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(Reference retrieved automatically from SciELO through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
A.C. Morales [1] ; S.R. Nozawa [2] ; G. Thedei Jr. [3] ; W. Maccheroni Jr. [4] ; A. Rossi [5]
Total Authors: 5
Affiliation:
[1] Universidade de São Paulo - Brasil
[2] Universidade de São Paulo - Brasil
[3] Universidade de Uberaba - Brasil
[4] Universidade de São Paulo - Brasil
[5] Universidade de São Paulo - Brasil
Total Affiliations: 5
Document type: Journal article
Source: Brazilian Journal of Medical and Biological Research; v. 33, n. 8, p. 905-912, 2000-08-00.
Abstract

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. (AU)