Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies
Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies
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DOI：
10.1042/bj3160353
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发表时间：
1996-05-15
影响因子：
4.1
通讯作者：
Penniston, JT
中科院分区：
文献类型：
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作者：
Caride, AJ;Filoteo, AG;Penniston, JT
利用在COS-1细胞中表达的抗人质膜钙泵(HPMCA)单抗JA9、5F10和JA3的合成肽及其全长亚型hPMCA4b、hPMCA4a和hPMCA1b，分析了它们的表位定位和特异性。对这些多肽的实验表明，JA9与含有hPMCA4(a或b)残基51-75的区域发生特异性反应，但不与同种异构体1、2或3的相同区域发生反应。JA3与残基1156-1180发生反应，这是hPMCA4b所特有的区域。5F10在719-738残基区域发生反应，该区域在所有PMCA亚型中都高度保守。事实上，5F10识别COS-1细胞中表达的所有三种异构体。相比之下，JA9与hPMCA4的a和b变体都有反应，但不与hPMCA1反应，JA3只能识别hPMCA4b。我们使用这些抗体来区分hPMCA4a和hPMCA4b在人脑、心、肾和肺中的分布。在人脑样本的Western blotts中，我们可以同时识别hPMCA4a和hPMCA4b。心脏组织也显示出4b，可能是4a的异构体。而肾和肺则以hPMCA4b为主。在大脑中，检测到与hPMCA4的任一变体都不对应的重叠条带，在肾脏中观察到一条与hPMCA1b在同一位置迁移的条带。通过这些抗体分析，hPMCA4的a和b型在蛋白质水平上的分布与关于hPMCA亚型mRNAs丰度的现有数据一致。所有样品中hPMCA4b的存在支持该同工酶作为泵的构成形式的拟议作用。
The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51-75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156-1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719-738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, we could identify both hPMCA4a and hPMCA4b. Heart tissue also showed isoform 4b, and probably 4a. In contrast, kidney and lung showed primarily hPMCA4b. In brain, overlapping bands that did not correspond to either variant of hPMCA4 were detected, and in kidney a band migrating in the same position as hPMCA1b was observed. The distribution of the a and b forms of hPMCA4 at the protein level, as analysed by these antibodies, is consistent with the available data about the abundance of mRNAs for the hPMCA isoforms. The presence of hPMCA4b in all the samples supports the proposed role of this isoenzyme as a constitutive form of the pump.