Academic literature on the topic 'Insulin chain-B'

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.

In a previous study [Muir, Offord & Davies (1986) Biochem. J. 237, 631-637] the chromatographic and electrophoretic behaviour of a major labelled fragment in the degradation of tritiated insulins by insulin proteinase were used to locate the probable sites of cleavage which had produced this fragment. In order to define these cleavage sites more precisely, authentic markers for the fragments which would be produced by cleavages at, or adjacent to, the most likely sites have now been synthesized. These markers were compared with labelled fragments of the A- and B-chains of insulin produced by insulin proteinase. The results, together with those of our previous study, show that in order to produce the observed major labelled fragment, the enzyme must have cleaved the insulin A-chain between leucine-A13 and tyrosine-A14 and the insulin B-chain between serine-B9 and histidine-B10. In addition, a minor component was observed in the labelled B-chain fragment which corresponded to a cleavage either between histidine-B10 and leucine-B11 or between leucine-B11 and valine-B12.

In this study, we investigated the folding pathway of insulin precursor and compared it with that of insulin-like growth factor I (IGF-I). The intra-A chain disulphide bond was found to form early in insulin precursor folding, whereas the corresponding disulphide bond in IGF-I formed late. Intra-A chain disulphide-bond deleted [A6, A11-Ser] proteins, including proinsulin, insulin, and A chain, were employed for this investigation. Under the same conditions the recombination yield of insulin from S-sulphonates of native A and B chains was 22%, while the yield of [A6, A11-Ser] insulin from S-sulphonates of [A6, A11-Ser] A chain and native B chains was only approx. 7%. This indicated that the intra-A chain disulphide bond may serve to stabilize the A chain folding intermediate so as to facilitate the correct recognition and pairing with the B chain. Time courses of oxidation of reduced insulin A chains, reduced A and B chains, and reduced proinsulins showed that the intra-A chain disulphide bond formed first during insulin precursor folding. The formation of intra-A chain disulphide bond further accelerated the formation of the other two inter-chain disulphide bonds. The time course of helix structure formation of insulin A chains also indicated that the intra-A chain disulphide bond formed first, and could stabilize partially folded A chain helix structure. The rate of intra-A chain disulphide bond formation was almost the same as that for both helix structure formation and insulin molecule formation, indicating that the formation of the intra-A chain disulphide bond was the rate limiting step for the folding of insulin precursor.

The B chain of mammalian insulins contains appropriately spaced amino acids that predict recognition by T cells. However, all T cell clones from an HLA-DR1, Dw6 diabetic donor recognize epitopes associated with the A chain, and the B chain was found to inhibit these responses. Effective intramolecular competition at the level of the APC, not a direct effect on the T cell, is responsible for the inhibition. Insulin B chain contains two clusters of amino acid homology with the TCR beta chain and B chain peptides lacking these clusters do not compete for antigen presentation. A hole in the repertoire for T cells that recognize this portion of the insulin molecule may arise in the thymus by deletion of T cells that recognize similar peptides.

Glargine became the first long-acting insulin analogue. Glargine was designed to meet basal insulin requirements throughout the day with a single injection. Pharmacokinetics of insulin glargine is characterized by biotransformation into metabolites M1 and M2 that transforms the B chain of glargine so it is similar to the B chain of human insulin. Plasma concentrations of active M1 and M2 metabolites have no pronounced peaks during the day, resulting in lower glucose variability and hypoglycaemia risk when compared with NPH insulin. The metabolic activities of M1 and M2 metabolites are similar to the effect of glargine, whereas the mitogenic effects of these metabolites do not exceed the effect of human insulin. Insulin glargine shows a higher affinity for the insulin-like growth factor-1 (IGF-1) receptor when compared with human insulin. Glargine has no proliferative effect in vivo owing to its rapid conversion into metabolites. Pharmacokinetic and pharmacodynamic variability of glargine is comparable to other insulins. These characteristics are important for the clinical efficacy and safety of glargine.

Insulin and insulin-like growth factor 1 (IGF-1) are closely related hormones involved in the regulation of metabolism and growth. They elicit their functions through activation of tyrosine kinase–type receptors: insulin receptors (IR-A and IR-B) and IGF-1 receptor (IGF-1R). Despite similarity in primary and three-dimensional structures, insulin and IGF-1 bind the noncognate receptor with substantially reduced affinity. We prepared [d-HisB24, GlyB31, TyrB32]-insulin, which binds all three receptors with high affinity (251 or 338% binding affinity to IR-A respectively to IR-B relative to insulin and 12.4% binding affinity to IGF-1R relative to IGF-1). We prepared other modified insulins with the aim of explaining the versatility of [d-HisB24, GlyB31, TyrB32]-insulin. Through structural, activity, and kinetic studies of these insulin analogs, we concluded that the ability of [d-HisB24, GlyB31, TyrB32]-insulin to stimulate all three receptors is provided by structural changes caused by a reversed chirality at the B24 combined with the extension of the C terminus of the B chain by two extra residues. We assume that the structural changes allow the directing of the B chain C terminus to some extra interactions with the receptors. These unusual interactions lead to a decrease of dissociation rate from the IR and conversely enable easier association with IGF-1R. All of the structural changes were made at the hormones' Site 1, which is thought to interact with the Site 1 of the receptors. The results of the study suggest that merely modifications of Site 1 of the hormone are sufficient to change the receptor specificity of insulin.

The use of electromagnetic devices such as microwave ovens and mobile phones has certainly brought convenience to our lives. At the same time, the proliferation of said devices has increased public awareness of the potential health hazards. It is generally assumed that there is little or no risk associated with the use of electromagnetic devices, based on the small amount of power associated with those devices. However, case studies on animals indicate that the risk cannot be entirely ruled out. It has long been known that proteins are sensitive to stress, arising from various sources such as temperature, chemical, pressure, and changes in pH condition. In all of these cases, the protein exhibits clear signs of damage and distress, which range from slight unfolding to complete loss of structure. Frequently, the damage to the protein is alleviated by refolding, either by itself or by the aid of molecular chaperones. However, if the damage to the protein is too great, the protein will generally undergo proteolysis. Opinion has been divided over the implication of prolonged use of electromagnetic devices to human health. Studies conducted on animals so far have given conflicting results. The studies on the separate components, electric and magnetic fields, also give inconclusive results. This indicates that our understanding on how electric and magnetic fields interact with biological matter is incomplete. In this project, we use molecular dynamics to explore the behaviour of two forms of insulin chain-B, isolated and monomeric (in the presence of chain-A with all disulfide bonds intact), at ambient conditions and under the influence of various stress. Specifically, we focus our attention to thermal stress and electric field stress. The electric field stress considered in this study takes several forms: static and oscillating with three different frequencies. These fields have strength ranging from 1806 V/m to 109 V/m. By performing molecular dynamics simulations totalling over 500 ns, we have gained valuable insights into the effects of elevated temperature and electric field on insulin chain-B. We observed differences in the damage mechanisms by the application of static electric field and oscillating field. The application of static fields restricts the conformational freedom of a protein, whereas the application of oscillating fields increases the mobility and flexibility of the protein, similar to the effect of thermal stress. Both of these interfere with the normal behaviour of a protein. We have also observed frequency-dependent effects, with low frequency fields having static field-like characteristics in damage mechanism.

In Chapter I is a general description of novel metal complexes which hydrolytically cleave peptides, proteins, DNA, and other biological molecules. These reagents are becoming the more important as potential therapeutic agents. A panel of ligands was investigated for coordination to ZrIV and other metals in groups 4, 5, and 6 to effect the greatest degree of hydrolysis. Chapter II describes a ZrIV complex which is capable of hydrolyzing a 30 amino acid peptide, insulin chain B, with amino acid specificity. Oxidized insulin chain B peptide was hydrolyzed after only 4 h of treatment at pH 7.0 and 60 °C using ZrCl4 in the presence of 4,13-diaza-18-crown-6. MALDI-TOF and ESI LC-MS mass spectra indicated that insulin chain B was hydrolyzed by ZrIV at the Gly8-Ser9, Ser9-His10, and Gly20-Glu21 amide bonds within the oligopeptide. To our surprise, the cysteine sulfonic acid sequences Cys(SO3H)7-Gly8 and Cys(SO3H)19-Gly20 were also cleaved. To the best of our knowledge, this constitutes the first example of metal-assisted hydrolysis of a Cys(SO3H)-Xaa amide bond. This is significant in light of the fact that cysteine sulfonic acid formation in proteins is triggered by oxidative stress and has been associated with amyloid fibril formation, Parkinson’s disease, and other deleterious, physiological processes. Chapter III describes the metal-assisted hydrolysis of sphingomyelin which is a principle phospholipid component of animal cell membranes. The sphingomyelin assays showed evidence of metal-assisted hydrolysis after 20 h of treatment at lysosomal pH 4.8 and cytosolic pH 7.0 at both physiological temperature 37 °C and 60 °C. The metal ion CeIV was the most reactive, followed by ZrIV, and then HfIV. The goal of this work is to develop metal-based reagents to reverse the lethal build-up of sphingomyelin that occurs in lysosomes of patients suffering from Niemann-Pick disease.

La cartographie peptidique est une méthode qui permet entre autre d’identifier les modifications post-traductionnelles des protéines. Elle comprend trois étapes : 1) la protéolyse enzymatique, 2) la séparation par électrophorèse capillaire (CE) ou chromatographie en phase liquide à haute performance (HPLC) des fragments peptidiques et 3) l’identification de ces derniers. Cette dernière étape peut se faire par des méthodes photométriques ou par spectrométrie de masse (MS). Au cours de la dernière décennie, les enzymes protéolytiques immobilisées ont acquis une grande popularité parce qu’elles peuvent être réutilisées et permettent une digestion rapide des protéines due à un rapport élevé d’enzyme/substrat. Pour étudier les nouvelles techniques d’immobilisation qui ont été développées dans le laboratoire du Professeur Waldron, la cartographie peptidique par CE est souvent utilisée pour déterminer le nombre total de peptides détectés et leurs abondances. La CE nous permet d’avoir des séparations très efficaces et lorsque couplée à la fluorescence induite par laser (LIF), elle donne des limites de détection qui sont 1000 fois plus basses que celles obtenues avec l’absorbance UV-Vis. Dans la méthode typique, les peptides venant de l’étape 1) sont marqués avec un fluorophore avant l’analyse par CE-LIF. Bien que la sensibilité de détection LIF puisse approcher 10-12 M pour un fluorophore, la réaction de marquage nécessite un analyte dont la concentration est d’au moins 10-7 M, ce qui représente son principal désavantage. Donc, il n’est pas facile d’étudier les enzymes des peptides dérivés après la protéolyse en utilisant la technique CE-LIF si la concentration du substrat protéique initial est inférieure à 10-7 M. Ceci est attribué à la dilution supplémentaire lors de la protéolyse. Alors, afin d’utiliser le CE-LIF pour évaluer l’efficacité de la digestion par enzyme immobilisée à faible concentration de substrat,nous proposons d’utiliser des substrats protéiques marqués de fluorophores pouvant être purifiés et dilués. Trois méthodes de marquage fluorescent de protéine sont décrites dans ce mémoire pour étudier les enzymes solubles et immobilisées. Les fluorophores étudiés pour le marquage de protéine standard incluent le naphtalène-2,3-dicarboxaldéhyde (NDA), la fluorescéine-5-isothiocyanate (FITC) et l’ester de 6-carboxyfluorescéine N-succinimidyl (FAMSE). Le FAMSE est un excellent réactif puisqu’il se conjugue rapidement avec les amines primaires des peptides. Aussi, le substrat marqué est stable dans le temps. Les protéines étudiées étaient l’-lactalbumine (LACT), l’anhydrase carbonique (CA) et l’insuline chaîne B (INB). Les protéines sont digérées à l’aide de la trypsine (T), la chymotrypsine (CT) ou la pepsine (PEP) dans leurs formes solubles ou insolubles. La forme soluble est plus active que celle immobilisée. Cela nous a permis de vérifier que les protéines marquées sont encore reconnues par chaque enzyme. Nous avons comparé les digestions des protéines par différentes enzymes telles la chymotrypsine libre (i.e., soluble), la chymotrypsine immobilisée (i.e., insoluble) par réticulation avec le glutaraldéhyde (GACT) et la chymotrypsine immobilisée sur billes d’agarose en gel (GELCT). Cette dernière était disponible sur le marché. Selon la chymotrypsine utilisée, nos études ont démontré que les cartes peptidiques avaient des différences significatives selon le nombre de pics et leurs intensités correspondantes. De plus, ces études nous ont permis de constater que les digestions effectuées avec l’enzyme immobilisée avaient une bonne reproductibilité. Plusieurs paramètres quantitatifs ont été étudiés afin d’évaluer l’efficacité des méthodes développées. La limite de détection par CE-LIF obtenue était de 3,010-10 M (S/N = 2,7) pour la CA-FAM digérée par GACT et de 2,010-10 M (S/N = 4,3) pour la CA-FAM digérée par la chymotrypsine libre. Nos études ont aussi démontrées que la courbe d’étalonnage était linéaire dans la région de travail (1,0×10-9-1,0×10-6 M) avec un coefficient de corrélation (R2) de 0,9991.
Peptide mapping is a routine method for identifying post-translational modifications of proteins. It involves three steps: 1) enzymatic proteolysis, 2) separation of the peptide fragments by capillary electrophoresis (CE) or high performance liquid chromatography (HPLC), 3) identification of the peptide fragments by photometric methods or mass spectrometry (MS). During the past decade, immobilized enzymes for proteolysis have been gaining in popularity because they can be reused and they provide fast protein digestion due to the high ratio of enzyme-to-substrate. In order to study new immobilization techniques developed in the Waldron laboratory, peptide mapping by CE is frequently used, where the total number of peptides detected and their abundance are related to enzymatic activity. CE allows very high resolution separations and, when coupled to laser-induced fluorescence (LIF), provides excellent detection limits that are 1000 times lower than with UV-Vis absorbance. In the typical method, the peptides produced in step 1) above are derivatized with a fluorophore before separation by CE-LIF. Although the detection sensitivity of LIF can approach 10 12 M for a highly efficient fluorophore, a major disadvantage is that the derivatization reaction requires analyte concentrations to be approx. 10 7 M or higher. Therefore, it is not feasible to study enzymes using CE-LIF of the peptides derivatized after proteolysis if the initial protein substrate concentration is <10-7 M because additional dilution occurs during proteolysis. Instead, to take advantage of CE-LIF to evaluate the efficiency of immobilized enzyme digestion of low concentrations of substrate, we propose using fluorescently derivatized protein substrates that can be purified then diluted. Three methods for conjugating fluorophore to protein were investigated in this work as a means to study both soluble and immobilized enzymes. The fluorophores studied for derivatization of protein standards included naphthalene-2,3-dicarboxaldehyde (NDA), fluoresceine-5-isothiocyanate (FITC) and 6-carboxyfluorescein N-succinimide ester (FAMSE). The FAMSE was found to be an excellent reagent that conjugates quickly with primary amines and the derivatized substrate was stable over time. The studied substrates were -lactalbumin (LACT), carbonic anhydrase (CA) and insulin chain-B (INB). The CE-LIF peptide maps were generated from digestion of the fluorescently derivatized substrates by trypsin (T), chymotrypsin (CT) or pepsin (PEP), either in soluble or insoluble forms. The soluble form of an enzyme is more active than the immobilized form and this allowed us to verify that the conjugated proteins were still recognized as substrates by each enzyme. The digestion of the derivatized substrates with different types of chymotrypsin (CT) was compared: free (i.e., soluble) chymotrypsin, chymotrypsin cross-linked with glutaraldehyde (GACT) and chymotrypsin immobilized on agarose gel particles (GELCT), which was available commercially. The study showed that, according to the chymotrypsin used, the peptide map would vary in the number of peaks and their intensities. It also showed that the digestion by immobilized enzymes was quite reproducible. Several quantitative parameters were studied to evaluate the efficacy of the methods. The detection limit of the overall method (CE-LIF peptide mapping of FAM-derivatized protein digested by chymotrypsin) was 3.010-10 M (S/N = 2.7) carbonic anhydrase using insoluble GACT and 2.010-10 M (S/N = 4.3) CA using free chymotrypsin. Our studies also showed that the standard curve was linear in the working region (1.0×10-9-1.0×10-6 M) with a correlation coefficient (R2) of 0.9991.