Lazarus L H, Perrin M H, Brown M R
J Biol Chem. 1977 Oct 25;252(20):7174-9.
Neurotensin was iodinated at equimolar concentrations of peptide, iodide, and chloramine-T, producing a labeled peptide with a specific activity of 1000 to 2000 Ci/mmol. Rat mast cells specifically and reversibly bound 1.27 pmol of neurotensin/10(6) cells with a reversible affinity, KD, of 154 nM. Optimum specific binding occurred betwen pH 6.8 and 7.2 under hypotonic conditions and dropped sharply as buffer concentration increased beyond 10 mM. The divalent cations Ca2+ and Mg2+ prevented binding with 50% inhibition at 1.5 and 4 mM, respectively. Binding was strongly and equally inhibited by the sodium and potassium salts of chloride, bromide, and iodide, and to a lesser degree by LiCl. Maximum binding of 125I-neurotensin occurred within 10 min at 0 degrees, and within 1.5 to 2 min binding was reduced to half-maximum in the presence of excess unlabeled neurotensin or upon 20-fold dilution in buffer. Both CaCl2 and NaCl were able to dissociate 60% of the total bound neurotensin: half the label bound was removed in 4 to 6 min. EDTA inhibited the binding only at high concentrations and no requirement was found for sulfhydryl groups, ATP, or a glycoprotein in the binding of neurotensin.
在肽、碘化物和氯胺 -T等摩尔浓度下对神经降压素进行碘化，产生比活为1000至2000 Ci/mmol的标记肽。大鼠肥大细胞以154 nM的可逆亲和力特异性且可逆地结合1.27 pmol神经降压素/10⁶个细胞。在低渗条件下，pH 6.8至7.2之间出现最佳特异性结合，当缓冲液浓度超过10 mM时，结合急剧下降。二价阳离子Ca²⁺和Mg²⁺分别在1.5 mM和4 mM时以50%的抑制率阻止结合。氯离子、溴离子和碘离子的钠盐和钾盐强烈且同等程度地抑制结合，LiCl的抑制程度较小。¹²⁵I -神经降压素在0℃下10分钟内出现最大结合，在存在过量未标记神经降压素或在缓冲液中稀释20倍后，1.5至2分钟内结合降至最大结合量的一半。CaCl₂和NaCl都能够使总结合神经降压素的60%解离：结合的标记物一半在4至6分钟内被去除。EDTA仅在高浓度时抑制结合，并且在神经降压素的结合中未发现对巯基、ATP或糖蛋白有需求。
