Purification and characterization of three low- molecular-weight acid phosphatases from peanut (Arachis hypogaea) seedlings

Abstract

th day of germination. At least, three acid phosphatases were identified and purified by successive chromatography separations on DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, Sephacryl S-200 HR, and PhenylSepharose HP to apparent homogeneity from developing five days old peanut seedlings. These enzymes designated acid phosphatase PI, PIIa and PIIb had native molecular weights of approximately 25.3, 22.4 and 24 kDa, respectively by gel permeation. SDS-PAGE of the purified acid phosphatase PI resolved two closely protein bands that migrated to approximately 14 and 12 kDa. Thus, this acid phosphatase likely functions as a heterodimer. Acid phosphatases PIIa and PIIb migrated as single band (each) with a similar molecular weight estimated to 21 kDa. The three enzymes had a similar optima pH (5.0) and temperature (55°C), and appeared to be stable in the presence of non-ionic detergents such as Triton X-100, Nonidet P 40 as well as Na + and K + . Substrate specificity indicated that the three acid phosphatases hydrolyzed a broad range of phosphorylated substrates. However, natural substrates such as ADP and ATP were the compounds with highest rate of hydrolysis for acid phosphatase PI, while acid phosphatase PIIa exhibited phytasic activity. These results indicate that each purified acid phosphatase from peanut seedlings played a peculiar role during germination.