Properties of a constitutive alkaline phosphatase from strain 74 A of the mold Neurospora crassa

A constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30oC. The molecular mass was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. This monomeric enzyme shows an apparent optimum pH ranging from 9.5 to 10.5 and Michaelis kinetics for the hydrolysis of p-nitrophenyl phosphate (the Km and Hill coefficient values were 0.35 mM and 1.01, respectively), a-naphthyl phosphate (the Km and Hill coefficient values were 0.44 mM and 0.97, respectively), ß-glycerol phosphate (the Km and Hill coefficient values were 2.46 mM and 1.01, respectively) and L-histidinol phosphate (the Km and Hill coefficient values were 0.47 mM and 0.94, respectively) at pH 8.9. The purified enzyme is activated by Mg2+, Zn2+ and Tris-HCl buffer, and is inhibited by Be2+, histidine and EDTA. Also, 0.3 M Tris-HCl buffer protected the purified enzyme against heat inactivation at 70oC (halflife of 19.0 min, k = 0.036 min-1) as compared to 0.3 M CHES (halflife of 2.3 min, k = 0.392 min-1) in the same experiment. Correspondence A. Rossi Departamento de Química FFCLRP-USP Av. Bandeirantes, 390


Introduction
The mold Neurospora crassa synthesizes three mycelial alkaline phosphatases (APase) when grown at pH 5.4.While two of them are expressed irrespective of the concentration of inorganic phosphate (Pi) in the culture medium a third one is only expressed when the medium is growth-limiting in Pi (1)(2)(3).Only one gene encoding the Pi-repressible APase has been identified (pho-2), cloned and characterized at the molecular level (4).The pho-2 gene is a component of the phos-phate acquisition system of N. crassa and is expressed irrespective of the growth pH, except that at alkaline pH the enzyme is largely secreted into the growth medium (5,6).Also, the molecular properties of the Pi-repressible APase expressed at both pH 5.4 and pH 7.8 have been extensively characterized (7)(8)(9)(10).On the other hand, much remains to be clarified about the molecular properties and physiological role of these constitutive APases of N. crassa (1,11,12).Thus, in an attempt to further investigate its properties, the major APase synthesized by strain 74A of N. crassa grown on high-Pi medium supplemented with acetate as the sole carbon source at pH 7.1 was purified to apparent homogeneity as determined by SDS-PAGE.Some properties of the purified enzyme such as molecular mass, relative heat stability and hydrolysis of L-histidinol-Pi, which is part of the pathway of histidine biosynthesis in N. crassa (13), were also determined.

Material
Except where otherwise stated, all chemicals were of analytical grade and supplied by Merck (Darmstadt, Germany) or Sigma Chemical Co.(St.Louis, MO, USA).

Strain and growth conditions
The wild type strain St. L. 74A of N. crassa (Fungal Genetic Stock Center, University of Kansas Medical Center, Kansas City, KS, USA) was used in the present study.Stock cultures were maintained on slants of Vogels medium (1.6% agar) (14).Conidial suspensions (2.5 ml containing about 10 8 cells/ml) were grown for 72 h at 30 o C in an orbital shaker (160 rpm) in Erlenmeyer flasks (500 ml) containing 100 ml of high (10 mM)-Pi medium, supplemented with 44 mM of the desired carbon source as follows: non-buffered sucrose adjusted to pH 5.4, acetate buffered at pH 5.4 with 100 mM sodium maleate, non-buffered acetate adjusted to pH 7.1 and sucrose buffered with 50 mM Tris-HCl and prepared as described previously (5,15).

Preparation of extracts and assays
The mycelium harvested by filtration was extracted with sand and 50 mM Tris-HCl buffer, pH 8.8 (10 ml buffer/g mycelium), containing 40 mM MgSO 4 , 1 mM benzamidine and 1 mM PMSF (final concentrations).After shaking for 15 min at 4 o C the supernatant (crude extract) was collected by centrifugation (20 min at 17,000 g) and used for enzyme assays or enzyme purification.Except where otherwise stated, constitutive APase activity was determined as described previously (1) in 0.3 M Tris-HCl buffer, pH 8.9, containing 1 mM MgSO 4 , using 6 mM pnitrophenyl phosphate (PNP-P) as substrate at 37 o C. L-Histidinol-Pi, ß-glycerol-Pi and a-naphthyl-Pi hydrolysis was carried out in 0.3 M Tris-HCl buffer as described by Kuo and Blumenthal (1).The liberated Pi was measured by the method of Heinonen and Lahti (16).Incubations were carried out at 37 o C, and all enzyme activities were measured in duplicate for at least two time intervals.One unit of APase activity was defined as one µmol substrate hydrolyzed min -1 .Protein concentration was estimated by the method of Folin as described by Hartree (17), with BSA (fraction V) as the standard.
The protein content of fractions obtained by column chromatography was monitored by measuring absorbance at 220 nm.

Enzyme purification
The procedure used for the purification to apparent homogeneity as determined by PAGE of a constitutive alkaline APase synthesized by the mold N. crassa was a modification of previously described methods (1,3).Mycelium extract (crude extract) of the wild-type strain 74A grown on high-Pi medium, pH 7.1, was incubated at 50 o C for 45 min and centrifuged at 17,000 g for 20 min at 4 o C and the supernatant was fractionated by (NH 4 ) 2 SO 4 precipitation.The APase activity recovered in the 40-60% salt saturation step was suspended in a small volume of 10 mM Tris-HCl buffer, pH 8.8, containing 30 mM MgSO 4 , dialyzed for 24 h against 4 l of the same buffer (3 changes of buffer) and centrifuged if necessary (14,000 g for 10 min).The dialyzed enzyme was applied to a column (1.1 x 50 cm) of DEAE-cellulose previously equilibrated with the buffer used for dialysis.Non-absorbed proteins were eluted with about 200 ml of the same buffer and showed little phosphatase activity.Enzyme elution was performed with a noncontinuous concentration gradient (12.5 and 50 mM KCl in 10 mM Tris-HCl buffer, pH 8.8, containing 40 mM MgSO 4 ) at a flow rate of about 150 ml/h (10-ml fractions).The tubes representing the enzyme peak were pooled and concentrated by ultrafiltration through Amicon (YM 10) membranes.This concentrate was applied to a column (1.5 x 130 cm) of Sephacryl S-200-HR previously equilibrated with 10 mM Tris-HCl buffer, pH 8.8, containing 30 mM MgSO 4 and 200 mM NaCl.Elution was performed with this buffer at a flow rate of 12 ml/h (2.5-ml fractions).The tubes representing the enzyme peak were pooled, concentrated by ultrafiltration through Amicon (YM 10) mem-branes and applied to a column (1.5 x 9.2 cm) of phenyl-Sepharose CL-4B previously equilibrated with 10 mM Tris-HCl buffer, pH 8.8, containing 10 mM MgSO 4 and 2 M NaCl (final concentrations).Enzyme elution was performed with a discontinuous concentration gradient (10 mM Tris-HCl buffer containing 10 mM MgSO 4 , and 5 mM Tris-HCl buffer containing 5 mM MgSO 4 and 5 mM Tris-HCl buffer, pH 8.8, respectively) at a flow rate of 60 ml/h (1-ml fractions).The effluent containing the enzyme peak was pooled and concentrated by ultrafiltration through Amicon (YM 10) membranes and stored at 4 o C.

Results and Discussion
Confirming earlier reports (3), chromatography of crude extracts on DEAE-cellulose, irrespective of the carbon source used, revealed the presence of two enzymatic fractions showing APase activity when the mold N. crassa was grown on high-Pi medium at pH 5.4 (Figures 1 and 2).It was also observed at pH 5.4 that fraction II was more abundant when the mold was grown on acetate (Table 1) and was poorly recovered when the mold was grown on sucrose (Table 2).Furthermore, fraction II was not detected or was poorly recovered at alkaline pH after growth on sucrose (Table 3) or acetate (Table 4), respectively.Although speculative, the above results (at least three independent experiments, done using each carbon source at both acid and alkaline pH, gave almost the same results) indicate that the expression of these two enzyme forms may be under the effect of the pH regulatory circuit (6,23,24).
The purification procedure described in this paper and summarized in Table 4 provided optimal conditions for the purification of the major constitutive APase (fraction I) synthesized by the wild-type strain 74A of the mold N. crassa grown in high-Pi medium supplemented with acetate at pH 7.1.As
Relative heat stability was determined by incubating the enzyme in 0.03 M, 0.15 M and 0.30 M CHES or Tris-HCl buffers containing 1 mM MgSO 4 (final concentrations), pH 8.9, at 70 o C, in the same experiment.At appropriate times, samples were removed to measure the remaining APase activity using the standard procedure.
Limiting velocities (V max ) and Michaelis constants (K m ) were determined as Lineweaver and Burk plots ( 18) by incubating the enzyme in 30 mM CHES buffer containing 1 mM MgSO 4 , pH 8.9, at 37 o C, using substrate concentrations in the range of 0.5 to 6.67 mM.The interaction constant for the substrate (n) was determined by the Hill procedure as described by Koshland Jr. (19).The kinetic constants reported here were obtained by linear-square analysis calculated from the data obtained in at least three independent experiments.
PAGE was carried out at pH 8.3 by the method of Davis as described by Han et al. (9) using 7.5% (w/v) polyacrylamide slab gels (10 x 10 x 0.1 cm).The phosphomonoesterase activity bands were developed by the method of Dorn as described by   already described (5,15), the pH of the medium increased during growth on acetate, reaching a value of 8.7 after 72-h incubation.At least five independent preparations of the enzyme appeared homogeneous as determined by 7.5% PAGE at pH 8.3, with the protein band being superimposable on APase activity.All of these preparations also essentially showed the same electrophoretic mobility, the R f value being 0.53.Overall, the constitutive APase was purified about 131fold with a yield of 5.2% (specific activity of 223 units/mg).Furthermore, the presence of Mg 2+ in all steps of the purification procedure was necessary for the maintenance of the enzyme activity, since activity was not restored when Mg 2+ was added a posteriori.
The molecular mass of the purified constitutive APase was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE (Figure 3), respectively, demonstrating that the enzyme is a monomer.This enzyme also showed no deviation from Michaelis kinetics for the hydrolysis of PNP-P, a-naphthyl-Pi, ß-glycerol-Pi and Lhistidinol-Pi (Table 5).The purified enzyme is activated by Mg 2+ and Zn 2+ and is inhibited by histidine, EDTA and 1 mM Be 2+ (Figure 4).Beryllium sensitivity was not observed for the hydrolysis of Lhistidinol-Pi because of the very low concentration of Be 2+ previously used in the assay (0.1 mM) (1), i.e., this concentration is about 9 times lower than the K m value (0.91 mM) earlier determined for the hydrolysis of L-histidinol-Pi (1), which makes its effect almost experimentally undetectable.
Determination of the pH activity profile showed an apparent optimum ranging from 9.5 to 10.5 for the purified APase (Figure 5).Also, the hydrolytic activity of the enzyme was enhanced at pH ranging from 7.5 to 8.5 in the presence of Tris-HCl buffer (Figure 5).Furthermore, Tris-HCl buffer had a protective effect when the enzyme was incubated at 70 o C, pH 8.9, as compared to the effect observed for the incubation in CHES buffer (Figure 6).Taken together, the above results indicate that the expression of a constitutive APase by the mold N. crassa, which is also probably responsible for the hydrolysis of L-histidinol-Pi in vivo (Table 5), in spite of its beryllium

Figure 2 -Figure 4 -
Figure 2 -Stepwise elution from DEAE-cellulose of constitutive alkaline phosphatases synthesized by the wild 74A strain of N. crassa grown in high-Pi medium at 30 o C, and supplemented with 44 mM acetate (A), pH 7.1, or 44 mM sucrose (B), pH 7.8, as the carbon source.After elution of non-absorbed proteins, enzyme elution was performed by the stepwise addition of 12.5 and 50.0 mM KCl in 10 mM Tris-HCl buffer, pH 8.8, containing 40 mM MgSO 4 .For details, see Material and Methods.

Figure 5 -
Figure 5 -pH activity profile of purified constitutive alkaline phosphatase synthesized by strain 74A of N. crassa grown in high-Pi medium at 30 o C, pH 7.1.Filled circles, triangles, squares and lozenges represent the enzyme activity in the presence of 0.3 M PIPES buffer, pH 6.5-7.5, 0.3 M TAPS buffer, pH 7.5-8.5,0.3 M CHES buffer, pH 8.5-10.0,and 0.3 M CAPES buffer, pH 10.0-11.0,respectively.Open circles represent the enzyme activity in the presence of the mixture containing the proper buffer and 0.3 M Tris-HCl, pH 7.5-10.5.For details, see Material and Methods.

Figure 3 -
Figure3-SDS-PAGE of purified alkaline phosphatase synthesized by N. crassa mycelium grown for 72 h on high-Pi medium.About 10 µg of purified protein was loaded onto lane A and stained with silver after electrophoresis.About 1 µg of protein markers (lane B) was stained with Coomassie blue after electrophoresis.

Figure 6 -
Figure 6 -Heat inactivation of purified constitutive alkaline phosphatase at 70 o C, pH 8.9, synthesized by strain 74A of N. crassa grown in high-Pi medium at 30 o C, pH 7.1.A, Triangles, circles, and squares represent the enzyme incubated in 0.03, 0.15 and 0.30 M Tris-HCl buffer, pH 8.9, respectively.B, Triangles, circles, and squares represent the enzyme incubated in 0.03, 0.15 and 0.30 M CHES buffer, pH 8.9, respectively.For details, see Material and Methods.

Table 1 -
Partial purification of the constitutive alkaline phosphatases synthesized by the wild 74A strain of N. crassa grown in high-Pi medium at 30 o C, supplemented with 44 mM acetate as the carbon source and buffered at pH 5.4 with 100 mM sodium maleate.

Table 3 -
Partial purification of a constitutive alkaline phosphatase synthesized by the wild 74A strain of N. crassa grown in high-Pi medium at 30 o C, supplemented with 44 mM sucrose as the carbon source and buffered at pH 7.8 with 50 mM Tris-HCl.n.d.,Not detected.For details, see Material and Methods.

Table 4 -
Purification of the major constitutive alkaline phosphatase synthesized by the wild 74A strain of N. crassa grown in non-buffered high-Pi medium at 30 o C, supplemented with 44 mM acetate as the carbon source and adjusted to pH 7.1.Maccheroni Jr. et al. (20)using sodium a-

Table 5 -
Summary of kinetic constants for the enzymatic hydrolysis of some organic esters at 37 o C, pH 8.9.