Isolation, Purification And Properties Of Alkaline Phosphatase From Sperm Of Pig(PIC344)

Alkaline phosphatase(AKP) was isolated and purified from the sperm of pig(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 48.41KD with SDS-PAGE, moleculer weight was 90.12KD with gel filtrition on Sephacryl S-300(AKTA FPLC). The optimum pH value for the enzyme was 9.5.The optimum temperature for the enzyme was about 40?.The Michealis-Menton constant(Km) was 为 3.98 X 10"4mol/L with disodium phenyl phosphate as its substrate. Ni2+,Ca2+,Mg2+,Sn2+can activate the enzyme determined from lmmol/L to 8mmol/L, Cu2+ ,Cd2+ can inhibitor the enzyme from lmmol/L to 3mmol/L.CuSO4 Cd(NO3)2 was selected for determing types of inhibition and the result showed that CUSO4 was a noncompetitive inhibitor, the inhibitor constant being 1.32 X 10'3mol/L, Cd(NO3)2 was a competitive inhibitor, the inhibitor constani being 3.33 X 10"4mol/L.Amino acid composition analysis showed that one subunit of AKP has about 450 amino acids and there are plentiful of Met, Glu, Val, Arg, Leu, Phe in it.The enzyme was modified by NBS,PMSF,TNBS,DTT and AcBr and the changes in their activities and absorption spectra were measured. It was found that the reactions of AKP from PCI344 with NBS,PMSF,TNBS and AcBrresulted in a strong inhibition of enzyme activities. The activities of AKP decreased steadily with the increase of modifier's concentration. These results predict that Trp, Lys, Ser and 2-SH group are essential for the activities of AKP from PCI344. It has also been shown that His was indifferent to the activities of AKP from PCI344.