Two insulins from channel catfish: purification, structures, receptor-binding and cDNA sequences

Authors: Mommsen T.P.^{1, 2}; Silverstein J.T.^{3, 4}; Plisetskaya E.M.⁵; Whittaker L.J.⁶; Whittaker J.⁶; Conlon J.M.⁷

Source: Fish Physiology and Biochemistry, Volume 25, Number 1, 2001 , pp. 61-71(11)

Publisher: Springer

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Abstract:

Two insulins were isolated from an extract of the Brockmann bodies of channel catfish (Ictalurus punctatus). The primary structure of insulin I is: A-Chain: GIVELCCHKP¹⁰ CSLHDLQNYC²⁰ N; B-Chain: GAPQHLCGSH¹⁰ LVDALYLVCG²⁰ PNGFFYNPK. Insulin II has three amino-acid substitutions compared with insulin I: A14His to Gln, B1Gly to Val, and B13Asp to Glu. Despite some unusual amino acid substitutions in the catfish insulins compared with human insulin, such as A5Gln to Leu, B21Glu to Pro and B22Arg to Asn, those residues believed to constitute the receptor-binding domain are conserved. Consistent with this, catfish insulins I and II were equipotent in inhibiting the binding of [3-[¹²⁵I] iodotyrosine-A14] human insulin to the soluble human insulin receptor and were only 3-fold less potent than human insulin in the same assays. An analysis of mRNA expression in Brockmann bodies by reverse-transcriptase PCR identified two proinsulin sequences for the channel catfish containing a single, highly conserved C-peptide whose deduced amino acid sequence is REVDPLLGFL¹⁰ PPKSAPEGEL²⁰ AEYPYKEYSE³⁰ LMVKR. PCR of genomic DNA with specific proinsulin primers spanning the intron II interrupting the C-peptide of all vertebrate insulins, produced two introns of 105 and 104 bp, respectively. The nucleotide sequences of the introns differ in 13 positions. Because of the high degree of conservation in insulin and C-peptide and the large variability in the small intron, we conclude that the two insulins isolated are the products of different genes and do not simply represent different alleles. The channel catfish is a diploid species that may have undergone gene or chromosome duplication and therefore we propose that multiple insulin genes may not be restricted to polyploid species such as salmonids or sturgeons.

Keywords: channel catfish; insulin; primary structure; receptor-binding; cDNA; Brockmann body; alleles

Language: English

Document Type: Research article

Affiliations: 1: Department of Microbiology and Biochemistry, PO Box 3055, University of Victoria, Victoria, B.C. Canada V8W 3P6 (Phone: 1-250-721-6508; Fax: 1-250-721-8855 2: E-mail: tpmom@uvic.ca) 3: USDA/ARS Catfish Genetics Research Unit, Thad Cochran National Warmwater Aquaculture Center, PO Box 38, Stoneville, MS, 38776, USA 4: Present address: USDA/ARS, National Center for Cool and Cold Water Aquaculture, 11876 Leetown Road, Kearneysville, WV 25430, USA 5: School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195-5100, USA 6: Hagedorn Research Institute, 2820 Gentofte, Denmark 7: Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University Medical School, Omaha NE68178-0405