The biological relevance of HPLC-purified vasoactive intestinal polypeptide monoiodinated at tyrosine 10 or tyrosine 22.
Three major forms of monoiodinated VIP (M125I-VIP) were isolated after chloramine-T iodination and HPLC purification. The iodinated tyrosine residue was located in each form of M125I-VIP using arginase C and trypsin digestion for obtaining defined fragments containing only one tyrosine residue. The HPLC isolated iodinated fragments thus obtained were used for HPLC comigration studies with iodinated synthetic C and N terminal VIP fragments and for amino acid analysis. The first two eluting peaks 1 and 2 are (M125I-Tyr10-VIP); peak 1 has an oxidized methionine; peak 3 is a (M125I-Tyr22-VIP) which also has an oxidized methionine. A reduced counterpart of peak 3 named peak 4 was isolated by further HPLC analysis. The ability of the different species of M125I-VIP to stimulate adenosine cyclic 3',5'-phosphate (cAMP) production in transformed colonic cells in culture (HT-29) was compared to that of native VIP. The mean potencies of the M125I-VIP species expressed as a percentage relative to the potency of native VIP were, peak (1): 0.98; (2): 0.84; (3): 1.38; (4): 1.48, in the range of concentrations tested (2-60 pM). The M125I-Tyr22-VIP are significantly more active than native VIP (P less than 0.01). Oxidation of methionine or iodination of tyrosine 10 does not significantly modify the biological activity of VIP. We conclude that iodination of Tyr-22 located in the apolar helical COOH-terminal of VIP increases the effectiveness of VIP interaction with its receptors. Thus the tyrosyl residue and the localized hydrophobic features of VIP are critically involved in the function of this neurotransmitter.
D000230:Adenocarcinoma; D001119:Arginase; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D010446:Peptide Fragments / Q000032:analysis; D011865:Radioisotope Dilution Technique; D013329:Structure-Activity Relationship; D014357:Trypsin; D014660:Vasoactive Intestinal Peptide / Q000302:isolation & purification / Q000494:pharmacology*
D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP; D014357:Trypsin; D001119:Arginase