Gibb G M, Reid G P, Lindsay J G
Biochem J. 1986 Sep 1;238(2):543-51. doi: 10.1042/bj2380543.
The phosphate transport protein was purified from rat liver mitochondria by extraction in an 8% (v/v) Triton X-100 buffer followed by adsorption chromatography on hydroxyapatite and Celite. SDS/polyacrylamide-gel electrophoresis (10%, w/v) demonstrated that the purified polypeptide was apparently homogeneous when stained with Coomassie Blue and had a subunit Mr of 34,000. However, lectin overlay analysis of this gel with 125I-labelled concanavalin A demonstrated the presence of several low- and high-Mr glycoprotein contaminants. To overcome this problem, mitochondria were pre-extracted with a 0.5% (v/v) Triton X-100 buffer as an additional step in the purification of phosphate transport protein. SDS/polyacrylamide gradient gel electrophoresis (14-20%, w/v) of the hydroxyapatite and Celite eluates revealed one major band of Mr 34,000 when stained with Coomassie Blue. The known thiol group sensitivity of the phosphate transporter was employed to characterize the isolated polypeptide further. Labelling studies with N-[2-3H]ethylmaleimide showed that only the 34,000-Mr band was labelled in both the hydroxyapatite and Celite fractions, when purified from rat liver mitochondria. Further confirmation of its identity has been provided with an antiserum directed against the 34,000-Mr protein. Specific partial inhibition of phosphate uptake, as measured by iso-osmotic swelling in the presence of (NH4)2HPO4, was achieved when mitoplasts (mitochondria minus outer membrane) were incubated with this antiserum. Finally, amino acid analysis of the rat liver mitochondrial phosphate/hydroxyl ion antiport protein indicates that it is similar in composition to the equivalent protein isolated from ox heart.
通过在8%（v/v）的 Triton X-100缓冲液中提取，随后在羟基磷灰石和硅藻土上进行吸附色谱法，从大鼠肝脏线粒体中纯化出磷酸转运蛋白。SDS/聚丙烯酰胺凝胶电泳（10%，w/v）表明，用考马斯亮蓝染色时，纯化的多肽显然是均一的，亚基分子量为34,000。然而，用125I标记的伴刀豆球蛋白A对该凝胶进行凝集素覆盖分析，显示存在几种低分子量和高分子量的糖蛋白污染物。为克服这个问题，作为磷酸转运蛋白纯化的额外步骤，先用0.5%（v/v）的 Triton X-100缓冲液对线粒体进行预提取。用考马斯亮蓝染色时，羟基磷灰石和硅藻土洗脱物的SDS/聚丙烯酰胺梯度凝胶电泳（14 - 20%，w/v）显示出一条分子量为34,000的主要条带。利用已知的磷酸转运体对巯基的敏感性进一步表征分离出的多肽。用N-[2-3H]乙基马来酰亚胺进行的标记研究表明，从大鼠肝脏线粒体中纯化时，在羟基磷灰石和硅藻土组分中只有分子量为34,000的条带被标记。用针对分子量为34,000的蛋白质的抗血清进一步证实了其身份。当线粒体（去除外膜的线粒体）与这种抗血清一起孵育时，通过在(NH4)2HPO4存在下的等渗肿胀测量，实现了对磷酸盐摄取的特异性部分抑制。最后，对大鼠肝脏线粒体磷酸/羟基离子反向转运蛋白的氨基酸分析表明，其组成与从牛心脏分离的等效蛋白相似。
