Preparation of iron-deficient tissue culture medium by deferoxamine-sepharose treatment and application to the differential actions of apotransferrin and diferric transferrin.
Preparation of iron-deficient tissue culture medium by deferoxamine-sepharose treatment and application to the differential actions of apotransferrin and diferric transferrin.
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去铁胺-琼脂糖处理制备缺铁组织培养基及其在脱铁转铁蛋白和二铁转铁蛋白差异作用中的应用。
DOI：
10.1016/0003-2697(92)90319-3
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发表时间：
1992
影响因子：
2.9
通讯作者：
Sirbasku,DA
中科院分区：
文献类型：
--
作者：
Eby,JE;Sato,H;Sirbasku,DA
我们已经证明，三碘甲状腺原氨酸依赖的GH 1大鼠垂体细胞在无血清确定培养中的生长需要脱铁转铁蛋白（apoTf）（D. A. Sirbasku等人，Biochemistry 30，295-304，7466-7477，1991）。这些研究是在没有Fe（III）Fe（II）盐的“低Fe”介质中进行的。尽管如此，铁的显着浓度可能是由其他组件，使这种介质不适合研究的差异影响的apoTf和二铁转铁蛋白（2Fe·Tf）。测量培养基中的残留铁一直是麻烦的，因为最灵敏的方法（即，原子吸收）仅检测到超过10 ng/ml的水平，并且不能区分存在的铁的形式。为了估计可用于结合apoTf的Fe（III），我们开发了一种更灵敏和特异的方法。尿素-聚丙烯酰胺凝胶电泳（PAGE）分离apoTf、两种单铁转铁蛋白和2Fe·Tf。将[125 I]apoTf与培养基或组分一起孵育，并通过尿素-PAGE/放射自显影监测[125 I]-2Fe统计Tf的形成。通过这种方法，低铁培养基中的Fe（III）的浓度估计为8.4至20 ng/ml，并确定了来源。我们接下来试图去除Fe（III）。标准螯合剂无效或具有细胞毒性。相比之下，使用去铁胺-琼脂糖凝胶的亲和方法消耗了≥90%的Fe（III）。在此培养基中，apoTf和2Fe·Tf对GH 11细胞和MCF-7、MTW 9、PL 2、MDCK细胞的作用不同。利用本文所述的方法，可以分别研究apoTf和2Fe·Tf对生长的影响。
We have shown that triiodothyronine-dependent GH1rat pituitary cell growth in serum-free defined culture required apotransferrin (apoTf) (D. A. Sirbasku, et al., Biochemistry 30, 295–304, 7466–7477, 1991). These studies were done in “low-Fe” medium without Fe(III) Fe(II) salts. Nonetheless, significant concentrations of iron may have been contributed by other components, making this medium unsuitable for study of the differential effects of apoTf and diferric transferrin (2Fe·Tf). Measuring residual iron in culture medium has been troublesome because the most sensitive method (i.e., atomic absorption) detected levels only in excess of 10 ng/ml and did not distinguish between the forms of iron present. To estimate the Fe(III) available to bind to apoTf, we developed a more sensitive and specific method. Urea-polyacrylamide gel electrophoresis (PAGE) separates apoTf, the two monoferric transferrins, and 2Fe·Tf. [125I]apoTf was incubated with medium, or components, and the formation of [125I]-2Fe·Tf was monitored by urea-PAGE/autoradiography. By this method, the concentration of Fe(III) in low-Fe medium was estimated at 8.4 to 20 ng/ml and the sources were identified. We next sought to remove the Fe(III). Standard chelators were ineffective or cytotoxic. In contrast, an affinity method with deferoxamine-Sepharose depleted ≥90% of the Fe(III). In this medium, apoTf and 2Fe·Tf showed differential effects with GH1cells and with MCF-7, MTW9 PL2 , and MDCK cells. With the methods described here, the effects of apoTf and 2Fe·Tf on growth can be studied separately.