Pascolo L, Del Vecchio S, Koehler R K, Bayon J E, Webster C C, Mukerjee P, Ostrow J D, Tiribelli C
Centro Studio Fegato, University of Trieste, Italy.
Biochem J. 1996 Jun 15;316 ( Pt 3)(Pt 3):999-1004. doi: 10.1042/bj3160999.
Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.
我们使用高度纯化的未结合[3H]胆红素（UCB），在不存在和存在内向正膜电位的情况下，测量了UCB与脱脂人血清白蛋白（HSA）的结合及其被大鼠肝基底外侧质膜囊泡的摄取。通过用于摄取研究的相同溶液经Centricon - 10装置（密理博公司）进行五个超滤循环来测定UCB - HSA亲和常数（K'f），并由此计算游离UCB浓度（[Bf]）。在HSA浓度为12至380 microM时，无论[Bf]/[HSA]比值如何，K'f（升/摩尔）与[HSA]呈负相关。K'f为2.066×10(6)+(3.258×10(8)/[HSA])。当用50 mM KCl等渗替代蔗糖时，K'f值显著降低{2.077×10(6)+(1.099×10(8)/[HSA])}。转运发生在对渗透压敏感的空间内。在饱和浓度以下（[Bf]≤65 nM），电中性和电生成成分相对于[Bf]均遵循饱和动力学，其K(m)值分别为28±7和57±8 nM（平均值±标准差，n = 3，P<0.001）。电生成成分的Vmax大于电中性成分（112±12对45±4 pmol UCB·mg-1蛋白质·15 s-1，P<0.001）。磺溴酞可反刺激电生成（61%）和电中性（72%）的UCB摄取。这些数据表明：(a)随着[HSA]增加，K'f降低，从而增加游离UCB的浓度。这可能解释了随着[HSA]增加观察到的有机阴离子肝细胞摄取增强的大部分原因。(b)UCB在肝细胞基底外侧膜通过两个系统摄取，其K(m)值在血浆中生理游离UCB水平范围内。电生成成分的亲和力较低但容量高于电中性成分。(c)使用在用于摄取研究的相同实验条件（培养基和[HSA]）下测定的K'f值来计算实际[Bf]很重要。
