- Email: [email protected]
Purification and properties of pi-repressible acid phosphatases from Aspergillus nidulans
PII: S0031-9422(98)00205-2
Phytochemistry Vol. 49, No. 6, pp. 1517±1523, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0031-9422/98/$ - see front matter
PURIFICATION AND PROPERTIES OF PI-REPRESSIBLE ACID PHOSPHATASES FROM ASPERGILLUS NIDULANS SEÂRGIO R. NOZAWA, WALTER MACCHERONI JR*, RODRIGO G. STAÂBELI, GERALDO THEDEI JR{ and ANTONIO ROSSI{ Departamento de QuõÂ mica, FFCLRP-USP, 14040-901 RibeiraÄo Preto, Brazil. (Received 24 February 1998)
Key Word IndexÐAspergillus nidulans; Fungi; enzyme secretion; acid phosphatase; phosphate regulation. AbstractÐTwo forms of the pacA-encoded acid phosphatase (designated acid phosphatases I and II) secreted by the mold Aspergillus nidulans grown in low-Pi medium at 378, pH 5.0, were puri®ed to apparent homogeneity by PAGE. The Mr of the puri®ed enzyme forms were ca 115 000 (60 000) and 113 000 (62 000) respectively for forms I and II secreted by strain biA1 and ca 118 000 (60 000) and 121 000 (61 000) respectively for forms I and II secreted by strain biA1 pacA1, as determined by exclusion chromatography (number between brackets are the Mr as determined by SDS-PAGE). All of these puri®ed enzyme forms showed an apparent optimum pH ranging from 6.0 to 6.5 and no deviation from Michaelis kinetics for the hydrolysis of both p-nitrophenylphosphate and a-naphthylphosphate. Heat inactivation at 608 and at pH 6.0 showed half-lives of 14 min (k = 0.033 minÿ1) and 10 min (k = 0.069 minÿ1), respectively, for the puri®ed acid phosphatases I and II secreted by biA1 strain and half-lives of 0.8 min (k = 0.92 minÿ1) and 0.6 min (k = 0.95 minÿ1), respectively, for the puri®ed forms I and II secreted by the biA1 pacA1 strain. The neutral sugar content of puri®ed acid phosphatases I and II secreted by strain biA1 was 48% and 37% (w/w), respectively, whereas the content of forms I and II secreted by strain biA1 pacA1 was 18% and 11%, respectively. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
In fungi, acquisition of nutrients from the environment involves the secretion of an array of hydrolytic enzymes acting speci®cally on dierent resources. The phosphatases, a generic designation for nonspeci®c phosphoesterases, belong to a family of enzymes responsible for supplying Pi to the cell. Four major phosphatases and their corresponding structural genes have been identi®ed in the ascomycetes fungus Aspergillus nidulans [1, 2]. Two of these, the palD-encoded alkaline phosphatase and the pacA-encoded acid phosphatase, are synthesized exclusively under Pi-limiting conditions and therefore are called Pi-repressible phosphatases. The study of these enzymes is a powerful tool in the elucidation of gene expression regarding not only the synthesis but also the secretion of enzymes by *Present address: Departamento de GeneÂtica, ESALQUSP, 13400-970 Piracicaba, SP, Brazil. {Present address: Instituto de CieÃncias da SauÂde, UNIUBE, 38065-500 Uberaba, MG, Brazil. {Author to whom correspondence should be addressed.
eukaryotic cells in response to ambient factors such as pH and carbon, nitrogen, sulfur and phosphorus sources [3, 4]. The intracellular form of the pacA-encoded acid phosphatase has been puri®ed and partially characterized [5], whereas the structural and steady state kinetic properties of its form secreted into the culture medium remain to be determined. Even though knowledge of these properties is not sucient per se to elucidate the mechanism of acid phosphatase secretion, this type of study may reveal a few important aspects concerning the regulation of the enzymes and permeases involved in Pi acquisition in A. nidulans, up to now studied only at the level of transcription of its structural genes ( [6, 7], and references therein). With this in mind, we puri®ed both forms I and II [8] of the major Pi-repressible acid phosphatases secreted into the growth medium by A. nidulans at pH 5.0 and compared their properties with those of the corresponding enzyme forms secreted by a pacA1 strain. Our results show that Pi-repressible acid phosphatases I and II secreted by A. nidulans are encoded by a
1517
1518
S. R. NOZAWA et al.
single gene (pacA) and that the pacA1 mutation resulted in the partial glycosylation of the enzyme forms secreted by the mutant strain. RESULTS AND DISCUSSION
Con®rming earlier reports [2], the levels of active Pi-repressible acid phosphatase secreted by the biA1 pacA1 strain of A. nidulans were reduced when the temperature of fungal culture was increased from 228 to 378, compared with the values shown by the biA1 strain (Fig. 1). Although only slightly, the mycelial yield of both strains also decreased with increasing culture temperature (Fig. 1). Nevertheless, the stepwise fractionation on DEAE-cellulose of the enzyme secreted by the biA1 pacA1 strain grown at 378 practically showed the same enzyme fractions as those secreted by the biA1 strain (Fig. 2). Not only the identi®cation but also the increased yield of each fraction were possible because the eluted fractions were collected in tubes
containing 0.1 M NaOAc buer, pH 4.7. This because the secreted acid phosphatase is highly labile at alkaline pH. In an attempt to further characterize the acid phosphatases secreted by A. nidulans we puri®ed to apparent homogeneity by PAGE forms I and II (Fig. 2) of the acid phosphatases secreted by the mold grown in low-Pi medium. The procedures summarized in Table 1 provided optimal conditions for the puri®cation of both forms secreted by strains biA1 and biA1 pacA1. All enzyme preparations (at least four independent preparations of each enzyme fraction) appeared to be homogeneous by 7.5% PAGE at pH 8.3, with the protein band being superimposable on acid phosphatase activity. All of these preparations also essentially showed the same electrophoretic mobility for each enzyme form secreted by both strains, i.e., the Rf values were 0.51 and 0.59 for acid phosphatases I and II, respectively. When mixed, the two enzyme forms combined in
Fig. 1. Production of acid phosphatase in cultures of A. nidulans grown in low-Pi medium, adjusted to pH 5.0, and buered with 50 mM Na citrate. A and C represent mycelial yield and acid phosphatase secreted by biA1 strain grown at 228 and 378, respectively. B and D represent mycelial yield and acid phosphatase secreted by biA1 pacA1 strain grown at 228 and 378 respectively.
Phosphatases from A. nidulans
1519
Fig. 2. Stepwise fractionation on DEAE-cellulose of acid phosphatases secreted by the biA1 (A) and biA1 pacA1 (B) strains of A. nidulans grown in low-Pi medium at 378, pH 5.0. After elution of nonabsorbed proteins with ca 100 ml of the equilibrating buer, proteins were eluted by the stepwise addition of 0.05, 0.10 M (fraction I), 0.15 M (fraction II), 0.30 M and 0.50 M NaCl dissolved in 10 mM MOPS, pH 7.2. 6 ml fractions were collected in tubes containing 1 ml of 0.10 M NaOAc buer, pH 4.7, at a ¯ow rate of 120 ml hrÿ1.
a stable manner since they migrated as a single band, the Rf value being 0.56. Furthermore, acid phosphatase I secreted by the biA1 pacA1 strain was puri®ed 120-fold with a low yield (3.8%) as compared with the value shown by the biA1 strain (Table 1). The neutral sugar content of puri®ed acid phosphatases I and II secreted by strain biA1 were 48% and 37% (w/w), respectively, whereas the content of forms I and II secreted by strain biA1 pacA1 was 18% and 11%, respectively, suggesting that these enzymatic forms are glycoproteins and that the pacA1 mutation possibly altered a glycosylation site or the molecule conformation, resulting in partial glycosylation of the enzyme forms secreted by the
mutant strain. Determination of the pH activity pro®le showed an apparent optimum ranging from 6.0 to 6.5 for puri®ed acid phosphatases I and II secreted by strains biA1 and biA1 pacA1 (Fig. 3). Also, the activity of acid phosphatases I and II secreted by strain biA1 pacA1 was inhibited at pH higher than 6.5 (Fig. 3B) as compared with the relative activity shown by the enzyme forms secreted by the biA1 strain (Fig. 3A), an eect probably due to the lower glycosylation level of these enzyme forms (see above). The Mr of the puri®ed enzyme forms were ca 115 000 (60 000) and 113 000 (62 000) respectively for forms I and II secreted by strain biA1 and ca 118 000 (60 000) and 121 000 (61 000) respectively
1520
S. R. NOZAWA et al. Table 1. Puri®cation of acid phosphatases I and II.
Fraction
Yield (%)
Puri®cation (fold)
100 62.1
1 21.8
18.5 15.0
40.1 37.7
12.2 7.4
88.4 64.3
pacA1 strain of A. nidulans grown in low-Pi medium at 378, pH 5.0. 1850 97.7 130 1.33 12 1.8 58.8 32.7
100 45.2
1 24.6
2.5 2.5
0.064 0.16
8 10
125 61.5
6.1 7.7
94.0 46.2
3 4
0.03 0.12
5 8
166.7 66.7
3.8 6.1
125.3 50.2
Volume (ml)
Total protein (mg)
Total activity (Units)
Speci®c activity (Units mgÿ1)
(A) Secreted by the biA1 strain of A. nidulans grown in low-Pi medium at 378, pH 5.0. Culture medium 1800 576 486 0.84 10 16.5 302 18.3 (NH4)2SO4 (65±95%) DEAE-cellulose Fraction I 3.3 2.67 89.9 33.7 Fraction II 2.8 2.3 72.8 31.7 Sephadex G-200 Fraction I 2.7 0.80 59.4 74.3 Fraction II 3.2 0.67 36.2 54.0 (B) Secreted by the biA1 Culture medium (NH4)2SO4 (65±95%) DEAE-cellulose Fraction I Fraction II Sephadex G-200 Fraction I Fraction II
for forms I and II secreted by strain biA1 pacA1, as determined by exclusion chromatography (number between brackets are the Mr as determined by SDSPAGE). Although these methods are not accurate for glycoproteins [9, 10] these results suggest that both the intracellular (Mr of ca 100 000 as estimated by Sephadex G100 gel ®ltration [5]) as the secreted enzyme forms are dimers. The puri®ed
enzyme forms I and II secreted by strains biA1 and biA1 pacA1 showed no deviation from Michaelis kinetics for the hydrolysis of both PNP-P and anaphthylphosphate (Table 2). The Km values of the two substrates were quite similar, except that form II secreted by both strains had a higher anity for the hydrolysis of a-naphthylphosphate (low Km value).
Fig. 3. pH activity pro®le of puri®ed acid phosphatases secreted by A. nidulans grown in low-Pi medium at 378, pH 5.0. (A) *, Q represent puri®ed acid phosphatases I and II secreted by the biA1 strain, respectively. (B) *, Q represent puri®ed acid phosphatases I and II secreted by the biA1 pacA1 strain, respectively. Table 2. Summary of kinetic constants for the enzymatic hydrolysis of PNP-P at 378, pH 6.0. Strains
Substrate PNP-P
pabaA1(I) pabaA1(II) biA1 (I) biA1 (II) pacA1 biA1(I) pacA1 biA1(II)
a-naphthylphosphate
Km
n
Km
n
0.46 20.05 0.40 20.08 0.53 20.07 0.47 20.04 0.44 20.04 0.38 20.06
0.97 20.04 1.10 20.05 1.04 20.03 0.97 20.05 1.06 20.03 1.08 20.04
0.402 0.07 0.252 0.06 0.462 0.05 0.222 0.07 0.522 0.04 0.242 0.07
1.032 0.04 0.972 0.02 0.992 0.06 1.062 0.04 1.002 0.04 0.952 0.04
For details see Experimental.
Phosphatases from A. nidulans
Fig. 4. Thermal inactivation of puri®ed acid phosphatases at 608, pH 6.0, secreted by A. nidulans grown in low-Pi medium at 378, pH 5.0. Q, * represent the enzyme forms I and II secreted by strain biA1. q, w represent the enzyme forms I and II secreted by strain biA1 pacA1.
Heat inactivation at 608 and at pH 6.0 showed half-lives of 14 min (k = 0.033 minÿ1) and 10 min (k = 0.069 minÿ1), respectively, (Fig. 4), for the puri®ed acid phosphatases I and II secreted by biA1 strain and half-lives of 0.8 min (k = 0.92 minÿ1) and 0.6 min (k = 0.95 minÿ1), respectively, (Fig. 4), for the puri®ed forms I and II secreted by the biA1 pacA1 strain. The above results indicate that Pi-repressible acid phosphatases I and II secreted by A. nidulans are encoded by a single gene (pacA). Although several mechanisms may be responsible for the dierential expression of these enzyme forms [11, 12], they could be generated by the glycosylation needed for the ecient transport of the enzyme from one compartment to another through the secretory pathway [13]. Such a mechanism does not imply a priori that all of the enzyme molecules will be modi®ed at the same time, and that the unprocessed acid phosphatase will not be translocated and subsequently modi®ed until its ®nal destination, i.e. the culture medium. EXPERIMENTAL
Materials Except where otherwise stated, all chemicals were of analytical grade and supplied by Merck or Sigma. Growth conditions and strains The solid complete and minimal liquid media of Ref. [14] were used. The complete medium is Pi-suf®cient (11 mM) and its pH is 6.5. Low-Pi medium,
1521
prepd by adding 200 mM KH2PO4, 10 mM KCl and 50 mM Na citrate to Pi-free minimal medium (®nal concn), was adjusted to pH 5.0. Both media contained (®nal concns) 1% (w/v) D-glucose as carbon source, 70 mM NaNO3 as nitrogen source and all nutrient requirements. Unless otherwise stated, a growth temp. of 378 was used. The following strains of A. nidulans were used in the present study: biA1 (biotin requiring), pabaA1 (p-aminobenzoic acid requiring) and biA1 pacA1 (FGSC A241). The pacA1 strain was identi®ed as carrying mutation in the structural gene for Pirepressible acid phosphatase V [2]. All of these strains are isogenic and mutations carried by A. nidulans have been described previously ( [15], and references therein) and are available from the Fungal Genetic Stock Center, University of Kansas Medical Center, Kansas City, Kansas (USA).
Assays Pi-repressible acid phosphatase activity was determined at pH 6.0 (100 mM Na maleate, 2 mM EDTA) using 6 mM p-nitrophenylphosphate (PNP-P) as substrate at 378 [3]. One unit of acid phosphatase activity was de®ned as one nmol substrate hydrolysed minÿ1. Mycelial speci®c activities were expressed as units per mg dry wt mycelium. Protein concn was estimated as in Ref. [16], with BSA (fraction V) as the standard. The protein content of frs from the column chromatographic separations were monitored by measuring the A at 280 nm.
Puri®cation of secreted acid phosphatases The procedure used for the puri®cation to apparent homogeneity by PAGE of acid phosphatases I and II secreted by strains biA1 and biA1 pacA1 of A. nidulans was a modi®cation of that described in Ref. [5]. All puri®cation procedures were carried out at 0±48 unless otherwise stated. p-Nitrophenylphosphatase activity was determined at each stage of puri®cation. Conidia of each strain grown on solid complete medium were harvested into sterile saline (0.9% NaCl) containing 0.01% Tween 80, ®ltered through a layer of glass wool to remove mycelia and adjusted to 109 cells mlÿ1. One ml of this conidial suspension was inoculated into 500 ml conceive ¯asks containing 100 ml low-Pi medium and grown for 14 hr (pabaA1 and biA1 strains) or 20 hr (biA1 pacA1 strain) in an orbital incubator (160 rpm). An appropriate vol. of the harvested culture medium was conc ca 10 times by ultra®ltration through AMICON (YM10) membranes, dialysed for 24 hr against 8 l of H2O (with 2 changes) and
1522
S. R. NOZAWA et al.
fractionated by (NH4)2SO4 pptn. The acid phosphatase activity, recovered in the 65% to 95% salt satd fr, was suspended in a small vol. of 50 mM NaOAc buer (pH 5.5), dialysed for 24 hr against 4 l of this (with 4 changes), centrifuged if necessary and the pH was adjusted to 7.2 with solid Tris. This enzyme fr was applied to a column (1.4 45 cm) of DEAEcellulose previously equilibrated with 10 mM MOPS (pH 7.2). After elution of non-absorbed proteins with ca 100 ml of the equilibrating buer, enzymes were eluted with a discontinuous gradient from 50 to 500 mM NaCl in 10 mM MOPS (pH 7.2) at a ¯ow rate of 120 ml hrÿ1 and 6 ml frs were collected into tubes containing 1 ml of 100 mM NaOAc buffer, pH 4.7. The tubes representing each enzyme peak were pooled, dialysed for 24 hr against 8 l of 50 mM NaOAc buer, pH 5.5, containing 100 mM NaCl (with 3 changes), conc by ultra®ltration through AMICON (YM 10) membranes and independently chromatographed on a Sephadex G200 column (1.2 120 cm) previously equilibrated with the dialysing buer. Elution was performed with this same buer at a ¯ow rate of 9 ml hrÿ1 and 3 ml frs were collected. The tubes representing the enzyme peak were pooled, dialysed for 24 hr against 8 l of H2O (with 2 changes), conc by ultra®ltration through AMICON (YM 10) membranes and stored at 48. Characterization of the puri®ed enzymes The buers used to cover the pH range required were 0.1 M citric acid/NaOH (pH 4.0±6.0) [17], 0.1 M maleic acid/NaOH (pH 6.0±7.5) and 0.1 M Tris-HCl (pH 7.5±8.0). Relative heat stability was determined by incubating the enzyme from dierent sources in 10 mM Na maleate buer, pH 6.0, at 608, in the same experiment. At appropriate times, samples were removed to measure the remaining phosphatase activities using the standard procedure. Limiting velocities (Vm) and Michaelis constants (Km) were determined from Lineweaver and Burk plots [18]. The interaction constant for the substrate (n) was determined by the Hill procedure as described in Ref. [19]. The kinetic constants reported here were obtained by linear-square analysis calculated from the data obtained in at least 3 independent experiments. PAGE was carried out at pH 8.3 by the method of Davis as described in Ref. [20] using 7.5% (w/v) polyacrylamide slab gels (10 10 0.1 cm). The phosphomonoesterase activity bands were developed by the method of Dorn as described in ref. [8] using Na a-naphthylphosphate as the substrate. SDS-PAGE was carried out as in Ref. [21] using polyacrylamide slab gels, and the protein bands were visualized with Coomassie blue. Prior to loading, all samples were incubated in the
presence of 1% (w/v) SDS and 100 mM b-mercaptoethanol for 3 min at 1008. When necessary, the protein bands were stained with Ag as described in Ref. [22]. The ratio of the distance covered by the enzyme bands to the distance covered by bromophenol blue (relative electrophoretic mobility, Rf) was measured. Mr were measured by exclusion chromatography under standard conditions (see above) and by SDSPAGE as described in Ref. [21], using appropriate Mr markers. Neutral sugars were measured as described in Ref. [23] with glucose as standard. Acknowledgements ÐThis work was supported by grants from FAPESP and CNPq. W.M. Jr was supported by a Postdoctoral fellowship from FAPESP. We thank Mrs Elettra Greene for revising the English text and Newton R. Alves for technical assistance.
REFERENCES
1. Dorn, G., Genetical Research (Cambridge), 1965, 6, 13. 2. Caddick, M. X. and Arst, H. N., Jr., Genetical Research (Cambridge), 1986, 47, 83. 3. Caddick, M. X., Brownlee, A. G. and Arst, H. N., Jr., Genetical Research (Cambridge), 1986, 47, 93. 4. Caddick, M. X., Brownlee, A. G. and Arst, H. N., Jr., Molecular and General Genetics, 1986, 203, 346. 5. Harsanyi, Z. and Dorn, G. L., Journal of Bacteriology, 1972, 110, 246. 6. Negrete-Urtasun, S., Denison, S. H. and Arst, H. N., Jr., Journal of Bacteriology, 1997, 179, 1832. 7. Maccheroni, W. M., Jr., May, G. S., MartinezRossi, N. M. and Rossi, A., Gene, 1997, 197, 163. 8. Maccheroni, W. M., Jr., Martinez-Rossi, N. M. and Rossi, A., Brazilian Journal of Medical and biological Research, 1995, 28, 31. 9. Odds, F. C. and Hierholzer, J. C., Journal of Bacteriology, 1973, 114, 257. 10. Frank, R. N. and Rodbard, D., Archives of Biochemistry and Biophysics, 1975, 171, 1. 11. Thedei, G., Jr., Nozawa, S. R., SimoÄes, A. L. and Rossi, A., World Journal of Microbiology & Biotechnology, 1997, 13, 609. 12. Danpure, C. J., Trends in Cell Biology, 1995, 5, 230. 13. Haguenauer-Tsapis, R., Molecular Microbiology, 1992, 6, 573. 14. Cove, D. J., Biochimica et Biophysica Acta, 1966, 113, 51. 15. Clutterbuck, A. J., Genetic Maps. Locus Maps of Complex Genomes. Cold Spring Harbor Laboratory Press, New York, 1993.
Phosphatases from A. nidulans
16. Hartree, E. F., Analytical Biochemistry, 1972, 48, 422. 17. Nozawa, S. R., Rigoli, I. C., Thedei, G., Jr and Rossi., A., Fungal Genetics Newsletter, 1995, 42, 56. 18. Lineweaver, H. and Burk, D., Journal of the American Chemical Society, 1934, 56, 658. 19. Koshland Jr, D. E., The Enzymes. Academic Press, New York, 1970.
1523
20. Han, S. W., Michelin, M. A., Barbosa, J. E. and Rossi, A., Phytochemistry, 1994, 28, 3281. 21. Thedei, G., Jr and Rossi., A., Plant and Cell Physiology, 1994, 35, 837. 22. Morrissay, J. H., Analytical Biochemistry, 1981, 117, 307. 23. Dubois, B., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F., Analytical Chemistry, 1965, 28, 350.
Phytochemistry Vol. 49, No. 6, pp. 1517±1523, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0031-9422/98/$ - see front matter
PURIFICATION AND PROPERTIES OF PI-REPRESSIBLE ACID PHOSPHATASES FROM ASPERGILLUS NIDULANS SEÂRGIO R. NOZAWA, WALTER MACCHERONI JR*, RODRIGO G. STAÂBELI, GERALDO THEDEI JR{ and ANTONIO ROSSI{ Departamento de QuõÂ mica, FFCLRP-USP, 14040-901 RibeiraÄo Preto, Brazil. (Received 24 February 1998)
Key Word IndexÐAspergillus nidulans; Fungi; enzyme secretion; acid phosphatase; phosphate regulation. AbstractÐTwo forms of the pacA-encoded acid phosphatase (designated acid phosphatases I and II) secreted by the mold Aspergillus nidulans grown in low-Pi medium at 378, pH 5.0, were puri®ed to apparent homogeneity by PAGE. The Mr of the puri®ed enzyme forms were ca 115 000 (60 000) and 113 000 (62 000) respectively for forms I and II secreted by strain biA1 and ca 118 000 (60 000) and 121 000 (61 000) respectively for forms I and II secreted by strain biA1 pacA1, as determined by exclusion chromatography (number between brackets are the Mr as determined by SDS-PAGE). All of these puri®ed enzyme forms showed an apparent optimum pH ranging from 6.0 to 6.5 and no deviation from Michaelis kinetics for the hydrolysis of both p-nitrophenylphosphate and a-naphthylphosphate. Heat inactivation at 608 and at pH 6.0 showed half-lives of 14 min (k = 0.033 minÿ1) and 10 min (k = 0.069 minÿ1), respectively, for the puri®ed acid phosphatases I and II secreted by biA1 strain and half-lives of 0.8 min (k = 0.92 minÿ1) and 0.6 min (k = 0.95 minÿ1), respectively, for the puri®ed forms I and II secreted by the biA1 pacA1 strain. The neutral sugar content of puri®ed acid phosphatases I and II secreted by strain biA1 was 48% and 37% (w/w), respectively, whereas the content of forms I and II secreted by strain biA1 pacA1 was 18% and 11%, respectively. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
In fungi, acquisition of nutrients from the environment involves the secretion of an array of hydrolytic enzymes acting speci®cally on dierent resources. The phosphatases, a generic designation for nonspeci®c phosphoesterases, belong to a family of enzymes responsible for supplying Pi to the cell. Four major phosphatases and their corresponding structural genes have been identi®ed in the ascomycetes fungus Aspergillus nidulans [1, 2]. Two of these, the palD-encoded alkaline phosphatase and the pacA-encoded acid phosphatase, are synthesized exclusively under Pi-limiting conditions and therefore are called Pi-repressible phosphatases. The study of these enzymes is a powerful tool in the elucidation of gene expression regarding not only the synthesis but also the secretion of enzymes by *Present address: Departamento de GeneÂtica, ESALQUSP, 13400-970 Piracicaba, SP, Brazil. {Present address: Instituto de CieÃncias da SauÂde, UNIUBE, 38065-500 Uberaba, MG, Brazil. {Author to whom correspondence should be addressed.
eukaryotic cells in response to ambient factors such as pH and carbon, nitrogen, sulfur and phosphorus sources [3, 4]. The intracellular form of the pacA-encoded acid phosphatase has been puri®ed and partially characterized [5], whereas the structural and steady state kinetic properties of its form secreted into the culture medium remain to be determined. Even though knowledge of these properties is not sucient per se to elucidate the mechanism of acid phosphatase secretion, this type of study may reveal a few important aspects concerning the regulation of the enzymes and permeases involved in Pi acquisition in A. nidulans, up to now studied only at the level of transcription of its structural genes ( [6, 7], and references therein). With this in mind, we puri®ed both forms I and II [8] of the major Pi-repressible acid phosphatases secreted into the growth medium by A. nidulans at pH 5.0 and compared their properties with those of the corresponding enzyme forms secreted by a pacA1 strain. Our results show that Pi-repressible acid phosphatases I and II secreted by A. nidulans are encoded by a
1517
1518
S. R. NOZAWA et al.
single gene (pacA) and that the pacA1 mutation resulted in the partial glycosylation of the enzyme forms secreted by the mutant strain. RESULTS AND DISCUSSION
Con®rming earlier reports [2], the levels of active Pi-repressible acid phosphatase secreted by the biA1 pacA1 strain of A. nidulans were reduced when the temperature of fungal culture was increased from 228 to 378, compared with the values shown by the biA1 strain (Fig. 1). Although only slightly, the mycelial yield of both strains also decreased with increasing culture temperature (Fig. 1). Nevertheless, the stepwise fractionation on DEAE-cellulose of the enzyme secreted by the biA1 pacA1 strain grown at 378 practically showed the same enzyme fractions as those secreted by the biA1 strain (Fig. 2). Not only the identi®cation but also the increased yield of each fraction were possible because the eluted fractions were collected in tubes
containing 0.1 M NaOAc buer, pH 4.7. This because the secreted acid phosphatase is highly labile at alkaline pH. In an attempt to further characterize the acid phosphatases secreted by A. nidulans we puri®ed to apparent homogeneity by PAGE forms I and II (Fig. 2) of the acid phosphatases secreted by the mold grown in low-Pi medium. The procedures summarized in Table 1 provided optimal conditions for the puri®cation of both forms secreted by strains biA1 and biA1 pacA1. All enzyme preparations (at least four independent preparations of each enzyme fraction) appeared to be homogeneous by 7.5% PAGE at pH 8.3, with the protein band being superimposable on acid phosphatase activity. All of these preparations also essentially showed the same electrophoretic mobility for each enzyme form secreted by both strains, i.e., the Rf values were 0.51 and 0.59 for acid phosphatases I and II, respectively. When mixed, the two enzyme forms combined in
Fig. 1. Production of acid phosphatase in cultures of A. nidulans grown in low-Pi medium, adjusted to pH 5.0, and buered with 50 mM Na citrate. A and C represent mycelial yield and acid phosphatase secreted by biA1 strain grown at 228 and 378, respectively. B and D represent mycelial yield and acid phosphatase secreted by biA1 pacA1 strain grown at 228 and 378 respectively.
Phosphatases from A. nidulans
1519
Fig. 2. Stepwise fractionation on DEAE-cellulose of acid phosphatases secreted by the biA1 (A) and biA1 pacA1 (B) strains of A. nidulans grown in low-Pi medium at 378, pH 5.0. After elution of nonabsorbed proteins with ca 100 ml of the equilibrating buer, proteins were eluted by the stepwise addition of 0.05, 0.10 M (fraction I), 0.15 M (fraction II), 0.30 M and 0.50 M NaCl dissolved in 10 mM MOPS, pH 7.2. 6 ml fractions were collected in tubes containing 1 ml of 0.10 M NaOAc buer, pH 4.7, at a ¯ow rate of 120 ml hrÿ1.
a stable manner since they migrated as a single band, the Rf value being 0.56. Furthermore, acid phosphatase I secreted by the biA1 pacA1 strain was puri®ed 120-fold with a low yield (3.8%) as compared with the value shown by the biA1 strain (Table 1). The neutral sugar content of puri®ed acid phosphatases I and II secreted by strain biA1 were 48% and 37% (w/w), respectively, whereas the content of forms I and II secreted by strain biA1 pacA1 was 18% and 11%, respectively, suggesting that these enzymatic forms are glycoproteins and that the pacA1 mutation possibly altered a glycosylation site or the molecule conformation, resulting in partial glycosylation of the enzyme forms secreted by the
mutant strain. Determination of the pH activity pro®le showed an apparent optimum ranging from 6.0 to 6.5 for puri®ed acid phosphatases I and II secreted by strains biA1 and biA1 pacA1 (Fig. 3). Also, the activity of acid phosphatases I and II secreted by strain biA1 pacA1 was inhibited at pH higher than 6.5 (Fig. 3B) as compared with the relative activity shown by the enzyme forms secreted by the biA1 strain (Fig. 3A), an eect probably due to the lower glycosylation level of these enzyme forms (see above). The Mr of the puri®ed enzyme forms were ca 115 000 (60 000) and 113 000 (62 000) respectively for forms I and II secreted by strain biA1 and ca 118 000 (60 000) and 121 000 (61 000) respectively
1520
S. R. NOZAWA et al. Table 1. Puri®cation of acid phosphatases I and II.
Fraction
Yield (%)
Puri®cation (fold)
100 62.1
1 21.8
18.5 15.0
40.1 37.7
12.2 7.4
88.4 64.3
pacA1 strain of A. nidulans grown in low-Pi medium at 378, pH 5.0. 1850 97.7 130 1.33 12 1.8 58.8 32.7
100 45.2
1 24.6
2.5 2.5
0.064 0.16
8 10
125 61.5
6.1 7.7
94.0 46.2
3 4
0.03 0.12
5 8
166.7 66.7
3.8 6.1
125.3 50.2
Volume (ml)
Total protein (mg)
Total activity (Units)
Speci®c activity (Units mgÿ1)
(A) Secreted by the biA1 strain of A. nidulans grown in low-Pi medium at 378, pH 5.0. Culture medium 1800 576 486 0.84 10 16.5 302 18.3 (NH4)2SO4 (65±95%) DEAE-cellulose Fraction I 3.3 2.67 89.9 33.7 Fraction II 2.8 2.3 72.8 31.7 Sephadex G-200 Fraction I 2.7 0.80 59.4 74.3 Fraction II 3.2 0.67 36.2 54.0 (B) Secreted by the biA1 Culture medium (NH4)2SO4 (65±95%) DEAE-cellulose Fraction I Fraction II Sephadex G-200 Fraction I Fraction II
for forms I and II secreted by strain biA1 pacA1, as determined by exclusion chromatography (number between brackets are the Mr as determined by SDSPAGE). Although these methods are not accurate for glycoproteins [9, 10] these results suggest that both the intracellular (Mr of ca 100 000 as estimated by Sephadex G100 gel ®ltration [5]) as the secreted enzyme forms are dimers. The puri®ed
enzyme forms I and II secreted by strains biA1 and biA1 pacA1 showed no deviation from Michaelis kinetics for the hydrolysis of both PNP-P and anaphthylphosphate (Table 2). The Km values of the two substrates were quite similar, except that form II secreted by both strains had a higher anity for the hydrolysis of a-naphthylphosphate (low Km value).
Fig. 3. pH activity pro®le of puri®ed acid phosphatases secreted by A. nidulans grown in low-Pi medium at 378, pH 5.0. (A) *, Q represent puri®ed acid phosphatases I and II secreted by the biA1 strain, respectively. (B) *, Q represent puri®ed acid phosphatases I and II secreted by the biA1 pacA1 strain, respectively. Table 2. Summary of kinetic constants for the enzymatic hydrolysis of PNP-P at 378, pH 6.0. Strains
Substrate PNP-P
pabaA1(I) pabaA1(II) biA1 (I) biA1 (II) pacA1 biA1(I) pacA1 biA1(II)
a-naphthylphosphate
Km
n
Km
n
0.46 20.05 0.40 20.08 0.53 20.07 0.47 20.04 0.44 20.04 0.38 20.06
0.97 20.04 1.10 20.05 1.04 20.03 0.97 20.05 1.06 20.03 1.08 20.04
0.402 0.07 0.252 0.06 0.462 0.05 0.222 0.07 0.522 0.04 0.242 0.07
1.032 0.04 0.972 0.02 0.992 0.06 1.062 0.04 1.002 0.04 0.952 0.04
For details see Experimental.
Phosphatases from A. nidulans
Fig. 4. Thermal inactivation of puri®ed acid phosphatases at 608, pH 6.0, secreted by A. nidulans grown in low-Pi medium at 378, pH 5.0. Q, * represent the enzyme forms I and II secreted by strain biA1. q, w represent the enzyme forms I and II secreted by strain biA1 pacA1.
Heat inactivation at 608 and at pH 6.0 showed half-lives of 14 min (k = 0.033 minÿ1) and 10 min (k = 0.069 minÿ1), respectively, (Fig. 4), for the puri®ed acid phosphatases I and II secreted by biA1 strain and half-lives of 0.8 min (k = 0.92 minÿ1) and 0.6 min (k = 0.95 minÿ1), respectively, (Fig. 4), for the puri®ed forms I and II secreted by the biA1 pacA1 strain. The above results indicate that Pi-repressible acid phosphatases I and II secreted by A. nidulans are encoded by a single gene (pacA). Although several mechanisms may be responsible for the dierential expression of these enzyme forms [11, 12], they could be generated by the glycosylation needed for the ecient transport of the enzyme from one compartment to another through the secretory pathway [13]. Such a mechanism does not imply a priori that all of the enzyme molecules will be modi®ed at the same time, and that the unprocessed acid phosphatase will not be translocated and subsequently modi®ed until its ®nal destination, i.e. the culture medium. EXPERIMENTAL
Materials Except where otherwise stated, all chemicals were of analytical grade and supplied by Merck or Sigma. Growth conditions and strains The solid complete and minimal liquid media of Ref. [14] were used. The complete medium is Pi-suf®cient (11 mM) and its pH is 6.5. Low-Pi medium,
1521
prepd by adding 200 mM KH2PO4, 10 mM KCl and 50 mM Na citrate to Pi-free minimal medium (®nal concn), was adjusted to pH 5.0. Both media contained (®nal concns) 1% (w/v) D-glucose as carbon source, 70 mM NaNO3 as nitrogen source and all nutrient requirements. Unless otherwise stated, a growth temp. of 378 was used. The following strains of A. nidulans were used in the present study: biA1 (biotin requiring), pabaA1 (p-aminobenzoic acid requiring) and biA1 pacA1 (FGSC A241). The pacA1 strain was identi®ed as carrying mutation in the structural gene for Pirepressible acid phosphatase V [2]. All of these strains are isogenic and mutations carried by A. nidulans have been described previously ( [15], and references therein) and are available from the Fungal Genetic Stock Center, University of Kansas Medical Center, Kansas City, Kansas (USA).
Assays Pi-repressible acid phosphatase activity was determined at pH 6.0 (100 mM Na maleate, 2 mM EDTA) using 6 mM p-nitrophenylphosphate (PNP-P) as substrate at 378 [3]. One unit of acid phosphatase activity was de®ned as one nmol substrate hydrolysed minÿ1. Mycelial speci®c activities were expressed as units per mg dry wt mycelium. Protein concn was estimated as in Ref. [16], with BSA (fraction V) as the standard. The protein content of frs from the column chromatographic separations were monitored by measuring the A at 280 nm.
Puri®cation of secreted acid phosphatases The procedure used for the puri®cation to apparent homogeneity by PAGE of acid phosphatases I and II secreted by strains biA1 and biA1 pacA1 of A. nidulans was a modi®cation of that described in Ref. [5]. All puri®cation procedures were carried out at 0±48 unless otherwise stated. p-Nitrophenylphosphatase activity was determined at each stage of puri®cation. Conidia of each strain grown on solid complete medium were harvested into sterile saline (0.9% NaCl) containing 0.01% Tween 80, ®ltered through a layer of glass wool to remove mycelia and adjusted to 109 cells mlÿ1. One ml of this conidial suspension was inoculated into 500 ml conceive ¯asks containing 100 ml low-Pi medium and grown for 14 hr (pabaA1 and biA1 strains) or 20 hr (biA1 pacA1 strain) in an orbital incubator (160 rpm). An appropriate vol. of the harvested culture medium was conc ca 10 times by ultra®ltration through AMICON (YM10) membranes, dialysed for 24 hr against 8 l of H2O (with 2 changes) and
1522
S. R. NOZAWA et al.
fractionated by (NH4)2SO4 pptn. The acid phosphatase activity, recovered in the 65% to 95% salt satd fr, was suspended in a small vol. of 50 mM NaOAc buer (pH 5.5), dialysed for 24 hr against 4 l of this (with 4 changes), centrifuged if necessary and the pH was adjusted to 7.2 with solid Tris. This enzyme fr was applied to a column (1.4 45 cm) of DEAEcellulose previously equilibrated with 10 mM MOPS (pH 7.2). After elution of non-absorbed proteins with ca 100 ml of the equilibrating buer, enzymes were eluted with a discontinuous gradient from 50 to 500 mM NaCl in 10 mM MOPS (pH 7.2) at a ¯ow rate of 120 ml hrÿ1 and 6 ml frs were collected into tubes containing 1 ml of 100 mM NaOAc buffer, pH 4.7. The tubes representing each enzyme peak were pooled, dialysed for 24 hr against 8 l of 50 mM NaOAc buer, pH 5.5, containing 100 mM NaCl (with 3 changes), conc by ultra®ltration through AMICON (YM 10) membranes and independently chromatographed on a Sephadex G200 column (1.2 120 cm) previously equilibrated with the dialysing buer. Elution was performed with this same buer at a ¯ow rate of 9 ml hrÿ1 and 3 ml frs were collected. The tubes representing the enzyme peak were pooled, dialysed for 24 hr against 8 l of H2O (with 2 changes), conc by ultra®ltration through AMICON (YM 10) membranes and stored at 48. Characterization of the puri®ed enzymes The buers used to cover the pH range required were 0.1 M citric acid/NaOH (pH 4.0±6.0) [17], 0.1 M maleic acid/NaOH (pH 6.0±7.5) and 0.1 M Tris-HCl (pH 7.5±8.0). Relative heat stability was determined by incubating the enzyme from dierent sources in 10 mM Na maleate buer, pH 6.0, at 608, in the same experiment. At appropriate times, samples were removed to measure the remaining phosphatase activities using the standard procedure. Limiting velocities (Vm) and Michaelis constants (Km) were determined from Lineweaver and Burk plots [18]. The interaction constant for the substrate (n) was determined by the Hill procedure as described in Ref. [19]. The kinetic constants reported here were obtained by linear-square analysis calculated from the data obtained in at least 3 independent experiments. PAGE was carried out at pH 8.3 by the method of Davis as described in Ref. [20] using 7.5% (w/v) polyacrylamide slab gels (10 10 0.1 cm). The phosphomonoesterase activity bands were developed by the method of Dorn as described in ref. [8] using Na a-naphthylphosphate as the substrate. SDS-PAGE was carried out as in Ref. [21] using polyacrylamide slab gels, and the protein bands were visualized with Coomassie blue. Prior to loading, all samples were incubated in the
presence of 1% (w/v) SDS and 100 mM b-mercaptoethanol for 3 min at 1008. When necessary, the protein bands were stained with Ag as described in Ref. [22]. The ratio of the distance covered by the enzyme bands to the distance covered by bromophenol blue (relative electrophoretic mobility, Rf) was measured. Mr were measured by exclusion chromatography under standard conditions (see above) and by SDSPAGE as described in Ref. [21], using appropriate Mr markers. Neutral sugars were measured as described in Ref. [23] with glucose as standard. Acknowledgements ÐThis work was supported by grants from FAPESP and CNPq. W.M. Jr was supported by a Postdoctoral fellowship from FAPESP. We thank Mrs Elettra Greene for revising the English text and Newton R. Alves for technical assistance.
REFERENCES
1. Dorn, G., Genetical Research (Cambridge), 1965, 6, 13. 2. Caddick, M. X. and Arst, H. N., Jr., Genetical Research (Cambridge), 1986, 47, 83. 3. Caddick, M. X., Brownlee, A. G. and Arst, H. N., Jr., Genetical Research (Cambridge), 1986, 47, 93. 4. Caddick, M. X., Brownlee, A. G. and Arst, H. N., Jr., Molecular and General Genetics, 1986, 203, 346. 5. Harsanyi, Z. and Dorn, G. L., Journal of Bacteriology, 1972, 110, 246. 6. Negrete-Urtasun, S., Denison, S. H. and Arst, H. N., Jr., Journal of Bacteriology, 1997, 179, 1832. 7. Maccheroni, W. M., Jr., May, G. S., MartinezRossi, N. M. and Rossi, A., Gene, 1997, 197, 163. 8. Maccheroni, W. M., Jr., Martinez-Rossi, N. M. and Rossi, A., Brazilian Journal of Medical and biological Research, 1995, 28, 31. 9. Odds, F. C. and Hierholzer, J. C., Journal of Bacteriology, 1973, 114, 257. 10. Frank, R. N. and Rodbard, D., Archives of Biochemistry and Biophysics, 1975, 171, 1. 11. Thedei, G., Jr., Nozawa, S. R., SimoÄes, A. L. and Rossi, A., World Journal of Microbiology & Biotechnology, 1997, 13, 609. 12. Danpure, C. J., Trends in Cell Biology, 1995, 5, 230. 13. Haguenauer-Tsapis, R., Molecular Microbiology, 1992, 6, 573. 14. Cove, D. J., Biochimica et Biophysica Acta, 1966, 113, 51. 15. Clutterbuck, A. J., Genetic Maps. Locus Maps of Complex Genomes. Cold Spring Harbor Laboratory Press, New York, 1993.
Phosphatases from A. nidulans
16. Hartree, E. F., Analytical Biochemistry, 1972, 48, 422. 17. Nozawa, S. R., Rigoli, I. C., Thedei, G., Jr and Rossi., A., Fungal Genetics Newsletter, 1995, 42, 56. 18. Lineweaver, H. and Burk, D., Journal of the American Chemical Society, 1934, 56, 658. 19. Koshland Jr, D. E., The Enzymes. Academic Press, New York, 1970.
1523
20. Han, S. W., Michelin, M. A., Barbosa, J. E. and Rossi, A., Phytochemistry, 1994, 28, 3281. 21. Thedei, G., Jr and Rossi., A., Plant and Cell Physiology, 1994, 35, 837. 22. Morrissay, J. H., Analytical Biochemistry, 1981, 117, 307. 23. Dubois, B., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F., Analytical Chemistry, 1965, 28, 350.