vasoactive-intestinal-peptide and methionine-sulfoxide

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.

Topics: Animals; Carboxypeptidases; Chloramines; Chromatography, High Pressure Liquid; Cross Reactions; Cyanogen Bromide; Electrophoresis, Polyacrylamide Gel; Glycogen; Hydrolysis; Iodine Radioisotopes; Kinetics; Leucyl Aminopeptidase; Methionine; Mice; Monoiodotyrosine; Peptide Fragments; Pronase; Radioimmunoassay; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Synaptic Membranes; Tosyl Compounds; Trypsin; Vasoactive Intestinal Peptide

During the isolation of cholecystokinin from natural sources, as well as during its bioassay, inactivation by oxidation can cause problems. We have attempted to reactivate oxidized CCK by reduction at room temperature with N-methylmercaptoacetamide, recently stated to be the reducing agent of choice for the reduction of methionine sulfoxide to methionine [22]. We have not yet been unequivocally successful in these attempts, but the results seem promising. In the case of oxidized VIP and of oxidized tetragastrin, reduction with N-methylmercaptoacetamide does seem to result in reconversion of the peptides to their preoxidation states, as evidenced by thin layer chromatography on silica gel. We have, together with A. Holmgren and A. Ehrnberg, made observations suggesting the presence in rate liver cytosol of an enzyme which catalyzes the reductive reactivation of oxidized CCK with reduced thioredoxin as the immediate hydrogen donor. In collaboration with A. Light, Purdue University, we have found that enterokinase cleaves 39-CCK and 33-CCk with release of 8-CCK and the tetrapeptide immediately preceding it in the peptide chain. The conversion of 39-CCK to 33-CCK by the action of dipeptidyl amino-peptidase I has been confirmed.

Topics: Animals; Cats; Cholecystokinin; Enteropeptidase; Gastrointestinal Hormones; Guinea Pigs; Liver; Methionine; Methionine Sulfoxide Reductases; NADP; Oxidation-Reduction; Oxidoreductases; Peptide Fragments; Sincalide; Tetragastrin; Thioacetamide; Vasoactive Intestinal Peptide