Purification of Alkaline Phosphatase from Rat Dental Pulp.
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概要
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Alkaline phosphatase (EC3.1.3.1) was purified from dental pulp tissues (4.5g) in rat incisors. The enzyme in dental pulps was initially extracted with n-butanol, followed by column chromatographies, such as DEAE-Sepharose CL-6B, Concanavalin A-Sepharose, Sephacryl S-300 HR and L-Histidyldiazobenzylphosphonic Acid Agarose. Finally, the enzyme was purified 95-fold higher than that found in the nbutanol extract and its recovery was 15% of the total. The final specific activity of the enzyme toward p-nitrophenylphosphate as the substrate was 1, 425U/mg protein.<BR>Purified alkaline phosphatase was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions with or without 5% β-mercaptoethanol and heating. Under non-reducing conditions, the purified enzyme had a molecular weight corresponding to 155k which was indicated on SDS-PAGE by both silver staining and enzyme activity staining with 5-bromo-3-indolylphosphate p-toluidine salt. However, the activity of crude enzyme in n-butanol extracts was found at 130k as well as 155k on the non-reduced type of SDS-PAGE. However, on the reduced type of SDS-PAGE, only one band corresponding to 77k was observed. Therefore, our findings showed that the alkaline phosphatase of rat dental pulp tissues is 155k and is composed of two subunits with identical molecular weights of 77k.<BR>Furthermore, using this purification method, the molecular weights of alkaline phosphatase from pig dental pulp, rat bone marrow, kidney and mouse osteoblasts-like cells (MC3T3-E1) were found to be 185k, 155k, 158k, and 157k, respectively.
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