Primary structure of a potent endogenous dopa-containing inhibitor of phenol oxidase from Musca domestica.
The complete amino acid sequence of a low molecular weight peptide from the hemolymph of the housefly Musca domestica L., which had been determined to competitively inhibit phenol oxidase (PO; monophenol, dihydroxy-phenylalanine:oxygen oxidoreductase; EC 1.14.18.1) in the nM range, was unambiguously established by employing both automatic Edman degradation and mass spectrometry. The physiologically active peptide, which was designated phenol oxidase inhibitor (POI), has an observed molecular weight of 4213.1 +/- 0.2 by electrospray ionization mass spectrometry. The relatively short and structurally dense peptide contained 38 amino acid residues rich in cysteine and lysine. Comparison of the observed and calculated molecular mass indicates that apparently all six cysteine residues form disulfide bridges. Interestingly, sequence analyses of both the intact and protease-digested S-pyridylethylated POI showed that one of the two tyrosine residues (Tyr-32) is hydroxylated to a 3,4-dihydroxyphenylalanine (dopa) residue. This agreed with the increase of 16 mass units observed in mass spectrometric measurements. This was further verified by submission of free L-dopa to the sequencer, which gave a retention time consistent with the atypical peak observed at the Edman cycle of the peptide containing dopa. This study demonstrates the existence of a biologically active, dopa-containing peptide among the insects. Since the POI activity was most prominent in aged pupae, especially pharate adults, the POI may play an important role in smoothing the way of adult emergence through hindering excessive melanization, as well as hardening, of cuticular proteins under the epicuticle.
Daquinag
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AC
Department of Applied Biological Sciences, Saga University, Japan.
Nakamura
S
S
Takao
T
T
Shimonishi
Y
Y
Tsukamoto
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004295:Dihydroxyphenylalanine / Q000032:analysis*; D006458:Hemolymph / Q000737:chemistry; D006793:Houseflies; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D011679:Pupa; D016339:Spectrometry, Mass, Fast Atom Bombardment
D000596:Amino Acids; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010455:Peptides; C092980:phenol oxidase inhibitor, housefly; D004295:Dihydroxyphenylalanine; D014442:Monophenol Monooxygenase