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 30 degrees C. 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 K(m) and Hill coefficient values 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-glycerol 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 values were 0.47 mM and 0.94, respectively) at pH 8.9. The purified enzyme is activated by Mg(2+), Zn(2+) and Tris-HCl buffer, and is inhibited by Be(2+), histidine 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.
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
A C
AC
Morales
S R
SR
Nozawa
G
G
Thedei
W
W
Maccheroni
A
A
Rossi
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006642:Histidinol-Phosphatase / Q000737:chemistry / Q000302:isolation & purification; D006868:Hydrolysis; D009492:Neurospora crassa / Q000201:enzymology*
D005656:Fungal Proteins; D000469:Alkaline Phosphatase; D006642:Histidinol-Phosphatase