Role of Tyr and Trp in membrane responses of Pyrularia thionin determined by optical and NMR spectra following Tyr iodination and Trp modification.
Pyrularia thionin is a strongly basic and bioactive 47 amino acid peptide which contains two Tyr residues at positions 13 and 45 and one Trp at position 8. Limited iodination does not have a significant effect, but prolonged iodination of the peptide leads to progressive inactivation for all known cellular responses (Evans, J. et al. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 5849-5853). 1H NMR spectra of the native Pyrularia thionin show four Tyr bands, two arising from each Tyr residue. One resonance band for the epsilon hydrogens of Tyr 45 disappears early during limited iodination and the band from the delta hydrogens shifts to low field. The two bands corresponding to Tyr 13 remain during limited iodination, but both decrease in intensity during prolonged iodination, with the epsilon hydrogen band decreasing somewhat more. The resonance bands arising from Trp disappear during prolonged iodination. This sequence of reactions is verified by the optical absorbance properties of two small peptide fragments obtained by Staphylococcal V8 protease hydrolysis of thionin which had been iodinated to varying degrees. Limited iodination did not significantly inhibit the thionin's biological activity, yet the fragment from the -COOH terminus showed the conversion of Tyr 45 to diiodoTyr. This treatment did not significantly modify the Tyr 13 or Trp 8 located in the -NH2 terminal fragment. More extensive iodination resulted in a disappearance of Trp 8 absorbance with an accompanying conversion of Tyr 13 to the monoiodo form. Extensive iodination yielded two atoms of iodine in the Tyr 45-containing fragment, and only one atom in the Tyr 13 fragment. The data indicate that Tyr 45 of the native thionin is more readily iodinated, proceeding to the diiodo form without significant loss of activity. Prolonged iodination does not lead to the formation of any diiodoTyr 13, but does lead to modification of Trp 8 and probably formation of monoiodoTyr 13. Modification of Trp 8 with N-bromosuccinimide inhibits the hemolytic activity of the thionin, showing that Trp 8 is necessary for Pyrularia thionin activity. It is most likely Trp 8 modification during prolonged iodination which results in the loss of biological activity.
Fracki
W S
WS
Department of Chemistry, Brigham Young University, Provo, UT 84602.
Li
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D
Owen
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N
Perry
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Naisbitt
G H
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Vernon
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LP
D023181:Antimicrobial Cationic Peptides; D004912:Erythrocytes / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007455:Iodine / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy; D008566:Membranes / Q000502:physiology; D010940:Plant Proteins / Q000494:pharmacology*; D012697:Serine Endopeptidases; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000032:analysis / Q000502:physiology*; D014443:Tyrosine / Q000032:analysis / Q000502:physiology*
D023181:Antimicrobial Cationic Peptides; D010940:Plant Proteins; C044942:THI1 protein, Pyrularia pubera; D014443:Tyrosine; D014364:Tryptophan; D007455:Iodine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase