Alkaline phosphatase(AKP),from the succus entericus of silkworm,was purified using 10%-50%ammonium sulfate fractions,ion exchange chromatography of DEAE-Sepharose,and size exclusion chromatography of Sephacryl S-200.T...Alkaline phosphatase(AKP),from the succus entericus of silkworm,was purified using 10%-50%ammonium sulfate fractions,ion exchange chromatography of DEAE-Sepharose,and size exclusion chromatography of Sephacryl S-200.The purification fold was 464 times and specified activity was 3936 U/mg.Optimum pH value of the phosphatase was 10.5,and was stable between pH 7.5 and 11.The optimum temperature of the phosphatase was 40℃ and it was unstable over 50℃.Km value of the phosphatase was 1.25 mmol/L.In a given condition,the phosphatase was selectively modified by PCMB,NBS,PMSF,TNBS,SUAN,DTT,BrAc,and IAc,the results indicate that PMSF,SUA,BrAc,IAc,and TNBS could obviously inhibit the activity of the phosphatase,and the degree of inhibition depended on the concentration of these reagents.There was little effect on the activity of phosphatase after treatment by PMSF,DTT,and NBT.We primarily conclude that mercapto and imidazole are essential for AKP from silkworm.Also,Lys residue and disulfide bands are necessary to protect the catalysis of the AKP.展开更多
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\|cell...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.展开更多
文摘Alkaline phosphatase(AKP),from the succus entericus of silkworm,was purified using 10%-50%ammonium sulfate fractions,ion exchange chromatography of DEAE-Sepharose,and size exclusion chromatography of Sephacryl S-200.The purification fold was 464 times and specified activity was 3936 U/mg.Optimum pH value of the phosphatase was 10.5,and was stable between pH 7.5 and 11.The optimum temperature of the phosphatase was 40℃ and it was unstable over 50℃.Km value of the phosphatase was 1.25 mmol/L.In a given condition,the phosphatase was selectively modified by PCMB,NBS,PMSF,TNBS,SUAN,DTT,BrAc,and IAc,the results indicate that PMSF,SUA,BrAc,IAc,and TNBS could obviously inhibit the activity of the phosphatase,and the degree of inhibition depended on the concentration of these reagents.There was little effect on the activity of phosphatase after treatment by PMSF,DTT,and NBT.We primarily conclude that mercapto and imidazole are essential for AKP from silkworm.Also,Lys residue and disulfide bands are necessary to protect the catalysis of the AKP.
文摘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.
