Purification And Enzymetic Characteristics Analysis Of Protein Phosphatases Of Liver From Trionyx Sinensis

The method of purification and characteristics of protein phosphatase of Trionyx Sinensis' liver have been studied in this article.An effective isolating and purifying process has been worked out on the basis of former researchers' performance.The data in key point of each step were determined:homogenized tissue,centrifuging with high-speed refrigerated centrifuge.The best method of salting out is:1.pH 8.5,use 30%saturated ammonium sulfate to remove impurity alkaline protein;2.pH 6.7-6.9, use 30-70%saturated ammonium sulfate to precipitate PP2A,dialysis ion exchange chromatography on DEAE-cellulose with a Tris buffer(pH8.1,contain 0.2mol/mL NaCl),column 1.5×5.0 cm,flow rate 0.60mL/min,at the collecting rate 3mL/tube.4 protein summits were collected,summitⅠis impurity protein with higher PI than PP2A,summitⅡand summitⅢhave high enzyme activity.SummitⅣis impurity protein with lower PI than PP2A.Ultra filtrating to remove small molecules protein in summitⅡand summitⅢ, PP2A was collected.The purification multiples of protein phosphates was 18.75 and the specific activity was 159U/mg.Analyzing the enzyme characteristics,it can be concluded:PP2A is stable at pH6.0-8.0 and temperature below zero,its PI is less than 7,which is a weak acid protein.But keep the PP2A in the acid condition(pH＜4)or alkali condition(pH＞10) for 2,4,6 h,or the temperature above 45℃for 10 min,the activity of the enzyme can reduce quickly.The results showed that the activity of enzyme was poor in thermostabilization and acid proof alkali.Use pNPP as substrate,the optimal reaction condition is as following:pH7.0,temperature 30℃,substrate concentration 28-29μmol/mL.pNPP has obvious inhibited effect to PP2A,when the concentration is above 29μmol/mL,the enzyme activity decreased rapidly.Above 48μmol/mL,the rate decreased slightly.As the substrate of PP2A,pNPP does not meet the condition of Michaelis-Menten equation.Michaelis-Menten equation can not be used to validate enzymatic reaction kinetics.The enzyme activity was activated by the ion of Co²⁺, Mn²⁺,Ca²⁺and Mg²⁺greatly.The increase rate is 2132%,1463%,1390%and 411%, respectively.