Purification and characterization of two acid phosphatases from germinating peanut [arachis hypogaea] seed

ABSTRACT

The maximum acid phosphatasic activity was detected in peanut seed at the 5th day of germination. At least, two acid phosphatases were purified by successive chromatography separations on DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, Sephacryl S-100 HR, and Phenyl-Sepharose HP to apparent homogeneity from five days old cotyledon of peanut after germination. These isoenzymes, designated peanut cotyledon acid phosphatase 1 and 2 [PCAP 1 and PCAP 2], had native molecular weights of approximately 27.5 and 24 kDa by gel permeation, respectively. SDS-PAGE [Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis] of PCAP 1 and PCAP 2 resolved a single protein band [each] that migrated to approximately 27 and 29 kDa, respectively. Thus, these acid phosphatases likely function as a monomer. The two isoenzymes had a similar optimum temperature [55°C], two closely optima pH [5.6 and 5.0], and appeared to be stable in the presence of some detergents such as Triton X-100, Nonidet P-40, Taurocholic acid sodium salt, Polyoxyethylene-9-lauryl ether as well as Mg2[+], Sr2[+], Fe3[+] and Ba2[+]. Substrate specificity indicated that PCAP 1 and PCAP 2 hydrolyzed a broad range of phosphorylated substrates. However, natural substrates such as ADP, ATP and phenylphosphate had the highest rate of hydrolysis for the two isoenzymes