Purification and kinetic study of bone and liver alkaline phosphatase isoenzymes in the dog

To study bone and liver alkaline phosphatases (ALP) in the dog, tissue samples were obtained immediately after death from dogs apparently involved in road traffic accidents. Tissue samples were homogenized, and extracted protein was subjected to diethylaminoethyl cellulose-Sephadex column chromatography equilibrated previously with 0.01 M Tris-hydrochloric acid buffer (pH 7.2) and eluted with linear gradient of ionic strength from 0 to 0.4 M NaCl. Fractions with peak activity of ALP were considered a source of purified enzyme. ALP activity was measured by a kinetic method using p-nitrophenylphosphate as a substrate. The kinetic study was performed using a constant concentration of purified isoenzymes and different concentrations of the substrate. Precise Km values for liver and bone ALP were identified using direct linear plot (Eisenthal-Cornish-Bowden plot). Our study showed that the Km value of liver isoenzyme was 13 times greater than that of bone isoenzyme (3.3 mmol and 0.25 mmol, respectively). This may be due to the physiological role of bone ALP in preparing phosphate for bone mineralization and the retention of phosphates in mineral complexes with calcium in bone tissues.