Aspergillus ficuum extracellular pH 6.0 optimum acid phosphatase: purification, N-terminal amino acid sequence, and biochemical characterization.
An extracellular acid phosphatase, pH optimum 6.0 from crude culture filtrate of Aspergillus ficuum was purified to homogeneity using cation exchange chromatography and chromatofocusing steps. SDS-PAGE of the purified enzyme exhibited two stained bands at approximately 82-KDa and 70-KDa. The mobility of the active enzyme in gel permeation chromatography indicated the molecular mass to be about 85-KDa. In the concentrated form the enzyme appeared to be purple, the visible absorption spectrum shows a lambda max at 580 nm. On the basis of molecular mass of 82-KDa, the molar extinction coefficient of the enzyme at 280 nm and 580 nm was estimated to be 1.2 x 10(5) M-1 cm-1 and 1.3 x 10(3) M-1 cm-1 respectively. Judging by chromatofocusing, the isoelectric point of the enzyme was about 4.9. The purified enzyme was unstable at 70 degrees C. The enzyme was catalytically very active from 55 degrees to 65 degrees C with a maximum activity at 63 degrees C. The Michaelis constant of the enzyme for p-nitrophenylphosphate was 200 microM with a computed Kcat of 260 per sec. Although the enzyme was insensitive to fluoride, tartrate, and N-ethylmaleimide (NEM), it was competitively inhibited by phosphomycin (Ki = 1.00 mM) and inorganic orthophosphate (Ki = 165 microM). While the enzyme was relatively insensitive to Mn++, Cu++ and Zn++ inhibited the activity 540 fold at a concentration of 100 microM. The enzyme showed positive PAS staining and hence is a glycoprotein (28% glycosylation); the sugar composition suggests the presence of N-linked high mannose-oligosaccharides and galactose. A partial N-terminal amino acid sequence up to the thirty-fourth residue was elucidated.
Ullah
A H
AH
Southern Regional Research Laboratory, ARS, USDA, New Orleans, Louisiana 70124.
Cummins
B J
BJ
D000135:Acid Phosphatase / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001230:Aspergillus / Q000201:enzymology*; D002850:Chromatography, Gel; D003470:Culture Media / Q000032:analysis; D005690:Galactose / Q000032:analysis; D008358:Mannose / Q000032:analysis; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D003470:Culture Media; D009844:Oligosaccharides; D000135:Acid Phosphatase; D008358:Mannose; D005690:Galactose