Resynthesis of insulin from its A and B chains in the presence of denaturants

Reoxidation of the reduced insulin A and B chains in 8 M urea leads to a recovery of native insulin of 2-7% whereas in 6 M guanidine or 0.5 mM dodecylsulfate very little, if any, resynthesis of insulin could be detected. Considering all the possibilities of different oligomeric forms containing one or both of the chains, the yield in 8 M urea is well over the yield as calculated from random joining of the chains and the yield in guanidine or dodecylsulfate is to be expected. It is concluded that some interaction and pairing of the chains occur even in the presence of 8 M urea.     

Interaction and reconstitution of carboxyl-terminal-shortened B chains with the intact A chain of insulin

With the S-(thiomethyl)-A chain and despentapeptide (26-30) and desoctapeptide (23-30) S-(thiomethyl)-B chains of insulin at pH 10.8 and a molar ratio of A/B = 1.5, difference spectra of the mixed against the separated chains with negative peaks at 245 and 295 nm and a weak positive peak at 278 nm indicate interaction of the chains leading to Tyr environmental changes as in the case for the intact chains. With the shortened B chains, freshly dissolved from lyophilized powders, it takes some 2 h for the difference spectra to approach completion whereas with the solutions of the shortened B chains left standing overnight at pH 10.8 and 4 degrees C the difference spectra, similar in shape to that described above, appear almost immediately after mixing. Solvent perturbation with 20% ethylene glycol suggests some ordered structure for the despentapeptide but not for the desoctapeptide B chain. The interactions of the A chain with the shortened B chains appear to be weaker as compared to that with the intact B chain as shown by decreasing reconstitution yields for the intact, despentapeptide, and desoctapeptide B chains respectively with the A chain. The above results indicate that the C-terminal portion of the B chain is important not only for the activity of insulin but also for the correct pairing of the chains.     

The insulin A and B chains contain sufficient structural information to form the native molecule

Refolding studies show that native insulin can be reformed from A and B chains only. This suggests that the A and B chains contain the necessary structural information and that the C peptide is not required for this process.     

Influence of imprinting with A and B chains of insulin on binding and functional changes in tetrahymena

Insulin and its A and B chain increased the quantity of intracellular PAS-positive material (glycogen) in tetrahymena, whereas the combined A+B chains decreased it. Imprinting—previous interaction—with insulin, its A and B chains in themselves and with the A+B chain increased the hormone binding capacity of tetrahymena, but the functional effect of imprinting (storage or breakdown of glycogen) showed a different tendency with insulin and A+B chain on the one hand, and A chain and B chain on the other. Since the imprinting potential of a molecule promotes the induction of receptor formation, the fact remains that both component chains of insulin were able to act as potential imprinters, although the A chain was superior to the B chain in this respect throughout, and combined treatment with the A+B chain ultimately induced the formation of a similar binding site as insulin itself.     

Interaction and combination of separate A and B chains of insulin effects of D-and L-tryptophan in position A1

The S-thiomethyl derivatives of insulin A chain with A1-Gly replaced by D- or L-Trp have been prepared and their respective interaction and combination with the S-thiomethyl B chain studied. The UV difference spectra of the mixed against the separated [Trp1]A chains with the B chain at pH 10.8 are similar to those obtained for the unmodified chains except that the 295-nm-negative peak for ionized Tyr residue appears to be less marked. Fluorescence studies show very little environmental changes at the A1-Trp residues when mixed with the B chain. The intact hormone with A1-Gly replaced by D-Trp is known to be considerably more active than the analog with L-Trp replacement. However, for both derivatives the resynthesis of the whole molecules correctly joined by disulfide bridges starting from the separated reduced chains, gives similar low yields as shown by HPLC analysis and by receptor-binding assay. The replacement of A1-Gly by D-Trp appears to affect the separated A chain more than the intact hormone and replacements at A1 by both D- and L-Trp probably lead to significant conformational changes of the A chain so as to prevent its correct pairing with the B chain.     

Fibrillation of human insulin B-chain by pulsed hydrogen-deuterium exchange mass spectrometry

Insulin forms amyloid fibrils under slightly destabilizing conditions, and B-chain residues are thought to play an important role in insulin fibrillation. Here, pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS), far-UV circular dichroism spectroscopy, thioflavin T (ThioT) fluorescence, turbidity, and soluble fraction measurements were used to monitor the kinetics and mechanisms of fibrillation of human insulin B-chain (INSB) in acidic solution (1 mg/mL, pH 4.5) under stressed conditions (40°C, continuous shaking). Initially, INSB rapidly formed β-sheet-rich oligomers that were protected from HD exchange and showed weak ThioT binding. Subsequent fibril growth and maturation was accompanied by even greater protection from HD exchange and stronger ThioT binding. With peptic digestion of deuterated INSB, HDX-MS suggested early involvement of the N-terminal (1–11, 1–15) and central (12–15, 16–25) fragments in fibril-forming interactions, whereas the C-terminal fragment (25–30) showed limited involvement. The results provide mechanistic understanding of the intermolecular interactions and structural changes during INSB fibrillation under stressed conditions and demonstrate the application of pulsed HDX-MS to probe peptide fibrillation.     

The pairing of the separated A and B chains of insulin and its derivatives, FTIR studies.

From the amide I bands of their deconvolved FTIR spectra, the S-thiomethyl derivatives of the insulin A, B, despentapeptide(26-30) B and desoctapeptide(23-30) B chains all contain significant amounts of ordered secondary structure. The intact B chain is considerably more ordered than either the A or the truncated B chains. Comparison of the spectra of the separated and mixed intact chains of insulin suggests further folding upon mixing of the chains leading to significant increases in ordered secondary structures, presumably because of stabilization by interaction of the chains. The interactions of the A chain with the DPI B chain appear to be weaker as compared to that with the intact B chain. The above results suggest that only the intact A and B chains contain sufficient structural information to recognize each other and interact to form a native-like structure which make the correct formation of the disulfide linkages possible.     

Purification and characterization of a rat liver cytosol neutral thiol peptidase that degrades glucagon, insulin, and isolated insulin A and B chains

A thiol peptidase that catalyzes at near neutral pH the hydrolysis of insulin, the isolated A and B chains of insulin, and glucagon was purified from rat liver cytosol by fractionation on Sephadex G-200, Affi-Gel Blue, and Spherogel TSK-G 3000 SW. The purified enzyme showed a single component by chromatography on a Spherogel TSK column and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 180,000 and consists of two subunits having pI's of 5.9 and 6.3. Studies on its substrate specificity showed that the purified enzyme degrades glucagon, insulin, insulin B chain, and insulin A chain, but it does not degrade proinsulin, ACTH, or denatured hemoglobin. Kinetic analyses were performed on three substrates. The Km values were: 34 nM for insulin, 276 nM for insulin B chain, and 3.5 microM for glucagon. The kcat and Vm/Km values were glucagon greater than B chain greater than insulin. Thus, the enzyme has the highest affinity/lowest efficiency for insulin, an intermediate affinity/intermediate efficiency for B chain of insulin and the lowest affinity/highest efficiency for glucagon. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. The enzyme activity was markedly inhibited by N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and was partially inhibited by dithiothreitol, by the chelating agents EDTA and EGTA, and by phenylmethylsulfonyl fluoride (PMSF). Bacitracin inhibited the activity of the enzyme, but the protease inhibitors aprotinin, leupeptin, pepstatin, and phosphoramidon had little or no effect. Reduced glutathione, iodoacetate, and N alpha,p-tosyl-L-lysine chloromethyl ketone (TLCK) also had little or no effect on the enzyme activity.     

The self-association of zinc-free human insulin and insulin analogue B13-glutamine

The self-association of Zn-free human insulin, Zn-free insulin analogue B13-glutamine, 2-Zn insulin and cobalt(III) human insulin in the millimolar concentration range has been investigated by measuring the osmotic pressure at pH 7.5 in 0.05 M NaCl, 25 degrees C. The pH dependence of association has been measured in the pH range 6.8-9. For all insulins, except Zn-free human insulin, the major association state has been found to be the hexamer. Maximal association of hexamer has been observed for Zn-free human insulin at high concentration (2-7 mM) and physiological pH. At concentrations less than 1 mM and pH greater than 7.0, dissociation to a lower state than the hexamer is found. The conclusion has been drawn that, in the absence of metal ions, human insulin and insulin analogue B13-glutamine associate to the hexamer in the physiological pH range at concentrations in the millimolar range.     

Synthesis of the human insulin gene. Part II. Further improvements in the modified phosphotriester method and the synthesis of seventeen deoxyribooligonucleotide fragments constituting human insulin chains B and mini-CDNA.

The purification of protected deoxyribooligonucleotides containing phosphotriester internucleotidic linkages has been improved by developing a deactivated silica gel chromatographic technique. The efficiency of this technique as applied in the modified phosphotriester approach has been demonstrated in the rapid synthesis of seventeen pure fragments constituting the sequence of human insulin B and mini-C DNA. The sequence of each oligomer was confirmed by the two-dimensional mobility shift method of fingerprinting.     

Synergy between immunotoxins prepared with native ricin A chains and chemically-modified ricin B chains

Ricin B chains treated with chloramine-T in the presence or absence of NaI show a 100-fold to 200-fold reduction in their ability to bind to the galactose-containing protein asialofetuin. Such treated B chains do not form covalently associated homodimers with treated B chains or heterodimers with native ricin A chains. Furthermore, they cannot enhance the toxicity of a ricin A chain-containing rabbit anti-human immunoglobulin (RAHIg-A) for Daudi cells. However, when such B chains are coupled to goat anti-rabbit Ig (GARIg), they potentiate the killing of RAHIg-A-treated Daudi cells only slightly less effectively than GARIg coupled to native B chains. Furthermore, if GARIg-B chain conjugates are treated with chloramine-T after coupling, they fail to bind to asialofetuin but enhance the killing of Daudi cells treated with RAHIg-A. These results demonstrate that the ability of ricin B chains to bind to galactose and to enhance the toxicity of ricin A chains (in the form of an antibody-A chain) can be operationally separated. Thus, the two functions of the B chain may reside on separate domains of the molecule.     

Activation of phosphatidylinositol-3-kinase by insulin is mediated by both A and B human insulin receptor types.

Activation of a phosphatidylinositol-3-kinase (PI-3-kinase) is one of the earliest consequences of insulin binding to the receptor. The human insulin receptor exists in two isoforms which differ in the length of the alpha-subunit (HIR-A = 719 aa, HIR-B = 731 aa). To test whether both isoforms transduce an insulin signal on PI-3-kinase we used rat-1-fibroblasts expressing HIR-A or HIR-B. We found that insulin stimulates 32P incorporation into PIP through both HIR-A and HIR-B to a similar extent (approx. 8-10 fold).     

人胰岛素A,B链基因的合成,克隆及其在大肠杆菌中的表达

Human insulin A and B chain genes were designed and synthesized by using a rapid and simple method. The synthesized A and B chain genes were cloned separately. The expression (plasmids) pWR 590-HIA and pWR 590-HIB were constructed, and the two plasmids can direct the synthesis of the approximately 590 amino acid-long truncated beta-galactosidases fused to human insulin A or B chains. The fused A or B chain proteins were isolated from the fermented cells and cleaved with BrCN. The resulting mixtures were sulfonated and the sulfonated A and B chains were purified. Human insulin was obtained by using an A and B chain combination method.