Porcine proinsulin

Figure 3.11. Top. Single-residue hydrophobicity average) for proinsulin. The T,-based hydrophobicity plot of porcine proinsulin. At each residue position scale is derived in Chapter 5 and utilized in Chapters the hydrophobicity of the residue is given as derived 7 and 8 to understand the hydrophobic associations from the T,-based hydrophobicity scale. Bottom, attending function for selected protein systems. Mean residue hydrophobicity plot (11-residue...

Phe(J)B ]insulm Porcine proinsulin Acetamidino-insulin Bic(Boc) insulin Des-Al-amino-insulin Destripeptide B - bovine insulin 58-Unit Peptides Bovine basic pancreatic trypsin inhibitor... 

We decided to investigate the self-association of human proinsulin, produced by recombinant DNA techniques, using large zone SEC. Due to the homology of human and porcine proinsulins, they would be expected to behave similarly under similar solution conditions. We repeated the experiment with human growth hormone described in the preceding section... 

Progress in the Insulin field has been rapid in these two years, particularly in the axea, of the blos thesis of the hormone. It is now well established that Insulin, which has two peptide chains (A, 21 amino acids and B, 30 amino acids) cross-linked by two disulfide bridges, is synthesized as a single peptide chain, prolnsulin. in which the A and B chains of inmUn are connected by a "connecting peptide" (C-peptide) chain of 33 (porcine) or 30 (bovine) amino acids. Work on prolnsulin has be reviewed, 1, 10 and the amino acid sequences of bovine and porcine proinsulins have been published. Hie amino acid composition of cod proinsulin has a o appeared Two different proinsulins have been demonstrated in the rat, 8 aod proinsulin has been Isolated from human islet cell tumor tissue cultures. The structures of porcine and bovine prolnsulins are as follows ... 

Extracted porcine C-peptide (21), porcine glucagon (24), porcine monocomponent insulin (25), porcine pancreatic polypeptide (36), porcine proinsulin (30), somatostatin (24), vasoactive intestinal polypeptide (19)... 

Several kinetically controlled processes are already used on an industrial scale, such as the conversion of porcine or recombinant proinsulin to human insuhn and the conversion... 

Insulin Preparations. Since diabetes mellitus is a defect of one or more of insulin production, secretion, or action, the administration of insulin replacement as a treatment for diabetes in the 1920s was a landmark discovery. Historically, most commercial insulin came from either bovine or porcine sources. Beef insulin differs from human insulin by three amino acid substitutions pork insulin differs by only one residue. For many years, standard insulin preparations were 70% beef and 30% porcine. However, the biosynthesis of human insulin has now displaced the animal insulins, especially bovine insulin which was more antigenic. Mass production of human insulin by recombinant DNA methods is achieved by inserting the human proinsulin gene into either E. coli or yeast and treating the resulting proinsulin to yield the human insulin molecule. Insulin preparations may be divided into four major types ... 

Fig. 15. The gradient reversed-phase separation of bovine and porcirte insulin with 0.05 M tetramethylammonium phosphate, pH 3, as the mobile phase, and acetonitrile as the organic modifier. The column used was a LiChrosorb RP18 and the flow rate was 1 ml/min. The following peaks were identified 1, hydroxybenzoic acid 2,/7-hydroxymethyl benzoate 3, bovine insulin 4, monodesamido bovine insulin 5, porcine insulin 6, monodesamido porcine insulin 7, proinsulin. Adapted from Fig. 5 of Biemond et al. (1979).

Human insulin (1980) is made either by enzyme modification of porcine insulin, or by using recombinant DNA to synthesise the proinsulin, precursor molecule for insulin. This is done by artificially introducing the DNA into either Escherichia coli or yeast. 

The insulin molecule consists of two chains, the A-chain with 21 amino acids and the B-chain with 30 amino acids (Fig. 12). They are interconnected by two intermolecular disulphide bridges between amino acids A7 and B7 and A20 and B19. A third disulphide bridge connects amino acids 6 and 11 on chain A, giving an intramolecular loop. It is synthesized as a single-chain precursor, preproinsulin, which is converted to proinsulin after the molecule has been translocated to the endoplasmic reticulum. There, the C-peptide, which connects the A- and B-chains, is cut away forming the active insulin (Briggs and Gierasch, 1986 Bailyes etal., 1993). The most often used insulins in therapeutics (Fig. 12), bovine, porcine and human insulin, exhibit differences in their amino acid sequences bovine insulin contains Ala instead of Thr in position 8 and Val instead of lie in position 10 of the A-chain, and both bovine and porcine insulin differ from human insulin by an Ala instead of Thr in position 30 of the B-chain. 

Structures of human proinsulin and insulin. Insulin is derived from proinsulin by cleavage at the dipeptides Arg-Arg and Lys-Arg to give A and B chains held together by disulfide bonds. In the pig, B30 is Ala. In the cow, A8 is Ala, AlO is Val, and B30 is Ala. Bovine and porcine insulins are used extensively in clinical practice. 

Fig. 6.13. The primary structure of human insuhn. The substituted amino acids in bovine (beef) and porcine (pork) insulin are shown in blue. Threonine 30 at the carboxy terminal of the B chain is replaced by alanine in both beef and pork insulin. In beef insulin, threonine 8 on the A chain is also replaced with alanine, and isoleucine 10 with vahne. The cysteine residues, which form the disulfide bonds holding the chains together, are invariant. In the bioengineered insulin Humalog (hspro insulin), the position of proline at B28 and lysine at B29 is switched. Insulin is synthesized as a longer precursor molecule, proinsulin, which is one polpeptide chain. Proinsulin is converted to insulin by proteolytic cleavage of certain peptide bonds (squiggly lines in the figure). The cleavage removes a few amino acids and the 31-amino acid C-peptide that connects the A and B chains. The active insulin molecule, thus, has two nonidentical chains.

Evidence assembled in human islet tumors, in rat islet cells, and in fetal bovine slices has established the existence of a proinsulin, a precursor of insulin. The amino acid sequences of porcine and bovine proinsulin are known NH2B chain Arg-Arg-C peptide Lys-Arg-A chain COOH (see Fig. 8-31). The C peptide forms the connecting unit in human proinsulin and is composed of 31 amino acids. Proinsulin is synthesized in the endoplasmic reticulum, transferred to the cisterna, and from there to the Golgi. Where the proinsulin is converted to insulin is unknown, but it has been suggested that the precursor is broken down into separate chains in the Golgi apparatus. After proteolysis of proinsulin, both proinsulin and the C peptide are stored in the zymogen granule. 

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