Purification and characterization of acid phosphatase from cotyledons of germinating soybean seeds.
Soybean acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) was completely separated from phytase (EC 3.1.3.8) isolated from cotyledons of germinating seeds and purified to homogeneity. A four-step purification regimen consisting of ammonium sulfate fractionation, and ion-exchange, affinity, and chromatofocusing gel chromatographies was employed to achieve a homogeneous preparation. Acid phosphatase activity appeared as a major band of the three forms of acid phosphatase identified on native gels. The purified enzyme had a molecular weight of 53,000 when electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular weight of 53,000 from its mobility in a Fracto-gel TSK HW-50F gel permeation column. The molar extinction coefficient of the enzyme at 278 nm was estimated to be 4.2 X 10(4) M-1 cm-1. The isoelectric point of the protein, as revealed by chromatofocusing, was about 6.7. The optimal pH for activity, like other plant acid phosphatases, was 5.0. While the enzyme failed to accommodate phytate as a substrate, the enzyme did exhibit a broad substrate selectivity. The affinity of the enzyme for p-nitrophenyl phosphate was high (Km = 70 microM), and activity was competitively inhibited by orthophosphate (Ki = 280 microM). The estimated catalytic turnover number (Kcat) of the enzyme for p-nitrophenyl phosphate was about 430 per second. Although the purified enzyme was stable at 0 degrees C and exhibited maximum catalytic activity at 60 degrees C, thermal inactivation studies indicated that the enzyme lost 100% activity after treatment at 68 degrees C for 10 min.
Ullah
A H
AH
Southern Regional Research Center, U.S. Department of Agriculture, New Orleans, Louisiana 70179.
Gibson
D M
DM
D010832:6-Phytase / Q000302:isolation & purification / Q000378:metabolism; D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D012639:Seeds / Q000201:enzymology*; D013025:Glycine max / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature
D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010710:Phosphates; C008644:nitrophenylphosphate; D000135:Acid Phosphatase; D010832:6-Phytase