Improved detection of the histamine receptor on human peripheral blood mononuclear cells by crosslinking using tritiated as compared with radioiodinated histamine.
In order to detect histamine receptors on the surface of human peripheral blood mononuclear 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 3H and 125I-radiolabelled ligands bound to a 16 kDa band, to be defined although a much clearer and obviously unequivocal signal was obtained with 3H-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 3H, fluorography with Entensify was required to obtain an autoradiographic signal. Although 3H took much longer to give a signal than 125I, the considerable background, artefacts and heavy lane trailing seen with [125I] histamine were completely abrogated when [3H]histamine was used. In addition, the distinction between specific and nonspecific binding was more clearly seen using [3H]histamine. The modifications reported here which improve signal detection for 3H 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.
Warlow
R S
RS
Department of Immunology, Concord Repatriation Hospital, N.S.W., Australia.
Dempsey
S
S
Carroll
R
R
Gibson
S
S
Bernard
C C
CC
D001345:Autoradiography; D002846:Chromatography, Affinity / Q000379:methods; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006632:Histamine / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007963:Leukocytes, Mononuclear / Q000032:analysis*; D011968:Receptors, Histamine / Q000032:analysis* / Q000378:metabolism; D013388:Succinimides; D014316:Tritium
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D011968:Receptors, Histamine; D013388:Succinimides; D014316:Tritium; D006632:Histamine; C019358:disuccinimidyl suberate