Purification and characterization of insulin from the Australian lungfish,Neoceratodus forsteri (Dipnoi)

The Australian lungfish Neoceratodus forsteri, a facultative air breather, is considered to be the most primitive of the extant Dipnoi and so occupies a uniquely important evolutionary position in the transition from fish to tetrapods. Insulin was isolated from an extract of the pancreas of N. forst eri and its primary structure established as: A-Chain, Gly-Ile-Val-Glu-Gln- Cys-Cys-His-Thr-Pro(10)-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys(20)-Asn-Glu -Thr-Glu; B-Chain, Ala-Ala-Val-Asn-Gln-His-Leu-Cys-Gly-Ser(10)-His-Leu-Val- Glu-Ala-Leu-Tyr-Phe-Val-Cys(20)-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Leu-Pro-Lys(30) -Gly. This amino acid sequence is more similar to that of human insulin tha n to insulins from present-day amphibians. All the residues in human insuli n that are considered to be important in receptor binding, dimerization, an d hexamerization are conserved in lungfish insulin except for the substitut ion (Leu --> Phe) at the position corresponding to B17 in human insulin. Co nsistent with the assertion that the Dipnoi is a monophyletic group, insuli ns from N. forsteri and from the African lungfish Protopterus annectens con tain extensions to the C-terminus of the A-chain and to the N-terminus of t he B-chain that have not been found in other sarcopterygian species. Howeve r, the unusual amino acid substitutions found in insulin from P. annectens (e.g., GlyB21 --> Ala, GluB22 --> Asp, and ArgB23 --> Asn) are not present in N. forsteri insulin, suggesting that they occurred in the Protopterus li neage after divergence of the genera, (C) 1999 Academic Press.