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

为了高灵敏度地检测非还原型SDS-或非还原型（0.1%Nonidet P-40）-聚丙烯酰胺凝胶电泳（PAGE）后的碱性磷酸酶（ALP）活性，采用靛蓝染料活性染色法，分析了甘氨酸缓冲液对ALP活性的影响。ALP在SDS-和本地-PAGEs与192 mM甘氨酸的活动下降到一半的PAGEs与40 mM硼酸盐。然而，当192 mM甘氨酸与Zn^<2+>预混合时，PAGEs中的ALP活性随锌浓度的增加而增加，0.1 mM Zn^<2+>诱导的最大酶活性等于或高于硼酸盐缓冲液诱导的最大酶活性（MC 3 T3-E1）或大鼠牙髓中，并在非还原条件下通过native-PAGE或SDS-PAGE进行分析，在native-PAGE上酶被分为ALP-N1和ALP-N2，在SDS-PAGE上，ALP-N1和-N2分别对应于130和155 k， ...更多信息 离子-PAGE。在37℃下孵育ALP浸提液后，ALP-N1（130 k）变为ALP-N2（155 k）。采用DEAE-SepharoseCL-6 B、ConcanavalinA-Sepharose、SephacrylS-300 HR和L-HistidyldiazobenzylphosphonicAcidAgarose等柱层析技术，从MC 3 T3-E1和大鼠牙髓中分离纯化ALP，并对纯化后的ALP进行形态学观察。纯化的ALP-N1对ALP-N2抗体具有与纯化的ALP-N2相同的pI（4.3）和结合亲和力。然而，通过气相色谱法仅在纯化的ALP-N1中检测到脂肪酸（C14-C18），而在纯化的ALP-N2中未检测到。与ALP-N1结合的SDS水平显著高于不含脂肪酸的ALP-N2。本研究表明，MC 3 T3-E1和大鼠牙髓中的ALP均为77 k-亚基的同源二聚体，GPI-PLD在提取组织中ALP的过程中产生了含脂肪酸和不含脂肪酸的两种形式。少

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