Characterization of a leucokinin binding protein in Aedes aegypti (Diptera: Culicidae) Malpighian tubule.
Characterization of a leucokinin binding protein in Aedes aegypti (Diptera: Culicidae) Malpighian tubule.
复制标题
埃及伊蚊（双翅目：库蚊科）马氏小管中白细胞激肽结合蛋白的表征。
DOI：
10.1016/s0965-1748(00)00091-6
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发表时间：
2000
影响因子：
3.8
通讯作者：
Hayesa,TK
中科院分区：
文献类型：
--
作者：
Pietrantonio,PV;Gibsona,GE;Streya,AA;Petzel,D;Hayesa,TK
昆虫肌激肽(Leucokinin-like)神经肽家族包括具有不同生理作用的多肽，如诱导后肠肌力活动和刺激尿液产生等。肌激肽的C端五聚体Phe-X-(Ser/Pro/Ala)-Trp-Gly-amide[X=Phe，His，Asn，Ser或Tyr]，已被确定为能够引起功能反应的最小片段。这些昆虫神经肽的受体(S)尚未确定。为了研究埃及伊蚊马氏管亮激素样多肽受体(S)的结构和活性，根据亮激肽的构效关系，设计了一个序列为Dala-dTyr-BPA-dLys-Phe-Phe-Ser-Trp-Gly-酰胺的亮激素光亲和类似物(LPA)。LPA引起雌性马氏管跨上皮电压(TEV)的去极化，证实了LPA的活性。达到半数最大去极化的有效浓度(EC50)为17 nM。然后用~(125)I-LPA鉴定雌性马氏小管膜上的亮激肽结合蛋白。通过十二烷基硫酸钠-PAGE/放射自显影和与过量的未标记的亮氨酸激动素类似物竞争实验显示，125I-LPA与54 kDa的蛋白(S)结合，Kd值为13±3 nM，与TEV生物测定的EC50一致。总之，这些数据表明，54 kDa的蛋白是一种伊蚊-亮氨酸受体。这是首次对昆虫亮氨酸受体进行表征，并揭示了LPA是标记昆虫肌激肽受体的有力工具。
The insect myokinin (leucokinin-like) neuropeptide family includes peptides that have different physiological effects such as the induction of hindgut myotropic activity and stimulation of urine production. The C-terminal pentamer of myokinins Phe-X-(Ser/Pro/Ala)-Trp-Gly-amide [X=Phe, His, Asn, Ser or Tyr], had been previously determined as the minimum fragment able to elicit a functional response. The receptor(s) for these insect neuropeptides has not yet been identified. In order to characterize the Malpighian tubule leucokinin-like peptide receptor(s) from the yellow fever mosquito (Aedes aegypti), a leucokinin photoaffinity analogue (LPA) of sequence dAla-dTyr-Bpa-dLys-Phe-Phe-Ser-Trp-Gly-amide was designed based on structure/activity relationships for leucokinins. LPA caused depolarization of the transepithelial voltage (TEV) in female Malpighian tubule, confirming the activity of the peptide. The effective concentration to give half the maximum depolarization (EC50) was 17 nM. The125I-LPA was then used to characterize leucokinin binding proteins in female Malpighian tubule membranes. It specifically labeled and saturated a protein(s) of about 54 kDa as shown by SDS-PAGE/autoradiography and by competition experiments with excess unlabeled leucokinin analogues.125I-LPA bound to the 54 kDa protein(s) with a Kdvalue of 13±3 nM in agreement with the EC50for the TEV bioassay. Altogether these data suggest that the 54 kDa protein is an Aedes-leucokinin receptor. This is the first characterization of an insect leucokinin receptor and reveals that LPA is a powerful tool to label insect myokinin receptors.