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

小鼠成骨细胞样细胞 (MC3T3-E1) 和牙髓中碱性磷酸酶的分子种类

基本信息

批准号：
11671911
负责人：
HASHIMOTO Shuichi
金额：
$ 0.7万
依托单位：
The Nippon Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

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

通过用靛蓝染料进行活性染色来分析甘氨酸缓冲液对碱性磷酸酶 (ALP) 活性的影响，以便在非还原型 SDS 或天然 (0.1% Nonidet P-40)-聚丙烯酰胺得到后，以高灵敏度检测凝胶中的 ALP 活性使用甘氨酸缓冲液进行电泳 (PAGE) 时，含有 192 mM 甘氨酸的 SDS 和天然 PAGE 中的 ALP 活性降低至一半。然而，当192 mM 甘氨酸与Zn^<2+> 预混合时，PAGE 中的ALP 活性随着锌浓度的增加而增加，并且0.1 mM Zn^<2+> 诱导最大酶活性。等于或高于用硼酸盐缓冲液进行 PAGE 后的结果。当从小鼠成骨细胞样细胞 (MC3T3-E1) 或大鼠牙髓中提取 ALP 并通过在非还原条件下进行native-PAGE或SDS-PAGE，酶在native-PAGE上分为ALP-N1和ALP-N2，或者在SDS-PAGE上分为130k和155k。ALP-N1和-N2对应于130和 155k 分别在两个尺寸的 ional-PAGE 上更改为 ALP-N2。 (155k) 37℃孵育ALP提取物后，已证明ALP的转化不是由ALP结合蛋白引起的，而是由ALP提取物样品中的糖基磷脂酰肌醇特异性磷脂酶D（GPI-PLD）引起的。通过柱色谱法（例如 DEAE-Sepharose）从 MC3T3-E1 或大鼠牙髓中纯化 ALP CL-6B、伴刀豆球蛋白 A-琼脂糖、Sephacryl S-300 HR 和 L-组氨酰重氮苯甲基膦酸琼脂糖纯化的 ALP-N1 与纯化的 ALP-N2 具有相同的 p1 (4.3) 和对 ALP-N2 抗体的结合亲和力。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-N1 中未检测到脂肪酸 (C14-C18)。 ALP-N2。与 ALP-N1 结合的 SDS 水平显着高于不含脂肪酸的 ALP-N2。这些结果表明，ALP-N1 (13Ok) 在 SDS 上的移动速度比 ALP-N2 (155k) 更快。 -聚丙烯酰胺凝胶。本研究证明 MC3T3-E1 和大鼠牙髓中的 ALP 是由 77k 亚基组成的同源二聚体，并且两个ALP 形式（含或不含脂肪酸）由 GPI-PLD 在从组织中提取 ALP 的过程中产生。