); o - - o coniferin specific /Lglucosidase from Glycine max. (H6sel and Todenhagen 1980); zz--zx ~-Glucosidase from almonds (Boehringer)

glucosidase II, which reacts very well with both antisera. This /?-glucosidase has only been observed in very small a m o u n t s in glucosidase purification procedures o f large a m o u n t s o f chick pea cell cultures, but never with intact plants. Interestingly e n o u g h it hydrolyses b o t h coniferin and isoflavone 7-glucosides very well, which is in contrast to the coniferinand isoflavone glucosidases which show preference for only one type o f substrate. We are tempted to speculate that this "coniferin glucosidase I I " might be an artifact originating f r o m the two glucosidases either in the cell cultures or during the purification procedure. But since this glucosidase has never been observed in chick pea seedlings it does n o t interfere with the i m m u n o f l u o r o m e t r i c a l studies with seedling tissue. Results which give rise to similar conclusions were obtained by O u c h t e r l o n y double diffusion tests and immunoelectrophoresis. Figure 2 illustrates the re-

Fig. 2a, b.Ouchterlony double immunodiffusion (a) and immunoelectrophoresis (b) of Is- and C-antisera and different proLein preparations from Cicer arielinum L. 1, Sephadex G 25 protein extract from stem; 2, Isoflavone 7-glucoside specific fl-glucosidase; 3, Sephadex G 25 protein extract from roots; 4, Coniferin specific /~glucosidase; 5, Mixture of 1 and 3

suits obtained with the two sera, the purified i m m u n o gens and crude protein extracts from the stem and roots of chick pea seedlings. It can be seen that the C-antiserum p r o d u c e d a strong precipitin line with its i m m u n o g e n in the O u c h t e r l o n y test and in immunoelectrophoresis, respectively. Yet the isoflavone glucosidase also gave a weak but unequivocal precipitation in the Ouchterlony test (see Fig. 2a, C-serum, well 2). W e are therefore unable to determine which of the two/3-glucosidases caused the weak precipitations obtained with the crude protein extracts from chick pea stems and roots, because the isoflavone glucosidase is present in m u c h larger a m o u n t s in these tissues than the coniferin glucosidase (Surholt and H6sel 1981). The Is-antiserum, on the other hand, revealed three different precipitin bands with its purified i m m u n o g e n (Is-serum, well 2 in Fig. 2a). These three bands are also present in the stem and r o o t protein extracts (see wells 1 and 5 in Fig. 2 a,

582

G. Burmeister and W. H6sel: Immunohistochemical localization of fl-glucosidases in Cicer

Is-serum). R o o t extract obviously displays an additional precipitin line (see wells 3 and 5 in Fig. 2a, Is-serum), which exhibited no glucosidase activity on incubation with 4-methylumbelliferyl-glucoside. All other bands obtained in Fig. 2 possessed fi-glucosidase activity. The Is-antiserum also formed a precipitin line with coniferin glucosidase, but a corresponding band is not present in the crude protein extracts. Since the two antibody preparations displayed these kinds of cross reactivities we decided to purify them by immunoadsorption to the more thoroughly purified immunogens.

[mM]

A

0,2

A 2BC

2OO

0,6 0,1

04.

I00

0,2

:~

8

II

Purification of the 7-globulins by immunoadsorption. As the two fi-glucosidases were known to be glycoproteins they were purified by c h r o m a t o g r a p h y on concanavalin A Sepharose. The results are shown in Fig. 3. It can be seen that a small amount of non-binding protein which did not show glucosidase activity could be separated in both cases from the fi-glucosidases. The bound fl-glucosidases were not eluted as a homogenous peak by the e-methylmannoside gradient, but rather in the patterns illustrated in Fig. 3. The indicated fractions were pooled and tested with the Cand Is-7-globulins. The fractions which revealed the lowest crossreactivity were bound to CNBr-activated Sepharose 4B. These were fractions III and IV in Fig. 3a and fraction II in Fig. 3 b (data not shown). The 7-globulins were then c h r o m a t o g r a p h e d on the respective columns as outlined in Fig. 4 for the C-7globulins. It can be seen that most C-7-globulins did not bind to the coniferin glucosidase containing column. The four fractions indicated in Fig. 4 were tested with both fi-glucosidases. All of them showed nearly the same reactivity with coniferin glucosidase. But, surprisingly enough, the 7-globulins which did not bind to the column displayed a much weaker reaction with the isoflavone glucosidase than the original C-7globulins. The procedure depicted in Fig. 4 was repeated with the non-binding fraction and after another four runs the non-binding fraction no longer reacted with the isoflavone glucosidase (data not shown). Having seen this result we modified the purification of the Is-7-globulins slightly. All bound antibodies were eluted in one step using the glycine-HC1 buffer, pH 2.5, containing 10% dioxane instead of three separate steps. The 50% precipitation titres for isoflavone- and coniferin glucosidase were determined for the bound and unbound 7-globulins after each run. The ratio of the starting IS-7-globulins was 10:1 in favor of the isoflavone glucosidase. After five runs the non-binding 7-globulin fraction revealed a ratio of 23 : 1. This shows that the purification worked in the same way as outlined above for the C-7-globulins,

8 0.3-

o.,

iq 10

30

50

0,2 70

9D

FRACTIONS

110

130

150

1"/0

[3,B ml]

Fig. 3 a , b. C h r o m a t o g r a p h y o f c o n i f e r i n g l u c o s i d a s e (a) a n d isofla-

vone glucosidase (h) on concanavalin A-Sepharose 4B. For the previous purification steps of the glucosidases and the conditions see methods. /,--z~ 4-nitrophenyl-fi-glucoside; 9 coniferin; e--e protein absorption at 280 nm; - - e-methylmannosidegradient

g o,9 N

g E tn < z ,'5, 0,5

#,

d._ox

O

O

~a

Immunohistochemical localization of β-glucosidases in lignin and isoflavone metabolism in Cicer arietinum L. seedlings.

Coniferin specific- and isoflavone 7-glucoside specific β-glucosidases have been localized in stem and root sections of chick pea (Cicer arietinum L.)...
2MB Sizes 0 Downloads 0 Views