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

采用原生染料活性染色法分析甘氨酸缓冲液对碱性磷酸酶（ALP）活性的影响，以甘氨酸缓冲液检测非还原型SDS-或原生（0.1% Nonidet P-40）聚丙烯酰胺get电泳（PAGE）后凝胶中的ALP活性。当甘氨酸浓度为192 mM时，SDS- page和天然page的ALP活性下降到40 mM硼酸盐的一半。而当192 mM甘氨酸与Zn^<2+>预混后，随着锌浓度的增加，PAGEs的ALP活性也随之增加，且0.1 mM Zn^<2+>诱导的最大酶活性等于或高于添加硼酸盐缓冲液后的酶活性。从小鼠成骨样细胞（MC3T3-E1）或大鼠牙髓中提取ALP，在非还原条件下用native-PAGE或SDS-PAGE进行分析，native-PAGE将酶分为ALP- n1和ALP- n2， SDS-PAGE将酶分为130k和155k。ALP-N1和-N2在两个维度上分别对应130和155k。37℃孵育ALP提取物后，ALP- n1 （130k）变为ALP- n2 （155k）。已经证明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）特异性引起的。采用柱层析法从mc3l3 - e1或大鼠牙髓中纯化ALP，如DEAE-Sepharose CL-6B、Concanavalin A-Sepharose、Sephacryl S-300 HR和l -组氨酸二氮苯基膦酸琼脂糖。纯化的ALP-N1与纯化的ALP-N2抗体具有相同的pl（4.3）和结合亲和力。然而，脂肪酸（C14-C18）仅在纯化的ALP-N1中检测到，而在纯化的ALP-N2中未检测到。与ALP-N1联合的SDS水平显著高于未加脂肪酸的ALP-N2。这些结果表明，在sds -聚丙烯酰胺凝胶上，ALP-N1 （13Ok）比ALP-N2 （155k）移动更快。本研究表明，MC3T3-E1和大鼠牙髓中的ALP是由77k-亚基组成的同型二聚体，GPI-PLD在提取组织ALP的过程中产生带脂肪酸和不带脂肪酸的两种ALP形态。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