Alkaline phosphatase(AKP)was isolated and purified from the skin of Tylototriton taliangensis Liu and its kinetic property was examined.The partially purified alkaline phosphatase was purified by salting\|out,CM\|cellulose ion\|exchange column and gel filtration with Sephadex G\|150.The purified enzyme from skin moved as a single electrophoretic band in PAGE.The specific activity was 90.26 units/mg protein.Its subunit weight was 42.0 kD as determined with SDS\|PAGE.The optimum pH value for the enzyme was pH9.0.The optimum temperature was about 34℃.The Michealis\|Menton constant( K \-m)was 0.83 mmol/L on the disodium phenyl phosphate.Activity differences of the enzymes were determined when metal ions effected on the AKP.The results showed that K + was found to have no inhibition of AKP activity.Mg 2+ ,Ca 2+ ,Cu 2+ could activate the AKP and the higher the metal ions concentration were,the more the activity of AKP increased.When 3.0 mmol/L Cu 2+ was used,the activity of AKP could rise to 187%.Ni 2+ ,Zn 2+ and Ag + could inhibit the AKP and the higher the metal ions concentration were,the more the activity of AKP decreased.When 3.0 mmol/L Ag + was used,the activity of AKP retained 23% only.
Acid phosphatase (ACPase, E.C.3.1.3.2) was isolated and purified from Apis cerana, and properties of the enzyme had been studied. The fresh bee pupa,honey,royal jelly and Apis cerana with wings cut were homogenized, acid treated to pH 5.0, then compared the ACPase activities of the four kinds of the homogenate. The Acid phosphatase was partially obtained Apis cerana by homogenate, ammunium sulfate fractionation and gel filtration with Sephadex G 150.The purified enzyme moved as a single electrophoretic band in PAGE. The purification multiple was 16.54,and the specific activity was 3.47U/mg.Pr with pNPP as its substrate. The optimum pH value for the enzymes was pH 4.1. The optomum temperature was about 50℃. The Michaelis Menten constant ( K m ) was 2.32×10 -4 mol/L on the pNPP. The ACPase was activated by Zn 2+ ,Mg 2+ and K +,while inhibited by ions of Cu 2+ ,Fe 3+ and Cr 3+ . Pb 2+ in low concentration activated the enzyme and in high concentration inhibited it.The ACPase is inactivated by urea.
