Japanese Marsh Shrimp Hepatopancreas Alkaline Phosphatase Purification And Characterization

Study of alkaline phosphatase from hepatopancreas of Macro brachium nipponensis collected from the lake of BaiYang, HeBei province. The alkaline phosphatase was partially purified from the mantle by homogenation, n-butanol extraction and ammonium sulfate fractionation. Gained alkaline phosphatase was filtrated by exchanging chromatography on DEAE-cellulose column (DEAE-52), CM-cellulose column and gel filtration chromatography on Sephadex G-200. The results were shown that the purification multiple of DEAE-cellulose column was the highest. Some properties of the enzyme were undertaken. The results are as follows: The optimum pH of alkaline phosphatase for PNPP is 8.5, and the optimum temperature is 45 0C. The Michaelis-Menten constant (Kin) is 2.8 X i03 molIL. The alkaline phosphatase is activated by Mg2~, Mn2~ and Zn2~ ion, but is inhibited by Cu2~ ion, formaldehyde, ethanol, methanol, oxalic acid and tartaric acid. The alkaline phosphatase was selectively modified by PMSF, NBS, ClAc, formaldehyde, ME and acetyl acetone, and changes in the activities of the enzyme have been detected. The reaction of alkaline phosphatase with ME, formaldehyde, PMSF and C1Ac resulted in a strong inhibition of the enzyme activities which decreased with the increase of modifier concentration. The acetyl acetone and NBS were found without any inhibition effect. Studies of inactivation of alkaline phosphatase by chemical modification demonstrated that Lys, Ser and His residues are indirpensble function groups of the alkaline phosphatase. Partial disulfide bones are also for the function of this enzyme.