Insulin, modification

Advances in polymer synthesis, characterization and conjugation chemistry have stimulated the development of drug macromolecularization technology. In this regard, a variety of studies on insulin modification with polysaccharides, polyvinyls and polyethylene glycols have been carried out. 

Once a gene is cloned it is necessary to convert the information contained in it into a functional protein. There are a number of steps in gene expression (i) transcription of DNA into mRNA (ii) translation of the mRNA into a protein sequence and (iii) in some instances, post-translational modification of the protein. In discussing these steps in more detail, expression of a cloned insulin gene will be used as an example. 

Lifestyle modifications are always in order in patients who have developed or those who are at increased risk of developing NODAT.74 Insulin therapy and oral hypoglycemic agents are used often (Table 52-8) in patients in whom lifestyle modifications alone have not controlled blood glucose levels. See Chapter 40 for appropriate treatment regimens for diabetes. 

Metz B, Kersten GFA, Baart GJ, et al. Identification of formaldehyde-induced modifications in proteins reactions with insulin. Bioconjug. Chem. 2006 17 815-822. 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

Triglyceride and fatty acid synthesis are promoted by insulin stimulation of liver and adipose tissues by causing the phosphorylation of the first and controlling enzyme in the pathway acetyl-CoA carboxylase (see Section 6.3.2). This enzyme catalyses the formation of malonyl-CoA and requires both allosteric activation by citrate and covalent modification for full activity. 

The disposition of insulin was shown to be susceptible to non-absorptive losses to metabolism and mucociliary clearance. Modification of the deposition profile of insulin in the lung showed that higher absorption rates were obtained for more peripheral deposition and co-administration of a metabolic inhibitor reduced losses to exopeptidase metabolism [101], It is acknowledged by the investigators that the IPL technique and the dosing technique of Byron and coworkers are not widely accessible and have therefore not been widely adopted [119], Active absorption has also been studied in this system as described in Sect. 6.2.43. 

M. Hashizume, T. Douen, M. Murakami, A. Yamamoto, K. Takada, S. Muranishi, Improvement of Large Intestinal Absorption of Insulin by Chemical Modification with Palmitic Acid in Rats , J. Pharm. Pharmacol. 1992, 44, 555-559. 

Cysteine disulfide formation is one of the most important posttranslational modifications involved in protein structure. Disulfides play a crucial role in maintaining the structure of many proteins including insulin, keratin, and many other structurally important proteins. While the cytoplasm and nucleus are reducing microenvironments, the Golgi and other organelles can have oxidizing environments and process proteins to contain disulfide bonds (Scheme 5). 

Protein modification Proinsulin is cleaved to form active insulin... 

Prenylation of proteins (a posttranslational modification) that need to be held in the cell membrane by a lipid tail. An example is the p21 G protein in the insulin and growth factor pathways. 

Now we can ask what is likely to happen to the three-dimensional structure of a protein if we make a conservative replacement of one amino acid for another in the primary structnre. A conservative replacement involves, for example, substitution of one nonpolar amino acid for another, or replacement of one charged amino acid for another. Intnitively, one would expect that conservative replacements would have rather little effect on three-dimensional protein structure. If an isoleucine is replaced by a valine or leucine, the structnral modification is modest. The side chains of all of these amino acids are hydrophobic and will be content to sit in the molecnlar interior. This expectation is borne out in practice. We have noted earlier that there are many different molecnles of cytochrome c in nature, all of which serve the same basic function and all of which have similar three-dimensional structnres. We have also noted the species specificity of insulins among mammalian species. Here too we find a number of conservative changes in the primary structure of the hormone. Although there are exceptions, as a general rule conservative changes in the primary structnre of proteins are consistent with maintenance of the three-dimensional structures of proteins and the associated biological functions. 

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