Persico M, Sottocasa G L
Biochim Biophys Acta. 1987 Sep 14;930(2):129-34. doi: 10.1016/0167-4889(87)90023-1.
A spectrophotometric technique is described for the continuous recording of sulfobromophthalein uptake by isolated hepatocytes. The technique is based on the principle that sulfobromophthalein behaves as a pH-indicator and may be followed photometrically when moving from the medium at pH 7.8 into the interior of the cell. Data show that upon addition of cells to a sulfobromophthalein solution, an absorbance change can be recorded. The kinetics of the process is biphasic and the initial rate is linearly related to the amount of cells added. By this technique it was confirmed that the substrate dependence of the initial velocity of transport is a compound function including a saturable portion with an apparent Km in the mu molar region. Experiments carried out either in the presence of valinomycin or of high concentrations of potassium chloride indicate that the presence of a membrane potential opposes the entry of sulfobromophthalein into isolated hepatocytes. This finding is in agreement with previous observations in isolated plasma membrane vesicles and in liposomes reconstituted with purified bilitranslocase which indicate a rheogenic type of transport for the dye. Low concentrations of nicotinate (1.6 microM) efficiently inhibit the saturable transport. It is suggested, in addition, that the sensitivity of the transport to valinomycin could be used as an early indication of the functional integrity of cell preparations.
本文描述了一种用于连续记录分离的肝细胞摄取磺溴酞的分光光度技术。该技术基于磺溴酞作为pH指示剂的原理，当它从pH 7.8的培养基进入细胞内部时，可以通过光度法进行跟踪。数据表明，将细胞加入磺溴酞溶液后，可以记录到吸光度变化。该过程的动力学是双相的，初始速率与添加的细胞量呈线性关系。通过该技术证实，转运初始速度对底物的依赖性是一种复合函数，包括在微摩尔区域具有表观Km的可饱和部分。在缬氨霉素存在或高浓度氯化钾存在下进行的实验表明，膜电位的存在会阻碍磺溴酞进入分离的肝细胞。这一发现与先前在分离的质膜囊泡和用纯化的胆红素转运酶重构的脂质体中的观察结果一致，这些观察结果表明该染料的转运是生电型的。低浓度的烟酸（1.6 microM）有效抑制可饱和转运。此外，有人提出，转运对缬氨霉素的敏感性可作为细胞制剂功能完整性的早期指标。
