Malaba L, Kindberg G M, Norum K R, Berg T, Blomhoff R
Institute for Nutrition Research, University of Oslo, Norway.
Biochem J. 1993 Apr 1;291 ( Pt 1)(Pt 1):187-91. doi: 10.1042/bj2910187.
Retinol-binding protein (RBP) was iodinated directly by radio-iodine substitution on the tyrosyl residues by the sodium hypochlorite (NaOCl) or the Enzymobead (EB) methods, or indirectly by linkage of 125I-tyramine-cellobiose (TC) or 125I-N-succinimidyl-3-(4- hydroxyphenyl)propionic acid ester (SHPP) adduct on to free amino residues of RBP. Binding, uptake and degradation of iodinated RBP were studied in isolated rat and rabbit liver parenchymal cells. The amount of ligand bound to cells at 4 degrees C was dependent on the type of labelling, in that the 125I-TC ligand was bound to a lesser extent than NaClO-labelled 125I-RBP, EB-labelled 125I-RBP and 125I-SHPP-RBP. At 37 degrees C, the 125I-SHPP-RBP and the EB-labelled 125I-RBP became cell-associated more rapidly than the other two ligands. The higher cell association at 37 degrees C than at 4 degrees C suggests that internalization of the ligand occurred at the higher temperature. The degradation of the ligands was also different. The EB-labelled 125I-RBP, the 125I-TC-RBP and the 125I-SHPP-RBP showed an apparent lag phase before a steady increase in acid-soluble radioactivity was observed. Much less of EB-labelled 125I-RBP and 125I-TC-RBP were degraded (about 6%) than of the other two ligands (about 16%) after 120 min. About 50% of the acid-soluble radioactivity in these experiments could be accounted for by degradation in the medium, suggesting that about half of the degradation observed was intracellular. The present study therefore shows that the different labelling techniques yield varying estimates of the cellular handling of RBP. In addition, a rapid release of RBP was observed in experiments where cells were pulsed with radioactive RBP at 4 degrees C, washed and incubated further at 37 degrees C. Between 50% and 70% was released after 5 min of incubation. By increasing the temperature during the pulse to 37 degrees C, or by lowering the temperature during the chase to 4 degrees C, much less RBP was released from the cells. These data suggest that the release process represents recycling of internalized ligand from an early endosome.
视黄醇结合蛋白（RBP）可通过次氯酸钠（NaOCl）法或酶珠（EB）法直接在酪氨酸残基上进行放射性碘取代而碘化，或者通过将125I-酪胺-纤维二糖（TC）或125I-N-琥珀酰亚胺基-3-（4-羟苯基）丙酸酯（SHPP）加合物连接到RBP的游离氨基上进行间接碘化。对碘化RBP在分离的大鼠和兔肝实质细胞中的结合、摄取和降解进行了研究。4℃时与细胞结合的配体量取决于标记类型，即125I-TC配体的结合程度低于次氯酸钠标记的125I-RBP、EB标记的125I-RBP和125I-SHPP-RBP。在37℃时，125I-SHPP-RBP和EB标记的125I-RBP比其他两种配体更快地与细胞结合。37℃时的细胞结合高于4℃，这表明配体在较高温度下发生内化。配体的降解也有所不同。EB标记的125I-RBP、125I-TC-RBP和125I-SHPP-RBP在观察到酸溶性放射性稳定增加之前显示出明显的延迟期。120分钟后，EB标记的125I-RBP和125I-TC-RBP的降解量（约6%）远低于其他两种配体（约16%）。在这些实验中，约50%的酸溶性放射性可归因于培养基中的降解，这表明观察到的降解约一半是细胞内的。因此，本研究表明，不同的标记技术对RBP细胞处理的估计不同。此外，在4℃用放射性RBP脉冲细胞、洗涤并在37℃进一步孵育的实验中观察到RBP快速释放。孵育5分钟后释放了50%至70%。通过将脉冲期间的温度提高到37℃，或通过将追踪期间的温度降低到4℃，从细胞中释放的RBP要少得多。这些数据表明，释放过程代表内化配体从早期内体的再循环。
