Fig 3 - uploaded by Teresa Kubiak
Content may be subject to copyright.
Primary structure of pig proinsulin (Show & Chance, 1968) The sequence of pig insulin is represented by the amino acids in dark circles. Thin unbroken arrows indicate the sequence that is deleted in DproL. Broken arrows show expected S. aureus proteinase cleavage sites in DOPI. 

Primary structure of pig proinsulin (Show & Chance, 1968) The sequence of pig insulin is represented by the amino acids in dark circles. Thin unbroken arrows indicate the sequence that is deleted in DproL. Broken arrows show expected S. aureus proteinase cleavage sites in DOPI. 

Source publication
Article
Full-text available
Incubation of pig desoctapeptide-(B23-30)-insulin with trypsin in solvent systems consisting of dimethyl sulphoxide, butane-1,4-diol and Tris buffer resulted in the formation of an extra peptide bond between Arg-B22 and Gly-A1 in the DOPI molecule. This DOPI derivative can also be regarded as pig des-(23-63)-proinsulin. The structure of the new, pr...

Context in source publication

Context 1
... above results show that the trypsin-catalysed peptide-bond formation between the Arg-B22 and Gly-A l residues took place leading to the conversion of DOPI into the unknown. This new DOPI derivative can also be regarded as a proinsulin analogue, namely Dprol (Fig. ...

Citations

... In addition to this single-chain des-(B30) insulin, there were other peptide bond-linked SCI analogues reported. Single-chain des-(23-63)-proinsulin, which is derived from two-chain pig des-(B23-30)-insulin, and contains an Arg B22 -Gly A1 peptide bond, exhibited only 20% of the IR-binding ability of pig des-(B23-30)-insulin and 0.02% of that of pig insulin (Kubiak and Cowburn 1986). InsΔC, containing a Thr B30 -Gly A1 peptide bond, can form an intact 3D structure almost identical to that of the native hormone in the absence of the Cdomain (Powell et al. 1988). ...
Article
Full-text available
Insulin therapy remains the most effective method to treat diabetes mellitus (DM), and the demand for this valuable hormone has exceeded that of any other protein-based medicine as a result of the dramatic increase in the number of diabetic patients worldwide. Understanding the structure of insulin and the interaction with its receptor is important for developing proper formulations. As a result of the relatively low thermal stability of native insulin and its two-chain analogues, the application of single-chain insulin (SCI) analogues, which can be obtained relatively easily by recombinant DNA technology or chemical synthetic methods, represents a promising alternative approach. In this review, the basic knowledge of insulin (discovery, biosynthesis, and structure) and the current model of the interaction with its receptor are outlined. Furthermore, we outline the strategies for the design and production of various SCI analogues and their reported applications.
... Fortunately, the C-terminus of the B-chain of insulin molecule is one of the most important segments, which is directly involved in the interaction of insulin with its receptor [15] and in the formation of insulin dimers [16]. The C-terminal octapeptide of the B-chain can be cleaved from insulin and attached again to desoctapeptide(B23-B30)insulin (DOI) with the use of trypsin [10,17,18]. This approach offers an extremely large variety of possible modifications that may produce interesting analogs. ...
... During semisynthesis not only the desired product (peak 3 in Figure 1(B)) was formed, but also various side products appeared as well (peaks 4 and 5 in Figure 1(B)), with the products of selfcondensation of DOI as the most frequent species. The coupling of the carboxyl group of ArgB22 either with the N -terminus of the same molecule results in SC-DOI (single-chain DOI) or with the N -terminus of another DOI molecule gives (DOI) 2 (covalent DOI dimer) [17,18,45]. The average yield of our semisyntheses of [TyrB25,N -MePheB26,Lys(Pac)B28,ProB29]insulin was about 11% calculated from the starting amount of DOI, which is a limiting reactant. ...
Article
In this paper, we present the detailed synthetic protocol and characterization of Fmoc-Lys(Pac)-OH, its use for the preparation of octapeptides H-Gly-Phe-Tyr-N-MePhe-Thr-Lys(Pac)-Pro-Thr-OH and H-Gly-Phe-Phe-His-Thr-Pro-Lys(Pac)-Thr-OH by solid-phase synthesis, trypsin-catalyzed condensation of these octapeptides with desoctapeptide(B23-B30)-insulin, and penicillin G acylase catalyzed cleavage of phenylacetyl (Pac) group from Nepsilon-amino group of lysine to give novel insulin analogs [TyrB25, N-MePheB26,LysB28,ProB29]-insulin and [HisB26]-insulin. These new analogs display 4 and 78% binding affinity respectively to insulin receptor in rat adipose membranes.
Article
Full-text available
Previous studies have suggested that the COOH-terminal pentapeptide of the insulin B-chain can play a negative role in ligand-receptor interactions involving insulin analogs having amino acid replacements at position B25 (Nakagawa, S. H., and Tager, H. S. (1986) J. Biol. Chem. 261, 7332-7341). We undertook by the current investigations to identify the molecular site in insulin that induces this negative effect and to explore further the importance of conformational changes that might occur during insulin-receptor interactions. By use of semisynthetic insulin analogs containing amino acid replacements or deletions and of isolated canine hepatocytes, we show here that (a) the markedly decreased affinity of receptor for insulin analogs in which PheB25 is replaced by Ser is apparent for analogs in which up to 3 residues of the insulin B-chain have been deleted, but is progressively reversed in the corresponding des-tetrapeptide and des-pentapeptide analogs, and (b) unlike the case for deletion of TyrB26 and ThrB27, replacement of residue TyrB26 or ThrB27 has no effect to reverse the decreased affinity of full length analogs containing Ser for Phe substitutions at position B25. Additional experiments demonstrated that introduction of a cross-link between Lys epsilon B29 and Gly alpha A1 of insulin decreases the affinity of ligand-receptor interactions whether or not PheB25 is replaced by Ser. We conclude that the negative effect of the COOH-terminal B-chain domain on insulin-receptor interactions arises in greatest part from the insulin mainchain near the site of the TyrB26-ThrB27 peptide bond and that multiple conformational perturbations may be necessary to induce a high-affinity state of receptor-bound insulin.
Chapter
Major aims of insulin chemistry are the large-scale production of the hormone for the treatment of diabetes mellitus as well as laboratory-scale syntheses of analogs for structure-function studies, of radioactive tracers, and of “tailormade” special derivatives. Further, one should include the detection and isolation of new native insulins and related compounds. The total synthesis, accomplished 25 years ago by the groups of ZAHN (MEIENHOFER et al. 1963), KATSOYANNIS (1964), and in China marked the advent of a new era in pep tide and protein chemistry. Remarkable progress has since been achieved through refinement of synthetic and semisynthetic procedures, the advances in recombinant DNA techniques, and high pressure liquid chromatography (HPLC).
Article
A series of dimethyl glutarates bearing basic substituents at C-3, and related monoesters, have been evaluated as substrates of trypsin in order to probe the asymmetric synthetic potential of the enzyme with respect to enantiotopic ester group, and enantiomer, discrimination. While none of the mono- or diesters proved to be a trypsin substrate, several of them accelerated trypsin-catalyzed hydrolysis of the standard reference substrate BAEE, in a manner consistent with an allosteric activation process. The results provide the first examples of allosteric activation of trypsin by modifiers that are sterically precluded from interacting effectively at the acive site.
Article
Full-text available
We have evaluated, by use of isolated canine hepatocytes, the importance of intramolecular hormone cross-linking (and of concomitant changes in molecular flexibility) to the interaction of insulin with its plasma membrane receptor. Cross-linked hormone analogs were prepared by reacting porcine insulin, N alpha A1-t-butyloxycarbonyl insulin or N alpha A1-t-butyloxycarbonyl [D-LysA1]insulin with various dicarboxylic acid active esters to obtain alpha-GlyA1/epsilon-LysB29-, alpha-PheB1/epsilon-LysB29-, and epsilon-D-LysA1/epsilon-LysB29-cross-linked insulins, respectively. In the aggregate, insulin analogs cross-linked by groups containing 2-12 atoms retained 1.4-35% of the receptor binding potency of native insulin. Analysis of our results suggests that: (a) loss of chemical functionality, steric interference, and restriction of potential intramolecular movement can all play roles in determining the receptor binding potencies of cross-linked insulin analogs; (b) restriction of intramolecular movement between residues A1 and B29 affects negatively the binding of insulin to its receptor (but accounts for only a fraction of the conformational change which insulin must undergo to achieve a high affinity state of ligand-receptor interaction); and (c) introduction of a cross-link between residues B1 and B29 (residues that are in fact in proximity in one crystalline form of the hormone) decreases markedly the receptor binding potencies of the corresponding analogs. The importance of these findings is discussed in relation to the potential structure of insulin when it is bound to its plasma membrane receptor.
Article
Full-text available
Unprotected porcine desoctapeptide(B23-30) insulin (DOPI) and the synthetic Gly-Phe-Phe were used as substrates for the trypsin-catalyzed synthesis of despentapeptide(B26-30) insulin (DPPI). The DPPI synthesis was accompanied by a moderate oligomerization and by the formation of a side produce which was identified as a DOPI derivative having an extra peptide bond between the Gly(A1) and Arg(B22) and which was named des(23-63) proinsulin (1). Despite side reactions, the conditions were found where the overall DPPI yields were comparable to those obtained via di-Boc DOPI, and these procedures were faster and simpler since the Boc protection and deprotection steps were omitted. The reaction progress was directly monitored by HPLC.