Isolation, purification, characterization and some kinetic properties of acid phosphatase from mungbean [vignaradiata] leaves

The present work was carried out on “Isolation, Purification, Characterization and Some Kinetic Properties of Acid Phosphatase from Mungbean [Vignaradiata] Leaves”.The acid phosphatase (EC. 3.1.3.2) has been noticed and purified from leaves of vignaradiatathrough ammonium sulphate precipitation, DEAE-Cellulose chromatography and concanavalin A-Sepharose 4B chromatography. The specific activity of 1291 nkat.mg-1of protein was obtained with recovery of nearly 1%. About 222 times purification was achieved. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) resolved two bands of acid phosphatase corresponding to molecular weights of 29 kilo Dalton (kDa) and 18 kDa. The molecular weights of native enzyme determined by gel filtration on calibrated Sephadex G-100 column were found to be 29 kDa and 19 kDa. The apparent Km value of 29 kDaisoenzyme with p-nitrophenyl phosphate (pNPP) as substrate was 0.3 mM and Vmax was 1336 nmol.sec-1.mg-1 of protein. 5.5 was the optimal pH for this enzyme and 4-7 was the pH stability. It had optimum temperature of 60oC and temperature stability was 0-50oC. Various phosphorylated compounds werehydrolysed non-specifically bythis enzyme. However, pNPP, phenyl phosphate, pyrophosphate, ATP, phosphotyrosine and ?- naphthyl phosphate seemed to be hydrolysed preferentially. It was competitively inhibited by phosphate, vanadate while fluoride and zinc showed non-competitive inhibitions, the molybdaterevealed an inhibition of mixed competitive and Mercury exhibited an inhibition of un-competitive type.It means that all these six elements are responsible for the inhibition of acid phosphatase of leaves of vignaradiata. Metal ions such as Cu+2, Hg+2, Zn+2 and Al+3 showed strong inhibition while other monovalent and divalent ions like Na+, K+, Pb+2, Ba+2, Cd+2, Mn+2,Ca+2, Mg+2 and Co+2 were determined that they had no effect on the enzyme activity.Modifiers, Reducing agents, Detergents and Anions were also established to have no effect on the enzyme activity.At the 4th day of seedlings formation its specific activity was found maximum.