Planta (1990)181:343-348
P l a n t a 9 Springer-Verlag 1990
Partial purification and immunological characterization of 1,3-fl-glucan synthase from suspension cells of Glycine m a x J. Fink, W. Jeblick, and H. Kauss Fachbereich Biologic, Universitfit Kaiserslautern, Postfach 3049, D-6750 Kaiserslautern, Federal Republic of Germany
Abstract. The plasma-membrane-localized 1,3-//-glucan synthase (EC 2.4.1.34) from suspension cultures of Glycine max (L.) Merr. was greatly enriched by a three-step purification procedure. Starting with a microsomal preparation, a six- to eightfold enrichment of the enzyme was achieved by isolating plasma-membrane vesicles in a polyethyleneglycol/dextran two-phase system. The enzyme was solubilized with the nonionic detergent digitonin and further purified 12-fold by successive centrifugations on two linear sucrose density gradients. The most purified enzyme preparation showed enrichment in a 31-kilodalton (kDa) polypeptide and was used to raise polyspecific antibodies which precipitated 1,3-//glucan synthase activity. These antibodies were purified by affinity chromatography against immobilized membrane protein fractions of lower molecular weight which were devoid of 1,3-fl-glucan synthase activity. The purified antibodies specifically labelled a single polypeptide of 31 kDa in the 1,3-//-glucan-synthase-containing heavy fractions of the first sucrose gradient indicating that this polypeptide represents part of the active enzyme complex. Key words: Callose - Cell culture (1,3-fl-glucan synthase) Digitonin - 1,3-fl-Glucan synthase (purification, immunology) - Glycine (1,3-//-glucan synthase)
Introduction Cellulose, a 1,4-fl-linked polyglucan, is the major cellwall component in higher plants. Although it was possible to detect protein complexes attached to growing cellulose microfibrils in freeze-fracture preparations of plant plasma membranes (Giddings et al. 1980; Robinson and Quader 1981), there has been no success in Abbreviations: CHAPS = 3-[(3-cholamidopropyl)-dimethylammon-
io]-l-propanesulfonic acid; SDS-PAGE=sodium dodecyl sulfatepolyacrylamide gel electrophoresis
creating an in vitro assay system for cellulose synthase of higher plants (Delmer 1987). The possibility exists that the minor amounts of 1,4-fl-linked glucans reported by various authors to be synthesized in vitro are unrelated to cellulose but reflect the biosynthesis of xyloglucan (Gordon and McLachlan 1989). Thus, any homogenization of plant cells which necessarily destroys the plasma membrane appears to result in a total loss of cellulose synthase activity. In contrast, the plasma-membrane-located 1,3-fl-glucan synthase becomes active on cell homogenization whereas in intact, undisturbed cells this enzyme is fully latent. The 1,3-fl-glucan synthase is likely to be involved in the production of callose, a polymer which is rapidly deposited in vivo as a response to mechanical injury or attack by pathogens (for citations see Kauss 1987). These observations led to the hypothesis that the two enzyme activities belong to the same enzyme complex which may change its specificity and regulatory properties on perturbation of the plasma membrane (Jacob and Northcote 1985; Delmer 1987). The mechanism of callose formation in vivo has been intensively studied by treating suspension-cultured cells with callose elicitors (K6hle et al. 1985; Kauss 1987; Waldmann et al. 1988; Kauss et al. 1990). This callose production can start about 5 min after elicitor addition and is accompanied by drastic changes in the ion concentration and pH of the surrounding medium. The concentration of Ca 2 + decreases while other electrolytes, mainly K +, are released from the cells. The induced Ca 2+ uptake may lead to a transient increase in cytoplasmic [Ca 2+]. As the plasma-membrane-located 1,3-fl-glucan synthase has an absolute requirement for Ca 2+ in the micromolar range (Kauss et al. 1983), this increase in cytoplasmic [Ca 2+] may be one of the signals initiating callose synthesis. Assay of the 1,3-fi-glucan synthase in sealed rightside-out plasma-membrane vesicles requires detergents such as digitonin to permeabilize the membrane (Fink et al. 1987). This indicates that the substrate-binding side of the enzyme is located at the cytoplasmic side of the plasma membrane. In contrast, the reaction product, cal-
344 lose, a p p e a r s t o be d e p o s i t e d o u t s i d e t h e p l a s m a m e m brane, indicating a vectorial transmembrane arrangement of the enzyme complex. T o i n v e s t i g a t e t h e f u n c t i o n o f t h e 1 , 3 - f l - g l u c a n synt h a s e c o m p l e x a n d to d e t e r m i n e w h e t h e r it is r e l a t e d to t h e c e l l u l o s e s y n t h a s e , it is n e c e s s a r y to p u r i f y the e n z y m e . W e r e p o r t h e r e o u r p r o g r e s s in this r e g a r d a n d show that a 31-kilodalton (kDa) polypeptide appears to be a s s o c i a t e d w i t h g r a d i e n t f r a c t i o n s c o n t a i n i n g a c t i v e 1,3-fl-glucan synthase.
J. Fink et al. : Partial purification of 1,3-fl-glucan synthase 4-ml portions onto linear sucrose density gradients ( 5 % 4 0 % , w/ v), in 50 mM Tes-NaOH (pH 7.0), 1 mM DTT and 0.015% (w/v) digitonin, and centrifuged for 1.5 h at 250000-g in a vertical rotor (TV 850). The gradients were fractionated into 2-ml fractions and 50-gl aliquots were immediately assayed for 1,3-fl-glucan synthase activity and protein concentration. The fractions containing 1,3-flglucan synthase activity were pooled as indicated in Fig. 1, concentrated in Amicon Centriprep vials and layered onto a second sucrose density gradient (20% 50%, w/v). After centrifugation for 1.75 h at 250000.g in the TV 850 rotor, the second gradient was fractionated into 1-ml fractions and assayed as for the first gradient. All fractions containing either enzyme activity or protein were stored at - 2 0 ~ C for further investigation.
Assay of 1,3-fl-glucan synthase. Enzyme activity was tested as de-
Material and methods Chemicals and radiochemicals. Polyethyleneglycol (PEG) 4000 was obtained from Merck (Darmstadt, FRG), dextran PL 500 VC from Pfeifer and Langen (Dormagen, FRG), digitonin from Serva (Heidelberg, FRG) and Sepharose CL-4B from Pharmacia (Heidelberg, FRG). All other chemicals were from Sigma Chemie (Miinchen, FRG) and UDP-U[14C]-glucose was purchased from Amersham (Braunschweig, FRG). Cell homogen&ation and microsomal preparation. Suspension cultures of soybean (Glycine max (L.) Merr. cv. Harasoy 63, supplied by J. Ebel, Freiburg, F R G ) were grown in darkness for 5-6 d as described by K6hle et al. (1985). Harvesting of cells and homogenization of small amounts of cells was carried out as described by Fink et al. (1987). Larger amounts of cells were homogenized in portions of 100 g in 100 ml of buffer (50 mM 2-{(hydroxy-l,1bis(hydroxymethyl)ethyl)-amino}ethanesulfonic acid (Tes)-NaOH, pH 7.0; 1 mM dithiothreitol (DTT); 0.25 M sucrose; 1 mM ethylenediamine tetraacetic acid (EDTA) by sonication in a Branson (Heinemann, Schw~ibisch Gmfind, FRG) Sonifier 250 for 2 min at maximum intensity. The microsomal preparation was obtained by three consecutive centrifugations at 500"g, 5000 g and 50 000"g as described by Fink et al. (1987).
scribed previously by Fink et al. (1987). All assays included 25 Ixg of poly-L-ornithine (44 kDa) and the concentration of free C a 2 + was about 70 ktM. Membrane-bound enzyme was measured in the presence of 0.02% (w/v) digitonin.
Protein determination. Protein was measured with Coomassie Brillant Blue G 250 by the method of Bradford (1976) with bovine serum albumin as a standard.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The pooled fractions containing protein and-or 1,3-fl-glucan synthase activity were analyzed for their polypeptide composition in a 10% gel in the presence of mercaptoethanol according to the method of Laemmli (1970). Prior to electrophoresis the proteins from the various fractions were precipitated from the detergent-containing solution according to Wessel and Fliigge (1984). Electrophoresis was carried out in a vertical mini-slab gel apparatus with 1-mm spaces at 2 mA per lane. Gels were stained with 0.25% (w/v) Coomassie Brillant Blue R250 in H20/methanol/acetic acid (5:4:1 ; by vol.).
Polyspecific antibodies to 1,3-fl-glucan synthase. The fraction with
Plasma-membrane isolation. Plasma-membrane vesicles were iso-
the highest specific 1,3-fl-glucan synthase activity (pool A, Fig. 1 B) was used to produce antibodies in a rabbit. The crude antiserum was precipitated by ammonium sulfate for isolation of the immunoglobulin fraction which was solubilized (same volume as serum used) in 100 mM Na2HPO4 (pH 7.2) containing 150 mM NaC1.
lated in a PEG/dextran two-phase system by the method of Kjellbom and Larsson (1984) as modified according to Fink et al. (1987).
Immunoprecipitation of l,3-fl-glucan synthase. Immunoprecipitation was carried out with Staphylococcus aureus cells (Pansorbin| Cal-
Solubilization of plasma-membrane proteins. Solubilization with digitonin was carried out by a two-step procedure. In the first step, each 10 ml of the plasma-membrane vesicle suspension was mixed with 10 mg of digitonin. The suspension was kept in a glass tube at 0 ~ C for 1 h and was sonicated every 10 min for 5 min in a sonifer bath filled with 0 ~ C water. The membrane material was pelleted in an AH 629 rotor at 150000"g in a Sorvall (Dupont, Bad Nauheim, F R G ) OTD 65B ultracentrifuge for 1 h. The supernatant containing part of the 1,3-fl-glucan synthase was stored at --20 ~ C. The pellet was resuspended in 10 ml of cold buffer (50 mM Tes-NaOH, pH 7.0; 1 mM DTT) and again 10 mg of digitonin were added. Furthermore, a stock solution of E D T A and ethyleneglycol-bis(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was added to result in a final concentration of 1 mM. This mixture was sonicated and centrifuged as described before and the supernatant of the second solubilization step containing additional enzyme was also stored at - 2 0 ~ C. Solubilization with 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propanosulfonic acid (CHAPS) was carried out by the method of Lawson et al. (1989) at an end concentration of 0.5% (w/v) of the detergent.
Sucrose density gradients. The solubilized enzyme fractions (200 ml, corresponding to I kg cells) were concentrated by centrifugation in Amicon (Witten, F R G ) Centriprep-vials (30 S) to a volume of 7 ml. This concentrated enzyme preparation was layered in 3- to
biochem, La Jolla, Calif., USA) as an adsorbent according to the method of Shimazaki and Pratt (1985) with the ammonium-sulfateprecipitated polyspecific antibody fraction. The same fraction from preimmune serum was used for controls.
Purification of antibodies by affinity chromatography. The bulk of the 1,3-fl-glucan synthase-free protein fractions from the first sucrose density gradient (4 mg, pool II, Fig. 1 A) was coupled to 10 ml of Sepharose CL-4B by the method of Bethell et al. (1979). The column (1.5 cm diameter) was loaded with 2 ml of a 1:10 diluted polyspecific immunoglobulin solution and washed with buffer. The protein-containing fractions (about 3 ml) which passed through the column were supplied with 0.1% NaN3. This solution of purified antibodies was stored at 0~ C for further investigation.
Immunoblot. Proteins were transferred from PAGE gels to nitrocellulose sheets in a wet-blot apparatus (Pharmacia-LKB; Freiburg F R G ; 2005 Transphor) for 2 h at 0.8 A. For immunological detection of peptides according to Ogata et al. (1983), the nitrocellulose sheets were incubated overnight at 4~ C either with the polyspecific antibody fraction in a 1:100 dilution in 100mM NazHPO4 (pH 7.2), 150 mM NaC1, 0.05% Tween 20, or with undiluted affinity-chromatography-purified antibodies. As a second antibody an anti-rabbit IgG-peroxidase conjugate was used with 4-chloro-1naphthol (0.2%, w/v) and 0.01% (v/v) H 2 0 2 as peroxidase substrates.
J. Fink et al. : Partial purification of 1,3-/%glucan synthase Results In the p l a s m a - m e m b r a n e fraction (upper phase) of the polyethyleneglycol-dextran system, the 1,3-/%glucan synthase activity was routinely enriched six- to eightfold over microsomes (Table 1). The first p l a s m a - m e m b r a n e containing upper phase showed a recovery of 4 0 % - 5 0 % of the enzyme activity. By adding a new upper phase to the first lower phase it was possible to increase the overall recovery to m o r e than 60%. Similar results were described by Kjellbom and Larsson (1984); they however, purified their p l a s m a - m e m b r a n e fraction by successive rewashing o f the first upper phase with two new lower phases. Solubilization of m e m b r a n e proteins with digitonin resulted in the loss of a considerable portion of 1,3-/% glucan synthase activity (Table 2 A). During the first step about 70% o f total protein was solubilized. In contrast, only about 12% of the 1,3-/%glucan synthase activity could be measured in the soluble fraction. The second solubilization step gave a further protein recovery o f 25% and another 12% of solubilized enzyme activity. The pellet after centrifugation of the second solubilization mixture included neither protein nor enzyme activity (data not shown). This indicates that the loss of 1,3-/3glucan synthase activity during the solubilization is not caused by insufficient solubilization of the enzyme molecules but by inactivation, presumably mainly by displacement of phospholipids and other activating compounds from the enzyme complex. Attempts to reactivate the solubilized enzyme by addition of phospholipids were not successful, in contrast to the experience of Wasserman and M c C a r t h y (1986) with red beet 1,3-/~-glucan synthase. Attempts to purify large volumes of dilute, solubilized 1,3-/~-glucan synthase using DEAE-Sepharose, Ca 2 +-loaded Chelating Sepharose (Pharmacia), Sepharose CL-4B and presumed affinity c h r o m a t o g r a p h y on immobilized spermine were unsuccessful. The latter method brought some increase in specific activity when small short columns were used (Kauss and Jeblick 1987) but scaling up was difficult due to the comparatively low capacity of the affinity material. In summary, chrom a t o g r a p h i c methods were probably not successful as they require the prolonged presence or the washing o f the bound enzyme with detergent-containing buffers. These problems were overcome by a purification protocol consisting only of rapidly performed bulk steps. Concentration of the pooled digitonin-solubilized fractions in Amicon Centriprep vials yielded a near total recovery of both protein and enzyme activity with less than 10% decrease in specific activity (data not shown). For further purification, this concentrated solubilized 1,3-/%glucan synthase was centrifuged in two successive linear sucrose density gradients (Fig. 1 A, B). In both gradients the enzyme activity appeared as a single sharp peak at approximately 30% (w/v) sucrose and was clearly separated from the bulk of solubilized m e m b r a n e protein. No second protein peak coincided with enzyme activity, indicating that the inactive proteins overlapped the 1,3-/%glucan synthase fractions. The solubilized 1,3-
345 Table 1. Enrichment of 1,3-fl-glucan synthase from soybean suspension-cultured cells in the plasma-membrane fraction of a polyethyleneglycol-dextran two-phase system. The microsomal suspension (5 ml) was added to 31 ml of a polyethyleneglycol-dextran solution to give a final concentration of 5.1% (w/w) for each of the two polymers. After 15 min centrifugation at 500.g the first upper phase was removed. A new upper phase without microsomes was added to the first lower phase to give a second two-phase system which was treated as before. The data are based on a cell fresh weight of 100 g Total activity Protein (nkat) (%) Microsomes 1. Upper phase 2. Upperphase Pool a 2. Lower phase
70.5 29.2 14.2 43.3 19.2
(mg) (%)
Specific Purifiactivity cation (nkat.mg -1) (-fold)
100 37 100 1.9 41.4 1.97 5.3 14.8 20.1 1.08 2.9 13.1 61.4 3.05 8.2 14.2 27.2 32.9 89 0.6
7.8 6.9 7.5 0.3
" The pooled first and second upper phases were used as the total plasma membrane preparation throughout this report
Table 2. Solubilization of 1,3-fl-glucan synthase from the plasmamembrane fraction using two different detergents. A) Two-step solubilization with digitonin. The first step was with 0.1% (w/v) digitonin and the second step with 0.1% (w/v) digitonin plus 1 mM EDTA and 1 mM EGTA. B) A one-step solubilization with CHAPS at a final concentration of 0.5% (w/v) in the presence of 1 mM EDTA and 1 mM EGTA
Membranes" A) Digitonin step 1 step 2 pool 1 + 2 b B) CHAPS
Total activity
Protein
(nkat)
(%)
(rag)
(%)
Specific activity (nkat. mg- 1)
43.3
100
3.05
100
14.1
2.19 0.73 2.92 3.32
71.7 23.8 95.5 109
5.3 5.2 10.5 7.8 c 13.8 d
12.2 12.0 24.2 18.0 31.8
2.4 7.1 3.6 2.3 b 4.2 c
" Total plasma-membrane fraction from line 4 of Table 1 b Although step 2 resulted in a higher specific activity the enzyme from both solubilization steps was pooled to increase the recovery, c Enzyme assayed without further addition of detergent d Same enzyme assayed in the presence of 0.02% (w/v) digitonin
/%glucan synthase was enriched approximately five fold in the first sucrose gradient (Table 3, pool I) and 12-fold after the second sucrose gradient (Table 3, pool A). Because of the inactivation of 1,3-/?-glucan synthase during digitonin solubilization (Table 2A), the total purification f r o m microsomes (Table 1) to the second density gradient (Table 2A) cannot be directly calculated from the respective specific activities. The enrichment can be estimated, however, by multiplying the purification factors from Table 3 (pool A) and from line 4 of Table 1 to be at least 90- to 100-fold. I f one considers that only about a quarter of the enzyme activity survived
346
J. Fink et al. : Partial purification of 1,3-fl-glucan synthase
i
Table 3. Enrichment of digitonin-solubilized1,3-/%glucan synthase in two successive sucrose-density-gradient centrifugation steps. Pools I and II of the first sucrose gradient refer to Fig. 1A and pools A, B and C of the second sucrose gradient to Fig. l B
/.,.
Total activity Protein
t
E
a;
I-,,
9
8
Tr
(nkat) (%)
JC c
d
~6
r" 13
~0
o
& /
_m'"
\
c)
20
++ 9
9
0 1
5
u~ o
n
2 9
i
10
15
§
20
Fraction number
B
i
, A
E El -Y
B
i1.~1
C
,,
Specific Purifiactivity cation (nkat.mg-~) (-fold)
i
Sol. enzyme" 90.0 1. Sucrose gradient Pool I 43.7 Pool II 0.7 2. Sucrose gradienP Pool A 17.3 Pool B 9.5 Pool C 2.8
100
(mg)
(%)
27.1
100
48.6 2.55 0.8 21.7 19.2 10.6 3.1
3.3
9.4 80
0.41 0.51 1.04
17.1 0.04
!.5 42.3 1.85 18.6 3.8 2.7
5.2 0.01 12.8 5.6 0.8
" Seven ml of concentrated 1,3-fl-glucan synthase from step 1 plus step 2 of the digitonin-solubilization,corresponding to the plasma membranes from 1 kg cell FW, were applied on two identical first sucrose gradients and the respective peak fractions pooled b Pool I concentrated and layered on the second gradient
i
0.8 6
El
0,6 k~ irll~ll
C
\
f,
\
u 3 (.9
&
++.~.
i
....'7
9247
= ----d:-" 10
15 2'0 Fraction number
-3"0
o,~ ~="
t,O |
0,2 9
20
0
§
Fig. 1 A, B. Distribution of digitonin-solubilized 1,3-fl-glucan synthase activity and membrane proteins from soybean cells in two successive sucrose density gradients. A First sucrose gradient. The concentrated solubilized enzyme (see Table 3) was layered on a linear 5 4 0 % (w/v) sucrose gradient and centrifuged for 1.5 h at 250000.g. B Second sucrose gradient. The concentrated pool I from the first sucrose gradient (A) was layered on a linear 20-50% (w/v) sucrose gradient and centrifuged for 1.75 h at 250000.g. Enzyme activities and enrichment in pools [ and II as well as A, B and C are reported in Table 3
the s o l u b i l i z a t i o n b u t all p r o t e i n was rendered soluble, a p u r i f i c a t i o n o f u p to 400-fold was achieved. F o r c o m p a r i s o n with the d i g i t o n i n - s o l u b i l i z a t i o n described above, the m e t h o d s o f L a w s o n et al. (1989) for the s o l u b i l i z a t i o n o f a 1,3-fl-glucan synthase from micros o m a l p r e p a r a t i o n s o f c a r r o t roots b y the detergent C H A P S were tested in s o y b e a n p l a s m a m e m b r a n e s (Table 2 B). W h e n assayed in the presence o f 0.02% digitonin, the recovery o f 1,3-/~-glucan synthase was slightly higher t h a n after d i g i t o n i n - s o l u b i l i z a t i o n b u t m e a s u r e d w i t h o u t f u r t h e r d e t e r g e n t it was lower. This C H A P S solubilized 1,3-/3-glucan synthase from s o y b e a n p l a s m a m e m b r a n e s was also subjected to the p u r i f i c a t i o n proced u r e described above. The positions o f the peaks for e n z y m e activity a n d p r o t e i n o n the two sucrose-density c e n t r i f u g a t i o n s were very similar to the results s h o w n
Table 4. Attempt to purify CHAPS-solubilized 1,3-fl-glucan synthase from soybean plasma membranes. The solubilized enzyme (similar to Table 2 B but from 1 kg of cells) was concentrated without considerable loss and centrifuged on two subsequent digitonincontaining sucrose density gradients as for Fig. 1. The positions of the enzyme-activity peaks and protein peaks were similar to those in Fig. 1 Enzyme activity (nkat) (%) Sol. enzyme a 193.3 100 First gradient Enzyme peak b 3 1 . 3 16.2 Protein peak 7.3 3.8 Second gradient c Enzyme peak d 5.8 3.0 Protein peak 3.0 1.6
Protein - (mg) (%) 31 5.5 21 0.75 0.63
100
Specific Purifiactivity cation (nkat.mg- 1) (-fold)
6.2
17.7 5.7 67.7 0.3
1.1 0.05
2.4 7.7 2.0 4.8
1.2 0.8
" Assayed in the presence of 0.02% (w/v) digitonin b Corresponding to peak I of Fig. 1A, all gradient fractions were assayed without further addition of digitonin as the gradients contained 0.015% (w/v) digitonin c Enzyme peak from the first gradient concentrated and layered on the second gradient d Corresponding in position to peak A of Fig. 1 B
for the d i g i t o n i n - s o l u b i l i z e d e n z y m e in Fig. 1. Table 4 d o c u m e n t s , however, that n o a p p a r e n t e n r i c h m e n t a n d a greater overall loss in e n z y m e activity occurred. F o r the p u r i f i c a t i o n o f the 1,3-fl-glucan synthase f r o m soyb e a n p l a s m a m e m b r a n e s , C H A P S - s o l u b i l i z a t i o n was obviously far less suitable t h a n the use of d i g i t o n i n (Table 3). We, therefore, p e r f o r m e d all further c h a r a c t e r i z a t i o n steps with the d i g i t o n i n - s o l u b i l i z e d enzyme. Analysis by S D S - P A G E o f the m o s t purified 1,3-flglucan synthase fraction from the second sucrose gradient showed a n e n r i c h m e n t in a 3 1 - k D a polypeptide (Fig. 2, lane A). In p r o t e i n fractions with little 1,3-fl-
J. Fink et al. : Partial purification of 1,3-fl-glucan synthase
347 ent fractions from the density gradients (Fig. 1) in a Western blot (Fig. 3). Comparing a protein fraction enriched in 1,3-fl-glucan synthase activity (lane A) with another devoid of this enzyme activity (lane B), we observed that a band at 31 kDa was missing in the latter. An affinity column made with immobilized proteins devoid of 1,3-fl-glucan synthase activity (pool II, Fig. 1 A) bound all polyspecific antibodies except those which labeled the 31-kDa polypeptide (lane C) also in the first sucrose density gradient.
Discussion
Fig. 2. Analysis by SDS-PAGE of peptides from 1,3-fl-glucan synthase fractions on a 10% gel. Each lane contained 25 ~tg of protein except lane B which contained 15 ~tg. The gel was stained with Coomassie Brillant blue. S, molecular weight standards; /, pool I from the first sucrose gradient (Fig. 1A); A, B and C refer to pool A, B and C of the second sucrose gradient (Fig. 1B)
Fig. 3. Immunological characterization of peptides from the 1,3-flglucan synthase preparations. The most highly purified fraction of 1,3-fl-glucan synthase (pool A from Fig. 1B, for proteins see lane A, Fig. 2) was used to raise polyspecific rabbit antibodies. Immunoblots with total serum are shown in lane A (proteins from the 1,3-fl-glucan synthase activity, pool I of Fig. 1A) and lane B (proteins devoid of 1,3-fl-glucan synthase activity, pool II of Fig. 1A). The proteins from pool II of Fig. 1A devoid of 1,3-flglucan synthase activity were immobilized on Sepharose CL-4B resulting in an affinity column. The antibodies that passed through this column were used to label lane C (fractions exhibiting 1,3-flglucan synthase activity, pool I of Fig. 1A) and lane D (proteins without 1,3-fl-glucansynthase activity, pool II of Fig. 1A)
glucan synthase activity this band was nearly absent (Fig. 2, lane C). Polyspecific antibodies against the most highly purified 1,3-fl-glucan synthase when used in a 1 : 16 dilution for immunoprecipitation gave a 20% reduction of enzyme activity in relation to control serum (data not shown). The same antibodies were taken to label differ-
The high loss in 1,3-fl-glucan synthase activity during solubilization with either digitonin or CHAPS (Table 2) again demonstrates the importance o f the hydrophobic environment o f the membrane-bound enzyme complex. Wasserman and McCarthy (1986) showed that phosphatidylcholine and similar phospholipids greatly restored 1,3-fl-glucan synthase activity which had been lost from microsomes by treatment with Triton X-100. Digitonin may partly exhibit a function similar to that of endogenous polar lipids as it could further increase the activity of CHAPS-solubilized 1,3-fl-glucan synthase (Table 2 B). The presence of digitonin was also essential in the sucrose density gradients used for the purification of the digitonin-solubilized enzyme (Fig. 1, Table 3) which resulted in an apparent 100-fold enrichment. A similar value was reported by Wasserman et al. (1989) for CHAPS-solubilized 1,3-fl-glucan synthase from red beets. Read and Delmer (1987) and Delmer and Soloman (1989) tried to identify membrane proteins related to 1,3-fl-glucan synthase by affinity-labelling with U D P [3H]pyridoxal and [32p]UDP-glucose. F r o m the various labelled bands, only two polypeptides with molecular weights o f about 34 kDa and 50 kDa were labelled in a Ca 2 +-dependent manner, indicating that they may be related to the 1,3-fl-glucan synthase known to be Ca 2 +activated. Only the 50-kDa protein co-purified with 1,3fl-glucan synthase activity in a sucrose density gradient. By contrast, a monoclonal antibody able to precipitate 1,3-fl-glucan synthase marked a double band at 6062 k D a which was, however, not enriched in the 1,3-flglucan synthase activity peak. Wasserman et al. (1989) performed similar experiments with membrane proteins from red beet and found that a 57-kD protein bound to 32p-labelled 5-azido-UDP-glucose in the presence of Ca z+ and Mg 2+, an effect reversed by E D T A and EGTA. This protein was enriched in a 1,3-fl-glucan synthase preparation purified by product-entrapment. Although these studies give some hints as to possible molecular components of the 1,3-fl-glucan synthase complex, final determination of subunit structure awaits purification. The present study with soybean suspension cells shows that a polypeptide of 31 kDa is enriched in protein fractions rich in 1,3-fl-glucan synthase activity (Fig. 2, lane A) and is almost absent in those fractions without enzyme activity (Fig. 2, lane C). Although a few
348 other protein b a n d s are also present in the m o s t purified preparation, their relationship to the 1,3-fl-glucan synthase remains unclear, as they a p p e a r also in the inactive gradient fractions (Fig. 2, lane C). In b o t h sucrose density gradients (Fig. 1) the enzyme activity is associated with a molecule p o p u l a t i o n migrating at a density greater t h a n that o f the majority o f proteins, indicating that the intact, 1,3-fl-glucan synthase c o m p l e x exhibits a higher molecular weight than the bulk o f the solubilized proteins. Nevertheless, the immunological characterization (Fig. 3) showed that the inactive protein peak in the first gradient contains antigenic determinants capable o f binding, f r o m the polyspecific antiserum, all antibodies except those specific for the 3 1 - k D a peptide. The 3 1 - k D a peptide was also enriched in the heavier fractions o f the second gradient which exhibited 1,3-fl-glucan synthase activity (Fig. 2). This might m e a n that the 1,3-fl-glucan synthase complex may, in the course o f purification, decrease in activity and b e c o m e c o n c o m i t a n t l y smaller by loosing constituents which a p p e a r in the protein peak devoid o f enzyme activity. This suggestion is admittedly based on correlations only. W h e t h e r the 3 1 - k D a peptide f r o m soybean is identical to the approx. 3 4 - k D a peptide f r o m c o t t o n and m u n g bean (Delmer a n d S o l o m o n 1989) and indeed represents an integrating part o f the active 1,3-fl-glucan synthase c o m p l e x awaits c o n f i r m a t i o n by affinity labelling with substrate analogs (Read and Delmer 1987; D e l m e r a n d S o l o m o n 1989; W a s s e r m a n et al. 1989), perf o r m e d in parallel with labelling by the antibodies specific for the 3 1 - k D a peptide. Financial support of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is acknowledged. We also thank T.J. Buckhout for many suggestions and for correcting the English manuscript. References Bethell, G.S., Ayers, J.S., Hancock, W.S. (1979) A novel method of activation of cross-linked agaroses with l,l'-carbonyldiimidazole which gives a matrix for affinity chromatography devoid of additional charged groups. J. Biol. Chem. 254, 2572-2574 Bradford, M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 Delmer, D.P. (1987) Cellulose biosynthesis. Annu. Rev. Plant Physiol. 38, 259 290 Delmer, D.P., Solomon, M. (1989) Identification of polypeptides involved in plant fl-glucan synthesis. In: Abstr., 5th Cell Wall Meeting, Edinburgh, Abstr. 36 Fink J., Jeblick, W., Blaschek, W., Kauss, H. (1987) Calcium ions and polyamines activated the plasma membrane-located 1,3-flglucan synthase. Planta 171, 130-135 Giddings, T.H., Brower, D.L., Staehelin, L.A. (1980) Visualization of particle complexes in the plasma membrane of Micrasterias denticulata associated with the formation of cellulose fibrils in primary and secondary walls. J. Cell. Biol. 84, 327-339 Gordon, R., McLachlan, G. (1989) Incorporation of UDP-
J. Fink et al. : Partial purification of 1,3-fl-glucan synthase [~4C]glucose into xyloglucan by pea membranes. Plant Physiol. 91,373-378 Jacob, S.R., Northcote, D.H. (1985) In vitro glucan synthesis by membranes of celery petioles: the role of the membrane in determining the type of linkage formed. J. Cell Sci. Suppl. 2, 1-11 Kauss, H. (1987) Some aspects of calcium-dependent regulation in plant metabolism. Annu. Rev. Plant Physiol. 38, 4272 Kauss, H., Jeblick, W. (1987) Solubilization, affinity chromatography and Ca z +/polyamine activation of the plasma membraneactivated 1,3-fl-o-glucan synthase. Plant Sci. 48, 63-69 Kauss, H., K6hle, H., Jeblick, W. (1983) Proteolytic activation and stimulation by Ca z + of the glucan synthase from soybean cells. FEBS Lett. 158, 84-88 Kauss, H., Waldmann, T., Quader, H. (1990) C a 2+ a s a signal in the induction of callose synthesis. In : Signal perception and transduction in higher plants, Ranjeva, R., Boudet, A., eds. Springer, Berlin Heidelberg New York, in press Kjellbom, P., Larsson, C. (1984) Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley. Physiol. Plant 62, 501-509 K6hle, H., Jeblick, W., Poten, F., Blaschek, W., Kauss, H. (1985) Chitosan-elicited callose synthesis in soybean cells as a C a 2 +dependent process. Plant Physiol. 77, 544-551 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 Lawson, S.G., Mason, T.L., Sabin, R.D., Sloan, M.E., Drake, R.R., Haley, B.E., Wasserman, B.P. (1989)UDP-glucose: (l,3)fl-glucan synthase from Daucus earota L. Plant Physiol. 90, 101-108 Ogata, K., Arakawa, M., Kasahara, T., Shiori-Nakano, K., Hiraoka, K. (1983) Detection of toxoplasma membrane antigens transferred from SDS-polyacrylamide gel to nictrocellulose with monoclonal antibody and avidin-biotin, peroxidase antiperoxidase and immunoperoxidase methods. J. Immunol. Methods 65, 75-82 Read, S.M., Delmer, DP (1987) Inhibition of mung bean UDPglucose: (1->3)-fl-glucan synthase by UDP-pyridoxal. Evidence for an active-site amino group. Plant Physiol. 85, 10081015 Robinson, D.G., Quader, H. (1981) Structure, synthesis and orientation of microfibrils. IX. A freeze-fracture investigation of the Oocystic plasma membrane after inhibitor treatment. Eur. J. Cell Biol. 25, 278-288 Shimazaki, Y., Pratt, L.H. (1985) Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Arena shoots. Planta 164, 333-334 Waldmann, T., Jeblick, W., Kauss, H. (1988) Induced net Ca 2+ uptake and callose biosynthesis in suspension-cultured plant cells. Planta 173, 88-95 Wasserman, B.P., McCarthy, K.J. (1986) Regulation of plasma membrane fl-glucan synthase from red beet root by phospholipids. Plant Physiol. 82, 396400 Wasserman, B.P., Frost, D.J., Wu, A., Reed, S.M. (1989) Identification of the UDPG-binding polypeptide of (1,3)-fl-glucan synthase of higher plants by photoaffinity labeling with 5-AzidoUDPG. In: Abstr., 5th Cell Wall Meeting, Edinburgh, Abstr. 35 Wessel, D., Flfigge, U.I. (1984) A method for the quantitative recovery of protein in dilute solutions in the presence of detergents and lipids. Anal. Biochem. 138, 141-143 Received 18 November 1989; accepted 5 January 1990
P l a n t a 9 Springer-Verlag 1990
Partial purification and immunological characterization of 1,3-fl-glucan synthase from suspension cells of Glycine m a x J. Fink, W. Jeblick, and H. Kauss Fachbereich Biologic, Universitfit Kaiserslautern, Postfach 3049, D-6750 Kaiserslautern, Federal Republic of Germany
Abstract. The plasma-membrane-localized 1,3-//-glucan synthase (EC 2.4.1.34) from suspension cultures of Glycine max (L.) Merr. was greatly enriched by a three-step purification procedure. Starting with a microsomal preparation, a six- to eightfold enrichment of the enzyme was achieved by isolating plasma-membrane vesicles in a polyethyleneglycol/dextran two-phase system. The enzyme was solubilized with the nonionic detergent digitonin and further purified 12-fold by successive centrifugations on two linear sucrose density gradients. The most purified enzyme preparation showed enrichment in a 31-kilodalton (kDa) polypeptide and was used to raise polyspecific antibodies which precipitated 1,3-//glucan synthase activity. These antibodies were purified by affinity chromatography against immobilized membrane protein fractions of lower molecular weight which were devoid of 1,3-fl-glucan synthase activity. The purified antibodies specifically labelled a single polypeptide of 31 kDa in the 1,3-//-glucan-synthase-containing heavy fractions of the first sucrose gradient indicating that this polypeptide represents part of the active enzyme complex. Key words: Callose - Cell culture (1,3-fl-glucan synthase) Digitonin - 1,3-fl-Glucan synthase (purification, immunology) - Glycine (1,3-//-glucan synthase)
Introduction Cellulose, a 1,4-fl-linked polyglucan, is the major cellwall component in higher plants. Although it was possible to detect protein complexes attached to growing cellulose microfibrils in freeze-fracture preparations of plant plasma membranes (Giddings et al. 1980; Robinson and Quader 1981), there has been no success in Abbreviations: CHAPS = 3-[(3-cholamidopropyl)-dimethylammon-
io]-l-propanesulfonic acid; SDS-PAGE=sodium dodecyl sulfatepolyacrylamide gel electrophoresis
creating an in vitro assay system for cellulose synthase of higher plants (Delmer 1987). The possibility exists that the minor amounts of 1,4-fl-linked glucans reported by various authors to be synthesized in vitro are unrelated to cellulose but reflect the biosynthesis of xyloglucan (Gordon and McLachlan 1989). Thus, any homogenization of plant cells which necessarily destroys the plasma membrane appears to result in a total loss of cellulose synthase activity. In contrast, the plasma-membrane-located 1,3-fl-glucan synthase becomes active on cell homogenization whereas in intact, undisturbed cells this enzyme is fully latent. The 1,3-fl-glucan synthase is likely to be involved in the production of callose, a polymer which is rapidly deposited in vivo as a response to mechanical injury or attack by pathogens (for citations see Kauss 1987). These observations led to the hypothesis that the two enzyme activities belong to the same enzyme complex which may change its specificity and regulatory properties on perturbation of the plasma membrane (Jacob and Northcote 1985; Delmer 1987). The mechanism of callose formation in vivo has been intensively studied by treating suspension-cultured cells with callose elicitors (K6hle et al. 1985; Kauss 1987; Waldmann et al. 1988; Kauss et al. 1990). This callose production can start about 5 min after elicitor addition and is accompanied by drastic changes in the ion concentration and pH of the surrounding medium. The concentration of Ca 2 + decreases while other electrolytes, mainly K +, are released from the cells. The induced Ca 2+ uptake may lead to a transient increase in cytoplasmic [Ca 2+]. As the plasma-membrane-located 1,3-fl-glucan synthase has an absolute requirement for Ca 2+ in the micromolar range (Kauss et al. 1983), this increase in cytoplasmic [Ca 2+] may be one of the signals initiating callose synthesis. Assay of the 1,3-fi-glucan synthase in sealed rightside-out plasma-membrane vesicles requires detergents such as digitonin to permeabilize the membrane (Fink et al. 1987). This indicates that the substrate-binding side of the enzyme is located at the cytoplasmic side of the plasma membrane. In contrast, the reaction product, cal-
344 lose, a p p e a r s t o be d e p o s i t e d o u t s i d e t h e p l a s m a m e m brane, indicating a vectorial transmembrane arrangement of the enzyme complex. T o i n v e s t i g a t e t h e f u n c t i o n o f t h e 1 , 3 - f l - g l u c a n synt h a s e c o m p l e x a n d to d e t e r m i n e w h e t h e r it is r e l a t e d to t h e c e l l u l o s e s y n t h a s e , it is n e c e s s a r y to p u r i f y the e n z y m e . W e r e p o r t h e r e o u r p r o g r e s s in this r e g a r d a n d show that a 31-kilodalton (kDa) polypeptide appears to be a s s o c i a t e d w i t h g r a d i e n t f r a c t i o n s c o n t a i n i n g a c t i v e 1,3-fl-glucan synthase.
J. Fink et al. : Partial purification of 1,3-fl-glucan synthase 4-ml portions onto linear sucrose density gradients ( 5 % 4 0 % , w/ v), in 50 mM Tes-NaOH (pH 7.0), 1 mM DTT and 0.015% (w/v) digitonin, and centrifuged for 1.5 h at 250000-g in a vertical rotor (TV 850). The gradients were fractionated into 2-ml fractions and 50-gl aliquots were immediately assayed for 1,3-fl-glucan synthase activity and protein concentration. The fractions containing 1,3-flglucan synthase activity were pooled as indicated in Fig. 1, concentrated in Amicon Centriprep vials and layered onto a second sucrose density gradient (20% 50%, w/v). After centrifugation for 1.75 h at 250000.g in the TV 850 rotor, the second gradient was fractionated into 1-ml fractions and assayed as for the first gradient. All fractions containing either enzyme activity or protein were stored at - 2 0 ~ C for further investigation.
Assay of 1,3-fl-glucan synthase. Enzyme activity was tested as de-
Material and methods Chemicals and radiochemicals. Polyethyleneglycol (PEG) 4000 was obtained from Merck (Darmstadt, FRG), dextran PL 500 VC from Pfeifer and Langen (Dormagen, FRG), digitonin from Serva (Heidelberg, FRG) and Sepharose CL-4B from Pharmacia (Heidelberg, FRG). All other chemicals were from Sigma Chemie (Miinchen, FRG) and UDP-U[14C]-glucose was purchased from Amersham (Braunschweig, FRG). Cell homogen&ation and microsomal preparation. Suspension cultures of soybean (Glycine max (L.) Merr. cv. Harasoy 63, supplied by J. Ebel, Freiburg, F R G ) were grown in darkness for 5-6 d as described by K6hle et al. (1985). Harvesting of cells and homogenization of small amounts of cells was carried out as described by Fink et al. (1987). Larger amounts of cells were homogenized in portions of 100 g in 100 ml of buffer (50 mM 2-{(hydroxy-l,1bis(hydroxymethyl)ethyl)-amino}ethanesulfonic acid (Tes)-NaOH, pH 7.0; 1 mM dithiothreitol (DTT); 0.25 M sucrose; 1 mM ethylenediamine tetraacetic acid (EDTA) by sonication in a Branson (Heinemann, Schw~ibisch Gmfind, FRG) Sonifier 250 for 2 min at maximum intensity. The microsomal preparation was obtained by three consecutive centrifugations at 500"g, 5000 g and 50 000"g as described by Fink et al. (1987).
scribed previously by Fink et al. (1987). All assays included 25 Ixg of poly-L-ornithine (44 kDa) and the concentration of free C a 2 + was about 70 ktM. Membrane-bound enzyme was measured in the presence of 0.02% (w/v) digitonin.
Protein determination. Protein was measured with Coomassie Brillant Blue G 250 by the method of Bradford (1976) with bovine serum albumin as a standard.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The pooled fractions containing protein and-or 1,3-fl-glucan synthase activity were analyzed for their polypeptide composition in a 10% gel in the presence of mercaptoethanol according to the method of Laemmli (1970). Prior to electrophoresis the proteins from the various fractions were precipitated from the detergent-containing solution according to Wessel and Fliigge (1984). Electrophoresis was carried out in a vertical mini-slab gel apparatus with 1-mm spaces at 2 mA per lane. Gels were stained with 0.25% (w/v) Coomassie Brillant Blue R250 in H20/methanol/acetic acid (5:4:1 ; by vol.).
Polyspecific antibodies to 1,3-fl-glucan synthase. The fraction with
Plasma-membrane isolation. Plasma-membrane vesicles were iso-
the highest specific 1,3-fl-glucan synthase activity (pool A, Fig. 1 B) was used to produce antibodies in a rabbit. The crude antiserum was precipitated by ammonium sulfate for isolation of the immunoglobulin fraction which was solubilized (same volume as serum used) in 100 mM Na2HPO4 (pH 7.2) containing 150 mM NaC1.
lated in a PEG/dextran two-phase system by the method of Kjellbom and Larsson (1984) as modified according to Fink et al. (1987).
Immunoprecipitation of l,3-fl-glucan synthase. Immunoprecipitation was carried out with Staphylococcus aureus cells (Pansorbin| Cal-
Solubilization of plasma-membrane proteins. Solubilization with digitonin was carried out by a two-step procedure. In the first step, each 10 ml of the plasma-membrane vesicle suspension was mixed with 10 mg of digitonin. The suspension was kept in a glass tube at 0 ~ C for 1 h and was sonicated every 10 min for 5 min in a sonifer bath filled with 0 ~ C water. The membrane material was pelleted in an AH 629 rotor at 150000"g in a Sorvall (Dupont, Bad Nauheim, F R G ) OTD 65B ultracentrifuge for 1 h. The supernatant containing part of the 1,3-fl-glucan synthase was stored at --20 ~ C. The pellet was resuspended in 10 ml of cold buffer (50 mM Tes-NaOH, pH 7.0; 1 mM DTT) and again 10 mg of digitonin were added. Furthermore, a stock solution of E D T A and ethyleneglycol-bis(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was added to result in a final concentration of 1 mM. This mixture was sonicated and centrifuged as described before and the supernatant of the second solubilization step containing additional enzyme was also stored at - 2 0 ~ C. Solubilization with 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propanosulfonic acid (CHAPS) was carried out by the method of Lawson et al. (1989) at an end concentration of 0.5% (w/v) of the detergent.
Sucrose density gradients. The solubilized enzyme fractions (200 ml, corresponding to I kg cells) were concentrated by centrifugation in Amicon (Witten, F R G ) Centriprep-vials (30 S) to a volume of 7 ml. This concentrated enzyme preparation was layered in 3- to
biochem, La Jolla, Calif., USA) as an adsorbent according to the method of Shimazaki and Pratt (1985) with the ammonium-sulfateprecipitated polyspecific antibody fraction. The same fraction from preimmune serum was used for controls.
Purification of antibodies by affinity chromatography. The bulk of the 1,3-fl-glucan synthase-free protein fractions from the first sucrose density gradient (4 mg, pool II, Fig. 1 A) was coupled to 10 ml of Sepharose CL-4B by the method of Bethell et al. (1979). The column (1.5 cm diameter) was loaded with 2 ml of a 1:10 diluted polyspecific immunoglobulin solution and washed with buffer. The protein-containing fractions (about 3 ml) which passed through the column were supplied with 0.1% NaN3. This solution of purified antibodies was stored at 0~ C for further investigation.
Immunoblot. Proteins were transferred from PAGE gels to nitrocellulose sheets in a wet-blot apparatus (Pharmacia-LKB; Freiburg F R G ; 2005 Transphor) for 2 h at 0.8 A. For immunological detection of peptides according to Ogata et al. (1983), the nitrocellulose sheets were incubated overnight at 4~ C either with the polyspecific antibody fraction in a 1:100 dilution in 100mM NazHPO4 (pH 7.2), 150 mM NaC1, 0.05% Tween 20, or with undiluted affinity-chromatography-purified antibodies. As a second antibody an anti-rabbit IgG-peroxidase conjugate was used with 4-chloro-1naphthol (0.2%, w/v) and 0.01% (v/v) H 2 0 2 as peroxidase substrates.
J. Fink et al. : Partial purification of 1,3-/%glucan synthase Results In the p l a s m a - m e m b r a n e fraction (upper phase) of the polyethyleneglycol-dextran system, the 1,3-/%glucan synthase activity was routinely enriched six- to eightfold over microsomes (Table 1). The first p l a s m a - m e m b r a n e containing upper phase showed a recovery of 4 0 % - 5 0 % of the enzyme activity. By adding a new upper phase to the first lower phase it was possible to increase the overall recovery to m o r e than 60%. Similar results were described by Kjellbom and Larsson (1984); they however, purified their p l a s m a - m e m b r a n e fraction by successive rewashing o f the first upper phase with two new lower phases. Solubilization of m e m b r a n e proteins with digitonin resulted in the loss of a considerable portion of 1,3-/% glucan synthase activity (Table 2 A). During the first step about 70% o f total protein was solubilized. In contrast, only about 12% of the 1,3-/%glucan synthase activity could be measured in the soluble fraction. The second solubilization step gave a further protein recovery o f 25% and another 12% of solubilized enzyme activity. The pellet after centrifugation of the second solubilization mixture included neither protein nor enzyme activity (data not shown). This indicates that the loss of 1,3-/3glucan synthase activity during the solubilization is not caused by insufficient solubilization of the enzyme molecules but by inactivation, presumably mainly by displacement of phospholipids and other activating compounds from the enzyme complex. Attempts to reactivate the solubilized enzyme by addition of phospholipids were not successful, in contrast to the experience of Wasserman and M c C a r t h y (1986) with red beet 1,3-/~-glucan synthase. Attempts to purify large volumes of dilute, solubilized 1,3-/~-glucan synthase using DEAE-Sepharose, Ca 2 +-loaded Chelating Sepharose (Pharmacia), Sepharose CL-4B and presumed affinity c h r o m a t o g r a p h y on immobilized spermine were unsuccessful. The latter method brought some increase in specific activity when small short columns were used (Kauss and Jeblick 1987) but scaling up was difficult due to the comparatively low capacity of the affinity material. In summary, chrom a t o g r a p h i c methods were probably not successful as they require the prolonged presence or the washing o f the bound enzyme with detergent-containing buffers. These problems were overcome by a purification protocol consisting only of rapidly performed bulk steps. Concentration of the pooled digitonin-solubilized fractions in Amicon Centriprep vials yielded a near total recovery of both protein and enzyme activity with less than 10% decrease in specific activity (data not shown). For further purification, this concentrated solubilized 1,3-/%glucan synthase was centrifuged in two successive linear sucrose density gradients (Fig. 1 A, B). In both gradients the enzyme activity appeared as a single sharp peak at approximately 30% (w/v) sucrose and was clearly separated from the bulk of solubilized m e m b r a n e protein. No second protein peak coincided with enzyme activity, indicating that the inactive proteins overlapped the 1,3-/%glucan synthase fractions. The solubilized 1,3-
345 Table 1. Enrichment of 1,3-fl-glucan synthase from soybean suspension-cultured cells in the plasma-membrane fraction of a polyethyleneglycol-dextran two-phase system. The microsomal suspension (5 ml) was added to 31 ml of a polyethyleneglycol-dextran solution to give a final concentration of 5.1% (w/w) for each of the two polymers. After 15 min centrifugation at 500.g the first upper phase was removed. A new upper phase without microsomes was added to the first lower phase to give a second two-phase system which was treated as before. The data are based on a cell fresh weight of 100 g Total activity Protein (nkat) (%) Microsomes 1. Upper phase 2. Upperphase Pool a 2. Lower phase
70.5 29.2 14.2 43.3 19.2
(mg) (%)
Specific Purifiactivity cation (nkat.mg -1) (-fold)
100 37 100 1.9 41.4 1.97 5.3 14.8 20.1 1.08 2.9 13.1 61.4 3.05 8.2 14.2 27.2 32.9 89 0.6
7.8 6.9 7.5 0.3
" The pooled first and second upper phases were used as the total plasma membrane preparation throughout this report
Table 2. Solubilization of 1,3-fl-glucan synthase from the plasmamembrane fraction using two different detergents. A) Two-step solubilization with digitonin. The first step was with 0.1% (w/v) digitonin and the second step with 0.1% (w/v) digitonin plus 1 mM EDTA and 1 mM EGTA. B) A one-step solubilization with CHAPS at a final concentration of 0.5% (w/v) in the presence of 1 mM EDTA and 1 mM EGTA
Membranes" A) Digitonin step 1 step 2 pool 1 + 2 b B) CHAPS
Total activity
Protein
(nkat)
(%)
(rag)
(%)
Specific activity (nkat. mg- 1)
43.3
100
3.05
100
14.1
2.19 0.73 2.92 3.32
71.7 23.8 95.5 109
5.3 5.2 10.5 7.8 c 13.8 d
12.2 12.0 24.2 18.0 31.8
2.4 7.1 3.6 2.3 b 4.2 c
" Total plasma-membrane fraction from line 4 of Table 1 b Although step 2 resulted in a higher specific activity the enzyme from both solubilization steps was pooled to increase the recovery, c Enzyme assayed without further addition of detergent d Same enzyme assayed in the presence of 0.02% (w/v) digitonin
/%glucan synthase was enriched approximately five fold in the first sucrose gradient (Table 3, pool I) and 12-fold after the second sucrose gradient (Table 3, pool A). Because of the inactivation of 1,3-/?-glucan synthase during digitonin solubilization (Table 2A), the total purification f r o m microsomes (Table 1) to the second density gradient (Table 2A) cannot be directly calculated from the respective specific activities. The enrichment can be estimated, however, by multiplying the purification factors from Table 3 (pool A) and from line 4 of Table 1 to be at least 90- to 100-fold. I f one considers that only about a quarter of the enzyme activity survived
346
J. Fink et al. : Partial purification of 1,3-fl-glucan synthase
i
Table 3. Enrichment of digitonin-solubilized1,3-/%glucan synthase in two successive sucrose-density-gradient centrifugation steps. Pools I and II of the first sucrose gradient refer to Fig. 1A and pools A, B and C of the second sucrose gradient to Fig. l B
/.,.
Total activity Protein
t
E
a;
I-,,
9
8
Tr
(nkat) (%)
JC c
d
~6
r" 13
~0
o
& /
_m'"
\
c)
20
++ 9
9
0 1
5
u~ o
n
2 9
i
10
15
§
20
Fraction number
B
i
, A
E El -Y
B
i1.~1
C
,,
Specific Purifiactivity cation (nkat.mg-~) (-fold)
i
Sol. enzyme" 90.0 1. Sucrose gradient Pool I 43.7 Pool II 0.7 2. Sucrose gradienP Pool A 17.3 Pool B 9.5 Pool C 2.8
100
(mg)
(%)
27.1
100
48.6 2.55 0.8 21.7 19.2 10.6 3.1
3.3
9.4 80
0.41 0.51 1.04
17.1 0.04
!.5 42.3 1.85 18.6 3.8 2.7
5.2 0.01 12.8 5.6 0.8
" Seven ml of concentrated 1,3-fl-glucan synthase from step 1 plus step 2 of the digitonin-solubilization,corresponding to the plasma membranes from 1 kg cell FW, were applied on two identical first sucrose gradients and the respective peak fractions pooled b Pool I concentrated and layered on the second gradient
i
0.8 6
El
0,6 k~ irll~ll
C
\
f,
\
u 3 (.9
&
++.~.
i
....'7
9247
= ----d:-" 10
15 2'0 Fraction number
-3"0
o,~ ~="
t,O |
0,2 9
20
0
§
Fig. 1 A, B. Distribution of digitonin-solubilized 1,3-fl-glucan synthase activity and membrane proteins from soybean cells in two successive sucrose density gradients. A First sucrose gradient. The concentrated solubilized enzyme (see Table 3) was layered on a linear 5 4 0 % (w/v) sucrose gradient and centrifuged for 1.5 h at 250000.g. B Second sucrose gradient. The concentrated pool I from the first sucrose gradient (A) was layered on a linear 20-50% (w/v) sucrose gradient and centrifuged for 1.75 h at 250000.g. Enzyme activities and enrichment in pools [ and II as well as A, B and C are reported in Table 3
the s o l u b i l i z a t i o n b u t all p r o t e i n was rendered soluble, a p u r i f i c a t i o n o f u p to 400-fold was achieved. F o r c o m p a r i s o n with the d i g i t o n i n - s o l u b i l i z a t i o n described above, the m e t h o d s o f L a w s o n et al. (1989) for the s o l u b i l i z a t i o n o f a 1,3-fl-glucan synthase from micros o m a l p r e p a r a t i o n s o f c a r r o t roots b y the detergent C H A P S were tested in s o y b e a n p l a s m a m e m b r a n e s (Table 2 B). W h e n assayed in the presence o f 0.02% digitonin, the recovery o f 1,3-/~-glucan synthase was slightly higher t h a n after d i g i t o n i n - s o l u b i l i z a t i o n b u t m e a s u r e d w i t h o u t f u r t h e r d e t e r g e n t it was lower. This C H A P S solubilized 1,3-/3-glucan synthase from s o y b e a n p l a s m a m e m b r a n e s was also subjected to the p u r i f i c a t i o n proced u r e described above. The positions o f the peaks for e n z y m e activity a n d p r o t e i n o n the two sucrose-density c e n t r i f u g a t i o n s were very similar to the results s h o w n
Table 4. Attempt to purify CHAPS-solubilized 1,3-fl-glucan synthase from soybean plasma membranes. The solubilized enzyme (similar to Table 2 B but from 1 kg of cells) was concentrated without considerable loss and centrifuged on two subsequent digitonincontaining sucrose density gradients as for Fig. 1. The positions of the enzyme-activity peaks and protein peaks were similar to those in Fig. 1 Enzyme activity (nkat) (%) Sol. enzyme a 193.3 100 First gradient Enzyme peak b 3 1 . 3 16.2 Protein peak 7.3 3.8 Second gradient c Enzyme peak d 5.8 3.0 Protein peak 3.0 1.6
Protein - (mg) (%) 31 5.5 21 0.75 0.63
100
Specific Purifiactivity cation (nkat.mg- 1) (-fold)
6.2
17.7 5.7 67.7 0.3
1.1 0.05
2.4 7.7 2.0 4.8
1.2 0.8
" Assayed in the presence of 0.02% (w/v) digitonin b Corresponding to peak I of Fig. 1A, all gradient fractions were assayed without further addition of digitonin as the gradients contained 0.015% (w/v) digitonin c Enzyme peak from the first gradient concentrated and layered on the second gradient d Corresponding in position to peak A of Fig. 1 B
for the d i g i t o n i n - s o l u b i l i z e d e n z y m e in Fig. 1. Table 4 d o c u m e n t s , however, that n o a p p a r e n t e n r i c h m e n t a n d a greater overall loss in e n z y m e activity occurred. F o r the p u r i f i c a t i o n o f the 1,3-fl-glucan synthase f r o m soyb e a n p l a s m a m e m b r a n e s , C H A P S - s o l u b i l i z a t i o n was obviously far less suitable t h a n the use of d i g i t o n i n (Table 3). We, therefore, p e r f o r m e d all further c h a r a c t e r i z a t i o n steps with the d i g i t o n i n - s o l u b i l i z e d enzyme. Analysis by S D S - P A G E o f the m o s t purified 1,3-flglucan synthase fraction from the second sucrose gradient showed a n e n r i c h m e n t in a 3 1 - k D a polypeptide (Fig. 2, lane A). In p r o t e i n fractions with little 1,3-fl-
J. Fink et al. : Partial purification of 1,3-fl-glucan synthase
347 ent fractions from the density gradients (Fig. 1) in a Western blot (Fig. 3). Comparing a protein fraction enriched in 1,3-fl-glucan synthase activity (lane A) with another devoid of this enzyme activity (lane B), we observed that a band at 31 kDa was missing in the latter. An affinity column made with immobilized proteins devoid of 1,3-fl-glucan synthase activity (pool II, Fig. 1 A) bound all polyspecific antibodies except those which labeled the 31-kDa polypeptide (lane C) also in the first sucrose density gradient.
Discussion
Fig. 2. Analysis by SDS-PAGE of peptides from 1,3-fl-glucan synthase fractions on a 10% gel. Each lane contained 25 ~tg of protein except lane B which contained 15 ~tg. The gel was stained with Coomassie Brillant blue. S, molecular weight standards; /, pool I from the first sucrose gradient (Fig. 1A); A, B and C refer to pool A, B and C of the second sucrose gradient (Fig. 1B)
Fig. 3. Immunological characterization of peptides from the 1,3-flglucan synthase preparations. The most highly purified fraction of 1,3-fl-glucan synthase (pool A from Fig. 1B, for proteins see lane A, Fig. 2) was used to raise polyspecific rabbit antibodies. Immunoblots with total serum are shown in lane A (proteins from the 1,3-fl-glucan synthase activity, pool I of Fig. 1A) and lane B (proteins devoid of 1,3-fl-glucan synthase activity, pool II of Fig. 1A). The proteins from pool II of Fig. 1A devoid of 1,3-flglucan synthase activity were immobilized on Sepharose CL-4B resulting in an affinity column. The antibodies that passed through this column were used to label lane C (fractions exhibiting 1,3-flglucan synthase activity, pool I of Fig. 1A) and lane D (proteins without 1,3-fl-glucansynthase activity, pool II of Fig. 1A)
glucan synthase activity this band was nearly absent (Fig. 2, lane C). Polyspecific antibodies against the most highly purified 1,3-fl-glucan synthase when used in a 1 : 16 dilution for immunoprecipitation gave a 20% reduction of enzyme activity in relation to control serum (data not shown). The same antibodies were taken to label differ-
The high loss in 1,3-fl-glucan synthase activity during solubilization with either digitonin or CHAPS (Table 2) again demonstrates the importance o f the hydrophobic environment o f the membrane-bound enzyme complex. Wasserman and McCarthy (1986) showed that phosphatidylcholine and similar phospholipids greatly restored 1,3-fl-glucan synthase activity which had been lost from microsomes by treatment with Triton X-100. Digitonin may partly exhibit a function similar to that of endogenous polar lipids as it could further increase the activity of CHAPS-solubilized 1,3-fl-glucan synthase (Table 2 B). The presence of digitonin was also essential in the sucrose density gradients used for the purification of the digitonin-solubilized enzyme (Fig. 1, Table 3) which resulted in an apparent 100-fold enrichment. A similar value was reported by Wasserman et al. (1989) for CHAPS-solubilized 1,3-fl-glucan synthase from red beets. Read and Delmer (1987) and Delmer and Soloman (1989) tried to identify membrane proteins related to 1,3-fl-glucan synthase by affinity-labelling with U D P [3H]pyridoxal and [32p]UDP-glucose. F r o m the various labelled bands, only two polypeptides with molecular weights o f about 34 kDa and 50 kDa were labelled in a Ca 2 +-dependent manner, indicating that they may be related to the 1,3-fl-glucan synthase known to be Ca 2 +activated. Only the 50-kDa protein co-purified with 1,3fl-glucan synthase activity in a sucrose density gradient. By contrast, a monoclonal antibody able to precipitate 1,3-fl-glucan synthase marked a double band at 6062 k D a which was, however, not enriched in the 1,3-flglucan synthase activity peak. Wasserman et al. (1989) performed similar experiments with membrane proteins from red beet and found that a 57-kD protein bound to 32p-labelled 5-azido-UDP-glucose in the presence of Ca z+ and Mg 2+, an effect reversed by E D T A and EGTA. This protein was enriched in a 1,3-fl-glucan synthase preparation purified by product-entrapment. Although these studies give some hints as to possible molecular components of the 1,3-fl-glucan synthase complex, final determination of subunit structure awaits purification. The present study with soybean suspension cells shows that a polypeptide of 31 kDa is enriched in protein fractions rich in 1,3-fl-glucan synthase activity (Fig. 2, lane A) and is almost absent in those fractions without enzyme activity (Fig. 2, lane C). Although a few
348 other protein b a n d s are also present in the m o s t purified preparation, their relationship to the 1,3-fl-glucan synthase remains unclear, as they a p p e a r also in the inactive gradient fractions (Fig. 2, lane C). In b o t h sucrose density gradients (Fig. 1) the enzyme activity is associated with a molecule p o p u l a t i o n migrating at a density greater t h a n that o f the majority o f proteins, indicating that the intact, 1,3-fl-glucan synthase c o m p l e x exhibits a higher molecular weight than the bulk o f the solubilized proteins. Nevertheless, the immunological characterization (Fig. 3) showed that the inactive protein peak in the first gradient contains antigenic determinants capable o f binding, f r o m the polyspecific antiserum, all antibodies except those specific for the 3 1 - k D a peptide. The 3 1 - k D a peptide was also enriched in the heavier fractions o f the second gradient which exhibited 1,3-fl-glucan synthase activity (Fig. 2). This might m e a n that the 1,3-fl-glucan synthase complex may, in the course o f purification, decrease in activity and b e c o m e c o n c o m i t a n t l y smaller by loosing constituents which a p p e a r in the protein peak devoid o f enzyme activity. This suggestion is admittedly based on correlations only. W h e t h e r the 3 1 - k D a peptide f r o m soybean is identical to the approx. 3 4 - k D a peptide f r o m c o t t o n and m u n g bean (Delmer a n d S o l o m o n 1989) and indeed represents an integrating part o f the active 1,3-fl-glucan synthase c o m p l e x awaits c o n f i r m a t i o n by affinity labelling with substrate analogs (Read and Delmer 1987; D e l m e r a n d S o l o m o n 1989; W a s s e r m a n et al. 1989), perf o r m e d in parallel with labelling by the antibodies specific for the 3 1 - k D a peptide. Financial support of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is acknowledged. We also thank T.J. Buckhout for many suggestions and for correcting the English manuscript. References Bethell, G.S., Ayers, J.S., Hancock, W.S. (1979) A novel method of activation of cross-linked agaroses with l,l'-carbonyldiimidazole which gives a matrix for affinity chromatography devoid of additional charged groups. J. Biol. Chem. 254, 2572-2574 Bradford, M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 Delmer, D.P. (1987) Cellulose biosynthesis. Annu. Rev. Plant Physiol. 38, 259 290 Delmer, D.P., Solomon, M. (1989) Identification of polypeptides involved in plant fl-glucan synthesis. In: Abstr., 5th Cell Wall Meeting, Edinburgh, Abstr. 36 Fink J., Jeblick, W., Blaschek, W., Kauss, H. (1987) Calcium ions and polyamines activated the plasma membrane-located 1,3-flglucan synthase. Planta 171, 130-135 Giddings, T.H., Brower, D.L., Staehelin, L.A. (1980) Visualization of particle complexes in the plasma membrane of Micrasterias denticulata associated with the formation of cellulose fibrils in primary and secondary walls. J. Cell. Biol. 84, 327-339 Gordon, R., McLachlan, G. (1989) Incorporation of UDP-
J. Fink et al. : Partial purification of 1,3-fl-glucan synthase [~4C]glucose into xyloglucan by pea membranes. Plant Physiol. 91,373-378 Jacob, S.R., Northcote, D.H. (1985) In vitro glucan synthesis by membranes of celery petioles: the role of the membrane in determining the type of linkage formed. J. Cell Sci. Suppl. 2, 1-11 Kauss, H. (1987) Some aspects of calcium-dependent regulation in plant metabolism. Annu. Rev. Plant Physiol. 38, 4272 Kauss, H., Jeblick, W. (1987) Solubilization, affinity chromatography and Ca z +/polyamine activation of the plasma membraneactivated 1,3-fl-o-glucan synthase. Plant Sci. 48, 63-69 Kauss, H., K6hle, H., Jeblick, W. (1983) Proteolytic activation and stimulation by Ca z + of the glucan synthase from soybean cells. FEBS Lett. 158, 84-88 Kauss, H., Waldmann, T., Quader, H. (1990) C a 2+ a s a signal in the induction of callose synthesis. In : Signal perception and transduction in higher plants, Ranjeva, R., Boudet, A., eds. Springer, Berlin Heidelberg New York, in press Kjellbom, P., Larsson, C. (1984) Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley. Physiol. Plant 62, 501-509 K6hle, H., Jeblick, W., Poten, F., Blaschek, W., Kauss, H. (1985) Chitosan-elicited callose synthesis in soybean cells as a C a 2 +dependent process. Plant Physiol. 77, 544-551 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 Lawson, S.G., Mason, T.L., Sabin, R.D., Sloan, M.E., Drake, R.R., Haley, B.E., Wasserman, B.P. (1989)UDP-glucose: (l,3)fl-glucan synthase from Daucus earota L. Plant Physiol. 90, 101-108 Ogata, K., Arakawa, M., Kasahara, T., Shiori-Nakano, K., Hiraoka, K. (1983) Detection of toxoplasma membrane antigens transferred from SDS-polyacrylamide gel to nictrocellulose with monoclonal antibody and avidin-biotin, peroxidase antiperoxidase and immunoperoxidase methods. J. Immunol. Methods 65, 75-82 Read, S.M., Delmer, DP (1987) Inhibition of mung bean UDPglucose: (1->3)-fl-glucan synthase by UDP-pyridoxal. Evidence for an active-site amino group. Plant Physiol. 85, 10081015 Robinson, D.G., Quader, H. (1981) Structure, synthesis and orientation of microfibrils. IX. A freeze-fracture investigation of the Oocystic plasma membrane after inhibitor treatment. Eur. J. Cell Biol. 25, 278-288 Shimazaki, Y., Pratt, L.H. (1985) Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Arena shoots. Planta 164, 333-334 Waldmann, T., Jeblick, W., Kauss, H. (1988) Induced net Ca 2+ uptake and callose biosynthesis in suspension-cultured plant cells. Planta 173, 88-95 Wasserman, B.P., McCarthy, K.J. (1986) Regulation of plasma membrane fl-glucan synthase from red beet root by phospholipids. Plant Physiol. 82, 396400 Wasserman, B.P., Frost, D.J., Wu, A., Reed, S.M. (1989) Identification of the UDPG-binding polypeptide of (1,3)-fl-glucan synthase of higher plants by photoaffinity labeling with 5-AzidoUDPG. In: Abstr., 5th Cell Wall Meeting, Edinburgh, Abstr. 35 Wessel, D., Flfigge, U.I. (1984) A method for the quantitative recovery of protein in dilute solutions in the presence of detergents and lipids. Anal. Biochem. 138, 141-143 Received 18 November 1989; accepted 5 January 1990
