Purification, N-terminal amino acid sequence and characterization of pH 2.5 optimum acid phosphatase (E.C. 3.1.3.2) from Aspergillus ficuum.
An acid phosphatase from crude culture filtrate of Aspergillus ficuum was purified to homogeneity using three ion exchange chromatographic steps. SDS-PAGE of the purified enzyme gave a single stained band at approximately 68-KDa. The mobility of the native enzyme in gel filtration chromatography, however, indicated that the molecular mass to be about 130-KDa implying the active form to be a dimer. On the basis of a molecular mass of 68-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 3.4 x 10(5) M-1 cm-1. The isoelectric point of the enzyme, as judged by chromatofocusing, was about 4.0. The purified enzyme is highly stable at 0 degree C. Thermal inactivation studies have indicated that the enzyme is unstable at 70 degrees C. The enzyme, however, exhibited a broad temperature optima with a maximum catalytic activity at 63 degrees C. The Km of the enzyme for p-nitrophenylphosphate is about 270 microM with an estimated turnover number of 2550 per sec. The enzyme is a glycoprotein as evidenced by the positive PAS staining; the sugar composition suggests the presence of N-linked high mannose-oligosaccharides. A partial N-terminal amino acid sequence up to the twenty-third residue was obtained. The enzyme was inhibited competitively by inorganic orthophosphate (Ki = 185 microM) and non-competitively by phosphomycin (Ki = 600 microM).
Southern Regional Research Laboratory, USDA, New Orleans, Louisiana 70124.
A H
AH
Ullah
B J
BJ
Cummins
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D002241:Carbohydrates / Q000032:analysis; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010503:Periodic Acid-Schiff Reaction; D012995:Solubility; D013057:Spectrum Analysis; D013696:Temperature
D002241:Carbohydrates; D000135:Acid Phosphatase