ArticlesIdentification and Characterization of a Myomodulin-Like Peptide in the Leech
Section snippets
Animals
Adult leeches, Hirudo medicinalis (4–6 g) were obtained from Leeches USA (Westbury, NY). Leeches were maintained in artificial leech pond water [0.5 g of Hirudo salt (Leeches USA) dissolved in 1 liter of dH2O] at 15°C with 12 h/12 h light/dark cycle.
Reagents
Synthetic Aplysia myomodulin-A PMSMLRL-NH2 was purchased from Peninsula Lab (Belmont, CA). Leech myomodulin-like peptide GMGALRL-NH2 was synthesized by Research Genetics (Huntsville, AL). The antisera for RIA and immunocytochemistry were generous
Identification of Myomodulin-Like Immunoreactivity by HPLC
The fractions resulting from the Sep-Pak separations of the ten batches of 100 dissected leech nerve cords were assayed for immunoreactivity and all the active fractions were pooled. After five sequential steps of reverse-phase HPLC (Fig. 1), two fused absorbance (210 nm) peaks were observed with the immunoreactivity peak associated with the first peak (indicated by arrowhead in Fig. 2A). Fraction 63 (indicated by arrowhead in Fig. 2B) was analyzed by automated sequencing and MALDItof mass
Discussion
We have isolated a novel myomodulin-like heptapeptide GMGALRL-NH2 from the leech, Hirudo medicinalis. The majority of the myomodulin family peptides identified so far are from molluscs and their effects appear to be modulation of neuromuscular transmission 6, 12, 26, 28, 29. The results presented here open up the possibility that myomodulin peptides are also involved in neuronal modulation underlying various animal behaviors.
In our final HPLC step, the immunoreactivity was associated with the
Acknowledgements
We thank Drs. J. Strong, S. Rossie, D. Ready and A. Bieber for critical reading of the manuscript. This work was supported by NSF Grant BIR 9512962 and NIMH Grant R01MH44789 (to C.L.S.).
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