Camel Liver Acid Phosphatases: Purificaion and Properties

Abstract

Three acid phosphatases were identified from the liver of a camel. Of these, two high molecular weight acid phosphatase forms were isolated and purified by successive chromatography on SP-Sephadex C-50, Sephadex G-75, CM-Cellulose, Sephacryl HR-200 and Reactive Blue 4Agarose columns. These were designated as P-I and P-II. The acid phosphatase, P-I was purified to homogeneity. A 1200 times purification was obtained with specific activity of 17U/mg of protein and a total yield of 3%. The SDS-PAGE showed a single band corresponding to the molecular weight of 66 kDa. Electrophoresis of the native enzyme resolved a single protein band that migrated to approximately 130 kDa, indicating the dimeric nature of protein. The acid phosphatase, P-II was purified 1000-fold with specific activity of 15 U/mg of protein and recovery of 1%. The SDS-PAGE revealed a single band around 48-50 kDa. Gel filtration chromatography estimated a native molecular mass to be 100 kDa. Thus, high molecular weight acid phosphatases likely function as a homodimer, consisting of two similar subunits. Low molecular weight acid phosphatase as P-III, was purified 3000-fold with specific activity of 45 U/mg of total protein and was found homogeneous on SDS-PAGE. Molecular weight of 18 kDa was obtained. The P-I, P-II and P-III enzymes were the most active over pH range 4.8-6.0 and at 55°C. The pH stability was found between pH 4 and 9 and appeared to be stable at temperature of 40°C. The Km values were found to be 0.31, 0.27 and 0.16 mM, respectively. These were further characterized with respect to thermal inactivation, inhibition, purine activation, substrate specificity and other kinetic parameters.