The arginine taste receptor. Physiology, biochemistry, and immunohistochemistry.
The arginine taste receptor. Physiology, biochemistry, and immunohistochemistry.
复制标题
精氨酸味觉受体。
DOI：
10.1111/j.1749-6632.1998.tb10555.x
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发表时间：
1998
影响因子：
5.2
通讯作者：
Brand,JG
中科院分区：
文献类型：
--
作者：
Grosvenor,W;Feigin,AM;Spielman,AI;Finger,TE;Wood,MR;Hansen,A;Kalinoski,DL;Teeter,JH;Brand,JG
氨基酸L-精氨酸(L-精氨酸)对斑点叉尾鱼有很强的味觉刺激作用。受体结合研究表明，L-精氨酸与可能的味觉受体结合具有高亲和力。用普通菜豆凝集素(PHA)和蓖麻凝集素I(Rca I)预先孵育组织可抑制这种结合。神经生理学研究表明，L-精氨酸受体是一种刺激门控型离子通道受体，其电导受L-精氨酸的兴奋和d-精氨酸的抑制。为了纯化该受体，我们用CHAPS溶解的触须上皮部分膜制剂，经Rca-I凝集素亲和层析。结合蛋白用D-半乳糖洗脱。当这些蛋白质被重组为脂质双层时，L-精氨酸激活了电导在45~85ps之间的单通道电流。洗脱后的蛋白质经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法在∼83 kDa处有一条明显的条带。针对这一83 kDa条带的多克隆抗体在豚鼠身上应用于固定的非通透性触须时，与每个味蕾味孔内的许多小的(∼1μm)部位发生反应。这一观察结果表明，抗体识别假定的Arg受体的一个面向外部的表位。在重构研究中，抗体还抑制L-精氨酸刺激的电流。亲和层析后的洗脱液经Sephacryl S-300HR层析，显示一高分子量峰(>；700 kDa)，该峰可被抗体识别。从这个峰到脂质双层的蛋白质重组导致了L-精氨酸刺激的通道，该通道可以被D-精氨酸抑制。这种高分子量组分可能是精氨酸味觉受体的聚集体。
The amino acid, l‐arginine (L‐Arg), is a potent taste stimulus for the channel catfish,Ictalurus punctatus. Receptor binding studies demonstrated a high‐affinity binding of L‐Arg to putative taste receptor sites. This binding could be inhibited by preincubation of the tissue in the lectins Phaseolus vulgaris agglutinin (PHA) andRicinus communis agglutinin I(RCA I). Neurophysiological studies demonstrated that the L‐Arg receptor is a stimulus‐gated ion channel type receptor whose conductance was stimulated by L‐Arg and inhibited by d‐arginine (D‐Arg). To purify the receptor we subjected CHAPS solubilized partial membrane preparation from barbel epithelium to RCA I lectin affinity chromatography. The bound proteins were eluted with d‐galactose. When these proteins were reconstituted into lipid bilayers, L‐Arg activated single channel currents with conductances between 45 and 85 pS. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) of the eluted protein showed a distinct band at ∼ 83 kDa. Polyclonal antibodies raised against this 83‐kDa band in guinea pigs reacted with numerous small (∼ 1μm) sites within the taste pore of every taste bud when applied to fixed nonpermeabilized barbels. This observation suggests that the antibodies recognize an externally‐facing epitope of the putative Arg receptor. The antibodies also inhibited L‐Arg‐stimulated currents in reconstitution studies. Sephacryl S‐300 HR chromatography of the eluant from the affinity column showed a high molecular weight peak (>700 kDa) which was recognized by the antibodies. Reconstitution of the protein from this peak into a lipid bilayer resulted in L‐Arg‐stimulated channels that could be inhibited by D‐Arg. This high molecular weight component may be aggregates of the arginine taste receptor.