Nye J S, Voglmaier S, Martenson R E, Snyder S H
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
J Neurochem. 1988 Apr;50(4):1170-8. doi: 10.1111/j.1471-4159.1988.tb10589.x.
Radioligand binding studies with the water-soluble cannabinoid [3H]5'-trimethylammonium delta 8-tetrahydrocannabinol ([3H]TMA) have revealed a saturable high-affinity site in brain that is specific for cannabinoids. To determine whether endogenous compounds of brain might act upon the site physiologically, we sought inhibitors in extracts of brain. An endogenous inhibitor has been purified to homogeneity by acid extraction of rat brain followed by adsorption to a reverse-phase matrix and gel filtration chromatography. The purified inhibitor has a subunit molecular mass of 14,500 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of [3H]TMA binding by the purified inhibitor occurs with a Ki of about 4 nM in a noncompetitive manner. The molecular weight, abundance, and extraction properties are the same as a species of myelin basic protein (MBP). The MBPs of rat, rabbit, pig, and cow also inhibit [3H]TMA binding noncompetitively with similar potencies. The purified inhibitor comigrates with rat MBP-small form on SDS-PAGE, has a similar amino acid composition, and is recognized by antibody directed against MBP. Studies of fragments of rabbit MBP suggest that the determinants of affinity for the [3H]TMA site are contained primarily within the C-terminal half of the rabbit MBP. Synthetic polycationic peptides such as polylysine and polyarginine mimic the effects of MBP, suggesting that the high-affinity cannabinoid binding site recognizes large polycations. The identification of the endogenous inhibitor of [3H]TMA binding as MBP suggests that MBP interacts physiologically with the high-affinity cannabinoid site.
使用水溶性大麻素[3H]5'-三甲基铵δ8-四氢大麻酚（[3H]TMA）进行的放射性配体结合研究表明，大脑中存在一个对大麻素具有特异性的可饱和高亲和力位点。为了确定大脑中的内源性化合物是否可能在生理上作用于该位点，我们在脑提取物中寻找抑制剂。通过对大鼠脑进行酸提取，然后吸附到反相基质上并进行凝胶过滤色谱，将一种内源性抑制剂纯化至同质。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），纯化后的抑制剂亚基分子量为14,500道尔顿。纯化后的抑制剂以非竞争性方式抑制[3H]TMA结合，其Ki约为4 nM。分子量、丰度和提取特性与一种髓鞘碱性蛋白（MBP）相同。大鼠、兔子、猪和牛的MBP也以相似的效力非竞争性抑制[3H]TMA结合。纯化后的抑制剂在SDS-PAGE上与大鼠MBP小形式共迁移，具有相似的氨基酸组成，并被针对MBP的抗体识别。对兔子MBP片段的研究表明，对[3H]TMA位点亲和力的决定因素主要包含在兔子MBP的C末端一半内。合成的聚阳离子肽如聚赖氨酸和聚精氨酸模拟了MBP的作用，表明高亲和力大麻素结合位点识别大的聚阳离子。将[3H]TMA结合的内源性抑制剂鉴定为MBP表明，MBP在生理上与高亲和力大麻素位点相互作用。
