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Action of Carica papaya latex on cell wall glycosidases from Lactuca sativa pith
Phytochemistry, Vol. 34, No. 6, pp. 1473-1475, 1993 Printedin Great Britain.
ACTION
OF CARICA
0031-9422/93 $6.00+0.00 @I 1993 PexgamonPress Ltd
PAPAYA LATEX ON CELL WALL GLYCOSIDASES FROM LACTUCA SATIVA PITH ROGER GIORDANI*
Centre de Biochimie et de Biologie Molkxlaire,
and
LAURENCE LAFON
CNRS, 31 Chemin Joseph Aiguier, 13009 Marseille, France
(Received in revised form 29 March 1993)
Key Word Index-Carica
papaya, Caricaceae;
Luctuca sativa; Compositae;
latex; cell wall glycosidases.
Abstract-The various glycosidase activities from Carica papaya latex and pith cell walls from Lactuca satiua were measured by using pNP-glycoside substrates. A decrease of enzymic activities was obtained by incubation of isolated cell walls with C. papaya latex. Thus parietal activities decreased by the following amounts: N-acetyl-fl-Dglucosaminidase by 83%, /?-D-glucosidase by 45%, fi-D-fucosidase by 53% and a-D-galactosidase by 25% after an incubation of 24 hr. These results indicate that C. papaya latex has no stimulatory effect on the cell wall enzymes tested. Addition of cysteine protease inhibitor to medium culture causes a smaller decrease of parietal glycosidic activities. Caricu papaya latex proteases and glycosidases are, therefore, responsible for the decrease of cell wall glycosidic activities after action of this latex. A comparison with the increase of glycosidic activities of Candida albicans cultured in a medium supplemented with latex indicates a possible stimulation of glycoprotein synthesis by Golgi apparatus for this enzymic activation.
INTRODUCTION
The action of latexes on Candida albicans cultured in vitro involves a cell wall degradation process and an increase of many glycosidase activities [l]. It could be thought that hydrolysis of latex involves lysis of glycosidic linkages between polysaccharidic residues thus facilitating the access of artificial substrates to cell wall glycosidases. Considering that cell wall glycosidases are of glycoproteic nature [Z] it is possible that these enzymes could be activated by a disruption of glycosidic linkages between the protein part and hemicellulose part (proenzyme-enzyme process). Finally, the increase of glycosidic activities after the action of latex could be explained by a stimulation of the Golgi apparatus involving the release of many vesicles which by coalescing with the cell wall would produce an increase in the secretion of fungal cell wall glycosidases. In order to demonstrate one or the other of these hypotheses it is necessary to examine the possible stimulation by latex of glycosidase activities contained in a cell wall preparation devoid of any membranous structure. The Carica papaya latex and the pith cell walls from LQch4ca satiua are used in this paper.
RESULTS AND DISCUSSION
The study of glycosidic activities in C. papaya latex indicates a very strong N-acetyl-/?-D-glucosaminidase activity (Table 1). On the other hand, isolated pith cell
*Author to whom correspondence should be addressed.
wall suspensions show high fl-D-glucosidase, t%-D-g&Wtosidase, a+arabinosidase and B-D-xylosidase activities (Table 1). These results may be compared with those obtained from isolated cell walls of cultured Conooloulus aruensis cells [3] which indicated a strong predominance of B-D-glucosidase activity. The action of C. papaya latex on isolated cell walls was measured by its effect on /?-D-glucosidase and E-Dgalactosidase, which are the enzymes showing the strongest activities in pith cell walls, whereas N-acetyl+Dglucosaminidase, a-D-mannosidase and /?-D-fucosidase show the weakest activities (Table 1). The effect of latex on parietal p-D-fucosidase activity was measured considering the major role of this enzyme in initiating the cell wall degradation process [4,5]. The effect of latex involves a significant fall of enzymic activities. Thus, 36% of N-acetyl-b-D-glucosaminidase was recovered by incubating the mixture of cell walls/latex for 2 hr and only 17% after an incubation for 24 hr (Fig. 1); a-D-mannosidase activity decreased very rapidly: 34% of initial activity after only 2 hr (Fig. 2). /?-D-Glucosidase, B-D-fucosidase and a-D-galactosidase activities decreased by 77,66 and 60%, respectively, after incubation for 4 hr 30 min and 45, 53 and 25% after incubation for 24 hr (Fig. 3). Other glycosidase activities decreased more slowly. Thus 72% of /?-D-fucosidase activity was recovered after incubation for 2 hr and 52% after 7 hr; 81 and 87% of U-Dgalactosidase and /I-D-glucosidase activities, respectively, were recovered by incubating for 2 hr, 33 and 42% after incubation for 16 hr. Latex and cell wall suspensions incubated separately under the same conditions of temperature, time and
1473
1474
R.
GIORDANI
and L. LAFON
Table 1. Substrate specificity for glycosidase activities at pH 5 in Curica papaya latex and in isolated pith cell walls of Lactuca s&vu
Substrate
C. papaya latex (U mg- * protein)
(U mg- ’ dry wt)
pNP-N-Acetyl-8-D-gtucosaminide pNP-~-L-Arabinoside pNP-~-~Fu~side pNP-a-D-Gafactoside pNP-j&o-Galactoside pNP-a-o_Glucoside pNP-/?-D-Glucoside pNP-u+Mannoside pNP-&DXyloside
28.00 0.34 0.44 1.35 0.12 0.08 0.23 5.00 0.11
IO.3 31.5 IO.9 46-O 15.1 15.7 78.8 10.9 18.1
CeII walls
Using pNP-glycosides as substrates, the enzymic activity is expressed in U mg “” protein (C. papaya latex) or U mg-’ dry matter (isolated cell walls).
.
t 12
2 Time
24
lhr)
Fig. 1. N-Acetyf-8-D-~ucosanidase activity vs time in isolated cell walls from L. satiua pith (30 ml containing 33 mg of dry matter; pH 6) incubated (30”) with C. papaya latex (3 ml containing 8 mg protein); pW of incubation mixture, 5.8.
stirring did not show any significant variation of initial glycosidic activities. Control experiments carried out with cell walls incubated with buffer or boiled latex did not show any variation of glycosidic activities. These results indicate that latex does not stimulate the cell wall glycosidases tested and rule out, therefore, the hypothesis of a proenzyme-enzyme process to explain the stimulation of glycosidase activities of C. a&cans cultured in presence of L. sariva and Asclepias curassavica latex Cl]. The decrease of enzymic activities may indicate that cell wall glycosidases are degraded by enzymes such as glycosidases revealed in this work and proteases whose presence in C. pupaya latex is well known [&?I. In order to examine these hypotheses we have carried out incubation of cell walls with a protease inhibitor. Conside~ng
L
I,
0 2
I
‘
12 lime
4
I
I4
4
1
24
(hr)
Fig. 2. ff-D-Mannosidase activity versus time in isolated cell walls from L. sat& pith (30 ml containing 33 mg dry matter; pH 6) incubated (30”) with C. papaya latex (3 ml containing 8 mg protein); pH of incubation mixture, 5.8.
that C. papaya latex contains four major proteases [8,9] which are all cysteine proteinases, we have used cystatin (2.3 pm), a well known cysteine protease inhibitor [lo] which is a low-molecular mass protein shown to be a potent inhibitor of lysosomal cysteine proteinase [ll, 123, like latex proteases. In these conditions parietal glycosidic activities decreased less: N-acetyl-/?-D-glucosaminidase, B-D-glucosidase, P-D-fucosidase and E-Dgalactosidase activities decreased by 30, t&26 and 11% respectively, after incubation for 24 hr. These decreases of enzymic activities indicate that cell wall glycosidases are degraded in part by latex glycosidases and that latex proteases are largety responsible for this degradation. These results support the idea that the stimuiatory effect of latex on cell wall glycosidases from C. Africans culture [ 11 may be caused by an increase of glycoprotein
Inhibition of cell wall glycosidases
-
0.6
7
E” a L .‘. > ._ c :
0.3
0.1
ol’~~-“~~,“‘, 0
2
Time
12
26
(hr)
Fig. 3. j-o-Glucosidase ( q), /3-D-fucosidase (0) and a-D-gahe tosidase (0) activities in isolated cell walls from L. sativa pith (30 ml containing 33 mg dry matter; pH 6) incubated (30”) with C. papayalatex (3 ml containing 8 mg protein); pH of incubation mixture, 5.8. from the Golgi apparatus. This is in good agreement with the limited cell wall degradation process which permits the tube-like constitution of secretory tissue (articulated laticifers), as the activatory effect of latex on cell wall glycosidases would involve a general degradation of the whole cell wall of articulated laticifers. Pith cell walls of L. sarioa and C. a&cans yeasts differ in their constituents in spite of the eukaryotic character of both types of cells [2]. In addition, in the fungistatic action of latex on C. albicans, the perforation process applies to the whole of the cell wall [l, 123 and not to specific sites as in articulated laticifers differentiation [13, 141. synthesis
EXPERIMENTAL Materials. The spray-dried
latex sap exuded from unripe fruit of C. papaya was purchased from Sigma. The latex was diluted 1: 80 with deionized H,O containing 2% NaN,, homogenized with an Ultra-Turrax T25 and centrifuged (10 min, 13 500 gmaX).The supernatant constituted the whole latex to be tested on isolated cell walls. Cell wall prepns were obtained from pith of mature L. s&vu plants. The fragments were thoroughly mixed (4 x 2 min) in 0.4 M sucrose in a blender. The cells were then broken in a French press at 2 T cm-‘. The suspension so obtained was centrifuged (1400 gmax, 15 min) and the supernatant was discarded_ The pellet was resuspended and centrifuged (13 500 g,,_, 15 min) in 0.6 M sucrose then in 1 M sucrose and finally in H,O (x4). The final cell wall prepn, devoid of any membranous structure, was kept at - 18” until use. Enzymic activities. Pith cell walls in aq. suspension (30 ml containing 33 mg dry matter) were incubated (30”) with 2.5 mM pNP-glycoside substrate in succinate buffer (0.1 M, pH 5) under stirring. The following activities were tested: a-D-glucosidase, (EC 3.2.1.20), /I-D-glucosidase (EC 3.2.1.21), a-D-galactosidase (EC 3.2.1.22), fl-D-galao
1475
tosidase (EC 3.2.1.23), a-D-mannosidase (EC 3.2.1.24), Nacetyl-/I-D-glucosaminidase (EC 3.2.1.36), fi-D-XylOSidase (EC 3.2.1.37), /I-D-fucosidase (EC 3.2.1.38) and a-Larabinosidase (EC 3.2.1.55). The, standard incubation medium contained 0.1 M succinate buffer pH 5.5,0.02% NaN,, 2.5 mM pNP-glycoside and 100 ~1 of cell walls suspension in a total vol. of 0.6 ml. The enzymic activity was measured at 30” by following the A at 400 nm of the p-nitrophenol formed after addition of 0.2 M Na,CO, to the incubation medium. Conditions of linearity of enzymic reaction were always selected and assays were done in triplicate. One unit(U) of the enzyme was defined as the amount which liberated one pm01 of p-nitrophenol per min. Otherwise several glycosidase activities were tested through time in aliquots of 100 ~1 of incubation (30”) mixture constituted with pith cell walls (30 ml containing 33 mg dry matter) and C. papaya latex (3 ml, 8 mg proteins). Control experiments were carried out with cell walls incubated with succinate buffer (0.1 M, pH 5; 3 ml) instead of latex, boiled latex (100” for 5 min; 3 ml containing 8 mg proteins) or latex (3 ml, 8 mg proteins) in the presence of cystatin (2.3 PM), a cysteine protease inhibitor. Protein determination. The proteins were estimated according to ref. [la with bovine serum albumin as standard. Acknowledgement-We thank Dr Maria Luiz Cardenas/ Cornish-Bowden (Laboratoire de Chimie Bactirienne, CNRS, Marseille) for critical reading of the manuscript. REFERENCES 1. Giordani, R., Moulin-Traffort, J. and Regli, P. (1991) Mycoses 34, 67. 2. Cassab, G. I. and Vamer, J. E. (1988) Ann. Rev. Plant Physiol. Plant Mol. Biol. 39, 321. 3. Pierrot, H. and Wielink, E. van (1977) Planta 137,
235. 4. Giordani,
R. and Noat, G. (1988) Eur. J. Biochem.
175, 619.
5. Giordani, R. (1992) C. R. Acad. SC., Paris, III 315, 189. 6. Brocklehurst, K., Baines, B. S. and Kierstan, M. P. J. (198 1) Biotechnology 5, 262. 7. Dubois, T., Jacquet, A., Schnek, A. and Looze, Y. (1988) Biol. Chem. Hoppe-Seyler 369, 733. 8. Robinson, G. W. (1975) Biochemistry 14, 3695. 9. Brocklehurst, K. and Salih, E. (1983) Biochem. J. 213, 559.
10. Anastasi, A., Brown, M. A., Kembhavi, A. A., Nicklin, M. J., Sayers, C. A., Sunter, D. C. and Barrett, A. J. (1983) Biochem. J. 211, 129. 11. Barrett, A. J., Davies, M. E. and Grubb, A. (1984) Biochem. Biophys. Res. Commun. lM, 631.
12. Giordani, R., Siepaio, M., Moulin-Traffort, J. and Regli, P. (1991) Mycoses 34,469. 13. Giordani, R. (1981) Biol. Cell 40, 217. 14. Nessler, C. and Mahlberg, P. (1977) Bot. Guz. 138, 402.
15. Bradford, M. M. (1976) Anal. Biochem. 72, 248.
ACTION
OF CARICA
0031-9422/93 $6.00+0.00 @I 1993 PexgamonPress Ltd
PAPAYA LATEX ON CELL WALL GLYCOSIDASES FROM LACTUCA SATIVA PITH ROGER GIORDANI*
Centre de Biochimie et de Biologie Molkxlaire,
and
LAURENCE LAFON
CNRS, 31 Chemin Joseph Aiguier, 13009 Marseille, France
(Received in revised form 29 March 1993)
Key Word Index-Carica
papaya, Caricaceae;
Luctuca sativa; Compositae;
latex; cell wall glycosidases.
Abstract-The various glycosidase activities from Carica papaya latex and pith cell walls from Lactuca satiua were measured by using pNP-glycoside substrates. A decrease of enzymic activities was obtained by incubation of isolated cell walls with C. papaya latex. Thus parietal activities decreased by the following amounts: N-acetyl-fl-Dglucosaminidase by 83%, /?-D-glucosidase by 45%, fi-D-fucosidase by 53% and a-D-galactosidase by 25% after an incubation of 24 hr. These results indicate that C. papaya latex has no stimulatory effect on the cell wall enzymes tested. Addition of cysteine protease inhibitor to medium culture causes a smaller decrease of parietal glycosidic activities. Caricu papaya latex proteases and glycosidases are, therefore, responsible for the decrease of cell wall glycosidic activities after action of this latex. A comparison with the increase of glycosidic activities of Candida albicans cultured in a medium supplemented with latex indicates a possible stimulation of glycoprotein synthesis by Golgi apparatus for this enzymic activation.
INTRODUCTION
The action of latexes on Candida albicans cultured in vitro involves a cell wall degradation process and an increase of many glycosidase activities [l]. It could be thought that hydrolysis of latex involves lysis of glycosidic linkages between polysaccharidic residues thus facilitating the access of artificial substrates to cell wall glycosidases. Considering that cell wall glycosidases are of glycoproteic nature [Z] it is possible that these enzymes could be activated by a disruption of glycosidic linkages between the protein part and hemicellulose part (proenzyme-enzyme process). Finally, the increase of glycosidic activities after the action of latex could be explained by a stimulation of the Golgi apparatus involving the release of many vesicles which by coalescing with the cell wall would produce an increase in the secretion of fungal cell wall glycosidases. In order to demonstrate one or the other of these hypotheses it is necessary to examine the possible stimulation by latex of glycosidase activities contained in a cell wall preparation devoid of any membranous structure. The Carica papaya latex and the pith cell walls from LQch4ca satiua are used in this paper.
RESULTS AND DISCUSSION
The study of glycosidic activities in C. papaya latex indicates a very strong N-acetyl-/?-D-glucosaminidase activity (Table 1). On the other hand, isolated pith cell
*Author to whom correspondence should be addressed.
wall suspensions show high fl-D-glucosidase, t%-D-g&Wtosidase, a+arabinosidase and B-D-xylosidase activities (Table 1). These results may be compared with those obtained from isolated cell walls of cultured Conooloulus aruensis cells [3] which indicated a strong predominance of B-D-glucosidase activity. The action of C. papaya latex on isolated cell walls was measured by its effect on /?-D-glucosidase and E-Dgalactosidase, which are the enzymes showing the strongest activities in pith cell walls, whereas N-acetyl+Dglucosaminidase, a-D-mannosidase and /?-D-fucosidase show the weakest activities (Table 1). The effect of latex on parietal p-D-fucosidase activity was measured considering the major role of this enzyme in initiating the cell wall degradation process [4,5]. The effect of latex involves a significant fall of enzymic activities. Thus, 36% of N-acetyl-b-D-glucosaminidase was recovered by incubating the mixture of cell walls/latex for 2 hr and only 17% after an incubation for 24 hr (Fig. 1); a-D-mannosidase activity decreased very rapidly: 34% of initial activity after only 2 hr (Fig. 2). /?-D-Glucosidase, B-D-fucosidase and a-D-galactosidase activities decreased by 77,66 and 60%, respectively, after incubation for 4 hr 30 min and 45, 53 and 25% after incubation for 24 hr (Fig. 3). Other glycosidase activities decreased more slowly. Thus 72% of /?-D-fucosidase activity was recovered after incubation for 2 hr and 52% after 7 hr; 81 and 87% of U-Dgalactosidase and /I-D-glucosidase activities, respectively, were recovered by incubating for 2 hr, 33 and 42% after incubation for 16 hr. Latex and cell wall suspensions incubated separately under the same conditions of temperature, time and
1473
1474
R.
GIORDANI
and L. LAFON
Table 1. Substrate specificity for glycosidase activities at pH 5 in Curica papaya latex and in isolated pith cell walls of Lactuca s&vu
Substrate
C. papaya latex (U mg- * protein)
(U mg- ’ dry wt)
pNP-N-Acetyl-8-D-gtucosaminide pNP-~-L-Arabinoside pNP-~-~Fu~side pNP-a-D-Gafactoside pNP-j&o-Galactoside pNP-a-o_Glucoside pNP-/?-D-Glucoside pNP-u+Mannoside pNP-&DXyloside
28.00 0.34 0.44 1.35 0.12 0.08 0.23 5.00 0.11
IO.3 31.5 IO.9 46-O 15.1 15.7 78.8 10.9 18.1
CeII walls
Using pNP-glycosides as substrates, the enzymic activity is expressed in U mg “” protein (C. papaya latex) or U mg-’ dry matter (isolated cell walls).
.
t 12
2 Time
24
lhr)
Fig. 1. N-Acetyf-8-D-~ucosanidase activity vs time in isolated cell walls from L. satiua pith (30 ml containing 33 mg of dry matter; pH 6) incubated (30”) with C. papaya latex (3 ml containing 8 mg protein); pW of incubation mixture, 5.8.
stirring did not show any significant variation of initial glycosidic activities. Control experiments carried out with cell walls incubated with buffer or boiled latex did not show any variation of glycosidic activities. These results indicate that latex does not stimulate the cell wall glycosidases tested and rule out, therefore, the hypothesis of a proenzyme-enzyme process to explain the stimulation of glycosidase activities of C. a&cans cultured in presence of L. sariva and Asclepias curassavica latex Cl]. The decrease of enzymic activities may indicate that cell wall glycosidases are degraded by enzymes such as glycosidases revealed in this work and proteases whose presence in C. pupaya latex is well known [&?I. In order to examine these hypotheses we have carried out incubation of cell walls with a protease inhibitor. Conside~ng
L
I,
0 2
I
‘
12 lime
4
I
I4
4
1
24
(hr)
Fig. 2. ff-D-Mannosidase activity versus time in isolated cell walls from L. sat& pith (30 ml containing 33 mg dry matter; pH 6) incubated (30”) with C. papaya latex (3 ml containing 8 mg protein); pH of incubation mixture, 5.8.
that C. papaya latex contains four major proteases [8,9] which are all cysteine proteinases, we have used cystatin (2.3 pm), a well known cysteine protease inhibitor [lo] which is a low-molecular mass protein shown to be a potent inhibitor of lysosomal cysteine proteinase [ll, 123, like latex proteases. In these conditions parietal glycosidic activities decreased less: N-acetyl-/?-D-glucosaminidase, B-D-glucosidase, P-D-fucosidase and E-Dgalactosidase activities decreased by 30, t&26 and 11% respectively, after incubation for 24 hr. These decreases of enzymic activities indicate that cell wall glycosidases are degraded in part by latex glycosidases and that latex proteases are largety responsible for this degradation. These results support the idea that the stimuiatory effect of latex on cell wall glycosidases from C. Africans culture [ 11 may be caused by an increase of glycoprotein
Inhibition of cell wall glycosidases
-
0.6
7
E” a L .‘. > ._ c :
0.3
0.1
ol’~~-“~~,“‘, 0
2
Time
12
26
(hr)
Fig. 3. j-o-Glucosidase ( q), /3-D-fucosidase (0) and a-D-gahe tosidase (0) activities in isolated cell walls from L. sativa pith (30 ml containing 33 mg dry matter; pH 6) incubated (30”) with C. papayalatex (3 ml containing 8 mg protein); pH of incubation mixture, 5.8. from the Golgi apparatus. This is in good agreement with the limited cell wall degradation process which permits the tube-like constitution of secretory tissue (articulated laticifers), as the activatory effect of latex on cell wall glycosidases would involve a general degradation of the whole cell wall of articulated laticifers. Pith cell walls of L. sarioa and C. a&cans yeasts differ in their constituents in spite of the eukaryotic character of both types of cells [2]. In addition, in the fungistatic action of latex on C. albicans, the perforation process applies to the whole of the cell wall [l, 123 and not to specific sites as in articulated laticifers differentiation [13, 141. synthesis
EXPERIMENTAL Materials. The spray-dried
latex sap exuded from unripe fruit of C. papaya was purchased from Sigma. The latex was diluted 1: 80 with deionized H,O containing 2% NaN,, homogenized with an Ultra-Turrax T25 and centrifuged (10 min, 13 500 gmaX).The supernatant constituted the whole latex to be tested on isolated cell walls. Cell wall prepns were obtained from pith of mature L. s&vu plants. The fragments were thoroughly mixed (4 x 2 min) in 0.4 M sucrose in a blender. The cells were then broken in a French press at 2 T cm-‘. The suspension so obtained was centrifuged (1400 gmax, 15 min) and the supernatant was discarded_ The pellet was resuspended and centrifuged (13 500 g,,_, 15 min) in 0.6 M sucrose then in 1 M sucrose and finally in H,O (x4). The final cell wall prepn, devoid of any membranous structure, was kept at - 18” until use. Enzymic activities. Pith cell walls in aq. suspension (30 ml containing 33 mg dry matter) were incubated (30”) with 2.5 mM pNP-glycoside substrate in succinate buffer (0.1 M, pH 5) under stirring. The following activities were tested: a-D-glucosidase, (EC 3.2.1.20), /I-D-glucosidase (EC 3.2.1.21), a-D-galactosidase (EC 3.2.1.22), fl-D-galao
1475
tosidase (EC 3.2.1.23), a-D-mannosidase (EC 3.2.1.24), Nacetyl-/I-D-glucosaminidase (EC 3.2.1.36), fi-D-XylOSidase (EC 3.2.1.37), /I-D-fucosidase (EC 3.2.1.38) and a-Larabinosidase (EC 3.2.1.55). The, standard incubation medium contained 0.1 M succinate buffer pH 5.5,0.02% NaN,, 2.5 mM pNP-glycoside and 100 ~1 of cell walls suspension in a total vol. of 0.6 ml. The enzymic activity was measured at 30” by following the A at 400 nm of the p-nitrophenol formed after addition of 0.2 M Na,CO, to the incubation medium. Conditions of linearity of enzymic reaction were always selected and assays were done in triplicate. One unit(U) of the enzyme was defined as the amount which liberated one pm01 of p-nitrophenol per min. Otherwise several glycosidase activities were tested through time in aliquots of 100 ~1 of incubation (30”) mixture constituted with pith cell walls (30 ml containing 33 mg dry matter) and C. papaya latex (3 ml, 8 mg proteins). Control experiments were carried out with cell walls incubated with succinate buffer (0.1 M, pH 5; 3 ml) instead of latex, boiled latex (100” for 5 min; 3 ml containing 8 mg proteins) or latex (3 ml, 8 mg proteins) in the presence of cystatin (2.3 PM), a cysteine protease inhibitor. Protein determination. The proteins were estimated according to ref. [la with bovine serum albumin as standard. Acknowledgement-We thank Dr Maria Luiz Cardenas/ Cornish-Bowden (Laboratoire de Chimie Bactirienne, CNRS, Marseille) for critical reading of the manuscript. REFERENCES 1. Giordani, R., Moulin-Traffort, J. and Regli, P. (1991) Mycoses 34, 67. 2. Cassab, G. I. and Vamer, J. E. (1988) Ann. Rev. Plant Physiol. Plant Mol. Biol. 39, 321. 3. Pierrot, H. and Wielink, E. van (1977) Planta 137,
235. 4. Giordani,
R. and Noat, G. (1988) Eur. J. Biochem.
175, 619.
5. Giordani, R. (1992) C. R. Acad. SC., Paris, III 315, 189. 6. Brocklehurst, K., Baines, B. S. and Kierstan, M. P. J. (198 1) Biotechnology 5, 262. 7. Dubois, T., Jacquet, A., Schnek, A. and Looze, Y. (1988) Biol. Chem. Hoppe-Seyler 369, 733. 8. Robinson, G. W. (1975) Biochemistry 14, 3695. 9. Brocklehurst, K. and Salih, E. (1983) Biochem. J. 213, 559.
10. Anastasi, A., Brown, M. A., Kembhavi, A. A., Nicklin, M. J., Sayers, C. A., Sunter, D. C. and Barrett, A. J. (1983) Biochem. J. 211, 129. 11. Barrett, A. J., Davies, M. E. and Grubb, A. (1984) Biochem. Biophys. Res. Commun. lM, 631.
12. Giordani, R., Siepaio, M., Moulin-Traffort, J. and Regli, P. (1991) Mycoses 34,469. 13. Giordani, R. (1981) Biol. Cell 40, 217. 14. Nessler, C. and Mahlberg, P. (1977) Bot. Guz. 138, 402.
15. Bradford, M. M. (1976) Anal. Biochem. 72, 248.