Isolation And Study On The Characteristics Of Tripolyphosphatase From Rabbit Psoas Major

The myosin has been isolated and purified from New Zealand rabbit psoas major by means of ion strength adjustment,ammonium sulfate precipitation,and ion-exchang chromatography on deithylaminoethyl-cellulose column.According to determine the tripolyphosphatase activity in main steps,the tripolyphosphatase activity was low in crude extract,which was higher after adjusting ion strength twice.The specific activity of TPPase in crude extract was 0.000374U/mg,and 0.003681U/mg in protein solution after adjusting ion strength twice.Protein precipitated within 35-48%saturation of ammonium sulfate had a 13.5-fold increase in fold purification of the enzyme.Approximately 28-fold purification was achieved after ion-exchange chromatography on DEAE-celulose column.Four bands had been found by SDS-PAGE,which were consistent with subunits of myosin.The molecular weights of these bands were 220KDa,20KDa,15KDa and 11KDa respectively. This indicated that myosin had the activity of tripolyphosphatase.The characteristics of tripolyphosphatase from rabbit psoas major had been studied by molybdenum blues colorimetry.The optimum pH of TPPase was 6.5,and its optimum temperature was 35℃.Mg²⁺ activated the TPPase,and the optimum concentration was around 3mM.Ca²⁺ also activated TPPase.TPPase activity increased with Ca²⁺ from 0mM to 6mM.Then the activity was stable when the concentration of Ca²⁺ was higher than 6mM. EDAT-Na₂ had no obvious effect on TPPase.However,EDAT-Na₄ and KIO₃ inhibited the TPPase,and the effect of KIO₃ on TPPase was better than EDAT-Na₄.This paper also studied the effect of TPPase activity on rheological properties of myosin from rabbit psoas major.The G′and G″of sample treated with hexametaphosphate were higher than the other samples.Hexametaphosphate promoted the crosslinking of myosin,while pyrophosphate and tripolyphosphate decreased the crosslinking degree in myosin.When the TPPase activity was inhibited by KIO₃,the G′and G″of sample treated with tripolyphosphate was lower.The more the TPPase was inhibited,the lower of G′.So the crosslinking of myosin had been affected when tripolyphosphate was not hydrolyzed.