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 th e presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30 degrees C. The molecular mass was 58 kDa and 56 kDa as determined by exclu sion 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 K-m and Hill coefficient va lues were 0.35 mM and 1.01, respectively), alpha-naphthyl phosphate (the K- m and Hill coefficient values were 0.44 mM and 0.97, respectively), beta-gl ycerol phosphate (the K-m and Hill coefficient values were 2.46 mM and 1.01 , respectively) and L-histidinol phosphate (the K-m and Hill coefficient va lues 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+, his tidine and EDTA. Also, 0.3 M Tris-HCl buffer protected the purified enzyme against heat inactivation at 70 degrees C (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.