Werner A, Moore M L, Mantei N, Biber J, Semenza G, Murer H
Department of Physiology, University of Zürich, Switzerland.
Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9608-12. doi: 10.1073/pnas.88.21.9608.
A cDNA library from rabbit kidney cortex was screened for expression of Na-dependent transport of phosphate (Pi) using Xenopus laevis oocytes as an expression system. A single clone was eventually isolated (designated NaPi-1) that stimulated expression of Na/Pi cotransport approximately 700-fold compared to total mRNA. The predicted sequence of the Na/Pi cotransporter consists of 465 amino acids (relative molecular mass, 51,797); hydropathy profile predictions suggest six (possibly eight) membrane-spanning segments. In vitro translation of NaPi-1/complementary RNA in the presence of pancreatic microsomes indicated NaPi-1 to be a glycosylated protein; four potential N-glycosylation sites are present in the amino acid sequence. Northern blot analysis demonstrated the presence of NaPi-1/mRNA in kidney cortex and liver; no hybridization signal was obtained with mRNA from other tissues (including small intestine). Kinetic analysis of Na/Pi cotransport expressed by NaPi-1/complementary RNA demonstrated characteristics (sodium interaction) similar to those observed in cortical apical membranes. The alignment of 5 amino acid residues (Gly342/Ala381-Xaa-Xaa-Xaa-Xaa-Leu386-Xaa-Xaa-Xaa-P ro390- Arg391) is consistent with a motif proposed for Na-dependent transport systems. We conclude that we have cloned a cDNA for a Na/Pi cotransport system present in rabbit kidney cortex.
以非洲爪蟾卵母细胞作为表达系统，对兔肾皮质的cDNA文库进行筛选，以寻找钠依赖性磷酸盐（Pi）转运体的表达情况。最终分离出一个单一克隆（命名为NaPi-1），与总mRNA相比，它能使钠/磷酸盐共转运体的表达增加约700倍。钠/磷酸盐共转运体的预测序列由465个氨基酸组成（相对分子质量为51,797）；亲水性图谱预测表明有六个（可能八个）跨膜区段。在胰腺微粒体存在的情况下对NaPi-1/互补RNA进行体外翻译，结果表明NaPi-1是一种糖基化蛋白；氨基酸序列中存在四个潜在的N-糖基化位点。Northern印迹分析表明在肾皮质和肝脏中存在NaPi-1/mRNA；未从其他组织（包括小肠）的mRNA中获得杂交信号。对由NaPi-1/互补RNA表达的钠/磷酸盐共转运体进行动力学分析，结果显示其特性（钠相互作用）与在皮质顶端膜中观察到的相似。5个氨基酸残基（Gly342/Ala381-Xaa-Xaa-Xaa-Xaa-Leu386-Xaa-Xaa-Xaa-Pro390-Arg391）的排列与为钠依赖性转运系统提出的基序一致。我们得出结论，我们已经克隆了兔肾皮质中存在的一种钠/磷酸盐共转运系统的cDNA。
