Indian Journal of Biochemistry & Biophysics Vol. 42, June 2005, pp. 182-185 Characterization and purification of alkaline phosphatase from Elephas trogontherii (Steppe elephant) bone Yaşar Demir, Safinur Yildirim and Nazan Demir* Atatürk University, Faculty of Science, Department of Chemistry, 25240 Erzurum, Turkey Received 9 December 2004; revised 25 April 2005 Four isozymes of alkaline phosphatase (AP) were purified from Elephas trogontherii (Steppe elephant) from different locations in the bone (outer and inner peripheral, cytosolic, and integral) using Sephadex G-200 gel filtration and TEAE-cellulose anion-exchange chromatography. The specimen was obtained from Erzurum Museum and its age was approx. 0.3-0.5 million years old. No fungi or bacteria were present in the bone sample. The enzyme activity was determined by using p- nitrophenylphosphate as a substrate. SDS-PAGE of all the isozymes gave a single band at the same location. The molecular mass of the four isozymes as determined by using gel filtration was about 60 kDa. Optimum pHs for the four isozymes were between 8-8.5. The optimum temperatures of the isozymes were: outer peripheral, 37.5°C, cytosolic, 37.5°C, inner peripheral, 35°C and integral, 40°C. The values of V max and K m , as well different optimum temperatures indicated that isozymes were structurally different. Keywords: Elephas trogontherii, elephant, bone, alkaline phos- phatase. IPC Code: A 01 N 63/00, C 12 N Alkaline phosphatase (AP) (E.C. 3.1.3.1; ortophosphoric monoester phosphohydrolase, alkaline optimum) is a zinc metalloenzyme which catalyses phosphate monoester hydrolysis 1,2 . It functions as an ectoenzyme attached to the cell membrane by a hydrophobic glycosyl-phophatidylinositol (GPI) anchor 2 . It also guides migratory cells and transports specific molecules such as fat and immunoglobulins across membranes 3 . In humans, AP consists of four isozymes, three are tissue-specific, namely intestinal, placental and germ cell APs and a tissue-non-specific AP 3 . Earlier, AP with molecular mass of 190 kDa with subunit mass 60 kDa was isolated from the mummy bones 4 . The molecular mass of AP, isolated from fresh human bones was found to be 200 kDa with subunit mass 60 kDa 4 . In another study, a similar molecular mass was reported from 3000-years-old human 5 . In addition, the molecular mass of bovine bone AP was found to be 216 kDa 1 . In the present study, we report the purification and characterization of four isozymes of AP from different locations of the bone (outer and inner peripheral, cytosolic, and integral) of Elephas trogontherii (Steppe elephant), an extinct animal. Materials and Methods Bone samples from Elephas trogontherii were obtained from Erzurum Museum and were believed to be approx. 0.3-0.5 million years old 6 . The samples were first broken into parts and then ground to a powder. Although, no fungi and bacteria were present, the samples were washed with alcohol (5%) several times to remove any possible bacterial contamination, followed by a saline wash, to remove the alcohol. Preparation of homogenates Outer peripheral AP The clean, powdered sample was placed in saline to allow swelling. The washed sample was mixed with KCl (1 M) for 2.5-3 hr at room temperature to remove the outer peripheral proteins. The mixture was then centrifuged, and the precipitant was saved for the second step. The supernatant was used for purification. It was subjected to extraction with CCl 4 , and the watery phase was dialyzed against 0.01 M Tris-HCl, containing 2 mM MgCl 2 (pH 7.5). The solution obtained was applied to a gel filtration column as described 7 . Cytosolic AP The precipitant obtained in step 1 was suspended in 0.01 M Tris-HCl containing 2 mM MgCl 2 (pH 7.5). The suspension was frozen and thawed using liquid nitrogen, to break the inorganic matrix of the bone and disrupt the cell membranes. The ultimate disruption was achieved by ultrasonic homogenization for 4 hr. The homogenate was centrifuged at 20,000 rpm for 1 hr. The precipitant was saved for the step 3. The supernatant was extracted with CCl 4 , to remove the lipids and was applied to the gel filtration column 7 . __________ *Corresponding author Tel: 442-231 4439; Fax: +442-2360948 Email: demirn@yahoo.com