Amino acid sequence of bovine insulin

FIGURE 23-5 Insulin. Mature insulin is formed from its larger precursor preproinsulin by proteolytic processing. Removal of a 23 amino acid segment (the signal sequence) at the amino terminus of preproinsulin and formation of three disulfide bonds produces proinsulin. Further proteolytic cuts remove the C peptide from proinsulin to produce mature insulin, composed of A and B chains. The amino acid sequence of bovine insulin is shown in Figure 3-24. 

Figure 3.22. Amino Acid Sequence of Bovine Insulin.

Figure 2.22 Amino acid sequence of bovine insulin.

FIGURE 3-24 Amino acid sequence of bovine insulin. The two polypeptide chains are joined by disulfide cross-linkages. The A chain is identical in human, pig, dog, rabbit, and sperm whale insulins. The B chains of the cow, pig, dog, goat, and horse are identical. 

Fig. 3. The amino acid sequence of bovine insulin and other mammalian insulins...

The amino acid sequence of bovine insulin (Fig. 24.7) was determined by Sanger in 1953 after 10 years of work. Bovine insulin has a total of 51 amino acid residues in two polypeptide chains, called the A and B chains. These chains are joined by two disulfide linkages. The A chain contains an additional disulfide linkage between cysteine residues at positions 6 and 11. 

With methods for the quantitative analysis of amino acids to hand, the way was now open for the determination of amino acid sequences. Purified bovine insulin was relatively freely available. On the basis of ultracentrifugal analysis (Gutfreund and Ogston), a molecular weight of 12,000 was assumed—as it later emerged, a factor of 2 too high. One of the advantages from the choice of insulin as the protein to sequence was that tryptophan is absent. A 100% recovery of the amino acids could therefore be obtained easily by simple hydrolysis with HC1. In 1948 Tristram reported the complete amino acid composition of the protein. 

Fig. 12. Differences in the amino acid sequence of bovine, porcine and human insulin.

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 ... 

The amino acid sequences of four avian insulins have been determined, namely domestic fowl, turkey, duck and goose. Those of domestic fowl and turkey are identical but differ from that of the duck and goose at three positions (Fig. 7.2). There are also differences in the size of the C-peptide, which is 28 residues in the domestic fowl and 26 in the duck. Another feature that is evident from the proinsulin sequence (Fig. 7.2) is that the C-peptide is less conserved than the A- and B-peptides. tW C-peptide simply acts as a link between the A- and B-peptides to enable the disulphide bridges to form. Domestic fowl insulin is more potent than bovine insulin in ellidting changes in metabolism in the domestic fowl. This has been attributed to the six differences in the amino acid sequences of the two proteins. 

Syntheses of human insulin B-chain S-sulfonate, the destripeptide B of human insulin, and the destripeptide B of bovine insulin were described by Katsoyannis and collaborators. Two advances noted which were not suitable for incorporation in the table concerned the complete amino acid sequence of ribonuclease Ti based on chymotryptic digests, and the photochemical modification of Gly-containing proteins such as collagen lysozyme and ribonuclease by photoalkylation with 1-butene. ... 

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. 

Biosynthetic Human Insulin from E. coli. Insulin [9004-10-8] a polypeptide hormone, stimulates anaboHc reactions for carbohydrates, proteins, and fats thereby producing a lowered blood glucose level. Porcine insulin [12584-58-6] and bovine insulin [11070-73-8] were used to treat diabetes prior to the availabiHty of human insulin [11061 -68-0]. AH three insulins are similar in amino acid sequence. EH LiHy s human insulin was approved for testing in humans in 1980 by the U.S. EDA and was placed on the market by 1982 (11,12). 

Sanger also determined the sequence of the A chain and identified the cysteine residues involved in disulfide bonds between the A and B chains as well as in the disulfide linkage within the A chain. The complete insulin structure is shown in Figure 27.11. The structure shown is that of bovine insulin (from cattle). The A chains of human insulin and bovine insulin differ in only two amino acid residues then B chains are identical except for the amino acid at the C terminus. 

Mature insulin consists of two polypeptide chains connected by two interchain disulfide linkages. The A-chain contains 21 amino acids, whereas the larger B-chain is composed of 30 residues. Insulins from various species conform to this basic structure, while varying slightly in their amino acid sequence. Porcine insulin (5777 Da) varies from the human form (5807 Da) by a single amino acid, whereas bovine insulin (5733 Da) differs by three residues. 

The significance of Sanger s work is immense. It proved for the first time that the structure of a protein is unique that is, aU molecules of bovine insulin, for example, possess the same sequence of amino acids along the polypeptide chains. This sequence has no obvious order, but it is unique. This singular finding requires that there is a genetic code information encoded in a molecule which specifies the sequence of amino acids in the insulin molecule and, for that matter, in all protein molecules. 

The formation of three disulfide bonds in bovine insulin brings parts of the chain that are distant in terms of amino acid sequence into close proximity in the three-dimensional structure of the protein. 

Fig. 1. Amino acid sequence for the A- and B-chains of human insulin [11061-68-0] where solid lines denote disulfide bonds. Porcine insulin [12584-58-6] differs by one amino acid in the B-chain where alanine replaces threonine at position 30. Bovine insulin [11070-73-8] differs by three amino acids. In the A-chain alanine replaces the threonine at position 8 and valine replaces the isoleucine at position 10. In the B-chain there is an alanine at position 30.

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