Biochemical and Biophysical Research Communications
Antimicrobial peptide defenses of the Tarahumara frog, Rana tarahumarae
Section snippets
Materials and methods
Animals. Rana tarahumarae were collected as an egg mass in Mexico by S. Hale. J. Rorabaugh of the US Fish and Wildlife Service raised the eggs to tadpoles and transferred some to Arizona State University. These animals were raised to maturity at Arizona State University and two frogs were transferred to the Rollins-Smith laboratory for peptide collection.
Collection of skin secretions. A single individual of R. tarahumarae (23.3 g) was injected bilaterally with 2 nmoles/gram body weight of
Purification of the peptides
The elution profile on a Vydac C-18 semipreparative column of the combined skin secretions and washings, after partial purification on Sep-Pak cartridges, is shown in Fig. 1. Analysis by electrospray mass spectrometry demonstrated that the fractions denoted by zone A contained a major component of mass 2898 (subsequently shown to be ranatuerin-2TRa). The fractions denoted by zone B contained a major component of mass 2614 (subsequently shown to be brevinin-1TRa). The fractions denoted by zone C
Discussion
The present study examined the activity of antimicrobial peptides from the Tarahumara frog against the chytrid fungus that has been associated with population declines in Australia, Central America, the Western US, and Europe [5], [6], [7], [27]. Our work illustrates the power of reversed-phase HPLC coupled with electrospray mass spectrometry for the separation and identification of peptide components in a complex mixture. The application of these techniques to analysis of the skin secretions
Acknowledgements
We thank Dr. Donald Babin, Creighton University, for amino acid analyses and Ms. Eva Lovas, Creighton University, for mass spectrometry measurements. Collection of R. tarahumarae eggs was supported by a grant to S. Hale from the US Fish and Wildlife Service. The current work was supported by a grant from Restoragen Inc., Lincoln, Nebraska (to J.M.C.), and by an NSF IRCEB Grant IBN- 9977063 (James Collins, P.I.) and NSF Grant IBN-0131184 (to L.R.-S.).
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