Characterization of naturally occurring peptides in the skin secretion of Rana pipiens frog reveal pipinin-1 as the novel insulin-releasing agent

L Marenah, Peter Flatt, DF Orr, C Shaw, Yasser Abdel-Wahab

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Abstract

Naturally occurring insulinotropic peptides were isolated from the skin secretions of Rana pipiens frogs. Crude secretions (50 mg; 5-10 frogs) obtained by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase high-performance liquid chromatography ( HPLC) yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11 cells, fractions 40-47 (band 1) and fractions 60-65 (band 2) showed significant 1.7-6.7-fold increases in insulin-releasing activity (P <0.001) compared with 5.6 mM glucose alone. Pooled fractions in bands 1 and 2 were rechromatographed yielding a total of seven peaks capable of subsequent 1.2-1.8-fold stimulation of insulin release. Final purification by HPLC to single homogenous peaks revealed one prominent peptide (peak 4.1) with insulin- releasing activity which lacked effects on cell viability. Electrospray mass spectrometric analysis of this peptide indicated molecular mass of 2562.6 Da. Determination of the primary amino acid sequence of this peptide revealed a 24-amino acid sequence: FLPIIAGVAAKVFPKIFCAISKKC. Database search showed a 100% homology to histamine-releasing pipinin-1. In conclusion, this study revealed the skin secretions of Rana pipiens to be a rich source of insulin- releasing peptides. The discovery of insulinotropic activity for pipinin-1, initially characterized as an antimicrobial is interesting and merits further investigation.
LanguageEnglish
Pages204-210
JournalJournal of Peptide Research
Volume66
Issue number4
DOIs
Publication statusPublished - Oct 2005

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Rana pipiens
Anura
Insulin
Skin
Peptides
High Pressure Liquid Chromatography
Glucose
Protein Sequence Analysis
Reverse-Phase Chromatography
Histamine
Electric Stimulation
Amino Acid Sequence
Cell Survival
Databases

Cite this

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title = "Characterization of naturally occurring peptides in the skin secretion of Rana pipiens frog reveal pipinin-1 as the novel insulin-releasing agent",
abstract = "Naturally occurring insulinotropic peptides were isolated from the skin secretions of Rana pipiens frogs. Crude secretions (50 mg; 5-10 frogs) obtained by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase high-performance liquid chromatography ( HPLC) yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11 cells, fractions 40-47 (band 1) and fractions 60-65 (band 2) showed significant 1.7-6.7-fold increases in insulin-releasing activity (P <0.001) compared with 5.6 mM glucose alone. Pooled fractions in bands 1 and 2 were rechromatographed yielding a total of seven peaks capable of subsequent 1.2-1.8-fold stimulation of insulin release. Final purification by HPLC to single homogenous peaks revealed one prominent peptide (peak 4.1) with insulin- releasing activity which lacked effects on cell viability. Electrospray mass spectrometric analysis of this peptide indicated molecular mass of 2562.6 Da. Determination of the primary amino acid sequence of this peptide revealed a 24-amino acid sequence: FLPIIAGVAAKVFPKIFCAISKKC. Database search showed a 100{\%} homology to histamine-releasing pipinin-1. In conclusion, this study revealed the skin secretions of Rana pipiens to be a rich source of insulin- releasing peptides. The discovery of insulinotropic activity for pipinin-1, initially characterized as an antimicrobial is interesting and merits further investigation.",
author = "L Marenah and Peter Flatt and DF Orr and C Shaw and Yasser Abdel-Wahab",
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T1 - Characterization of naturally occurring peptides in the skin secretion of Rana pipiens frog reveal pipinin-1 as the novel insulin-releasing agent

AU - Marenah, L

AU - Flatt, Peter

AU - Orr, DF

AU - Shaw, C

AU - Abdel-Wahab, Yasser

PY - 2005/10

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N2 - Naturally occurring insulinotropic peptides were isolated from the skin secretions of Rana pipiens frogs. Crude secretions (50 mg; 5-10 frogs) obtained by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase high-performance liquid chromatography ( HPLC) yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11 cells, fractions 40-47 (band 1) and fractions 60-65 (band 2) showed significant 1.7-6.7-fold increases in insulin-releasing activity (P <0.001) compared with 5.6 mM glucose alone. Pooled fractions in bands 1 and 2 were rechromatographed yielding a total of seven peaks capable of subsequent 1.2-1.8-fold stimulation of insulin release. Final purification by HPLC to single homogenous peaks revealed one prominent peptide (peak 4.1) with insulin- releasing activity which lacked effects on cell viability. Electrospray mass spectrometric analysis of this peptide indicated molecular mass of 2562.6 Da. Determination of the primary amino acid sequence of this peptide revealed a 24-amino acid sequence: FLPIIAGVAAKVFPKIFCAISKKC. Database search showed a 100% homology to histamine-releasing pipinin-1. In conclusion, this study revealed the skin secretions of Rana pipiens to be a rich source of insulin- releasing peptides. The discovery of insulinotropic activity for pipinin-1, initially characterized as an antimicrobial is interesting and merits further investigation.

AB - Naturally occurring insulinotropic peptides were isolated from the skin secretions of Rana pipiens frogs. Crude secretions (50 mg; 5-10 frogs) obtained by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase high-performance liquid chromatography ( HPLC) yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11 cells, fractions 40-47 (band 1) and fractions 60-65 (band 2) showed significant 1.7-6.7-fold increases in insulin-releasing activity (P <0.001) compared with 5.6 mM glucose alone. Pooled fractions in bands 1 and 2 were rechromatographed yielding a total of seven peaks capable of subsequent 1.2-1.8-fold stimulation of insulin release. Final purification by HPLC to single homogenous peaks revealed one prominent peptide (peak 4.1) with insulin- releasing activity which lacked effects on cell viability. Electrospray mass spectrometric analysis of this peptide indicated molecular mass of 2562.6 Da. Determination of the primary amino acid sequence of this peptide revealed a 24-amino acid sequence: FLPIIAGVAAKVFPKIFCAISKKC. Database search showed a 100% homology to histamine-releasing pipinin-1. In conclusion, this study revealed the skin secretions of Rana pipiens to be a rich source of insulin- releasing peptides. The discovery of insulinotropic activity for pipinin-1, initially characterized as an antimicrobial is interesting and merits further investigation.

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