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Improved detection of the histamine receptor on human peripheral blood mononuclear cells by crosslinking using tritiated as compared with radioiodinated histamine
Author(s) -
Warlow Robert S.,
Dempsey Susan,
Carroll Raeleen,
Gibson Steven,
Bernard Claude C. A.
Publication year - 1990
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150110611
Subject(s) - histamine , chemistry , receptor , sodium dodecyl sulfate , gel electrophoresis , ligand (biochemistry) , polyacrylamide gel electrophoresis , chromatography , histamine receptor , biochemistry , protein subunit , microbiology and biotechnology , biology , enzyme , endocrinology , gene , antagonist
In order to detect histamine receptors on the surface of human peripheral blood monouclear cells, the cells were incubated in the presence of radiolabelled histamine and then the bifunctional crosslinker disuccimidyl suberate was added in various concentrations. They were then solubilized with sodium dodecyl sulphate, boiled, reduced and the lysate separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Both 3 H and 125 I‐radiolabelled ligands bound to a 16 kDa band, to be defined although a much clearer and obviously unequivocal signal was obtained with 3 H‐labelled histamine. This molecule migrated with the same mass on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis as a 16 kDa subunit which had been purified on a histamine affinity column from Triton X‐100 solubilized mononuclear cells, indicating it to be the ligand‐binding subunit for the histamine receptor on these cells. For 3 H, fluorography with Entensify TM was required to obtain an autoradiographic signal. Although 3 H took much longer to give a signal than 125 I, the considerable background, artefacts and heavy lane trailing seen with [ 125 I] histamine were completely abrogated when [ 3 H]histamine was used. In addition, the distinction between specific and nonspecific binding was more clearly seen using [ 3 H]histamine. The modifications reported here which improve signal detection for 3 H should encourage the use of tritiated ligands in radioreceptor crosslinking, particularly those of low molecular weight which might otherwise undergo steric modification due to iodination, this having the potential for interfering with receptor ligand binding.