The Structure of Bovine Proinsulin

• Published in: The Journal of biological chemistry 1971-05, Vol.246 (9), p.2780-2795
• Main Authors: Nolan, C, Margoliash, E, Peterson, J D, Steiner, D F
• Format: Article
• Language: English
• Subjects: Alanine; Amino Acid Sequence; Amino Acids - analysis; Animals; Carboxypeptidases; Cattle; Chemical Phenomena; Chemistry; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, Ion Exchange; Chymotrypsin; Cysteine; Electrophoresis; Insulin - biosynthesis; Islets of Langerhans - cytology; Islets of Langerhans - metabolism; Pepsin A; Peptides; Polymers; Species Specificity; Sulfides - analysis; Swine; Trypsin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)62252-5

Several insulin-related substances, comprising 1 to 2% of the total protein, were isolated from commercial crystalline bovine insulin. The main components of this mixture were: the 81-residue, single chain proinsulin, two degradation products of proinsulin, termed Intermediate Forms I and II, and an insulin dimer, possibly linked by covalent bonds and apparently an artifact of the preparation. Treatment of the biologically inactive proinsulin or intermediate forms with trypsin yielded dealanylinsulin, which is fully biologically active. The amino acid sequence of the intact proinsulin was shown to be bovine insulin B chain-Arg-Arg-Glu-Val-Glu-Gly-Pro-Gln-Val-Gly-Ala-Leu-Glu-Leu-Ala-Gly-Gly-Pro-Gly-Ala-Gly-Gly-Leu-Glu-Gly-Pro-Pro-Gln-Lys-Arg-bovine insulin A chain. The only disulfide bonds in this protein are those in the insulin moiety. Intermediate Form I is a two-chain protein consisting of the insulin B chain extended to the glutaminyl residue preceding the Lys-Arg terminal sequence of the connecting peptide segment, and the A chain, these two chains being held together by the disulfide bonds found in insulin. Intermediate Form II is a similar protein, consisting of the insulin B chain and an A chain extended to the glutamyl residue following the Arg-Arg sequence at the amino terminus of the connecting segment. Proinsulin is the biosynthetic precursor of insulin and functions to facilitate the folding of the molecule to yield the correct pairing of cysteinyl residues required to form the disulfide bonds of insulin. The transformation of proinsulin to insulin occurs intracellularly in the β cells of the islets of Langerhans and insulin is the major storage form. The structures of the intermediate forms, the recovery from pancreas of connecting peptide lacking the amino- and carboxyl-terminal basic dipeptide sequences in molar quantities equal to those of insulin, and the variability of the residue at the carboxyl terminus of the B chain of the insulins of various species, all indicate that the proteolytic enzyme or enzymes responsible for the proinsulin to insulin transformation probably act by virtue of trypsin-like and carboxypeptidase B-like specificities.