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[mono[125I]iodo-Tyr10,MetO17]-vasoactive intestinal polypeptide. Preparation, characterization, and use for radioimmunoassay and receptor binding.
Journal of Biological Chemistry
Vasoactive intestinal polypeptide (VIP) was labeled with sodium [125I]iodide using the chloramine-T method and subsequently purified by reverse-phase high performance liquid chromatography.Three main 125I-labeled peaks designated A, B, and C resulted from the radioiodination and purification procedures. They were characterized by electrophoresis of tryptic fragments; Edman degradation (for Peaks A and C); enzymatic digestion to amino acids by leucine aminopeptidase, carboxypeptidase Y and Pronase; and treatment with cyanogen bromide. Peak A corresponds to VIP monoiodinated on Tyr10 and with the Met17 residue oxidized to methionine sulfoxide. This [mono[125I]iodo-Tyr10,MetO17]VIP displays the following characteristics. 1) It constitutes quantitatively the major product of the iodination procedure (62.5%); 2) it is well resolved from other labeled and unlabeled products; 3) it is stable (2 months at -20 degrees C); 4) it possesses a high specific activity (2050 Ci/mmol); 5) it maintains the biological activity of native VIP; and 6) it binds to antibody and membrane recognition sites in a specific, saturable, and reversible manner. Reduction of [mono[125I]iodo-Tyr10, Met-O17]VIP to [mono[125I]iodo-Tyr10]VIP does not improve the performance of the tracer in a radioimmunoassay. The method described in this article is simple and rapid and yields a molecular form of 125I-labeled VIP that has been fully characterized and is suitable for use in biological studies.
Animals, Carboxypeptidases/metabolism, Chloramines, Chromatography, High Pressure Liquid, Cross Reactions, Cyanogen Bromide/pharmacology, Electrophoresis, Polyacrylamide Gel, Glycogen/metabolism, Hydrolysis, Iodine Radioisotopes/metabolism, Kinetics, Leucyl Aminopeptidase/metabolism, Methionine/analogs & derivatives, Methionine/pharmacology, Mice, Monoiodotyrosine/metabolism, Peptide Fragments/analysis, Pronase/metabolism, Radioimmunoassay, Receptors, Cell Surface/metabolism, Receptors, Vasoactive Intestinal Peptide, Synaptic Membranes/metabolism, Tosyl Compounds, Trypsin/metabolism, Vasoactive Intestinal Peptide/analogs & derivatives, Vasoactive Intestinal Peptide/metabolism
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