[Purification and characterization of alkaline phosphatase obtained from bovine periodontal ligament].

Abstract

Alkaline phosphatase (ALP) activity has been demonstrated in periodontal ligament (PDL). On the basis of electron microscopic study, distribution of the enzyme in PDL tissue has also been indicated not only as a cell associated activity but also as an extracellular matrix associated activity. This study is concerned with the purification and characterization of the enzyme obtained from bovine PDL tissue. Purification of ALP extracted from the tissue included solubilization with 10 mM Tris-HCl buffer, pH 7.4, containing 0.2 mM MgCl2 and 0.1% Nonidet P-40 and fractionation by sequential chromatography utilizing DEAE-sephacel, Sepharose CL-6B and concanavalin A Sepharose 4B. Purity was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This was followed by staining for ALP activity first with 2 mM beta-naphthyl acid phosphate and 1 mM Fast Blue BB Salt and then the protein with Coomassie Brilliant Blue. SDS-PAGE of the crude enzyme preparations gave a broad band with apparent molecular weight of 110,000-130,000 dalton. ALP activities were separated into two major peaks using Sepharose CL-6B chromatography. The void volume peak showed a purified form of 110,000 dalton ALP (110K ALP) while the second peak contained 120,000-130,000 dalton ALP (120-130K ALP) and other proteins. Sequentially, 120-130K ALP was purified by chromatography on concanavalin A Sepharose 4B. A polyclonal antibody was raised against purified bovine PDL 110K ALP in a rabbit. Immunodiffusion analysis showed that a polyclonal antibody against 110K ALP recognized 120-130K ALP. Analytical affinity chromatography on concanavalin A Sepharose 4B indicated that 110K ALP and 120-130K ALP had distinct affinity to the column which may depend upon the sugar chain structure. Digestion of 110K ALP with phosphatidylinositol-specific phospholipase C affected electrophoretic mobility but 120-130K ALP had no effect. It is suggested that 110K ALP is attached to a cell membrane anchored by a phosphatidylinositol glycan. In conclusion, bovine PDL contains two types of alkaline phosphatase i.e. 110K ALP and 120-130K ALP. Both ALPs are immunologically related although they have different sugar chain moieties. Furthermore, 110K ALP has a membrane anchoring domain. These results suggest that 110K ALP would be localized on the surface of the cell membrane and 120-130K ALP may associated with the extracellular matrix.