Planta (1982)155:467-472
P l a n t a 9 Springer-Verlag 1982
Exoglucanases from Zea mays L. seedlings: their role in p-D-gluean hydrolysis and their potential role in extension growth* Donald J, Huber and Donald J. Nevins Vegetable Crops Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, and Department of Botany, Iowa State University, Ames, IA 50011, USA
Abstract. Exoglucanases of corn seedlings were examined and evaluated in terms of their participation in the hydrolysis of cell-wall fl-D-glucan and their possible role in extension growth. An exo-fl1,3-glucanase (EC 3.2.1.58), a component of the protein dissociated from isolated wall by use of high salt solutions, was purified using gel-filtration and ion-exchange chromatography. The purified enzyme hydrolyzed a number of polymeric and oligosaccharide substrates, including those of mixedlinkage, and their direct conversion to monosaccharide was evidence that the enzyme was capable of hydrolyzing both fll-4 and/71-3 linkages. The enzyme was considerably more active toward glucan that had been previously hydrolyzed by a cell-wall endo-/?-D-glucanase. Similarly, the capacity of the purified exo-fl-D-glucanase to degrade isolated wall was enhanced by more than 60% when the wall had been previously treated with the endoenzyme. The exo-fl-D-glucanase did not exhibit growth-promoting properties nor was its activity, measured in vivo, enhanced by auxin. Another glucanase was obtained from the soluble fraction of seedling homogenates. It functioned strictly as a fl-glucosidase and did not appear to participate in the hydrolysis of wall fl-D-glucan. Key words: Cell wall (autolysis) Glucan (t-D) Glucanase (exo-fl-D) - fl-Glucosidase - Zea.
Introduction
In monocotyledons perhaps the most apparent feature of cell-wall growth is the increased turnover or metabolism of cell-wall fl-D-clucan (Loescher * Journal Series Article 3370 of the Florida Agricultural Experiment Station, Gainesville, Fla., USA
and Nevins 1972; Sakurai and Masuda 1977). Auxin-induced growth of coleoptile tissue, when measured under conditions to minimize net wall synthesis, is accompanied by the loss of as much as 60% of the wall/LD-glucan, indicating that this wall polymer might play an important role in growth metabolism. However, direct evidence that the metabolism of this wall polymer actually affects the physical properties of the cell wall is lacking. The mechanism by which/?-D-glucan is metabolized in growing tissue is unknown. It is likely that glucanolytic enzymes, which have been reported in a number of monocotyledon sources (Katz and Ordin 1967; Tanimoto and Masuda 1968; Heyn 1969; Huber and Nevins 1981), participate in the turnover of this wall component. Huber and Nevins (1981) showed, for example, that enzymes associated with Zea cell wall hydrolyzed fl-D-glucan in a concerted reaction involving both endo- and exoglucanolytic activities. It has been primarily exoenzymes that have received attention as possible growth-promoting proteins. Masuda and cow0rkers (Masuda and Wada 1967; Masuda 1968 ; Tanimoto and Masuda 1968) reported growth-promoting properties of an exo-fl-l,3-glucanase from the fungus Sclerotinia libertiana, and Yamamoto and Nevins (1979, 1981) have more recently shown this enzyme to be rather unusual in a number of respects, most notably in its capacity to hydrolyze fl-D-glucan, a mixed-linkage polymer containing/?1~4 and/71-3 linkages. An exo-/?-l,3-glucanase derived from a basidiomycete (QM 806) was ineffective at degrading/?-Dglucan, presumably because of its inability to degrade the fll-4 linkages, and had no activity in coleoptile growth assays (Yamamoto and Nevins 1981). Information is lacking regarding the characteristics and physiological roles of exoglucanases 0032-0935/82/0155/0467/$01.20
468
D.J. Huber and D.J. Nevins: Properties of Zea exoglucanases
f r o m h o r m o n e - s e n s i t i v e tissue. E x o - f l - l , 3 - g l u c a n a s e ( H u b e r a n d N e v i n s 1981) a n d e x o c e l l u l a s e ( H e y n 1969) a c t i v i t i e s h a v e b e e n r e p o r t e d i n m o n o c o t y l e d o n s b u t it is n o t k n o w n h o w t h e s e e n z y m e s , e i t h e r a l o n e o r in c o m b i n a t i o n , m i g h t b e i n v o l v e d in t h e h y d r o l y s i s o f m i x e d - l i n k a g e f l - D - g l u c a n . I n t h e i n v e s t i g a t i o n d e s c r i b e d in t h i s p a p e r w e examined the characteristics and multiplicity of the Zea exoglucanases and attempted to evaluate their r o l e in t h e h y d r o l y s i s o f f l - D - g l u c a n a n d t h e i r p o s sible r o l e in e x t e n s i o n g r o w t h .
"Buffer-soluble" glucanase was derived from the wall-free filtrate from seedling homogenates (prepared in 10 mM Naphosphate buffer, pH 6.5) as described earlier (Huber and Nevins 1981). Briefly, after filtering the homogenate (prepared form 300 g seedling tissue), the filtrate was centrifuged for 20 min at 10,000 g, and the pellet discarded. The supernatant was adjusted to 80% saturation with NH~(SO4)2 and after 12 h at 4 ~ C the preparation was centrifuged for 20 min at 15,000 g. The pellet was resuspended in and dialyzed against Na-citratephosphate buffer (10 raM, pH 5.5) containing 0.02% NaN 3 and used for further study.
Material and methods Plant material. Imbided corn caryopses (Zea mays L., hybrid B73 X Mo17; Brayton Seed Co., Ames, Ia., USA) were sown on vermiculite, maintained under red light (0.9 W m-2, 7.5-W tungsten lamp; filtered through Rohm and Haas red plastic No. 2423. Corth Plastics, Redwood City, Cal., USA) for 72 h, and then transferred to darkness for an additional 12 h. After 84 h, the seedlings shoots consisting of all tissue above the coleoptilear node were excised and stored at --20 ~ C. For growth experiments and for measuring in-vivo glucanase activity, coleoptiles were excised after 84 h. Preparation and pur~'cation of Zea glucanases. Cell wall was prepared by homogenizing seedling tissue in buffer and filtering (Huber arid Nevins 1979) and the glucanases extracted from the wall fraction with 3M LiC1 (Huber and Nevins 1980). Approximately 14 nag of wall protein derived from 300 g Zea seedling tissue were dialyzed against Na-citrate-phosphate buffer (10 raM, pH 5.5) containing 0.02% NaN 3 and then concentrated to a volume of 10 ml employing a PM-10 ultrafiltration ceil (Amicon Corp., Lexington, Mass., USA). Initial purification of cell-wall protein was performed on a bed of Bio-Gel P-100 (Bio-Rad Laboratories, Richmond, Cal., USA) (65 cm high, 2.5 cm wide) as described in Huber and Nevins (1981). This step removed the major portion of endo-fl-D-glucanase present in the salt-dissociated wall protein. P-100 fractions containing exo-/~-D-glucanase (95-130 ml), determined using laminarin as substrate, were combined and concentrated to a volume of 10 ml (PM-10; Amicon). The concentrate, containing around 4 mg protein (Bio-Rad determination 1), was dialyzed against Na-acetate buffer (20 raM, pH 5.2) containing 20 mM NaC1 and 0.02% NaN 3. The exo-fl-D-glucanase was further purified on a bed (15 cm high, J.2 cm wide) of CM-Sephadex (Sigma Chemical Co., St. Louis, Mo., USA), equilibrated and packed in Na-acetate buffer (20raM, pH 5.2) containing 20 mM NaC1. The protein was applied to the column and eluted with the acetate buffer. After 90 ml were collected, a sodiumchloride gradient generated with 125 ml of the starting buffer and 125 ml of starting buffer containing 0.5 M NaC1 was passed through the column. Fractions of 3 ml were collected at a rate of 10 ml cm -2 h -1. Protein was monitored at 280 nm. Exo-flD-glucanase was assayed using laminarin (Koch-Light Laboratories, Colnbrook, Berks, UK) and Arena caryopsis glucan (Quaker Oats, Barrington, Ill., USA) as substrates. Laminarin (0.5 mg) or Arena glucan (1 rag) in 0.5 ml Na-citrate-phosphate buffer (10 mM, pH 5.5) and 0.1 ml of the CM-Sephadex fractions were incubated for 10 rain (laminarin) or 3 h (glucan) at 34~ C. Products were measured reductometrically using the Nelson-Somogyi procedure (Somogyi 1952). 1 See Instruction Manual, p. 8, Bio-Rad Laboratories, Richmond, Cal., USA 1979
Enzyme assays. Substrates were prepared in Na-citrate-phosphate buffer (10 mM, pH 5.5) containing 0.02% NaN 3. Substrates tested included laminarin (1 mg ml - x); Arena caryopsis glucan (1 mg ml- 1) ; lichenan from Cetraria islandica (1 mg ml- 1; Sigma) cellotetraose (200 gg ml- 1), prepared from acid-hydrolyzed cellulose paper (Yamamoto and Nevins 1979); p-nitrophenyl-fl-D-glucoside (7.5 m g m l - i ) ; and endoenzymegenerated cell-wall glucan (1 mg ml 1). Endoenzyme-generated glucan was prepared by permitting cell wall to autolyze in the presence of 100 gM HgC12 provided to inhibit wall-bound exoenzyme activity (Huber and Nevins 1980). Autolytically liberated glucan was recovered by filtration through Miracloth (Calbiochem-Behring Corp., LaJolla, Cal., USA) and then dialyzed against Na-citrate-phosphate buffer (10 mM, pH 5.5). To facilitate the removal of inorganic mercury, L-cysteine (Sigma) was added at a final concentration of 10 mM prior to dialysis. Assay mixtures contained 0.5 ml substrate along with 20 gg (0.1 ml) of protein prepared from a direct extraction of celt wall with 3 M LiC1 (" unpurified "), 5 gg (0.1 ml) of P-100 purified exo-fl-D-glucanase, i gg (0.1 ml) of the CM-Sephadex purified exo-fl-D-glucanase, or 11 gg of the "buffer-soluble" protein. Reaction mixtures were incubated at 34 ~ C for 5 rain (cellotetraose); 10rain (laminarin, p-NO2-phenyl-fl-D-glucoside, endoenzyme-generated glucan); 20 rain (lichenan) or 3 h (Arena glucan), p-NOz-phenylglucoside hydrolase activity was determined after adding 2 ml of 200 mM Na2CO3, and measuring free nitrophenol at 400 nm. Activity against all other substrates was measured reductometrically (Somogyi 1952). pH dependency of Zea glucanases. The pH characteristics of the purified exo-fl-D-glucanase were examine using laminarin (1 mg ml-1), endoenzyme-generated glucan (1 mg ml-1) and p-nitrophenyl-fl-D-glucoside(7.5 mg ml- 1) as substrates. Reaction mixtures consisted of 1.0 ml substrate (prepared in distilled H20), 0.2 ml Na-citrate-phosphate buffer prepared over the pH range 3-8, and 0.1 ml (1 gg protein) of the purified exo-fl-Dglucanase. These were incubated at 34~ C for 10 rain (laminarin and nitrophenyl glucoside) or 20 rain (glucan). Products generated were measured reductometricaUy (laminarin and glucan) or by measuring free nitrophenol at 400 nm. Buffer-soluble glucanase was assayed using p-nitrophenyl/?-D-glucoside. One ml of substrate (7.5 rag) along with 0.2 ml buffer and 0.1 ml enzyme (3 gg protein) were incubated for 10 min at 34 ~ C. pH dependency of cell-wall autolytic reactions. Cell wall for autolysis experiments was prepared as described in Huber and Nevins (1979). Approximately 50 mg of freshly prepared cell wall were placed in 10 ml of Na-citrate-phosphate buffer (20 raM, pH 3.0-8.0) and incubated 12 h at 34 ~ C. At intervals, samples were removed and filtered through glass fiber filter discs. The filtrate was analyzed for total and reducing sugars using the phenol-sulfuric acid (Hodge and Hofreitor 1962) and Nelson-Somogyi (Somogyi 1952) methods, respectively. In an experiment designed to measure autolytic reactions in the
D.J. Huber and D.J. Nevins : Properties of Zea exoglucanases
469
absence of exoenzyme activity, buffers were provided with 100 ~tM HgC12 (Hnber and Nevins 1980). After terminating the autolysis reactions the residual wall glucan was measured employing a B. subtilis fl-D-glucanase (Huber and Nevins 1979). Wall-hydrolyzing activity of the Zea glucanases. Cell wall was prepared as described in Huber and Nevins (1979) and then boiled to inactivate wall-bound enzymes. Endoenzyme-modifled wall was prepared by permitting wall to autolyze for 4 h in the presence of 100 gM HgCI2 prior to boiling. Approximately 30 mg of cell wall or endoenzyme-modified wall in 10 ml of Na-citrate-phosphate buffer (10 mM, pH 5.5) containing NaN 3 were incubated with 15 gg of the purified exo-fl-D-glucanase or with 200 Ltg of protein derived from the soluble fraction of seedling homogeuates. At intervals subsamples were removed, filtered through glass-fiber filter discs, and measured for total soluble sugars using the phenol-sulfuric acid method. Growth experiments and assay of in-vivo glucanase activity. Coleoptiles were excised and floated on distilled water for a period of 90 rain. Afterwards from each coleoptile one 5-mm section was prepared with the aid of a double-bladed cutter. For the growth experiments coteoptiles were incubated in 4 ml solutions of K-citrate buffer (1 raM, pH 5.8) containing exo-fl-D-glucanase protein recovered following CM-Sephadex chromatography at concentrations ranging from 0.1 to 50 gg ml-1. All manipulations were performed under a safelight (0.3 W m -z, 7.5-Wtungsten lamp; filtered through "Green 545", Carolina Biological Supply, Burlington, N.C., USA). In-vivo enzyme activity was determined with coloptiles incubated in 4 ml of K-citrate buffer (1 raM, pH 5.8) containing laminarin or endoenzyme-modified glncan, both provided at 1 mg m1-1. Auxin was provided at 5-10 -5 M. After 6h, aliquots of the bathing solutions were removed and measured using the Nelson-Somogyi reductometric procedure (Somogyi 1952).
Results and discussion
Purification of cell wall exo-fl-D-glucanase. The requirement in glucan hydrolysis for exoenzyme activity is a consequence of the somewhat unusual action pattern of the endo-/~-D-glucanase, an enzyme that is specific for mixed-linkage substrates but which has little affinity for polymers having a molecular weight of less than 104 (Huber and Nevins 1980). This limited hydrolytic capacity is in contrast to that of the endo-fl-D-glucanases from dicotyledons, which generate glucose and oligosaccharides from substrates of mixed-linkage (Wong and Maclachlan 1979). During autolytic reactions in Zea, the polymeric products generated by the endoenzyme are converted directly to glucose by an exoenzyme(s) that is sensitive to mercury and nojirimycin (Huber and Nevins 1980). Huber and Nevins (1981) employed gel-filtration chromatography in showing that at least one exoenzyme, an exo-fl-l,3-glucanase, participated in autolytic reactions. It was not demonstrated however that this enzyme degraded/%D-glucan without the assistance of an enzyme specific for the /3
3,8
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P l a n t a 9 Springer-Verlag 1982
Exoglucanases from Zea mays L. seedlings: their role in p-D-gluean hydrolysis and their potential role in extension growth* Donald J, Huber and Donald J. Nevins Vegetable Crops Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, and Department of Botany, Iowa State University, Ames, IA 50011, USA
Abstract. Exoglucanases of corn seedlings were examined and evaluated in terms of their participation in the hydrolysis of cell-wall fl-D-glucan and their possible role in extension growth. An exo-fl1,3-glucanase (EC 3.2.1.58), a component of the protein dissociated from isolated wall by use of high salt solutions, was purified using gel-filtration and ion-exchange chromatography. The purified enzyme hydrolyzed a number of polymeric and oligosaccharide substrates, including those of mixedlinkage, and their direct conversion to monosaccharide was evidence that the enzyme was capable of hydrolyzing both fll-4 and/71-3 linkages. The enzyme was considerably more active toward glucan that had been previously hydrolyzed by a cell-wall endo-/?-D-glucanase. Similarly, the capacity of the purified exo-fl-D-glucanase to degrade isolated wall was enhanced by more than 60% when the wall had been previously treated with the endoenzyme. The exo-fl-D-glucanase did not exhibit growth-promoting properties nor was its activity, measured in vivo, enhanced by auxin. Another glucanase was obtained from the soluble fraction of seedling homogenates. It functioned strictly as a fl-glucosidase and did not appear to participate in the hydrolysis of wall fl-D-glucan. Key words: Cell wall (autolysis) Glucan (t-D) Glucanase (exo-fl-D) - fl-Glucosidase - Zea.
Introduction
In monocotyledons perhaps the most apparent feature of cell-wall growth is the increased turnover or metabolism of cell-wall fl-D-clucan (Loescher * Journal Series Article 3370 of the Florida Agricultural Experiment Station, Gainesville, Fla., USA
and Nevins 1972; Sakurai and Masuda 1977). Auxin-induced growth of coleoptile tissue, when measured under conditions to minimize net wall synthesis, is accompanied by the loss of as much as 60% of the wall/LD-glucan, indicating that this wall polymer might play an important role in growth metabolism. However, direct evidence that the metabolism of this wall polymer actually affects the physical properties of the cell wall is lacking. The mechanism by which/?-D-glucan is metabolized in growing tissue is unknown. It is likely that glucanolytic enzymes, which have been reported in a number of monocotyledon sources (Katz and Ordin 1967; Tanimoto and Masuda 1968; Heyn 1969; Huber and Nevins 1981), participate in the turnover of this wall component. Huber and Nevins (1981) showed, for example, that enzymes associated with Zea cell wall hydrolyzed fl-D-glucan in a concerted reaction involving both endo- and exoglucanolytic activities. It has been primarily exoenzymes that have received attention as possible growth-promoting proteins. Masuda and cow0rkers (Masuda and Wada 1967; Masuda 1968 ; Tanimoto and Masuda 1968) reported growth-promoting properties of an exo-fl-l,3-glucanase from the fungus Sclerotinia libertiana, and Yamamoto and Nevins (1979, 1981) have more recently shown this enzyme to be rather unusual in a number of respects, most notably in its capacity to hydrolyze fl-D-glucan, a mixed-linkage polymer containing/?1~4 and/71-3 linkages. An exo-/?-l,3-glucanase derived from a basidiomycete (QM 806) was ineffective at degrading/?-Dglucan, presumably because of its inability to degrade the fll-4 linkages, and had no activity in coleoptile growth assays (Yamamoto and Nevins 1981). Information is lacking regarding the characteristics and physiological roles of exoglucanases 0032-0935/82/0155/0467/$01.20
468
D.J. Huber and D.J. Nevins: Properties of Zea exoglucanases
f r o m h o r m o n e - s e n s i t i v e tissue. E x o - f l - l , 3 - g l u c a n a s e ( H u b e r a n d N e v i n s 1981) a n d e x o c e l l u l a s e ( H e y n 1969) a c t i v i t i e s h a v e b e e n r e p o r t e d i n m o n o c o t y l e d o n s b u t it is n o t k n o w n h o w t h e s e e n z y m e s , e i t h e r a l o n e o r in c o m b i n a t i o n , m i g h t b e i n v o l v e d in t h e h y d r o l y s i s o f m i x e d - l i n k a g e f l - D - g l u c a n . I n t h e i n v e s t i g a t i o n d e s c r i b e d in t h i s p a p e r w e examined the characteristics and multiplicity of the Zea exoglucanases and attempted to evaluate their r o l e in t h e h y d r o l y s i s o f f l - D - g l u c a n a n d t h e i r p o s sible r o l e in e x t e n s i o n g r o w t h .
"Buffer-soluble" glucanase was derived from the wall-free filtrate from seedling homogenates (prepared in 10 mM Naphosphate buffer, pH 6.5) as described earlier (Huber and Nevins 1981). Briefly, after filtering the homogenate (prepared form 300 g seedling tissue), the filtrate was centrifuged for 20 min at 10,000 g, and the pellet discarded. The supernatant was adjusted to 80% saturation with NH~(SO4)2 and after 12 h at 4 ~ C the preparation was centrifuged for 20 min at 15,000 g. The pellet was resuspended in and dialyzed against Na-citratephosphate buffer (10 raM, pH 5.5) containing 0.02% NaN 3 and used for further study.
Material and methods Plant material. Imbided corn caryopses (Zea mays L., hybrid B73 X Mo17; Brayton Seed Co., Ames, Ia., USA) were sown on vermiculite, maintained under red light (0.9 W m-2, 7.5-W tungsten lamp; filtered through Rohm and Haas red plastic No. 2423. Corth Plastics, Redwood City, Cal., USA) for 72 h, and then transferred to darkness for an additional 12 h. After 84 h, the seedlings shoots consisting of all tissue above the coleoptilear node were excised and stored at --20 ~ C. For growth experiments and for measuring in-vivo glucanase activity, coleoptiles were excised after 84 h. Preparation and pur~'cation of Zea glucanases. Cell wall was prepared by homogenizing seedling tissue in buffer and filtering (Huber arid Nevins 1979) and the glucanases extracted from the wall fraction with 3M LiC1 (Huber and Nevins 1980). Approximately 14 nag of wall protein derived from 300 g Zea seedling tissue were dialyzed against Na-citrate-phosphate buffer (10 raM, pH 5.5) containing 0.02% NaN 3 and then concentrated to a volume of 10 ml employing a PM-10 ultrafiltration ceil (Amicon Corp., Lexington, Mass., USA). Initial purification of cell-wall protein was performed on a bed of Bio-Gel P-100 (Bio-Rad Laboratories, Richmond, Cal., USA) (65 cm high, 2.5 cm wide) as described in Huber and Nevins (1981). This step removed the major portion of endo-fl-D-glucanase present in the salt-dissociated wall protein. P-100 fractions containing exo-/~-D-glucanase (95-130 ml), determined using laminarin as substrate, were combined and concentrated to a volume of 10 ml (PM-10; Amicon). The concentrate, containing around 4 mg protein (Bio-Rad determination 1), was dialyzed against Na-acetate buffer (20 raM, pH 5.2) containing 20 mM NaC1 and 0.02% NaN 3. The exo-fl-D-glucanase was further purified on a bed (15 cm high, J.2 cm wide) of CM-Sephadex (Sigma Chemical Co., St. Louis, Mo., USA), equilibrated and packed in Na-acetate buffer (20raM, pH 5.2) containing 20 mM NaC1. The protein was applied to the column and eluted with the acetate buffer. After 90 ml were collected, a sodiumchloride gradient generated with 125 ml of the starting buffer and 125 ml of starting buffer containing 0.5 M NaC1 was passed through the column. Fractions of 3 ml were collected at a rate of 10 ml cm -2 h -1. Protein was monitored at 280 nm. Exo-flD-glucanase was assayed using laminarin (Koch-Light Laboratories, Colnbrook, Berks, UK) and Arena caryopsis glucan (Quaker Oats, Barrington, Ill., USA) as substrates. Laminarin (0.5 mg) or Arena glucan (1 rag) in 0.5 ml Na-citrate-phosphate buffer (10 mM, pH 5.5) and 0.1 ml of the CM-Sephadex fractions were incubated for 10 rain (laminarin) or 3 h (glucan) at 34~ C. Products were measured reductometrically using the Nelson-Somogyi procedure (Somogyi 1952). 1 See Instruction Manual, p. 8, Bio-Rad Laboratories, Richmond, Cal., USA 1979
Enzyme assays. Substrates were prepared in Na-citrate-phosphate buffer (10 mM, pH 5.5) containing 0.02% NaN 3. Substrates tested included laminarin (1 mg ml - x); Arena caryopsis glucan (1 mg ml- 1) ; lichenan from Cetraria islandica (1 mg ml- 1; Sigma) cellotetraose (200 gg ml- 1), prepared from acid-hydrolyzed cellulose paper (Yamamoto and Nevins 1979); p-nitrophenyl-fl-D-glucoside (7.5 m g m l - i ) ; and endoenzymegenerated cell-wall glucan (1 mg ml 1). Endoenzyme-generated glucan was prepared by permitting cell wall to autolyze in the presence of 100 gM HgC12 provided to inhibit wall-bound exoenzyme activity (Huber and Nevins 1980). Autolytically liberated glucan was recovered by filtration through Miracloth (Calbiochem-Behring Corp., LaJolla, Cal., USA) and then dialyzed against Na-citrate-phosphate buffer (10 mM, pH 5.5). To facilitate the removal of inorganic mercury, L-cysteine (Sigma) was added at a final concentration of 10 mM prior to dialysis. Assay mixtures contained 0.5 ml substrate along with 20 gg (0.1 ml) of protein prepared from a direct extraction of celt wall with 3 M LiC1 (" unpurified "), 5 gg (0.1 ml) of P-100 purified exo-fl-D-glucanase, i gg (0.1 ml) of the CM-Sephadex purified exo-fl-D-glucanase, or 11 gg of the "buffer-soluble" protein. Reaction mixtures were incubated at 34 ~ C for 5 rain (cellotetraose); 10rain (laminarin, p-NO2-phenyl-fl-D-glucoside, endoenzyme-generated glucan); 20 rain (lichenan) or 3 h (Arena glucan), p-NOz-phenylglucoside hydrolase activity was determined after adding 2 ml of 200 mM Na2CO3, and measuring free nitrophenol at 400 nm. Activity against all other substrates was measured reductometrically (Somogyi 1952). pH dependency of Zea glucanases. The pH characteristics of the purified exo-fl-D-glucanase were examine using laminarin (1 mg ml-1), endoenzyme-generated glucan (1 mg ml-1) and p-nitrophenyl-fl-D-glucoside(7.5 mg ml- 1) as substrates. Reaction mixtures consisted of 1.0 ml substrate (prepared in distilled H20), 0.2 ml Na-citrate-phosphate buffer prepared over the pH range 3-8, and 0.1 ml (1 gg protein) of the purified exo-fl-Dglucanase. These were incubated at 34~ C for 10 rain (laminarin and nitrophenyl glucoside) or 20 rain (glucan). Products generated were measured reductometricaUy (laminarin and glucan) or by measuring free nitrophenol at 400 nm. Buffer-soluble glucanase was assayed using p-nitrophenyl/?-D-glucoside. One ml of substrate (7.5 rag) along with 0.2 ml buffer and 0.1 ml enzyme (3 gg protein) were incubated for 10 min at 34 ~ C. pH dependency of cell-wall autolytic reactions. Cell wall for autolysis experiments was prepared as described in Huber and Nevins (1979). Approximately 50 mg of freshly prepared cell wall were placed in 10 ml of Na-citrate-phosphate buffer (20 raM, pH 3.0-8.0) and incubated 12 h at 34 ~ C. At intervals, samples were removed and filtered through glass fiber filter discs. The filtrate was analyzed for total and reducing sugars using the phenol-sulfuric acid (Hodge and Hofreitor 1962) and Nelson-Somogyi (Somogyi 1952) methods, respectively. In an experiment designed to measure autolytic reactions in the
D.J. Huber and D.J. Nevins : Properties of Zea exoglucanases
469
absence of exoenzyme activity, buffers were provided with 100 ~tM HgC12 (Hnber and Nevins 1980). After terminating the autolysis reactions the residual wall glucan was measured employing a B. subtilis fl-D-glucanase (Huber and Nevins 1979). Wall-hydrolyzing activity of the Zea glucanases. Cell wall was prepared as described in Huber and Nevins (1979) and then boiled to inactivate wall-bound enzymes. Endoenzyme-modifled wall was prepared by permitting wall to autolyze for 4 h in the presence of 100 gM HgCI2 prior to boiling. Approximately 30 mg of cell wall or endoenzyme-modified wall in 10 ml of Na-citrate-phosphate buffer (10 mM, pH 5.5) containing NaN 3 were incubated with 15 gg of the purified exo-fl-D-glucanase or with 200 Ltg of protein derived from the soluble fraction of seedling homogeuates. At intervals subsamples were removed, filtered through glass-fiber filter discs, and measured for total soluble sugars using the phenol-sulfuric acid method. Growth experiments and assay of in-vivo glucanase activity. Coleoptiles were excised and floated on distilled water for a period of 90 rain. Afterwards from each coleoptile one 5-mm section was prepared with the aid of a double-bladed cutter. For the growth experiments coteoptiles were incubated in 4 ml solutions of K-citrate buffer (1 raM, pH 5.8) containing exo-fl-D-glucanase protein recovered following CM-Sephadex chromatography at concentrations ranging from 0.1 to 50 gg ml-1. All manipulations were performed under a safelight (0.3 W m -z, 7.5-Wtungsten lamp; filtered through "Green 545", Carolina Biological Supply, Burlington, N.C., USA). In-vivo enzyme activity was determined with coloptiles incubated in 4 ml of K-citrate buffer (1 raM, pH 5.8) containing laminarin or endoenzyme-modified glncan, both provided at 1 mg m1-1. Auxin was provided at 5-10 -5 M. After 6h, aliquots of the bathing solutions were removed and measured using the Nelson-Somogyi reductometric procedure (Somogyi 1952).
Results and discussion
Purification of cell wall exo-fl-D-glucanase. The requirement in glucan hydrolysis for exoenzyme activity is a consequence of the somewhat unusual action pattern of the endo-/~-D-glucanase, an enzyme that is specific for mixed-linkage substrates but which has little affinity for polymers having a molecular weight of less than 104 (Huber and Nevins 1980). This limited hydrolytic capacity is in contrast to that of the endo-fl-D-glucanases from dicotyledons, which generate glucose and oligosaccharides from substrates of mixed-linkage (Wong and Maclachlan 1979). During autolytic reactions in Zea, the polymeric products generated by the endoenzyme are converted directly to glucose by an exoenzyme(s) that is sensitive to mercury and nojirimycin (Huber and Nevins 1980). Huber and Nevins (1981) employed gel-filtration chromatography in showing that at least one exoenzyme, an exo-fl-l,3-glucanase, participated in autolytic reactions. It was not demonstrated however that this enzyme degraded/%D-glucan without the assistance of an enzyme specific for the /3
3,8
o.6T
T
I,~,~ 'x ,~. ~
0 cO oJ
