Isolation,Purification and Properties of Alkaline Phosphatase from Sperm of PIC Pigs(PIC344)

Alkaline phosphatase(AKP) was isolated and purified from the sperm of PIC pigs(PIC344) and its kinetic property was examined.The partially purified AKP was purified by salting-out,DEAE-Sepharose FF ion-exchange column,and gel filtrition with sephacryl S-200.The purified enzyme moved as a single electrophoretic band in PAGE.The specific activity was 81.23U/mg.It's subunit weight was 48.41 KD with SDS-PAGE,moleculer weight was 90.12 KD with gel filtrition on Sephacryl S-300(AKTA FPLC).The optimun pH value for the enzyme was 9.5.The optimun temperature for the enzyme was about 40℃.The Michealis-Menton constant(Km) was 3.98×10~(-4)mol/L with disodium phenyl phosphate as its substrate.Ni~(2+),Ca~(2+),Mg~(2+),Sn~(2+) could activate the enzyme determined from 1 mmol/L to 8 mmol/L,Cu~(2+),Cd~(2+) could inhibit the enzyme from 1mmol/L to 3 mmol/L.CuSO_4,Cd(NO_3)_2 was selected for determing types of inhibition,and the result showed that CuSO_4 was a noncompetitive inhibitor,the inhibition constant being 1.32×10~(-3)mol/L,Cd(NO_3)_2 was a competitive inhibitor,with inhibition constant 3.33×10~(-4)mol/L.