Boyer C J, Baines A D, Beaulieu E, Béliveau R
Laboratoire d'Oncologie Moléculaire, Université du Québec à Montréal-Hôpital Ste-Justine, Canada.
Biochim Biophys Acta. 1998 Jan 5;1368(1):73-83. doi: 10.1016/s0005-2736(97)00159-4.
Polyclonal antibodies were raised in rabbits against a 14-amino acid portion of the gibbon ape leukemia virus human membrane receptor Glvr-1. This epitope also contained seven amino acids common to the receptor for the amphotropic murine retrovirus Ram-1. Antibody specificity and molecular size of Glvr-1/Ram-1-related proteins were assayed by Western blot. Using a standard Laemmli buffer system, under reducing conditions, a single band of approximately 85 kDa (designated p85) was immunodetected in membranes prepared from opossum kidney (OK) cells and in brain membranes from rat, rabbit and hamster. In mouse brain, p85 as well as a protein of 70-72 kDa were immunodetected. This protein was also present in several other mouse tissues. Limited proteolysis of p85 and the 70-72kDa-protein from mouse yielded similar peptide fragments, suggesting that both proteins are related. Fragments of the same molecular masses were also detected in OK cell membranes following proteolysis, showing that p85 in both models (mouse brain and OK cell) share a similar sequence. p85 is not N-glycosylated since an assay using endoglycosidase F/N-glycosidase F did not alter the electrophoretic mobility of p85. We also observed that regulation of phosphate transport by incubating OK cells without any phosphate or by PTH treatment occurs without any changes in the amount of p85. In conclusion, these data demonstrate for the first time a Western blot detection of a type III phosphate transporter using polyclonal antibodies. They also suggest that, conversely to type I and type II phosphate transporters which are localized in the kidney, this third type of transporter is ubiquitous and probably absorbs the readily available phosphate from interstitial fluid for normal cellular functions in many species and tissues, serving as a housekeeping Na+/Pi cotransport system. This is also the first report showing that p85 is not regulated in the same manner as type II phosphate transporters.
用长臂猿白血病病毒人类膜受体Glvr-1的一段14个氨基酸的片段对兔子进行免疫，制备多克隆抗体。该表位还包含嗜双性鼠逆转录病毒Ram-1受体共有的7个氨基酸。通过蛋白质印迹法检测Glvr-1/Ram-1相关蛋白的抗体特异性和分子大小。在还原条件下，使用标准的Laemmli缓冲系统，在负鼠肾（OK）细胞制备的膜以及大鼠、兔子和仓鼠的脑膜中免疫检测到一条约85 kDa的单带（命名为p85）。在小鼠脑中，免疫检测到p85以及一种70 - 72 kDa的蛋白质。这种蛋白质也存在于其他几种小鼠组织中。对小鼠的p85和70 - 72 kDa蛋白质进行有限的蛋白酶解，产生了相似的肽片段，表明这两种蛋白质相关。蛋白酶解后在OK细胞膜中也检测到相同分子量的片段，表明在两种模型（小鼠脑和OK细胞）中p85具有相似的序列。由于使用内切糖苷酶F/N-糖苷酶F的检测未改变p85的电泳迁移率，所以p85不是N-糖基化的。我们还观察到，在无磷酸盐条件下孵育OK细胞或用甲状旁腺激素处理来调节磷酸盐转运时，p85的量没有任何变化。总之，这些数据首次证明了使用多克隆抗体通过蛋白质印迹法检测III型磷酸盐转运体。它们还表明，与定位于肾脏的I型和II型磷酸盐转运体相反，这种第三类转运体无处不在，可能从组织液中吸收易于利用的磷酸盐，以维持许多物种和组织中正常的细胞功能，作为一种管家型Na⁺/Pi共转运系统。这也是第一份表明p85的调节方式与II型磷酸盐转运体不同的报告。
