Camel Liver Acid Phosphatases: Purificaion and Properties

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 4-Agarose 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 degrees C. The pH stability was found between pH 4 and 9 and appeared to be stable at temperature of 40 degrees C. The K-m 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.