Article

Gly-His-Thr-Asp-Amide, an Insulin-Activating Peptide from the Human Pancreas Is a Strong Cu(II) but a Weak Zn(II) Chelator

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Abstract

The Cu(II) and Zn(II) binding abilities of Gly-His-Thr-Asp-amide (GHTD-am), a tetrapeptide coreleased from the pancreas along with insulin, were studied using UV–vis and circular dichroism spectroscopies, potentiometry, and calorimetry. GHTD-am is a very strong Cu(II) chelator, forming a three-nitrogen complex with a conditional affinity constant ^CK at pH 7.4 of 4.5 × 10^(12) M^(–1). The fourth coordination site can be occupied by a solvent molecule or a ternary ligand, such as imidazole, with CK on the order of several hundred reciprocal molar. The Zn(II) binding ability of GHTD-am is relatively weak, with CK values at pH 7.4 of 3.0 × 10^4 and 2.0 × 10^3 M^(–1) for the first and second GHTD-am molecule coordinated, respectively. These results are discussed in light of the modes of interactions of Zn(II) and Cu(II) ions with insulin. A direct effect of GHTD-am on the Zn(II) interactions with insulin is unlikely, but its Cu(II) complex may have a biological relevance because of its high affinity and ability to form ternary complexes.

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... Such complex corresponds to CuH 2 (GHK) 2 stoichiometry. However, the observed changes in the electronic spectra are very similar to those seen previously in ternary complexes of other Xaa-His peptides (where Xaa is any amino acid residue except Pro) with imidazole (3+1N coordination, where three nitrogen atoms, NH 3 + (N-term) , N amide , and N Im , are from the first peptide molecule, and one nitrogen atom is N Im from the second peptide molecule) [20,21]. Therefore, we assign the 3+1N coordination to this complex and propose to call it "auto-ternary", with the actual Cu(GHK)(H 2 GHK) stoichiometry. ...
... Two additional deprotonations occur in the mono-complex at alkaline pH. The first probably corresponds to a water molecule (pK = 9.61) within the equatorial plane of the Cu(GHK) species [20,21]. Another is associated with the ε-amino nitrogen (pK = 10.77) from a side chain of Lys residue. ...
... Another is associated with the ε-amino nitrogen (pK = 10.77) from a side chain of Lys residue. The aforementioned assignment of the deprotonation to a water molecule instead of an imidazole N1 from another Cu(GHK) complex (with concomitant polynuclear species formation) is rationalized by the lack of drastic changes of CD parameters that would be expected upon formation of imidazole-bridged dimeric or tetrameric species [21][22][23]. ...
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... 14 Considering the labile equatorial site in the 3N coordination mode, the present work explores the ability of Cu(II)-Aβ 5-9 (K D = 0.17 pM at pH 7.4) to bind biologically relevant phosphate anions. In contrast to previous studies on ternary complexes with the His-2 motif, we focused mostly on the electrochemical behavior of Cu(II)-Aβ [5][6][7][8][9] in the presence of phosphate species using voltammetric techniques (CV, DPV). These experiments were complemented by UV-Vis and CD spectroscopic measurements of the interaction between Cu(II)-Aβ 5-9 and phosphates or other selected anions. ...
... The changes in the DPV trace were associated with the appearance of a new signal at around 1.0 V, already noticeable when adding 0.1 mM of phosphates. Its intensity kept increasing until the phosphate titration reached the level comparable to that of the binary Cu(II)-Aβ 5-9 at a three-fold ligand excess over Cu(II)-Aβ [5][6][7][8][9] . Cu(II) cation. ...
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Significant changes observed in the electrochemical response of the Cu( ii )-Aβ 5–9 complex upon phosphates addition provided a new insight into the design of a promising class of peptide-based molecular receptors selective for phosphate species.
... The first one, around 605 nm, appeared at pH above 3 (see Fig. 2, open symbols) and is characteristic of the 3N [N am , N im , N − ] square planar Cu(II) complex of the His2 peptides. 4,16,[24][25][26] What is important for this work is the presence of the 3N complex as the main Cu(II) coordination mode for all the studied complexes at physiological pH 7.4. The second d-d band around 520 nm was noticed at pH higher than 9 (see Fig. 2, full symbols) and corresponded to the formation of the 3N + OH − [N am , N im , N − , OH − ] complex due to the deprotonation of a water molecule. ...
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... All peptides, except for GGH purchased from Sigma, were synthesized in-house with standard Fmoc solid phase synthesis, as described before. 62 Crude synthesis products were purified with RP-HPLC on ACE C18-300 column 250 × 8 mm with a rising gradient of acetonitrile in water with 0.1% TFA. ...
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... Imidazole is also a relatively strong ternary ligand for XH peptides, as demonstrated recently. 12,19 In these experiments AAH and AH peptides were used as references representing pure 4N and 3N coordination modes, respectively. ...
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Isothermal titration calorimetry (ITC) is a powerful and widely used method to measure the energetics of macromolecular interactions by recording a thermogram of differential heating power during a titration. However, traditional ITC analysis is limited by stochastic thermogram noise and by the limited information content of a single titration experiment. Here we present a protocol for bias-free thermogram integration based on automated shape analysis of the injection peaks, followed by combination of isotherms from different calorimetric titration experiments into a global analysis, statistical analysis of binding parameters and graphical presentation of the results. This is performed using the integrated public-domain software packages NITPIC, SEDPHAT and GUSSI. The recently developed low-noise thermogram integration approach and global analysis allow for more precise parameter estimates and more reliable quantification of multisite and multicomponent cooperative and competitive interactions. Titration experiments typically take 1-2.5 h each, and global analysis usually takes 10-20 min.
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Insulin-secretory granules isolated from a pancreatic islet-cell tumour by centrifugation on Percoll density gradients exhibited a membrane-associated Mg2+-dependent ATPase activity. In granule suspensions incubated in iso-osmotic media, activity was increased 2–3-fold by carbonyl cyanide p -trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K2SO4 or by the addition of a detergent. Permeant anions also increased Mg2+-dependent ATPase activity under iso-osmotic conditions when combined with K+ and nigericin, or NH4+. It was deduced that a major component of the activity was coupled to the translocation of protons into the granule interior. The granule membrane appeared poorly permeable to H+, K+, NH4+ and SO42− but permeable, in increasing order, to phosphate or acetate, Cl−, I− and SCN−. Like the proton-translocating ATPase of mammalian mitochondria the granule enzyme when membrane-bound was inhibited by up to 85% by tributyltin or NN ′-dicyclohexylcarbodi-imide and was solubilized in a tributyltin-insensitive form after extraction with dichloromethane. It was clearly not a mitochondrial contaminant as evidence by the distribution of marker proteins on density gradients. Unlike mitochondrial activity it was insensitive to oligomycin, efrapeptin, atractyloside, azide and oxyanions. Its properties, however, were indistinguishable from those of the proton-translocating ATPase found in the chromaffin granules of the adrenal medulla. Moreover, insulin granules and chromaffin granules exhibited similar levels of activity. This indicated that in spite of the differences in their internal composition, granules from tissues involved in polypeptide and amine hormone secretion possess catalytic components in common. Only a minor role for the ATPase in amine transport in insulin granules was apparent. Rather, its presence here may relate to the process of secretory vesicle morphogenesis or to the exocytotic mechanism.
Article
Loss-of-function mutations protective against human disease provide in vivo validation of therapeutic targets, but none have yet been described for type 2 diabetes (T2D). Through sequencing or genotyping of ~150,000 individuals across 5 ancestry groups, we identified 12 rare protein-truncating variants in SLC30A8, which encodes an islet zinc transporter (ZnT8) and harbors a common variant (p.Trp325Arg) associated with T2D risk and glucose and proinsulin levels. Collectively, carriers of protein-truncating variants had 65% reduced T2D risk (P = 1.7 × 10−6), and non-diabetic Icelandic carriers of a frameshift variant (p.Lys34Serfs*50) demonstrated reduced glucose levels (−0.17 s.d., P = 4.6 × 10−4). The two most common protein-truncating variants (p.Arg138* and p.Lys34Serfs*50) individually associate with T2D protection and encode unstable ZnT8 proteins. Previous functional study of SLC30A8 suggested that reduced zinc transport increases T2D risk, and phenotypic heterogeneity was observed in mouse Slc30a8 knockouts. In contrast, loss-of-function mutations in humans provide strong evidence that SLC30A8 haploinsufficiency protects against T2D, suggesting ZnT8 inhibition as a therapeutic strategy in T2D prevention.
Article
The thermodynamics of formation of the insulin hexamer, which is stabilized by two Zn2+ ions, have been quantified by isothermal titration calorimetry (ITC). Since the insulin monomer is unstable to aggregation (fibrillation) during ITC measurements, an original method involving EDTA chelation of Zn2+ from the hexamer was employed. The two metal ions are chelated sequentially, reflecting step-wise Zn2+ binding and stabilization of the quaternary structure. Analysis of the ITC data reveals that 2-3 H+ bind to the hexamer upon its formation at pH 7.4, which is both enthalpically and entropically favored. The former is due to Zn2+ coordination to His residues from three subunits and the latter is associated with desolvation that accompanies the protonation and the packing of the subunits in the hexamer.
Article
Interest in the crystal structure of a copper complex of the growth factor, glycyl-L-histidyl-L-lysine has been stimulated by the tripeptide's ability to facilitate copper uptake in cultured hepatoma cells and by the copper complex's tendency to induce angiogenesis. The coordination polyhedron is a distorted square pyramid, CuN3O2, with the four basal ligating atoms bonded to the copper at about 2.00 Å and the apical ligating atom at 2.49 Å. One tripeptide furnishes three of the basal atoms, the glycine amino nitrogen atom, the peptide nitrogen atom of the histidine, and the imine nitrogen atom of the imidazole. A second tripeptide is involved via its terminal carboxyl oxygen atom while the fifth copper ligand is a carboxyl oxygen atom of a third tripeptide. The carboxyl oxygen atoms form bridges between copper centers and thus the system is polymeric in the solid state. The crystal structure can be used to propose a model for the first step in the transport of copper into cells via a copper-tripeptide complex.
Article
Zinc (Zn(2+)) is an essential element crucial for growth and development, and also plays a role in cell signaling for cellular processes like cell division and apoptosis. In the mammalian pancreas, Zn(2+) is essential for the correct processing, storage, secretion, and action of insulin in beta (β)-cells. Insulin is stored inside secretory vesicles or granules, where two Zn(2+) ions coordinate six insulin monomers to form the hexameric-structure on which maturated insulin crystals are based. The total Zn(2+) content of the mammalian pancreas is among the highest in the body, and Zn(2+) concentration reach millimolar levels in the interior of the dense-core granule. Changes in Zn(2+) levels in the pancreas have been found to be associated with diabetes. Hence, the relationship between co-stored Zn(2+) and insulin undoubtedly is critical to normal β-cell function. The advances in the field of Zn(2+) biology over the last decade have facilitated our understanding of Zn(2+) trafficking, its intracellular distribution and its storage. When exocytosis of insulin occurs, insulin granules fuse with the β-cell plasma membrane and release their contents, i.e., insulin as well as substantial amount of free Zn(2+), into the extracellular space and the local circulation. Studies increasingly indicate that secreted Zn(2+) has autocrine or paracrine signaling in β-cells or the neighboring cells. This review discusses the Zn(2+) homeostasis in β-cells with emphasis on the potential signaling role of Zn(2+) to islet biology.
Article
The complexes formed in aqueous solution by copper(II) with glycyl-L-histidylglycine (L–) have been extensively studied, by using potentiometry, calorimetry, visible spectrophotometry and circular dichroism techniques, with the aim of elucidating their type and probable structure. Only monomeric species are formed in the acidic range of pH ([CuL]+, [CuLH–1], [CuL2] and [CuL2H–1]–), while at higher pH (>8) polynuclear [Cu4L4H–6]4– is formed in addition to [CuLH–2]–. The interpretation of all the experimental data is strongly consistent with a structure involving three nitrogen atoms co-ordinated to copper(II) for [CuLH–1] and [CuL]+ and with the participation of a fourth nitrogen atom in [CuLH–2]– and [Cu4L4H–8]4–. In the tetrameric species very probably there is a dissociation of the N(1)-pyrrole hydrogen ion, with consequent formation of imidazolate bridges.
Article
Thermodynamic data for the proton and metal ion complexation equilibria of imidazole ( 1.3 - diazole, glyoxaline) and histamine ( 4-imidoazolethylamine, 4 - ( 2' - aminoethyl ) imidazole) have been collected and critically evaluated. The literature search has been performed by using IUPAC's ''Stability Constants Database'' and covers the literature until 1992. Important criteria for acceptance comprise specification of the essential reaction conditions, the correctness of calibration techniques and appropriate equilibrium analysis of experimental data. Recommended values with respect to proton - ligand and metal ion - ligand equilibria have been tabulated. Furthermore, the medium dependence of these reactions has been evaluated using an extended Debye - Huckel expression.
Article
Copper(II) complexes of the histidine-containing tripeptides glyclyl-L-histidylglycine (GlyHisGly), glycylglycyl-L-histidine (GlyGlyHis), and L-pyroglutamyl-L-histidyl-L-prolinamide (trf), and the dipeptide L-pyroglutamyl-L-histidine methyl ester (pghme), have been studied by pH-metric and spectrophotometric methods. It was found that, similarly as for glycyl-L-histidine (GlyHis), GlyHisGly forms a complex [CuAH–1], but bis complexes are also formed in low concentration. For GlyGlyHis, only the highly stable species [CuAH–2]– is formed. A complex [CuAH–2]– is also formed with trf and pghme which indicates that the prolinamide side-chain in trf does not take part in the co-ordination. The sequence of deprotonation of the N1H group of the imidazole side-chain is as follows: GlyHisGly > GlyHis > pghme > trf > GlyGlyHis.
Article
Complexes formed between H+, Cu2+, and Ni2+ and glycylhistidine, β-alanyihistidine, and histidylglycine have been studied potentiometrically at 25 °C and I= 0.10M(K[NO3]). The equilibria are very complicated but polynuclear species are insignificant below pH 10 in dilute solution (10–3M). Suggestions are made as to the probable co-ordination sites as a function of pH.
Article
Equilibrium constants at 37 °C and 0·15M(KNO3) are reported for zinc and cobalt(II) complexes of glycyl-L-histidine, glycyl-L-histidylglycine, L-histidylglycine, and carnosine from pH titration data, using a range of metal ion and ligand concentrations. Binuclear complexes are major species in solutions containing zinc ions and glycylhistidine. Related mixed complexes are formed in ternary mixtures of zinc ion, histidine, and glycylnistidine. Mixed-ligand complexes of the type MAL are also formed by zinc and cobalt(II) with histidine (HA) and the peptides. A comparison is made of the zinc-complexing abilities of the peptides in competition with α-amino-acids under physiological conditions.
Article
Serum albumin is the most abundant protein in the blood and cerebrospinal fluid and plays a fundamental role in the distribution of essential transition metal ions in the human body. Human serum albumin (HSA) is an important physiological transporter of the essential metal ions Cu(2+), and Zn(2+) in the bloodstream. Its binding of metals like Ni(2+), Co(2+), or Cd(2+) can occur in vivo, but is only of toxicological relevance. Moreover, HSA is one of the main targets and hence most studied binding protein for metallodrugs based on complexes with Au, Pt and V. We discuss i) the four metal-binding sites so far described on HSA, their localization and metal preference, ii) the binding of the metal ions mentioned above, i.e. their stability constants and association/dissociation rates, their coordination chemistry and their selectivity versus the four binding sites iii) the methodology applied to study issues of items i and ii and iv) oligopeptide models of the N-terminal binding site. Albumin has four partially selective metal binding sites with well-defined metal preferences. It is an important regulator of the blood transport of physiological Cu(II) and Zn(II) and toxic Ni(II) and Cd(II). It is also an important target for metal-based drugs containing Pt(II), V(IV)O, and Au(I). The thorough understanding of metal binding properties of serum albumin, including the competition of various metal ions for specific binding sites is important for biomedical issues, such as new disease markers and design of metal-based drugs. This article is part of a Special Issue entitled Serum Albumin.
Article
A series of aminoacids of general formula CH2:CH(CH2)nCH(NH2)CO2H has been synthesised and the formation constant of their complexes with H+, Cu2+, Ni2+, Zn2+, Co2+, Cd2+ and Ag+ measured at 25° and I = 0·10 M (KNO3). The olefinic bond does not appear to take part in bonding to any of the acceptors with the exception of Ag+ when it chelates extensively when n = 1 and decreasingly when n = 2 and 3. The high stability of the resulting “ membered” chelate ring demonstrates the readiness of Ag+ to accept non-linear co-ordination with soft acceptors.
Article
A new computer program has been developed in which formation constants are determined by minimisation of an error-square sum based on measured electrode potentials. The program also permits refinement of any reactant concentration or standard electrode potential. The refinement is incorporated into a new procedure which can be used for model selection.
Article
Formation constants for the various complexes formed between H+, Cu2+, and Ni2+ and a range of dipeptides which do not contain co-ordinating substituent groups have been measured potentiometrically at 25 °C and I= 0.10M(K[NO3]). The structures of the complexed species are discussed and conclusions drawn on the equilibria present in nickel–dipeptide systems.
Article
8-Hydroxyquinolines (8HQ) have found widespread application in chemistry and biology due to their ability to complex a range of transition metal ions. The family of 2-substituted 8HQs has been proposed for use in the treatment of Alzheimer's disease (AD). Most notably, the therapeutic PBT2 (Prana Biotechnology Ltd.) has been shown to act as an efficient metal chaperone, disaggregate metal-enriched amyloid plaques comprised of the Aβ peptide, inhibit Cu/Aβ redox chemistry, and reverse the AD phenotype in transgenic animal models. Yet surprisingly little is known about the molecular interactions at play. In this study, we show that the homologous ligand 2-[(dimethylamino)methyl]-8-hydroxyquinoline (HL) forms a CuL complex with a conditional (apparent) dissociation constant of 0.33 nM at pH 6.9 and is capable of forming ternary Cu(2+) complexes with neurotransmitters including histamine (HA), glutamic acid (Glu), and glycine (Gly), with glutathione disulfide (GSSG), and with histidine (His) side chains of proteins and peptides including the Aβ peptide. Our findings suggest a molecular basis for the strong metal chaperone activity of PBT2, its ability to attenuate Cu(2+)/Aβ interactions, and its potential to promote neuroprotective and neuroregenerative effects.
Article
The formation constants of Cd(II), Ni(II) and Zn(II) complexes of glycylglycine, glycylglycylglycine, tetraglycine and glycine amide have been determined. With each of these three metal cations, the glycine amide and the three peptide complexes are about equally stable. With Mg(II) cation, the glycylglycine and tetraglycine complexes are equally stable. It is shown that the three glycine peptides probably have common coordination sites, which are probably the terminal amino group and the immediately adjacent peptide group.
Article
Electronic spectra over the 50 000-20 000-cm-1 region are reported for well-characterized chromophores having Cu(II)-imidazole (ImH) and Cu(II)-imidazolate (Im-) units. For tetragonal Cu(II)-ImH chromophores, three ligand to metal charge-transfer (LMCT) absorptions originate from the σ-symmetry nitrogen donor lone pair and from two π-symmetry ring orbitals, one having primarily carbon character (π1) and the other having primarily nitrogen character (π2). These σ(ImH) →, π2(ImH) →, and π1(ImH) → Cu(II) LMCT absorptions occur at ∼220, ∼260, and ∼330 nm, respectively. Ligand rotation causes the π-symmetry absorptions to be broadened for solutions containing geometrically unconstrained Cu(II)-ImH complexes. The π-symmetry absorptions generally are well-resolved spectral features of crystalline complexes, and may be split when the ImH groups have nonequivalent orientations. The σ(ImH) → Cu(II) absorption at 220 nm is insensitive to ligand rotation about the Cu-N axis, and is well resolved from the ligand-localized absorption at ∼205 nm. The Cu(II)-Im- complexes exhibit an additional and characteristic broad absorption at ∼375 nm for which a tentative assignment has been suggested. Tetragonal type 2 and type 3 copper protein chromophores are expected to exhibit corresponding π(ImH) → Cu(II) LMCT transitions in the near-UV region. Such absorptions are expected to be red shifted for the approximately tetrahedral type 1 copper chromophores. The reported spectra of the above types of proteins briefly are reconsidered from this point of view.
Article
With a view to obtaining information of the structures and stabilities of the Cu(II) complexes of tyrosine (Tyr)-containing dipeptides, spectroscopic and potentiometric studies have been carried out with the peptides to L-Tyr-X, where X refers to L-/D-alanine, -arginine, -Tyr, -tryptophan (Trp), -histidine (His), L-phenylalanine (Phe) , L-/D-glutamic acid. The complex species and their stability constants have been determined by potentiometric titrations at 25°C and I = 0.1 (KNO3). All the peptides react with Cu(II) in the manner analogous to L-tyrosylglycine (L-Tyr·Gly), but the deprotonation of the peptide NH group is affected by the C-terminal side-chain groups. The dipeptides except L-Tyr·L-/D-His form at pH 8-11 a dimeric species, the maximum distribution of which occurs at pH ∼9.5 in the 1:1 Cu(II)-peptide systems with the dimer accounting for as much as 80% of the total Cu(II) in 5 mM Cu(II)-L-Tyr·L-Trp. The constants for 2(monomer) ⇌ dimer equilibria are in the range 2.04-3.70 log units. The absorption spectra of the 1:1 systems (∼2 mM) exhibit a d-d peak at 610-630 nm (ε 90-150) at pH >6 and in the presence of the dimeric complex an additional peak at ∼380 nm (ε 260-720), whose assignment to the charge transfer between Cu(II) and the phenolate group has been confirmed by the resonance Raman spectra of the isolated complexes, [Cu(L-Tyr·Gly)]·0.5H2O and Na2[Cu2(L-Tyr·Gly)2]·7.5H 2O. While the circular dichroism (CD) spectral magnitudes in the d-d region for the Cu(II)-dipeptide complexes with a C-terminal aliphatic amino acid are an additive function of those exhibited by the component amino acid complexes irrespective of the diastereoisomerism of the peptides, remarkable CD magnitude anomaly was observed for the active (L-L) peptides with a C-terminal aromatic amino acid, L-Tyr·L-X (X = Tyr, Trp, and Phe). The anomaly is diastereospecific and strictly coincident with the dimer formation, which is taken to imply distortion by the dimeric structure of the C-terminal side-chain orientation favoring the Cu(II)-aromatic ring interaction.
Article
The binding of zinc by the tripeptide glycyl-L-histidyl-L-lysine (GHL) has been studied by 1H NMR spectroscopy and by potentiometric titration. At physiological pH, resonances are observed for two kinetically stable complexes, and binding sites in these complexes have been identified from the chemical shifts of carbon-bonded protons of complexed GHL. Formation constants have been determined for Zn(II)-GHL complexes from pH titration data. Proton balance considerations indicate that an extra proton is titrated from the Zn(II)-GHL complexes, while the NMR results indicate that the extra proton is titrated from an amide group. It is concluded from chemical shift data that the GHL in one of the two kinetically stable complexes is tridentate, with Zn(II) coordinated by the deprotonated glycyl amino nitrogen, the deprotonated amide nitrogen of the glycyl-histidyl peptide bond, and the imidazole 1-nitrogen, while the amino group of the lysine side chain is protonated. The results are discussed with reference to the synergistic alteration of growth patterns of hepatoma cells by GHL and Zn(II).5
Article
Cells of Saccharomyces cerevisiae exhibiting the mating type excrete into the culture medium a low-molecular-weight substance, termed factor. This factor, which specifically inhibits DNA replication in cells of the opposite mating type a, has been purified more than 100000-fold from culture filtrates of cells. Purified factor appears to be homogeneous as judged from thin-layer chromatography and thin-layer electrophoresis in different systems. It shows a positive-ninhydrin reaction and, upon hydrolysis, gives rise to several ninhydrin-positive substances of which the amino acids leucine, glycine, proline, glutamic acid, tyrosine, tryptophan and possibly histidine have been identified. The presence of tryptophan and tyrosine is also confirmed by the ultraviolet absorption spectrum of the factor. In addition, purified factor contains cupric ions which can be separated from the ninhydrin-positive material by thin-layer electrophoresis at pH 3.6. Gel filtration on Sephadex G-25 in 8 M urea indicates a molecular weight in the range of 1400. The properties of the purified factor are consistent with those of a low-molecular- weight peptide.
Article
This review discusses the modes of coordination of oligopeptides by Cu(II) and Ni(II). Special attention is given to two general classes of peptides. The first part of the review deals with indirect effects introduced by special sequences of non-bonding side-chains. Unusual coordination modes resulting from the introduction of the break-point proline residues are also discussed. The second part of the review describes the binding properties of histidine peptides. The effects of the positioning of a His residue are discussed in the terms of cooperation and competition between potential metal anchoring sites. Special attention is given to His-3 peptides, modeling the biologically relevant albumin-like metal binding site. Finally, the coordination-related specific hydrolysis processes in histidine peptides are briefly discussed.
Article
The Gly-His-Lys (GHK) peptide and the Asp-Ala-His-Lys (DAHK) sequences are naturally occurring high-affinity copper(II) chelators found in the blood plasma and are hence of biological interest. A structural study of the copper complexes of these peptides was conducted in the solid state and in solution by determining their X-ray structures, and by using a large range of spectroscopies, including EPR and HYSCORE (hyperfine sub-level correlation), X-ray absorption and 1H and 13C NMR spectroscopy. The results indicate that the structures of [Cu II(DAHK)] in the solid state and in solution are similar and confirm the equatorial coordination sphere of NH 2, two amidyl N and one imidazole N. Additionally, a water molecule is bound apically to Cu II as revealed by the X-ray structure. As reported previously in the literature, [Cu II(GHK)], which exhibits a dimeric structure in the solid state, forms a monomeric complex in solution with three nitrogen ligands: NH 2, amidyl and imidazole. The fourth equatorial site is occupied by a labile oxygen atom from a carboxylate ligand in the solid state. We probe that fourth position and study ternary complexes of [Cu II(GHK)] with glycine or histidine. The Cu II exchange reaction between different DAHK peptides is very slow, in contrast to [Cu II(GHK)], in which the fast exchange was attributed to the presence of a [Cu II(GHK) 2] complex. The redox properties of [Cu II(GHK)] and [Cu II(DAHK)] were investigated by cyclic voltammetry and by measuring the ascorbate oxidation in the presence of molecular oxygen. The measurements indicate that both Cu II complexes are inert under moderate redox potentials. In contrast to [Cu II(DAHK)], [Cu II(GHK)] could be reduced to Cu I around -0.62 V (versus AgCl/Ag) with subsequent release of the Cu ion. These complete analyses of structure and redox activity of those complexes gave new insights with biological impact and can serve as models for other more complicated Cu II-peptide interactions.
Article
GHTD-amide is a tetrapeptide originally isolated from human urine that has hypoglycemic activity. Insulin occurs in secretory granules of beta cells as zinc-stabilized hexamers and must disperse to monomeric form in order to bind to its receptor. The aim of this study was to identify whether GHTD-amide and an analog called ISF402 (VHTD-amide) reduce blood glucose through enhancement of insulin activity by dispersing oligomers of insulin. Peptides containing the HTD-amide sequence and a free alpha-amino group were optimal at binding Zn(2+) and adopting secondary structure in the presence of Zn(2+). Binding was concentration dependent and resulted in a 1:1 Zn:peptide complex. In vitro the tetrapeptides dispersed hexameric insulin to dimers and monomers. GHTD-amide and ISF402 potentiated the activity of hexameric insulin when co-injected into insulin resistant Zucker rats. Injection of peptides with insulin caused reductions in blood glucose and C-peptide significantly larger than achieved with insulin alone, and serum insulin time profiles were also altered consistent with a reduced clearance or enhanced dispersal of the injected insulin. Insulin potentiation by ISF402 was reduced when lispro insulin, which does not form zinc-stabilized hexamers, was used in place of hexameric zinc insulin. In conclusion, GHTD-amide and ISF402 are zinc binding peptides that disperse hexameric insulin in vitro, and potentiate the activity of hexameric insulin more so than monomeric lispro insulin. These results suggest that dispersal of hexameric insulin through chelation of Zn(2+) contributes to the hypoglycemic activity of these tetrapeptides.
Article
Zinc is an essential trace element crucial for the function of more than 300 enzymes and it is important for cellular processes like cell division and apoptosis. Hence, the concentration of zinc in the human body is tightly regulated and disturbances of zinc homeostasis have been associated with several diseases including diabetes mellitus, a disease characterized by high blood glucose concentrations as a consequence of decreased secretion or action of insulin. Zinc supplementation of animals and humans has been shown to ameliorate glycemic control in type 1 and 2 diabetes, the two major forms of diabetes mellitus, but the underlying molecular mechanisms have only slowly been elucidated. Zinc seems to exert insulin-like effects by supporting the signal transduction of insulin and by reducing the production of cytokines, which lead to beta-cell death during the inflammatory process in the pancreas in the course of the disease. Furthermore, zinc might play a role in the development of diabetes, since genetic polymorphisms in the gene of zinc transporter 8 and in metallothionein (MT)-encoding genes could be demonstrated to be associated with type 2 diabetes mellitus. The fact that antibodies against this zinc transporter have been detected in type 1 diabetic patients offers new diagnostic possibilities. This article reviews the influence of zinc on the diabetic state including the molecular mechanisms, the role of the zinc transporter 8 and MT for diabetes development and the resulting diagnostic and therapeutic options.
Article
In the early 1970s, a peptide fraction with insulin potentiating activity was purified from human urine but the identity and origins of the active constituent remained unknown. Here we identify the active component and characterize its origins. The active peptide was identified as an alpha amidated tetrapeptide with the sequence GHTD-amide. The peptide was synthesized and tested for stimulation of glycogen synthesis and insulin potentiation by insulin tolerance testing in insulin-deficient rats, which confirmed GHTD-amide as the active peptide. Tissue localization using a peptide-specific anti-serum and epifluorescent and confocal microscopy showed decoration of pancreatic islets but not other tissues. Confocal microscopy revealed co-localization with insulin and immunogold and electron microscopy showed localization to dense core secretory granules. Consistent with these observations GHTD-amide was found in media conditioned by MIN6 islet beta cells. Sequence database searching found no annotated protein in the human proteome encoding a potential precursor for GHTD-amide. We conclude that the insulin potentiating activity originally described in human urine is attributable to the tetrapeptide GHTD-amide. GHTD-amide is a novel peptide produced by pancreatic beta cells and no precursor protein is present in the annotated human proteome. Stimulation of glycogen synthesis and co-localization with insulin in beta cells suggest that GHTD-amide may play a role in glucose homeostasis by enhancing insulin action and glucose storage in tissues.
Article
A new suite of 10 programs concerned with equilibrium constants and solution equilibria is described. The suite includes data preparation programs, pretreatment programs, equilibrium constant refinement and post-run analysis. Data preparation is facilitated by a customized data editor. The pretreatment programs include manual trial and error data fitting, speciation diagrams, end-point determination, absorbance error determination, spectral baseline corrections, factor analysis and determination of molar absorbance spectra. Equilibrium constants can be determined from potentiometric data and/or spectrophotometric data. A new data structure is also described in which information on the model and on experimental measurements are kept in separate files.
Article
Summary The structure of a growth-modulating tripeptide from human serum and plasma has been determined to be H-glycyl-histidyl-lysine-OH.
Article
The membrane potential (DeltaPsi) and the pH gradient (DeltapH) across the membrane of the insulin-secretory granule were determined in studies in vitro from the uptake of the permeant anion thio[(14)C]cyanate or the permeant base [(14)C]methylamine. Freshly prepared granules incubated in iso-osmotic medium containing sucrose and low concentrations of buffer salts exhibited an acidic internal pH and a DeltaPsi positive inside. Addition of MgATP(2-) under these conditions did not alter the DeltapH, but produced a marked increase in the DeltaPsi. Conversely, when a permeant anion was also included, ATP produced a marked increase in the DeltapH and a lesser increment in the DeltaPsi. NH(4) (+) salts reduced the DeltapH across granule membranes. In the presence of ATP this effect was accompanied by a reciprocal increase in the DeltaPsi. A similar reciprocity was evident when nigericin was added together with K(+) or on decreasing the medium pH, suggesting that these gradients were linked by a common electrogenic process. The effects of ATP were reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(+), and by the Mg(2+)-dependent ATPase inhibitor tributyltin. Uptakes of (14)C-labelled tracer molecules were also markedly reduced by cryogenic disruption of the granule membrane or hypo-osmotic incubation conditions. These results were readily interpreted within a chemiosmotic hypothesis, which proposed that the insulin granules possess an inwardly-directed electrogenic proton-translocating Mg(2+)-dependent ATPase with the additional postulate that the membrane has a low proton permeability. The intragranular pH was estimated as being between 5 and 6 in vivo. Such a value corresponds to optimal conditions for the crystallization of zinc-insulin hexamers. Several other functions related to chemiosmotic processes within insulin granules, however, may be envisaged.