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Solubilization of wall-bound peroxidases by limited proteolysis
0031-9422/93 $6 .00+0 .00
Phytochemistry, Vol. 33, No . 4 . pp. 765-767, 1993
Pergamon Press Ltd
Printed in Great Britain.
SOLUBILIZATION OF WALL-BOUND PEROXIDASES BY LIMI D PROTEOLYSIS GORDON
J. MCDOUGALL
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, U .K . (Received in revised form 1 October 1992)
Key Word Index- Linum usitatissimum ; Linaceae; flax; lignin; cell wall-bound peroxidases .
Abstract-The cell wall residue, enriched in lignin, which was obtained from non-xylem tissues of flax stems after extensive digestion with the glycanase mixture, Driselase, had appreciable peroxidase activity which was tightly bound to the wall fragments . A portion of this residual activity was solubilized by limited proteolysis using trypsin . Nondenaturing gel electrophoresis of the trypsin-solubilized extracts revealed the presence of at least one highly mobile anionic peroxidase isozyme.
due either to the removal of further peroxidases from the wall by the second Driselase treatment, although no peroxidase activity was detected in the soluble digest, or to the inactivation of wall-bound peroxidases by the proteolysic activity present in Driselase [5] . In any case, the imposition of these severe conditions effectively percludes the possibility that peroxidases remain attached to the wall-bound residue by bonds normally susceptible to Driselase . Driselase, although it contains a host of cell walldegrading enzymes, lacks ligninase activity [1] and the wall-bound residue is enriched in lignin relative to the original wall preparation (results not shown) . In fact, the wall-bound residue is prepared in a similar manner to `enzyme-liberated' lignin preparations [6] . A portion of the peroxidases remaining in the wall-bound residue can be solubilized by the action of trypsin (Table 1) . The use of non-denaturing gel electrophoresis has shown that the trypsin-solubilized extracts contain at least one highly anionic peroxidase isozyme (Fig. 1). The extracts contained no detectable cationic isoforms . There are two possible means by which these peroxidases may be attached to the wall and released by limited proteolysis . Firstly, the peroxidases may have been attached to other wall proteins. In this model, rather selective proteolysis of these wall proteins would be required to release the peroxidases . Alternatively, the trypsin-solubilized peroxidases may have been attached to the wall by lignin-tyrosine bonds . It has been shown that peroxidases used to polymerize cinnamyl alcohols in vitro become covalently attached, via lignin-tyrosine bonds, to the product lignin [7] . The insolubilized ligninbound peroxidases retain their activity . Trypsin could release peroxidases, linked in such a manner, if it cleaved the polypeptide backbone of the peroxidase at the site that separated the anchoring lignintyrosine residue from the rest of the enzyme. Such a site
INTRODUCTION
Cell wall-bound peroxidases can be routinely fractionated for study by methods that exploit their means of attachment to the wall matrix. The ionically bound peroxidases can be eluted from the wall by ion exchange using metal ions . Covalently bound wall peroxidases can be obtained after homogenizing plant tissue in buffers containing salts and detergents to produce an insoluble wall fraction, free of intracellular contaminants [1]. The covalently bound enzymes can then be subfractionated into those released by treatment with cell wall degrading glycanases or those which remain tightly bound to the wall residue [2] . It has been found that the levels of residual or intrinsic peroxidases that remain in the wall after glycanase digestion rise sharply during the lignification of tracheary elements [3] and of flax fibres [4]. This paper reports the solubilization of peroxidases from the glycanase-resistant wall residue by limited proteolysis using trypsin. The nature of the bonds through which those peroxidases are attached to the wall is discussed .
RESULTS AND DISCUSSION
The levels of peroxidase activity in the various cell wall fractions obtained in three separate preparations are shown in Table 1 . The peroxidase activity in the intrinsic wall-bound residue is much lower than previously reported [4]. This is due to two factors . Firstly, the wallbound residue was subjected to a more stringent washing procedure than before to remove all peroxidases not covalently bound to the wall . This is illustrated by the removal of peroxidases made soluble by Driselase treatment but which had remained ionically bound to the wall by using elution with Cal + ions . Secondly, the inclusion of a second Driselase digestion reduced the activity of the wall residue markedly (results not shown) . This may be 765
766
G . J. MCDOUGALL Table. 1. Peroxidase activity of wall-associated fractions Peroxidase activity (units g -' fr. wt)*
I II III
Ionically bound
Covalently bound
Driselase solubilized
Driselase Ca" solubilized-wash
Wall-bound Trypsin residue solubilized
768±12 786±9 747+37
51±1 65±4 61+4
75±3 112±7 102+9
12±1 10±2 17+2
2.4±0.1 3.1±0.2 4.4+0.1
3 .9±0.1 2.1±0.1 2.2+0.1
*One unit of peroxidase activity is defined as that amount of enzyme that causes an increase in absorbance of 0.1 units min ` in the standard assay . All peroxidase values are averages of four replicates ± s .e.
a
b
c
Al A2 A3
A4
Fig. 1 . Wall-associated peroxidase isozymes . The anode is at the bottom of the gel . Lane (a) contains 2 µg of wall protein from the ionically bound fraction . Lane (b) contains 10 pg of wall protein from the Driselase-solubilized fraction. Lane (c) contains 100 pg of wall protein from the trypsin-solubilized fraction . A, is the least mobile anionic isozyme.
may exist. Horseradish peroxidase C contains five tyrosine residues [8] but only one of these tyrosine residues is accessible to ionization studies in the native, active enzyme [9] . Taking into account the common tertiary domain structure shared by plant peroxidases [10], an examination of the sequences of plant peroxidases [8, 11-15] reveals that only one tyrosine residue is in a location on the polypeptide chain that would allow it to be involved in a linkage with lignin that retained the catalytic activity of the peroxidase . This specific tyrosine residue lies close to the amino terminus of the enzymes in a flexible region of the protein which would also be accessible to proteolytic attack by trypsin . Given the known stability of native plant peroxidases to trypsin degradation [8], it is possible to speculate how trypsin
could liberate peroxidase activity which had been linked to lignin in this manner . This model predicts that the trypsin-solubilized peroxidases would be structurally altered compared with other isoenzymes . Studies are in progress to purify and examine the putative lignin-bound enzymes . EXPERIMENTAL
Plant material . Flax (Linum usitatissimum var. Belinka) plants were cultivated as described previously [16]. At 8 weeks after seedling emergence 150 flax plants were harvested and the first 3 cm of stem above the cotyledonary node excised from each . The stem segments were split longitudinally and fibre-bearing tissue (cortex, fibres,
767
Cell wall-bound peroxidases parenchyma and epidermis) peeled from the central xylem core . The peeled tissue was kept on ice, washed, blotted dry then weighed . The extent of fibre lignification was estimated microscopically as before [16] at ca 20% . This figure means that about one in five fibres showed visible signs of lignin deposition . Isolation of wall-associated peroxidases . lonically bound and freely soluble proteins were collected selectively from the intercellular air and wall spaces by vacuum infiltration [3] . Briefly, the peeled tissue was submerged in 100 mM CaC12 and subjected to a reduced pressure ( max. at 15 mm Hg) for 10 min. Thereafter the vacuum was dissipated and the CaC1 2 soln allowed to infiltrate into the evacuated air spaces of the tissue . The infiltrate and excess bathing soln was collected by low speed centrifugation (1000 g, 5 min). This extract is called the ionically bound fraction . A cell wall prepn was obtained by repeatedly homogenizing the same tissue in 50 mM Tris-HC1 pH 8 containing 1 M NaCl, 0.5% (v/v) Triton X-100 and 0 .5% (w/v) bovine serum albumin at 4° . The insoluble material after 8-10 rounds of homogenization/centrifugation was washed 3 times with H 2 O then resuspended in H20 . This insoluble material is the covalently bound wall sample . Driselase treatment of cell walls . Partially purified Driselase [1] at a final concn of l0mgml - ' in 250 mM NaOAc pH 4 .5 was added to cell wall samples and incubated at room temp . for 16-18 hr. The Driselasesolubilized extract was collected by centrifugation at 3500 g for 10 min . The pellet was resuspended in a small vol. of 100 mM CaCl2 then re-centrifuged. The soluble material released by this treatment is called the Driselasesolubilized Ca t+ -wash and contains proteins which were solubilized by Driselase treatment but which continue to be associated with the wall by ionic interactions . The pellet was then subjected to a second Driselase treatment but for only 3 hr. No active peroxidase was solubilized by this second treatment . After washing with 3 x 25 ml of 100 mM CaCl2 and 4 x 25 ml of H 2O, the pellet, the wallbound residue, was resuspended in H20. Trypsin treatment of the wall-bound residue . Aliquots of the wall-bound residue were pre-equilibrated in 100 mM Tris-HCI, pH 8 .5 by resuspension and centrifugation . Chymotrypsin-free trypsin (Sigma; product no. T1005) was added to the wall-bound residue at 0 .5 mg ml - ' in the same buffer and incubated at room temp . for 15 min with orbital shaking at 100 rpm . Longer incubations and higher trypsin levels reduced the amount of peroxidase activity recovered, whereas very low levels (< 1 jig ml - ') or the absence of trypsin did not solubilize any activity . After centrifugation (3500 g, 5 min), the supernatant was decanted, added to excess soybean trypsin inhibitor linked to DITC glass beads (Sigma ; product no. T-9024) and incubated at room temp . for 10 min with intermittent inversion to prevent settling . This treatment effectively removes trypsin as the trypsin-solubilized extracts now
lacked detectable activity against benzoyl-L-arginine ethyl ester. Peroxidase assay . Peroxidase activity was measured in a continuous spectrophotometric assay using tetramethylbenzidine (TMB) by the method described before [16] . Electrophoresis . Peroxidase isozymes were separated by non-denaturing polyacrylamide gel electrophoresis in high pH [17] and low pH [18] buffer systems . Protein contents were estimated using the method of ref. [19]. Sample prepn, ultrafiltration and electrophoresis conditions were as before [16]. Gels were stained using odianisidine [20]. Acknowledgements-The author acknowledges funding from the Scottish Office Agriculture and Fisheries Department and thanks Mrs Fiona Carr for excellent technical help .
REFERENCES
1 . Fry, S. C. (1988) The Growing Plant Cell Wall . Longman, Essex, U.K. 2. Ridge, I . and Osborne, D . J. (1970) J. Exp. Botany 21, 843. 3. Masuda, H., Fukuda, H. and Komamine, A. (1983) Z . pflanzenphysiol . 112, 417 . 4 . McDougall, G. J. (1992) Phytochemistry 31, 3385. 5. McDougall, G. J. and Fry, S . C. (1993) in prep . 6. Monties, B. (1989) Methods Plant Biochem . 1, 113. 7. Evans, J. J . and Himmelsbach, D . S. (1991) J. Agric. Food Chem . 39, 830. 8. Welinder, K . G. (1979) Eur. J. Biochem . 96, 493 . 9. Phelps, C ., Forlani, L . and Antonini, E . (1971) Biochem. J. 124, 605. 10. van Huystee, R . B. (1987) A. Rev . Plant Physiol . 38, 205. 11 . Lagrimini, L . M ., Buckhart, W ., Moyer, M. and Rothstein, S . (1987) Proc . Natl Acad . Sci . U.S.A. 84, 7542. 12. Mazza, G . and Welinder, K. G. (1980) Eur. J . Biochem. 108, 481 . 13 . Roberts, E., Kutchan, T . and Kolattukudy, P . E. (1988) Plant Mol. Biol. 11, 15. 14. Buffard, D., Breda, C., van Huystee, R . B., Asemoto, 0., Pierre, M ., Danglta, D. B. and Esnault, R . (1990) Proc. Natl Acad . Sci. U.S.A. 87, 8874. 15. Morgens, P. H., Callahan, A . M., Dunn, L . J. and Abeles, F . B. (1990) Plant Mol. Biol. 14, 715 . 16. McDougall, G. J. (1991) J . Plant Physiol. 139, 182. 17 . Davis, B . J . (1964) Ann. N.Y. Acad . Sci. 121, 404. 18 . Reisfield, R . A ., Lewis, V . J . and Williams, D . E. (1962) Nature 195, 281 . 19. Bradford, M . M. (1976) Analyt. Biochem . 72, 248. 20. Church, D. L. and Galston, A . W. (1988) Plant Physiol. 88, 679.
Phytochemistry, Vol. 33, No . 4 . pp. 765-767, 1993
Pergamon Press Ltd
Printed in Great Britain.
SOLUBILIZATION OF WALL-BOUND PEROXIDASES BY LIMI D PROTEOLYSIS GORDON
J. MCDOUGALL
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, U .K . (Received in revised form 1 October 1992)
Key Word Index- Linum usitatissimum ; Linaceae; flax; lignin; cell wall-bound peroxidases .
Abstract-The cell wall residue, enriched in lignin, which was obtained from non-xylem tissues of flax stems after extensive digestion with the glycanase mixture, Driselase, had appreciable peroxidase activity which was tightly bound to the wall fragments . A portion of this residual activity was solubilized by limited proteolysis using trypsin . Nondenaturing gel electrophoresis of the trypsin-solubilized extracts revealed the presence of at least one highly mobile anionic peroxidase isozyme.
due either to the removal of further peroxidases from the wall by the second Driselase treatment, although no peroxidase activity was detected in the soluble digest, or to the inactivation of wall-bound peroxidases by the proteolysic activity present in Driselase [5] . In any case, the imposition of these severe conditions effectively percludes the possibility that peroxidases remain attached to the wall-bound residue by bonds normally susceptible to Driselase . Driselase, although it contains a host of cell walldegrading enzymes, lacks ligninase activity [1] and the wall-bound residue is enriched in lignin relative to the original wall preparation (results not shown) . In fact, the wall-bound residue is prepared in a similar manner to `enzyme-liberated' lignin preparations [6] . A portion of the peroxidases remaining in the wall-bound residue can be solubilized by the action of trypsin (Table 1) . The use of non-denaturing gel electrophoresis has shown that the trypsin-solubilized extracts contain at least one highly anionic peroxidase isozyme (Fig. 1). The extracts contained no detectable cationic isoforms . There are two possible means by which these peroxidases may be attached to the wall and released by limited proteolysis . Firstly, the peroxidases may have been attached to other wall proteins. In this model, rather selective proteolysis of these wall proteins would be required to release the peroxidases . Alternatively, the trypsin-solubilized peroxidases may have been attached to the wall by lignin-tyrosine bonds . It has been shown that peroxidases used to polymerize cinnamyl alcohols in vitro become covalently attached, via lignin-tyrosine bonds, to the product lignin [7] . The insolubilized ligninbound peroxidases retain their activity . Trypsin could release peroxidases, linked in such a manner, if it cleaved the polypeptide backbone of the peroxidase at the site that separated the anchoring lignintyrosine residue from the rest of the enzyme. Such a site
INTRODUCTION
Cell wall-bound peroxidases can be routinely fractionated for study by methods that exploit their means of attachment to the wall matrix. The ionically bound peroxidases can be eluted from the wall by ion exchange using metal ions . Covalently bound wall peroxidases can be obtained after homogenizing plant tissue in buffers containing salts and detergents to produce an insoluble wall fraction, free of intracellular contaminants [1]. The covalently bound enzymes can then be subfractionated into those released by treatment with cell wall degrading glycanases or those which remain tightly bound to the wall residue [2] . It has been found that the levels of residual or intrinsic peroxidases that remain in the wall after glycanase digestion rise sharply during the lignification of tracheary elements [3] and of flax fibres [4]. This paper reports the solubilization of peroxidases from the glycanase-resistant wall residue by limited proteolysis using trypsin. The nature of the bonds through which those peroxidases are attached to the wall is discussed .
RESULTS AND DISCUSSION
The levels of peroxidase activity in the various cell wall fractions obtained in three separate preparations are shown in Table 1 . The peroxidase activity in the intrinsic wall-bound residue is much lower than previously reported [4]. This is due to two factors . Firstly, the wallbound residue was subjected to a more stringent washing procedure than before to remove all peroxidases not covalently bound to the wall . This is illustrated by the removal of peroxidases made soluble by Driselase treatment but which had remained ionically bound to the wall by using elution with Cal + ions . Secondly, the inclusion of a second Driselase digestion reduced the activity of the wall residue markedly (results not shown) . This may be 765
766
G . J. MCDOUGALL Table. 1. Peroxidase activity of wall-associated fractions Peroxidase activity (units g -' fr. wt)*
I II III
Ionically bound
Covalently bound
Driselase solubilized
Driselase Ca" solubilized-wash
Wall-bound Trypsin residue solubilized
768±12 786±9 747+37
51±1 65±4 61+4
75±3 112±7 102+9
12±1 10±2 17+2
2.4±0.1 3.1±0.2 4.4+0.1
3 .9±0.1 2.1±0.1 2.2+0.1
*One unit of peroxidase activity is defined as that amount of enzyme that causes an increase in absorbance of 0.1 units min ` in the standard assay . All peroxidase values are averages of four replicates ± s .e.
a
b
c
Al A2 A3
A4
Fig. 1 . Wall-associated peroxidase isozymes . The anode is at the bottom of the gel . Lane (a) contains 2 µg of wall protein from the ionically bound fraction . Lane (b) contains 10 pg of wall protein from the Driselase-solubilized fraction. Lane (c) contains 100 pg of wall protein from the trypsin-solubilized fraction . A, is the least mobile anionic isozyme.
may exist. Horseradish peroxidase C contains five tyrosine residues [8] but only one of these tyrosine residues is accessible to ionization studies in the native, active enzyme [9] . Taking into account the common tertiary domain structure shared by plant peroxidases [10], an examination of the sequences of plant peroxidases [8, 11-15] reveals that only one tyrosine residue is in a location on the polypeptide chain that would allow it to be involved in a linkage with lignin that retained the catalytic activity of the peroxidase . This specific tyrosine residue lies close to the amino terminus of the enzymes in a flexible region of the protein which would also be accessible to proteolytic attack by trypsin . Given the known stability of native plant peroxidases to trypsin degradation [8], it is possible to speculate how trypsin
could liberate peroxidase activity which had been linked to lignin in this manner . This model predicts that the trypsin-solubilized peroxidases would be structurally altered compared with other isoenzymes . Studies are in progress to purify and examine the putative lignin-bound enzymes . EXPERIMENTAL
Plant material . Flax (Linum usitatissimum var. Belinka) plants were cultivated as described previously [16]. At 8 weeks after seedling emergence 150 flax plants were harvested and the first 3 cm of stem above the cotyledonary node excised from each . The stem segments were split longitudinally and fibre-bearing tissue (cortex, fibres,
767
Cell wall-bound peroxidases parenchyma and epidermis) peeled from the central xylem core . The peeled tissue was kept on ice, washed, blotted dry then weighed . The extent of fibre lignification was estimated microscopically as before [16] at ca 20% . This figure means that about one in five fibres showed visible signs of lignin deposition . Isolation of wall-associated peroxidases . lonically bound and freely soluble proteins were collected selectively from the intercellular air and wall spaces by vacuum infiltration [3] . Briefly, the peeled tissue was submerged in 100 mM CaC12 and subjected to a reduced pressure ( max. at 15 mm Hg) for 10 min. Thereafter the vacuum was dissipated and the CaC1 2 soln allowed to infiltrate into the evacuated air spaces of the tissue . The infiltrate and excess bathing soln was collected by low speed centrifugation (1000 g, 5 min). This extract is called the ionically bound fraction . A cell wall prepn was obtained by repeatedly homogenizing the same tissue in 50 mM Tris-HC1 pH 8 containing 1 M NaCl, 0.5% (v/v) Triton X-100 and 0 .5% (w/v) bovine serum albumin at 4° . The insoluble material after 8-10 rounds of homogenization/centrifugation was washed 3 times with H 2 O then resuspended in H20 . This insoluble material is the covalently bound wall sample . Driselase treatment of cell walls . Partially purified Driselase [1] at a final concn of l0mgml - ' in 250 mM NaOAc pH 4 .5 was added to cell wall samples and incubated at room temp . for 16-18 hr. The Driselasesolubilized extract was collected by centrifugation at 3500 g for 10 min . The pellet was resuspended in a small vol. of 100 mM CaCl2 then re-centrifuged. The soluble material released by this treatment is called the Driselasesolubilized Ca t+ -wash and contains proteins which were solubilized by Driselase treatment but which continue to be associated with the wall by ionic interactions . The pellet was then subjected to a second Driselase treatment but for only 3 hr. No active peroxidase was solubilized by this second treatment . After washing with 3 x 25 ml of 100 mM CaCl2 and 4 x 25 ml of H 2O, the pellet, the wallbound residue, was resuspended in H20. Trypsin treatment of the wall-bound residue . Aliquots of the wall-bound residue were pre-equilibrated in 100 mM Tris-HCI, pH 8 .5 by resuspension and centrifugation . Chymotrypsin-free trypsin (Sigma; product no. T1005) was added to the wall-bound residue at 0 .5 mg ml - ' in the same buffer and incubated at room temp . for 15 min with orbital shaking at 100 rpm . Longer incubations and higher trypsin levels reduced the amount of peroxidase activity recovered, whereas very low levels (< 1 jig ml - ') or the absence of trypsin did not solubilize any activity . After centrifugation (3500 g, 5 min), the supernatant was decanted, added to excess soybean trypsin inhibitor linked to DITC glass beads (Sigma ; product no. T-9024) and incubated at room temp . for 10 min with intermittent inversion to prevent settling . This treatment effectively removes trypsin as the trypsin-solubilized extracts now
lacked detectable activity against benzoyl-L-arginine ethyl ester. Peroxidase assay . Peroxidase activity was measured in a continuous spectrophotometric assay using tetramethylbenzidine (TMB) by the method described before [16] . Electrophoresis . Peroxidase isozymes were separated by non-denaturing polyacrylamide gel electrophoresis in high pH [17] and low pH [18] buffer systems . Protein contents were estimated using the method of ref. [19]. Sample prepn, ultrafiltration and electrophoresis conditions were as before [16]. Gels were stained using odianisidine [20]. Acknowledgements-The author acknowledges funding from the Scottish Office Agriculture and Fisheries Department and thanks Mrs Fiona Carr for excellent technical help .
REFERENCES
1 . Fry, S. C. (1988) The Growing Plant Cell Wall . Longman, Essex, U.K. 2. Ridge, I . and Osborne, D . J. (1970) J. Exp. Botany 21, 843. 3. Masuda, H., Fukuda, H. and Komamine, A. (1983) Z . pflanzenphysiol . 112, 417 . 4 . McDougall, G. J. (1992) Phytochemistry 31, 3385. 5. McDougall, G. J. and Fry, S . C. (1993) in prep . 6. Monties, B. (1989) Methods Plant Biochem . 1, 113. 7. Evans, J. J . and Himmelsbach, D . S. (1991) J. Agric. Food Chem . 39, 830. 8. Welinder, K . G. (1979) Eur. J. Biochem . 96, 493 . 9. Phelps, C ., Forlani, L . and Antonini, E . (1971) Biochem. J. 124, 605. 10. van Huystee, R . B. (1987) A. Rev . Plant Physiol . 38, 205. 11 . Lagrimini, L . M ., Buckhart, W ., Moyer, M. and Rothstein, S . (1987) Proc . Natl Acad . Sci . U.S.A. 84, 7542. 12. Mazza, G . and Welinder, K. G. (1980) Eur. J . Biochem. 108, 481 . 13 . Roberts, E., Kutchan, T . and Kolattukudy, P . E. (1988) Plant Mol. Biol. 11, 15. 14. Buffard, D., Breda, C., van Huystee, R . B., Asemoto, 0., Pierre, M ., Danglta, D. B. and Esnault, R . (1990) Proc. Natl Acad . Sci. U.S.A. 87, 8874. 15. Morgens, P. H., Callahan, A . M., Dunn, L . J. and Abeles, F . B. (1990) Plant Mol. Biol. 14, 715 . 16. McDougall, G. J. (1991) J . Plant Physiol. 139, 182. 17 . Davis, B . J . (1964) Ann. N.Y. Acad . Sci. 121, 404. 18 . Reisfield, R . A ., Lewis, V . J . and Williams, D . E. (1962) Nature 195, 281 . 19. Bradford, M . M. (1976) Analyt. Biochem . 72, 248. 20. Church, D. L. and Galston, A . W. (1988) Plant Physiol. 88, 679.