The solubilized locust neuronal 3H-mianserin binding site, a histamine 3H 1-like receptor molecule

Abstract

The 3H-mianserin binding site of the desert locust Schistocerca gregaria, which represents an insect histamine 3H 1-like receptor molecule, could be solubilized from its membrane-bound state with the detergent digitonin. Using this detergent more than 30% of the total specific binding sites could be solubilized. The binding parameters of 3H-mianserin remained unchanged (K D solubilized = 0.77 nM; K D membrane bound = 0.8 nM). Furthermore, the affinity of all substances tested, although they derived from different classes of compounds, varied only marginally between the solubilized and the membrane bound state. This indicates that the solubilized binding site is identical with the native locust H 1-like receptor molecule. Gelfiltration of the receptor detergent complex revealed a molecular weight of approx 500,000. Lectin affinity chromatography using a variety of different immobilized lectins could demonstrate that the 3H-mianserin binding site is glycosylated. Only lentil lectin and ConA, both with a specificity for mannose-like terminal sugars, bound the 3H-mianserin binding site in a reversible manner. In addition to these two immobilized lectins two other affinity matrices bound specifically the insect histamine H 1-like receptor molecule. The ligands demethylmianserin and SKF 94461, immobilized onto an activated gelmatrix, have high affinity properties for the 3H-mianserin binding site. Using these gelmatrices together with gelfiltration, an approx 400-fold purification was possible. Further experiments should enable the purification of this molecule in the near future.