Properties of a constitutive alkaline phosphatase from strain 74A 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), alpha-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(half-life of 19.0 min, k = 0.036 min-1) as compared to 0.3 M CHES (half-life of 2.3 min, k = 0.392 min-1) in the same experiment

Does gene palB regulate the transcription or the post-translational modification of Pi-repressible phosphatases of Aspergillus nidulans?

When grown on low-Pi medium, the chaA1 pabaA1 palB7 mutant of Aspergillus nidulans excretes an acid phosphatase with steady-state kinetic properties, temperature sensitivity and electrophoretic mobility different from those of the enzyme excreted by the pabaA1 strain. The enzyme excreted by the pabaA1 strain at pH 6.5 showed PNP-P activity with negative cooperativity (K0.5 = 0.87 +/- 0.06 mM, n = 0.68 +/- 0.03) whereas the enzyme excreted by the chaA1 pabaA1 palB7 mutant showed Michaelian kinetics (Km = 0.46 +/- 0.03 mM, n = 1.00 +/- 0.02). The apparent half-lives at 60 degrees C, pH 5.5, of acid phosphatase excreted by the pabaA1 and chaA1 pabaA1 palB7 strains were 58.6 +/- 4.9 min and 21.5 +/- 1.8 min, respectively. Furthermore, the electrophoretic mobility of acid phosphatases excreted by the palA1, palB7, palC4, palE11 and palF15 mutants of A. nidulans was altered and differed from the electrophoretic mobility of the enzyme excreted by the wild-type strain. Also, the palB7 mutation altered the electrophoretic pattern of acid phosphatases synthesized on high-Pi medium. These results are compatible with the post-translational modifications in the Pi-repressible phosphatases rather than with the action of gene palB in controlling the transcription of structural genes of these enzymes.

Does gene palB regulate the transcription or the post-translational modification of Pi-repressible phosphatases of Aspergillus nidulans?

When grown on low-Pi medium, the chaA1 pabaA1 palB7 mutant of Aspergillus nidulans excretes an acid phosphatase with steady-state kinetic properties, temperature sensitivity and electrophoretic mobility different from those of the enzyme excreted by the pabaA1 strain. The enzyme excreted by the pabaA1 strain at pH 6.5 showed PNP-P activity with negative cooperativity (K0.5 = 0.87 + or - 0.06 mM, n = 0.68 + or - 0.03) whereas the enzyme excreted by the chaA1 pabaA1 palB7 mutant showed Michaelian kinetics (Km = 0.46 + or - 0.03 mM, n = 1.00 + or - 0.02). The apparent half-lives at 60§C, pH 5.5, of acid phosphatase excreted by the pabaA1 and chaA1 pabaA1 palB7 strains were 58.6 + or - 4.9 min and 21.5 + or - 1.8 min, respectively. Furthermore, the electrophoretic mobility of acid phosphatases excreted by the palA1, palB7, palC4, palE11 and palF15 mutants of A. nidulans was altered and differed from the electrophoretic mobility of the enzyme excreted by the wild-type strain. Also, the palB7 mutation altered the electrophoretic pattern of acid phosphatases synthesized on high-Pi medium. These results are compatible with the post-translational modifications in the Pi-repressible phosphatases rather than with the action of gene palB in controlling the transcription of structural genes of these enzymes

The PHO-2A mutant of neurospora crassa which is deficient in PI-repressible alkaline phosphatase (EC 3.1.3.1) is also defective in PI-represssible acid phosphatase (EC 3.1.3.2)

The mycelial Pi-repressible acid phosphatase presented p-nitrophenylphosphatase activity with negative cooperativity and Michaelian behavior when synthesized by the wild-type and pho-2A mutant strains of Neurospora crassa, respectively. The major acid phosphatase present in cell extracts of the pho-2A mutant of N. crassa grown in low Pi medium is more thermolabile (t½= 4 min at 54 grade C, pH 5.4) than that of the wild strain (stable for at least 80 min at 54 grade C, pH 5.4). The pho-2A mutant of N. crassa secreted a more thermobabile acid phosphatase (t½=30 min at 50 grade C, pH 5.4) than the wild strain (t½ of at least 80 min at 50 grade C, pH 5.4). The pho-2A mutant of N. crassa synthesized a more thermolabile acid phosphatase (t½=37 min at 54 grade C, pH 5.4) than the wild strain in high Pi medium (t½=14 min at 54 grade C, pH 5.4). The pleiotropic nature of the pho-2 locus and its possible involement in the mechanism of phosphatase secretion by N. crassa are proposed