Amino Acid Sequence Of Insulin Research Articles

PH-Driven Polymorphism of Insulin Amyloid-Like Fibrils.

Prions are infective proteins, which can self-assemble into different strain conformations, leading to different disease phenotypes. An increasing number of studies suggest that prion-like self-propagation may be a common feature of amyloid-like structures. Thus it is important to unravel every possible factor leading to the formation of different amyloid strains. Here we report on the formation of two types of insulin amyloid-like fibrils with distinct infrared spectroscopic features grown under slightly different pH conditions. Similar to prion strains, both insulin fibril types are able to self-propagate their conformational template under conditions, favoring spontaneous formation of different type fibrils. The low-pH-induced insulin amyloid strain is structurally very similar to previously reported strains formed either in the presence of 20% ethanol, or by modification of the amino acid sequence of insulin. A deeper analysis of literature data in the context of our current findings suggests a shift of the monomer-dimer equilibrium of insulin as a possible factor controlling the formation of different strains.

An Isothermal Titration and Differential Scanning Calorimetry Study of the G-Quadruplex DNA–Insulin Interaction

The binding of insulin to the G-quadruplexes formed by the consensus sequence of the insulin-linked polymorphic region (ILPR) was investigated with differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The thermal denaturation temperature of insulin was increased by almost 4 °C upon binding to ILPR G-quadruplex DNA as determined by DSC. The thermodynamic parameters (K(D), ΔH, ΔG, and ΔS) of the insulin-G-quadruplex complex were further investigated by temperature-dependent ITC measurement over the range of 10-37 °C. The binding of insulin to the ILPR consensus sequence displays micromolar affinity in phosphate buffer at pH 7.4, which is mainly driven by entropic factors below 25 °C but by enthalpic terms above 30 °C. The interaction was also examined in several different buffers, and results showed that the observed ΔH is dependent on the ionization enthalpy of the buffer used. This indicates proton release upon the binding of G-quadruplex DNA to insulin. Additionally, the large negative change in heat capacity for this interaction may be associated with the dominant hydrophobicity of the amino acid sequence of insulin's β subunit, which is known to bind to the ILPR G-quadruplex DNA.

Insulin: moments in history

AbstractThe isolation of insulin by Banting and Best in 1922 was a historic event of life‐saving importance. Thousands of individuals doomed to die of type I diabetes were saved. The inspiring story of insulin continued to grow and evolve with the development of PZI, NPH, and lente insulins. The advent of production of human insulin from recombinant DNA brought an end to the extraction of insulin from animal pancreas. Modification of the amino acid sequences of insulin led to new forms of fast‐acting insulin, such as lispro, aspart, and glulisine, and to long‐acting insulins like glargine and detemir. The technology of insulin pumps advanced from large, cumbersome equipment to present‐day devices no bigger than a hand calculator. The accuracy of implantable glucose sensors improved progressively, culminating in the approval of devices that will soon exert reliable feedback control of insulin pumps. Transplantation of the pancreas offers a cure for type I diabetes, and the development of islet cell transplantation continues. It is now appreciated that islet cells regenerate in the normal pancreas, and islet cell neogenesis agents have been identified and may eventually contribute to the reversal of diabetes. Drug Dev Res 69:95–100, 2008. ©2008 Wiley‐Liss, Inc.

Islet Hormones from the African Bullfrog Pyxicephalus adspersus (Anura:Ranidae): Structural Characterization and Phylogenetic Implications

The African bullfrog Pyxicephalus adspersus is generally classified along with frogs of the genus Rana in the subfamily Raninae of the family Ranidae but precise phylogenetic relationships between species are unclear. Pancreatic polypeptide (PP), insulin, and glucagon-like peptide (GLP-1) were isolated from an extract of P. adspersus pancreas and characterized structurally. A comparison of the amino acid sequence of Pyxicephalus PP (APSEPQHPGG10QATPEQLAQY20YSDLYQYITF30ITRPRF · NH2) with those of the known amphibian PP molecules in a maximum parsimony analysis generates a single phylogenetic tree in which Pyxicephalus is the sister to the clade comprising the members of the genus Rana. The three orders of living amphibians form discrete clades with the representative of the Gymnophiona appearing as sister to the Caudata-Anura. In contrast, Pyxicephalus insulin (A chain, GIVEQCCHSA10CSLYDLENYC20N; B-chain, LANQHLCGSH10LVEALYMVCG20ERGFFYYPKS30) and and GLP-1 (HAEGTFTSDM10TSYLEEKAAK20EFVDWLIKGR30PK) resemble more closely the corresponding peptides from the cane toad Bufo marinus than the peptides from any species of Rana. Cladistic analysis based upon the amino acid sequences of insulin produced a polyphyletic assemblage with the Gymnophiona nesting within an unresolved clade containing the non-ranid frogs. The data support the assertion that the amino acid sequence of PP, but not those of the other islet hormones, is of value as a molecular marker for inferring phylogenetic relationships between early tetrapod species.

Molecular Evolution of Insulin in Non-Mammalian Vertebrates1

The traditional view, based primarily on X-ray crystallographic data, is that the amino acid residues at positions B12, B16, B23-B26, A1-A5, A19 and A21 in the insulin molecule comprise the receptor-binding domain. More recently, however, it has been proposed that the conformation adopted by insulin in the crystal structure is an inactive one. The results of alanine-scanning mutagenesis studies suggest that GlyB23, PheB24, IleA2, ValA3, and TyrA19 interact directly with the receptor with LeuB6, GlyB8, LeuB11, GluB13 and PheB25, although not part of the binding epitope, being important in maintaining the receptor-binding conformation. A comparison of the primary structures of insulins from a wide range of non-mammalian vertebrates, from hagfish to birds, provides support for this revised view by demonstrating that strong evolutionary pressure has acted to conserve those amino acids postulated to be important in the biologically active conformation. In addition to the cysteine residues, the amino acids at B6, B8, B11, B23, B24, A2, A3, and A19 are invariant in all species yet studied with only conservative substitutions (Glu → Asp) at B13 and (Phe → Tyr) at B25. In contrast, several insulins containing substitutions at positions B16, A5 and A21, sites of importance in maintaining the crystal structure conformation, have been identified. Although the amino acid sequences of insulin are not generally useful as molecular markers for inferring phylogenetic relationships between species, the presence of common structural features in insulins from closely related species may permit a valid inference. For example, the presence of an N-terminal pentapeptide extension to the B-chains of insulins isolated from both holarctic and southern hemisphere lampreys supports the monophyletic status of the Petromyzontiformes.

Gastric motor and cardiovascular effects of insulin in dorsal vagal complex of the rat.

Insulin-binding sites exist in the lower brain stem of the rat, raising the possibility that the circulating hormone may have cardiovascular and gastric effects at this site. Therefore, we investigated the autonomic effects of applying rat insulin to the surface of the dorsal medulla (0.3 and 3 microU/rat) or microinjecting it into the dorsal vagal complex (DVC) (0.1-10 nU/site) in anesthetized rats. Application of rat insulin to the surface (3 microU/rat) and its microinjection into the DVC (1 and 10 nU/site) both evoked marked, albeit transient, increases in intragastric pressure, pyloric and greater curvature contractile activity, and blood pressure. Much higher doses of human (100 mU) and porcine insulin (3 mU) were needed to evoke modest changes in gastric motor and cardiovascular function when applied to the surface of the dorsal medulla. In addition, a 1,000-fold higher dose of porcine insulin (10 microU) in the DVC was not enough to mimic the autonomic effects of rat insulin microinjected into the same site. The excitatory gastric motor effects of rat insulin in the lower brain stem were abolished by vagotomy, whereas spinal cord transection blocked insulin-evoked increases in blood pressure. To test whether the gastric motor effects of rat insulin in the lower brain stem were caused by potential contamination with pancreatic polypeptide, we microinjected rat pancreatic polypeptide into the DVC at a single dose of 2 pmol. Only a modest increase in intragastric pressure in response to the hormone was observed. Thus it is likely that insulin, through its action in the lower brain stem, may be implicated in the pathogenesis of gastrointestinal and cardiovascular complications in hyperinsulinemia. In addition, species variations in the amino acid sequence of insulin may affect its biological activity in the brain of different species.

Freeze tolerance in the wood frog Rana sylvatica is associated with unusual structural features in insulin but not in glucagon.

The wood frog Rana sylvatica utilises glucose, derived from hepatic glycogen, as a cryoprotectant in order to survive freezing during winter hibernation, and glycogenolysis is initiated by hormonal and/or neural stimuli. The primary structure of insulin was determined from R. sylvatica and from two species of freeze-intolerant Ranid frogs R. catesbeiana (American bullfrog) and R. ridibunda (European green frog). All three insulins contain a dipeptide (Lys-Pro) extension to the N-terminus of the A-chain. The amino acid sequences of insulins from R. catesbeiana and R. ridibunda differ by only one residue (Asp for Glu at B21) but R. sylvatica insulin differs from R. catesbeiana insulin at A12 (Thr-->Met), A23 (Asn-->Ser), B5 (Tyr-->His) and B13 (Glu-->Asp). The residue at A23 (corresponding to A21 in human insulin) has been otherwise fully conserved during evolution and the residue at B13 has been strongly conserved in tetrapods. Insulin isolated from specimens of R. sylvatica that had been frozen for 24 h and from control animals that had not been frozen had the same structure, showing that freezing did not alter the pathway of post-translational processing of proinsulin. R. sylvatica glucagon was isolated in two molecular forms. Glucagon-29 was identical to R. catesbeiana glucagon-29 and contains only one amino acid substitution (Thr-->Ser) compared with human glucagon. Glucagon-36 represents glucagon-29 extended from its C-terminus by Lys-Arg-Ser-Gly-Gly-Ile-Ser and is identical to R. catesbeiana glucagon-36. We speculate that selective changes in the structure of the insulin molecule may contribute to the anomalous regulation of glycogen phosphorylase in the wood frog.

Purification of a marsupial insulin: amino-acid sequence of insulin from the Eastern grey kangaroo Macropus giganteus

Insulin has been purified from kangaroo pancreas by acidic ethanol extraction, diethyl ether precipitation and gel filtration. The amino-acid sequence of this, the first marsupial insulin to be studied, is reported. It differs from human insulin by only four amino-acid substitutions, all in regions of the molecule previously known to be variable. However, it should be noted that one of these, asparagine for threonine at A8, has not been reported before. Computer comparisons of all 43 insulin sequences reported to date with kangaroo insulin show it to be most closely related to a group of mammalian insulins (dog, pig, cow, human) known to be of high biological potency. The measurement of blood glucose lowering in the rabbit by kangaroo insulin is consistent with this conclusion. Comparisons of amino-acid sequences of other proteins with their kangaroo counterparts show a greater difference, in line with the time of divergence of marsupials. The limited differences observed in insulin and cytochrome c suggest that their structures need to be closely conserved in order to maintain function.

Structure and function of proteins.

It is now many years since Linderstr6m-Lang suggested that the threedimensional conformation of a protein is the end result of its primary, secondary and tertiary structures. The protein's primary structure is represented by its amino acid sequence and the secondary structure by its tr-helices and E-sheets; its final three-dimensional conformation is its tertiary structure. Another fundamental insight came from Sanger, who showed--much to the surprise of many scientists at that time--that each protein possesses a well-defined amino acid sequence. He led the way by identifying the amino acid sequence of insulin, and as a result of his work and that of others there are now many proteins whose amino acid sequences are known. Linus Pauling deciphered the secondary structure of proteins by postulating the existence of helices and peptide sheets in fibriUar and globular proteins. Confirmation of this hypothesis came from X-ray analysis of different poly-oc-amino acids, the simple peptide polymers which were synthesized and studied in my laboratory in Rehovot. Perutz and Kendrew ,.successfully used X-ray techniques to elucidate the three-dimensional structure of two globular proteins, myobloglobin and haemoglobin. It was no easy task--Max Perutz devoted nearly 20 years of his life to it--but the effort proved to be worthwhile. The development of modern X-ray techniques represented a methodological breakthrough, which meant that it was not possible to determine the threedimensional structure of practically every protein or nucleic acid that could be oriented or crystallized. Newer techniques such as two-dimensional nuclear magnetic resonance (NMR) are gradually catching up, at least as far as low molecular weight proteins are concerned, and have already provided valuable information on the behaviour of biopolymers in solution.

The isolation, purification and amino-acid sequence of insulin from the teleost fish Cottus scorpius (daddy sculpin).

Insulin from the principal islets of the teleost fish, Cottus scorpius (daddy sculpin), has been isolated and sequenced. Purification involved acid/alcohol extraction, gel filtration, and reverse-phase high-performance liquid chromatography to yield nearly 1 mg pure insulin/g wet weight islet tissue. Biological potency was estimated as 40% compared to porcine insulin. The sculpin insulin crystallised in the absence of zinc ions although zinc is known to be present in the islets in significant amounts. Two other hormones, glucagon and pancreatic polypeptide, were copurified with the insulin, and an N-terminal sequence for pancreatic polypeptide was determined. The primary structure of sculpin insulin shows a number of sequence changes unique so far amongst teleost fish. These changes occur at A14 (Arg), A15 (Val), and B2 (Asp). The B chain contains 29 amino acids and there is no N-terminal extension as seen with several other fish. Presumably as a result of the amino acid substitutions, sculpin insulin does not readily form crystals containing zinc-insulin hexamers, despite the presence of the coordinating B10 His.

Amino acid sequences of insulins.

The amino acid sequences of insulins determined to date are summarized. Guinea pig, coypu and rat insulins are discussed in particular.

Insulin and proinsulin.

HALF a century has passed since Banting and Best were preparing extracts of pancreas that lowered blood glucose levels consistently in pancreatectomized diabetic dogs. Several years later, Abel crystallized the active principle, and in the next 20 years, other investigators were able to show that it consisted of two chains of amino acids attached by sulfur bridges. The complete structure and amino acid sequence of insulin was defined by Sanger in 1955. Over the next decade, three groups were able to synthesize the molecule, one led by Katsoyannis at Brookhaven National Laboratory, one led by Zahn in Aachen, Germany, and . . .

The chemistry of proteolytic enzymes

Abstract

Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias).

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11.5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.

Species variation in the amino acid sequence of insulin

T HE INITIAL differences in amino acid sequences of some mammalian insulins as determined by Sanger and his co-workers [ 7-51 were confined to positions 8, 9 and 10 of the A chain of the molecule. (Fig. 1, Table I.) These changes in sequence produced no apparent change in the biological activity of the molecule. The limited number of amino acids involved suggested that the insulin could have arisen from a small number of single step mutations, but with so few examples it was not possible to deduce the direction in which the changes had occurred. (Fig. 2.) It was decided to analyze a larger number of species in the hope that greater variations in both sequence and biological activity might be found. The methods used were similar to those described for the earlier species comparisons [4-61, together with subtractive Edman degradations as modified by Konigsberg and Hill [7]. The amino acid sequence of insulin from a number of mammals and chicken are compared with the pig insulin sequence in Table II. Also included are two species of whale insulin determined by other workers [8,9]. In the course of analyzing rat insulin isolated from a large number of rat pancreases it was found that three components were obtained after oxidation with performic acid and electrophoresis at pH 9 in 1 per cent ammonium

Immunochemical distinction between insulins with identical amino-acid sequences.

VIRTUALLY all human subjects treated with commercial mixtures of beef-pork insulin develop circulating insulin-binding antibodies that are readily detectable with insulin labelled with iodine-131 by means of paper or starch eleetrophoresis, paper chromatography or ultracentrifugation1. These antibodies react not only with the specific immunizing insulins but also with horse, sheep and human insulins2, and with dog, monkey, rabbit and whale insulins (unpublished work) in greater or lesser degree. Harris, Sanger and Naughton3 have shown that the amino-acid sequences of insulins from four different ungulate species (cattle, pig, horse and sheep) differ only in the 8–10 positions of the A chains. Although some human antisera react quite similarly with these four insulins, certain antisera can distinguish clearly among insulins from the different species, generally reacting much more strongly with beef and sheep insulins than with pork and horse insulins2. These differences in reaction of the ungulate insulins have been related2 to the known differences in the 8–10 sequence of the A-chain or to possible differences in secondary or tertiary structure.