NA-Selank Amidate Research & Studies

The vitamin C deficiency disease scurvy is characterised by musculoskeletal pain and recent epidemiological evidence has indicated an association between suboptimal vitamin C status and spinal pain. Furthermore, accumulating evidence indicates that vitamin C administration can exhibit analgesic properties in some clinical conditions. The prevalence of hypovitaminosis C and vitamin C deficiency is high in various patient groups, such as surgical/trauma, infectious diseases and cancer patients. A number of recent clinical studies have shown that vitamin C administration to patients with chronic regional pain syndrome decreases their symptoms. Acute herpetic and post-herpetic neuralgia is also diminished with high dose vitamin C administration. Furthermore, cancer-related pain is decreased with high dose vitamin C, contributing to enhanced patient quality of life. A number of mechanisms have been proposed for vitamin C's analgesic properties. Herein we propose a novel analgesic mechanism for vitamin C; as a cofactor for the biosynthesis of amidated opioid peptides. It is well established that vitamin C participates in the amidation of peptides, through acting as a cofactor for peptidyl-glycine α-amidating monooxygenase, the only enzyme known to amidate the carboxy terminal residue of neuropeptides and peptide hormones. Support for our proposed mechanism comes from studies which show a decreased requirement for opioid analgesics in surgical and cancer patients administered high dose vitamin C. Overall, vitamin C appears to be a safe and effective adjunctive therapy for acute and chronic pain relief in specific patient groups.

Computational investigations have implicated the amidate ligand in nickel superoxide dismutase (NiSOD) in stabilizing Ni-centered redox catalysis and in preventing cysteine thiolate ligand oxidation. To test these predictions, we have used an experimental approach utilizing a semisynthetic scheme that employs native chemical ligation of a pentapeptide (HCDLP) to recombinant S. coelicolor NiSOD lacking these N-terminal residues, NΔ5-NiSOD. Wild-type enzyme produced in this manner exhibits the characteristic spectral properties of recombinant WT-NiSOD and is as catalytically active. The semisynthetic scheme was also employed to construct a variant where the amidate ligand was converted to a secondary amine, H1*-NiSOD, a novel strategy that retains a backbone N-donor atom. The H1*-NiSOD variant was found to have only ∼1% of the catalytic activity of the recombinant wild-type enzyme, and had altered spectroscopic properties. X-ray absorption spectroscopy reveals a four-coordinate planar site with N2S2-donor ligands, consistent with electronic absorption spectroscopic results indicating that the Ni center in H1*-NiSOD is mostly reduced in the as-isolated sample, as opposed to 50:50 Ni(II)/Ni(III) mixture that is typical for the recombinant wild-type enzyme. The EPR spectrum of as-isolated H1*-NiSOD accounts for ∼11% of the Ni in the sample and is similar to WT-NiSOD, but more axial, with gz < gx,y. (14)N-hyperfine is observed on gz, confirming the addition of the apical histidine ligand in the Ni(III) complex. The altered electronic properties and implications for redox catalysis are discussed in light of predictions based on synthetic and computational models.

A gene named ltsA was earlier identified in Rhodococcus and Corynebacterium species while screening for mutations leading to increased cell susceptibility to lysozyme. The encoded protein belonged to a huge family of glutamine amidotransferases whose members catalyze amide nitrogen transfer from glutamine to various specific acceptor substrates. We here describe detailed physiological and biochemical investigations demonstrating the specific role of LtsA protein from Corynebacterium glutamicum (LtsACg) in the modification by amidation of cell wall peptidoglycan diaminopimelic acid (DAP) residues. A morphologically altered but viable ΔltsA mutant was generated, which displays a high susceptibility to lysozyme and β-lactam antibiotics. Analysis of its peptidoglycan structure revealed a total loss of DAP amidation, a modification that was found in 80% of DAP residues in the wild-type polymer. The cell peptidoglycan content and cross-linking were otherwise not modified in the mutant. Heterologous expression of LtsACg in Escherichia coli yielded a massive and toxic incorporation of amidated DAP into the peptidoglycan that ultimately led to cell lysis. In vitro assays confirmed the amidotransferase activity of LtsACg and showed that this enzyme used the peptidoglycan lipid intermediates I and II but not, or only marginally, the UDP-MurNAc pentapeptide nucleotide precursor as acceptor substrates. As is generally the case for glutamine amidotransferases, either glutamine or NH4(+) could serve as the donor substrate for LtsACg. The enzyme did not amidate tripeptide- and tetrapeptide-truncated versions of lipid I, indicating a strict specificity for a pentapeptide chain length.

Nickel superoxide dismutase (NiSOD) is a nickel-containing metalloenzyme that catalyzes the disproportionation of superoxide through a ping-pong mechanism that relies on accessing reduced Ni(II) and oxidized Ni(III) oxidation states. NiSOD is the most recently discovered SOD. Unlike the other known SODs (MnSOD, FeSOD, and (CuZn)SOD), which utilize "typical" biological nitrogen and oxygen donors, NiSOD utilizes a rather unexpected ligand set. In the reduced Ni(II) oxidation state, NiSOD utilizes nitrogen ligands derived from the N-terminal amine and an amidate along with two cysteinates sulfur donors. These are unusual biological ligands, especially for an SOD: amine and amidate donors are underrepresented as biological ligands, whereas cysteinates are highly susceptible to oxidative damage. An axial histidine imidazole binds to nickel upon oxidation to Ni(III). This bond is long (2.3-2.6 Å) owing to a tight hydrogen-bonding network. All of the ligating residues to Ni(II) and Ni(III) are found within the first 6 residues from the NiSOD N-terminus. Thus, small nickel-containing metallopeptides derived from the first 6-12 residues of the NiSOD sequence can reproduce many of the properties of NiSOD itself. Using these nickel-containing metallopeptide-based NiSOD mimics, we have shown that the minimal sequence needed for nickel binding and reproduction of the structural, spectroscopic, and functional properties of NiSOD is H2N-HCXXPC. Insight into how NiSOD avoids oxidative damage has also been gained. Using small NiN2S2 complexes and metallopeptide-based mimics, it was shown that the unusual nitrogen donor atoms protect the cysteinates from oxidative damage (both one-electron oxidation and oxygen atom insertion reactions) by fine-tuning the electronic structure of the nickel center. Changing the nitrogen donor set to a bis-amidate or bis-amine nitrogen donor led to catalytically nonviable species owing to nickel-cysteinate bond oxidative damage. Only the amine/amidate nitrogen donor atoms within the NiSOD ligand set produce a catalytically viable species. These metallopeptide-based mimics have also hinted at the detailed mechanism of SOD catalysis by NiSOD. One such aspect is that the axial imidazole likely remains ligated to the Ni center under rapid catalytic conditions (i.e., high superoxide loads). This reduces the degree of structural rearrangement about the nickel center, leading to higher catalytic rates. Metallopeptide-based mimics have also shown that, although an axial ligand to Ni(III) is required for catalysis, the rates are highest when this is a weak interaction, suggesting a reason for the long axial His-Ni(III) bond found in NiSOD. These mimics have also suggested a surprising mechanistic insight: O2(-) reduction via a "H(•)" tunneling event from a R-S(H(+))-Ni(II) moiety to O2(-) is possible. The importance of this mechanism in NiSOD has not been verified.

Nickel containing superoxide dismutase (NiSOD) is a metalloenzyme that catalyzes the disproportionation of O2(-). In its reduced state, the Ni(II) ion is coordinated by two cis-cysteinates, an amine nitrogen and an amidate nitrogen atom. It thus bears a resemblance to the distal bis-cysteinate bis-amidate ligated nickel center of acetyl coenzyme A synthase. Using metallopeptide based NiSOD mimics derived from the first 12 residues of the NiSOD sequence we demonstrate that altering the primary coordination sphere from a bis-thiolate amine/amidate motif to a bis-thiolate bis-amidate motif changes the O2 and ROS stability of the metallopeptide. Using FT-IR, ESI-MS and S K-edge XAS we show that the bis-amidate bis-thiolate ligated metallopeptide {Ni(II)(SOD(m1)-Ac)} (SOD(m1)-Ac=AcHN-HCDLPCGVYSPA-COOH) undergoes oxidation at one thiolate ligand in the presence of O2, converting it into a coordinated sulfinate. Upon exposure of {Ni(II)(SOD(m1)-Ac)} to O2(-) the metallopeptide undergoes extensive sulfur oxidation. This can be contrasted with the unacylated metallopeptide {Ni(II)(SOD(m1))} which does not undergo sulfur based oxidation under these conditions. The biological implications of these findings are discussed.

Secreted peptides, produced by enzymatic processing of larger precursor molecules, are found throughout the animal kingdom and play important regulatory roles as neurotransmitters and hormones. Many require a carboxy-terminal modification, involving the conversion of a glycine residue into an α-amide, for their biological activity. Two sequential enzymatic activities catalyze this conversion: a monooxygenase (peptidylglycine α-hydroxylating monooxygenase or PHM) and an amidating lyase (peptidyl-α-hydroxyglycine α-amidating lyase or PAL). In vertebrates, these activities reside in a single polypeptide known as peptidylglycine α-amidating monooxygenase (PAM), which has been extensively studied in the context of neuropeptide modification. Bifunctional PAMs have been reported from some invertebrates, but the phylogenetic distribution of PAMs and their evolutionary relationship to PALs and PHMs is unclear. Here, we report sequence and expression data for two PAMs from the coral Acropora millepora (Anthozoa, Cnidaria), as well as providing a comprehensive survey of the available sequence data from other organisms. These analyses indicate that bifunctional PAMs predate the origins of the nervous and endocrine systems, consistent with the idea that within the Metazoa their ancestral function may have been to amidate epitheliopeptides. More surprisingly, the phylogenomic survey also revealed the presence of PAMs in green algae (but not in higher plants or fungi), implying that the bifunctional enzyme either predates the plant/animal divergence and has subsequently been lost in a number of lineages or perhaps that convergent evolution or lateral gene transfer has occurred. This finding is consistent with recent discoveries that other molecules once thought of as "neural" predate nervous systems.

We report the fabrication of water-stable electrospun γ-polyglutamic acid (γ-PGA) nanofibers with morphology control for biomedical applications. In this study, the processing variables including polymer concentration, flow rate, applied voltage, collection distance, and ambient humidity were systematically optimized to generate uniform γ-PGA nanofibers with a smooth morphology. By changing the trifluoroacetic acid concentration in the electrospinning solution, the diameter of the γ-PGA nanofibers can be controlled within the range of 186-603 nm. To render the γ-PGA nanofibers with good water stability, cystamine was employed as a crosslinking agent to amidate the carboxyl groups of γ-PGA. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay in conjunction of cell morphology observation reveals that the obtained γ-PGA nanofibers have an excellent biocompatibility to promote the cell adhesion and proliferation. We anticipate that the fabricated electrospun γ-PGA nanofibers with controllable morphology and good water stability may find extensive applications in future development of tissue engineering scaffold materials, drug delivery systems, environmental remediation, and sensing.

The Sir2 enzyme family is responsible for a newly classified chemical reaction, NAD(+)-dependent protein deacetylation. New peptide substrates, the reaction mechanism, and the products of the acetyl transfer to NAD(+) are described for SIR2. The final products of SIR2 reactions are the deacetylated peptide and the 2' and 3' regioisomers of O-acetyl ADP ribose (AADPR), formed through an alpha-1'-acetyl ADP ribose intermediate and intramolecular transesterification reactions (2' --> 3'). The regioisomers, their anomeric forms, the interconversion rates, and the reaction equilibria were characterized by NMR, HPLC, 18O exchange, and MS methods. The mechanism of acetyl transfer to NAD(+) includes (1) ADP ribosylation of the peptide acyl oxygen to form a high-energy O-alkyl amidate intermediate, (2) attack of the 2'-OH group on the amidate to form a 1',2'-acyloxonium species, (3) hydrolysis to 2'-AADPR by the attack of water on the carbonyl carbon, and (4) an SIR2-independent transesterification equilibrating the 2'- and 3'-AADPRs. This mechanism is unprecedented in ADP-ribosyl transferase enzymology. The 2'- and 3'-AADPR products are candidate molecules for SIR2-initiated signaling pathways.

Novel oxorhenium and oxotechnetium complexes based on the tetradentate 1-(2-hydroxybenzamido)-2-(pyridinecarboxamido)benzene, H3L, ligand have been synthesized and characterized herein. Thus, by reacting equimolar quantities of the triply deprotonated ligand L3- with the suitable MO3+ precursor, the following neutral MOL complexes could be easily produced following similar synthetic routes: M = Re (1), M = 99gTc (2), and M = 99mTc (3). Complexes 1 and 2, prepared in macroscopic amounts, were chemically characterized and their structure determined by single-crystal X-ray analysis. They are isostructural metal chelates, adopting a distorted square pyramidal geometry around the metal. The N3O donor atom set of the tetradentate ligand defines the basal plane and the oxygen atom of the M = O core occupies the apex of the pyramid. Complex 3 forms quantitatively at tracer level by mixing the H3L ligand with Na99mTcO4 generator eluate in aqueous alkaline media and using tin chloride as reductant in the presence of citrate. Its structure was established by chromatographic comparison with prototypic complexes 1 and 2 using high-performance liquid chromatographic techniques. When challenged with excess glutathione in vitro, complex 3 is rapidly converted to hydrophilic unidentified metal species. Tissue distribution data after administration of complex 3 in vivo revealed a significant uptake and retention of this compound in brain tissue.

The synthesis and processing of the precursor for neuropeptide Y (NPY) were studied in 16 human and murine neuroendocrine cell lines. Eight of the cell lines, NS-20Y, PC12, LA-N-5, CHP-234, SMS-KCNR, SH-SY5Y, SMS-KCN, and BE(2)-M17, produced sufficient quantities to permit chromatographic characterization of the NPY immunoreactivity. Although the cell lines varied in the amount of NPY they produced, both within and between cell lines, they displayed a relatively constant pattern of posttranslational modifications. In contrast to observations in tumor extracts (M. M. T. O'Hare and T. W. Schwartz, Cancer Res., 49: 7010-7014, 1989), all cell lines studied contained a substantial amount of the intracellular NPY in the form of the unprocessed propeptide, 57% (range, 33-72%) as characterized by both gel filtrations (32 experiments in 8 cell lines) and "in vitro conversion" with endoproteinase Lys-C. In the majority, 4 of 6 cell lines studied, almost all of the NPY, which by size corresponded to the mature 36-amino acid form, was amidated as assessed by isoelectric focusing and by a radioimmunoassay specific for the COOH-terminal amide group of the peptide. Both the propeptide and smaller molecular forms of NPY were secreted from the cell cultures; however, proteolytic degradation in the tissue culture medium prevented a detailed, meaningful characterization of these peptides. It is concluded that many neuroendocrine cell lines, especially those derived from human neuroblastomas, express the NPY gene; the cells display a partly impaired dibasic processing capacity but they generally amidate the products efficiently.

Peptidylglycine alpha-amidating monooxygenase is a copper- and ascorbate-dependent enzyme that converts peptides with COOH-terminal glycine residues into the corresponding alpha-amidated product peptides. The relatively selective copper chelator N,N-diethyldithiocarbamate (DDC) and its disulfide dimer, disulfiram (Antabuse), were used to determine whether the availability of copper affects the production of two alpha-amidated pro-ACTH/endorphin-derived peptides, alpha-melanotropin (alpha MSH) and joining peptide. When mouse pituitary corticotropic tumor cells (AtT-20) were grown in medium containing micromolar concentrations of disulfiram or DDC, alpha-amidation of newly synthesized joining peptide was specifically inhibited in a dose-dependent manner. In rats injected twice with disulfiram or DDC, the ability of the intermediate pituitary to alpha-amidate newly synthesized alpha MSH and joining peptide was inhibited in a dose-dependent manner; at disulfiram doses equivalent to those used in alcohol abuse therapy (4 mg/kg/day), only about 10% of the newly synthesized peptides were correctly alpha-amidated. Chronic treatment of rats with DDC or disulfiram produced a dose-dependent increase in the pituitary content of glycine-extended alpha MSH and joining peptide; the total amount of pro-ACTH/endorphin-related material was unaltered. After 11 days of treatment with 4 mg/kg/day disulfiram, about one-third of the pituitary alpha MSH and joining peptide were present in the glycine-extended rather than the alpha-amidated form; pituitary extracts normally contain almost entirely alpha-amidated peptides.

In previous studies we have demonstrated a secretory granule-associated peptide alpha-amidation activity in rat anterior, intermediate, and posterior pituitary. This activity is capable of converting 125I-labeled synthetic D-Tyr-Val-Gly to labeled D-Tyr-Val-NH2, and requires ascorbic acid, CuSO4, and molecular oxygen for optimal activity. Because of the requirement for peptides with COOH-terminal glycine residues, and cofactor requirements similar to monooxygenases such as dopamine beta-monooxygenase, we have proposed that the alpha-amidating enzyme be named peptidylglycine alpha-amidating monooxygenase, or PAM. The present study focused on (i) verifying that PAM could utilize a physiologically relevant peptide substrate, and (ii) demonstrating the retention of the cofactor requirements with purification of PAM. PAM (Mr = 50,000) was partially purified from rat anterior pituitary secretory granules and was shown to be capable of converting alpha-N-acetyl-ACTH(1-14) to alpha-N-acetyl-ACTH(1-13)NH2 (alpha-melanocyte stimulating hormone) and ACTH(9-14) to ACTH(9-13)NH2. The optimal rates for these conversions were dependent on ascorbic acid and CuSO4. Kinetic analyses, using the model compound D-Tyr-Val-Gly as the peptide substrate, demonstrated that, compared to the crude granule extract, the partially purified enzyme displayed increased apparent affinities for both the peptide substrate and ascorbate. These analyses also showed that the Km for D-Tyr-Val-Gly was dependent on the concentration of ascorbate, while the Km for ascorbate was constant over a wide range of D-Tyr-Val-Gly concentrations. The results presented here indicate that PAM can alpha-amidate physiologically relevant peptides related to alpha MSH, and performs the reaction in an ascorbate-dependent fashion. Retention of the ascorbate and copper requirements with purification further support the hypothesis that these cofactors are important requirements for the COOH-terminal alpha-amidation of neuro and endocrine peptides.