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
介绍了一种设计、合成与克隆人造核酶的新方法.通过在“锤头结构”模型中非保守性区域引入 Bgl I 切点,不仅维持了核酶切割活性所必需的二级结构,而且为核酶克隆的鉴定提供了极为方便的手段.另外还通过在核酶模板两端引入限制性内切酶...介绍了一种设计、合成与克隆人造核酶的新方法.通过在“锤头结构”模型中非保守性区域引入 Bgl I 切点,不仅维持了核酶切割活性所必需的二级结构,而且为核酶克隆的鉴定提供了极为方便的手段.另外还通过在核酶模板两端引入限制性内切酶半切点,一步直接将核酶模板连接、聚合、克隆到体外转录载体上,大大简化了以往核酶克隆的繁琐操作,省时、省力,克隆效率也较高.展开更多
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.
文摘介绍了一种设计、合成与克隆人造核酶的新方法.通过在“锤头结构”模型中非保守性区域引入 Bgl I 切点,不仅维持了核酶切割活性所必需的二级结构,而且为核酶克隆的鉴定提供了极为方便的手段.另外还通过在核酶模板两端引入限制性内切酶半切点,一步直接将核酶模板连接、聚合、克隆到体外转录载体上,大大简化了以往核酶克隆的繁琐操作,省时、省力,克隆效率也较高.