Benyhe S, Simon J, Borsodi A, Wollemann M, Barnard E A
Institute of Biochemistry, Hungarian Academy of Sciences, Szeged.
Neuropeptides. 1994 May;26(5):359-64. doi: 10.1016/0143-4179(94)90121-x.
Opioid binding sites specific for [3H]dynorphin1-8 were characterized in the particulate membrane fraction of frog (Rana esculenta) brain. The degradation of the radioligand during the assay was prevented by the use of a broad spectrum of peptidase inhibitors. The binding of [3H]dynorphin1-8 to frog brain membranes was stereoselective, reversible, saturable, and displaceable by a series of opioid ligands including dynorphin1-13, bremazocine, levorphanol and naloxone. The specific binding of [3H]dynorphin1-8 can be significantly inhibited by Na+ ions and/or guanine nucleotides confirming the agonist property of the ligand in vitro. A single set of high affinity opioid binding sites with a Kd approximately 7.5 nM is present in the membranes. The maximum density of binding sites (Bmax approximately 1.1 pmol [3H]dynorphin1-8 per mg protein) was considerably higher than such sites in guinea-pig brain. In addition, comparison with binding of tritiated opioid peptides selective for the mu- and delta-types of opioid receptor showed that in the frog brain most of the sites labelled by [3H]dynorphin1-8 are kappa-sites and that this is a rich source of such sites.
在青蛙（食用蛙）脑的微粒体膜部分对[3H]强啡肽1 - 8特异性的阿片样物质结合位点进行了表征。在测定过程中，通过使用多种肽酶抑制剂来防止放射性配体的降解。[3H]强啡肽1 - 8与青蛙脑膜的结合具有立体选择性、可逆性、饱和性，并且可被一系列阿片样物质配体取代，包括强啡肽1 - 13、布马佐辛、左啡诺和纳洛酮。[3H]强啡肽1 - 8的特异性结合可被Na⁺离子和/或鸟嘌呤核苷酸显著抑制，这证实了该配体在体外的激动剂特性。膜中存在一组单一的高亲和力阿片样物质结合位点，其解离常数（Kd）约为7.5 nM。结合位点的最大密度（Bmax约为每毫克蛋白质1.1 pmol [3H]强啡肽1 - 8）明显高于豚鼠脑中的此类位点。此外，与对μ型和δ型阿片受体具有选择性的氚标记阿片肽的结合比较表明，在青蛙脑中，大多数被[3H]强啡肽1 - 8标记的位点是κ位点，并且这是此类位点的丰富来源。
