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

通过用土著染料进行主动染色，分析了甘氨酸缓冲液对醇磷酸酶（ALP）活性的影响，以便在非还原型SDS或本机（0.1％nonidet P-40）（0.1％nonidet P-40）中检测凝胶中高敏感性ALP活性的影响 - 丙烯酰胺 - 丙烯酰胺 - 丙烯酰胺（Page）使用gage Electophoresis（Page）使用glycine pageine pageine。 ALP在SDS和天然页面中具有192毫米甘氨酸的活性减少到40毫米硼酸盐的一半。但是，当将192 mM甘氨酸与Zn^<2+>预混合时，页面中的ALP活性根据锌浓度的增加而增加，而由0.1 mm Zn^<2+>诱导的最大酶活性等于或更高等于或更高，则与bote buffer的页面相当于buffer。在非还原条件下，本地页面或SDS-PAGE在天然页面上将酶分为ALP-N1和ALP-N2，在SDS-PAGE.ALP-N1和-N2上分为130K和155K，分别在二维上对应于130和155K…分别在二维上……更多。 ALP-N1（130K）在37℃下孵育ALP提取物后，更改为ALP-N2（155K）。 It has been proven 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 chromatography, such as DEAE-Sepharose CL-6B, Concavalin a- sepharose，sephacryl S-300小时和L----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------磷酸琼脂糖琼脂糖琼脂糖琼脂糖琼脂糖。纯化的ALP-N1具有与ALP-N2抗体相同的PL（4.3）和结合亲和力与纯化的ALP-N2。但是，仅在纯化的ALP-N1中通过气相色谱法检测到脂肪酸（C14-C18），而不是在纯化的ALP-N2中检测到。与没有脂肪酸的ALP-N2相比，与ALP-N1结合的SD水平明显高得多。这些发现表明，ALP-N1（13OK）的移动速度比SDS-N2（155K）在SDS-聚丙烯酰胺凝胶上的移动速度快。这项研究表明，MC3T3-E1和Rat Dental Pulps中的Alps是由77k-subunits组成的同型二聚体，以及在没有脂肪时的过程中，该过程是fat fly the fat fly the gp，而GP的过程是fallp fr的过程。组织。较少的