Evaluation of endothelin receptor populations using endothelin-1 biotinylated at lysine-9 sidechain.
Optimal conditions for biotinylation of endothelin-1 (Et-1) were determined using biotinylating reagents of variable linker arm length and mono- vs dual-biotinylated Et-1. Specific modification of lysine-9 sidechain with NHS-LC-biotin (Et-1[BtK9]) produced a derivative with maximal binding and retention of vascular smooth muscle contractile activity. The Et-1[BtK9] probe bound to Chinese hamster ovary cells transfected with EtA receptor cDNA (CHO[EtR]), but not untransfected cells. Binding to rat vascular smooth muscle cells (VSMC) was detectable at 0.01 nM with maximal binding at 1 nM. Displacement of 1 nM Et-1 [BtK9] binding by Et-1 indicated an IC50 value of 6 +/- 3 nM. Et-1 displaced Et-1[BtK9] binding to VSMC and CHO[EtR] to a greater extent than endothelin-3, indicating predominant expression of EtA receptor sub-type. Thus, biotinylation of Et-1 at the lysine-9 sidechain may be of general use for localization and typing of Et-receptor populations.
Magazine
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Department of Biochemistry & Molecular Biology, Albany Medical College, NY 12208.
Andersen
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Goligorsky
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Malik
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D000818:Animals; D001011:Aorta / Q000378:metabolism*; D001667:Binding, Competitive; D001710:Biotin; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D016232:Endothelins / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine; D008856:Microscopy, Fluorescence; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017466:Receptors, Endothelin; D014162:Transfection
D016232:Endothelins; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D001710:Biotin; D008239:Lysine