Structural and kinetic alterations of constitutive conidial alkaline phosphatase from the osmotically-sensitive mutant of Neurospora crassa.
The osmotically-sensitive os-1 mutant of Neurospora crassa overproduced conidial alkaline phosphatase. The enzyme was purified by Phenyl-Sepharose CL-4B chromatography and Sephadex G-200 gel filtration. PAGE analysis of the purified enzyme suggested the occurrence of aggregation and/or disaggregation phenomena. The enzyme is a glycoprotein containing 16% saccharide, with apparent molar mass of 137 kDa. Two protein bands (36 and 62 kDa) were observed in SDS-PAGE, suggesting that the native enzyme was a trimer. The pI was estimated to be 2.7, and optima of pH and temperature were 9.5 and 65 degrees C, respectively. The enzyme showed broad substrate specificity, hydrolyzing preferentially 4-nitrophenyl phosphate, O-phosphoamino-acids and 2-phosphoglycerate. The hydrolysis of 4-nitrophenyl phosphate was stimulated by Co(II) (26%), Ni(II) (23%) and Mg(II) ions (80%). The enzyme was stable for up to 6 months at 4 degrees C in 5 mmol/L Tris-HCl buffer and also upon storage at 25 degrees C for 10 d. The kinetic and structural properties of the conidial enzyme purified from the os-1 mutant were quite different from those of the wild type strain. The enzyme overproduction observed in the mutant may be related to cell wall alterations that affect the process of enzyme secretion.
Bogo
K R
KR
Departamento de Quimica, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, São Paulo, Brazil.
Masui
D C
DC
Leone
F A
FA
Jorge
J A
JA
Furriel
R P M
RP
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D009997:Osmotic Pressure; D013172:Spores, Fungal / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D000469:Alkaline Phosphatase