Molecular Species of Alkaline Phosphatase in Mouse Osteoblast-Like Cells (MC3T3-E1) and Dental Pulps

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The effects of glycine buffer on alkaline phosphatase (ALP) activity were analyzed by active staining with indigogenic dye in order to detect with high sensitivity ALP activity in gels after nonreduced-type SDS- or native (0.1% Nonidet P-40)-polyacrylamide get electrophoresis (PAGE) using glycine buffer. The activities of ALP in SDS- and native-PAGEs with 192 mM glycine decreased to one half those of PAGEs with 40 mM borate. However, when 192 mM glycine was premixed with Zn^<2+>, ALP activity in PAGEs increased according to the increment of zinc concentration and the maximal enzyme activity induced by 0.1 mM Zn^<2+> was equal to or higher than that after PAGEs with borate buffer.When ALP was extracted from mouse osteoblast-like cells (MC3T3-E1) or rat dental pulps and analyzed by native-PAGE or SDS-PAGE under a non-reducing condition, the enzyme was divided into ALP-N1 and ALP-N2 on native-PAGE, or into 130k and 155k on SDS-PAGE.ALP-N1 and -N2 corresponded to 130 and 155k on two dimens … More ional-PAGE, respectively. ALP-N1 (130k) changed to ALP-N2 (155k) after incubating the ALP extract at 37℃. It has been proved that the transformation of ALP is not caused by ALP-binding proteins, but is specifically caused by glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) in the ALP extract sample.ALP was purified from MC3T3-E1 or rat dental pulps by column chromatographies, such as DEAE-Sepharose CL-6B, Concanavalin A-Sepharose, Sephacryl S-300 HR and L-Histidyldiazobenzylphosphonic Acid Agarose. The purified ALP-N1 had the same pl (4.3) and binding affinity for ALP-N2 antibody as the purified ALP-N2. However, fatty acids (C14-C18) were detected only in the purified ALP-N1 by gas chromatography, and not in the purified ALP-N2. The level of SDS that combined with ALP-N1 was significantly more than with ALP-N2 without the fatty acids. These findings suggest that the ALP-N1 (13Ok) moves faster than the ALP-N2 (155k) on SDS-polyacrylamide gel.This study demonstrated that ALPs in MC3T3-E1 and rat dental pulps are a homo-dimer composed of 77k-subunits, and that two ALP-forms, with and without the fatty acids, are produced by GPI-PLD during the process of extracting ALP from the tissue. Less