PubMed Health⌕ Search

Biomedical subjects

W K Alderton

Publications and source records attributed to W K Alderton.

8 recordsLinked to original sources
PubMed2001

Kinetics of CO binding to the haem domain of murine inducible nitric oxide synthase: differential effects of haem domain ligands.

The binding of CO to the murine inducible nitric oxide synthase (iNOS) oxygenase domain has been studied by laser flash photolysis. The effect of the (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)) cofactor L-arginine and several Type I L-arginine analogues/ligands on the rates of CO rebinding has been evaluated. The presence of BH(4) in the iNOS active site has little effect on the rebinding of protein-caged haem-CO pairs (geminate recombination), but decreases the bimolecular association rates 2-fold. Addition of L-arginine to the BH(4)-bound complex completely abolishes geminate recombination and results in a further 80-fold decrease in the overall rate of bimolecular association. Three of the Type I ligands, S-ethylisothiourea, L-canavanine and 2,5-lutidine, displaced the CO from the haem iron upon addition to the iNOS oxygenase domain. The Type I ligands significantly decreased the rate of bimolecular binding of CO to the haem iron after photolysis. Most of these ligands also completely abolished geminate recombination. These results are consistent with a relatively open distal pocket that allows CO to bind unhindered in the active site of murine iNOS in the absence of L-arginine or BH(4). In the presence of BH(4) and L-arginine, however, the enzyme adopts a more closed structure that can greatly reduce ligand access to the haem iron. These observations are discussed in terms of the known structure of iNOS haem domain and solution studies of ligand binding in iNOS and neuronal NOS isoenzymes.

Animals↗
PubMed2001

Nitric oxide synthases: structure, function and inhibition.

This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

Alternative Splicing↗
PubMed1998

Nitroarginine and tetrahydrobiopterin binding to the haem domain of neuronal nitric oxide synthase using a scintillation proximity assay.

Nitric oxide synthases (NOS) have a bidomain structure comprised of an N-terminal oxygenase domain and a C-terminal reductase domain. The oxygenase domain binds haem, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin) and arginine, is the site where nitric oxide synthesis takes place and contains determinants for dimeric interactions. A novel scintillation proximity assay has been established for equilibrium and kinetic measurements of substrate, inhibitor and cofactor binding to a recombinant N-terminal haem-binding domain of rat neuronal NOS (nNOS). Apparent Kd values for nNOS haem-domain-binding of arginine and Nomega-nitro-L-arginine (nitroarginine) were measured as 1.6 microM and 25 nM respectively. The kinetics of [3H]nitroarginine binding and dissociation yielded an association rate constant of 1.3x10(4) s-1.M-1 and a dissociation rate constant of 1.2x10(-4) s-1. These values are comparable to literature values obtained for full-length nNOS, suggesting that many characteristics of the arginine binding site of NOS are conserved in the haem-binding domain. Additionally, apparent Kd values were compared and were found to be similar for the inhibitors, L-NG-monomethylarginine, S-ethylisothiourea, N-iminoethyl-L-ornithine, imidazole, 7-nitroindazole and 1400W (N-[3-(aminomethyl) benzyl] acetamidine). [3H]Tetrahydrobiopterin bound to the nNOS haem domain with an apparent Kd of 20 nM. Binding was inhibited by 7-nitroindazole and stimulated by S-ethylisothiourea. The kinetics of interaction with tetrahydrobiopterin were complex, showing a triphasic binding process and a single off rate. An alternating catalytic site mechanism for NOS is proposed.

Animals↗
PubMed1997

Cysteine-200 of human inducible nitric oxide synthase is essential for dimerization of haem domains and for binding of haem, nitroarginine and tetrahydrobiopterin.

Nitric oxide synthase (EC 1.14.13.39) is a homodimer. Limited proteolysis has previously shown that it consists of two major domains. The C-terminal or reductase domain binds FMN, FAD and NADPH. The N-terminal or oxygenase domain is known to bind arginine, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin) and haem. The exact residues of the inducible nitric oxide synthase (iNOS) protein involved in binding to these molecules have yet to be identified, although the haem moiety is known to be co-ordinated through a cysteine thiolate ligand. We have expressed two forms of the haem-binding domain of human iNOS (residues 1-504 and 59-504) in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The iNOS 1-504 and 59-504 fusion proteins bound similar amounts of haem, Nomega-nitro-l-arginine (nitroarginine) and tetrahydrobiopterin, showing that the first 58 residues are not required for binding these factors. Using site-directed mutagenesis we have mutated Cys-200, Cys-217, Cys-228, Cys-290, Cys-384 and Cys-457 to alanine residues within the iNOS 59-504 haem-binding domain. Mutation of Cys-200 resulted in a complete loss of haem, nitroarginine and tetrahydrobiopterin binding. Mutants of Cys-217, Cys-228, Cys-290, Cys-384 or Cys-457 showed no effect on the haem content of the fusion protein, no effect on the reduced CO spectral peak (444 nm) and were able to bind nitroarginine and tetrahydrobiopterin at levels equivalent to the wild-type fusion protein. After removal of the GST polypeptide, the wild-type iNOS 59-504 domain was dimeric, whereas the C200A mutant form was monomeric. When the mutated domains were incorporated into a reconstructed full-length iNOS protein expressed in Xenopus oocytes, only the Cys-200 mutant showed a loss of catalytic activity: all the other mutant iNOS proteins showed near wild-type enzymic activity. From this systematic approach we conclude that although Cys-217, Cys-228, Cys-290, Cys-384 and Cys-457 are conserved in all three NOS isoforms they are not essential for cofactor or substrate binding or for enzymic activity of iNOS, and that Cys-200 provides the proximal thiolate ligand for haem binding in human iNOS.

Animals↗
PubMed1995

Affinity labeling of recombinant ricin A chain with Procion blue MX-R.

Recombinant ricin A chain was irreversibly modified by Procion blue MX-R, a dichlorotriazinyl analogue of Cibacron blue F3G-A, at pH 7.5 and 4 degrees C in 90 h with over 95% loss of activity in an in vitro translation assay. The presence of total yeast RNA reduced the covalent attachment of Procion blue MX-R to ricin A chain. Quantitatively modified ricin A chain contained 2 mol Procion blue MX-R/mol 29-kDa subunit. Tryptic digestion and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a blue peptide corresponding to Gln5-Arg26 of ricin A chain. Thus, a likely dye-binding site on recombinant ricin A was identified. This region is removed from the active-site cleft of recombinant ricin A but may be involved in its substrate binding.

Affinity Labels↗
PubMed1995

Mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine against herpesviruses.

The activity, metabolism, and mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) were studied. Compared to acyclovir (ACV), H2G has superior activity against VZV (50% inhibitory concentration of 2.3 microM) and Epstein-Barr virus (50% inhibitory concentration of 0.9 microM), comparable activity against HSV-1, and weaker activity against HSV-2. The antiviral effect on HSV-1 showed persistence after removal of compound. H2G was metabolized to its mono-, di- and triphosphate derivatives in virus-infected cells, with H2G-triphosphate being the predominant product. Only small amounts of H2G-triphosphate were detected in uninfected cells (1 to 10 pmol/10(6) cells), whereas the level in HSV-1-infected cells reached 1,900 pmol/10(6) cells. H2G was a substrate for all three viral thymidine kinases and could also be phosphorylated by mitochondrial deoxyguanosine kinase. The intracellular half-life of H2G-triphosphate varied in uninfected (2.5 h) and infected (HSV-1, 14 h; VZV, 3.7 h) cells but was always longer than the half-life of ACV-triphosphate (1 to 2 h). H2G-triphosphate inhibited HSV-1, HSV-2, and VZV DNA polymerases competitively with dGTP (Ki of 2.8, 2.2, and 0.3 microM, respectively) but could not replace dGTP as a substrate in a polymerase assay. H2G was not an obligate chain terminator but would only support limited DNA chain extension. Only very small amounts of radioactivity, which were too low to be identified by high-performance liquid chromatography analysis of the digested DNA, could be detected in purified DNA from uninfected cells incubated with [3H]H2G. Thus, H2G acts as an anti-herpesvirus agent, particularly potent against VZV, by formation of high concentrations of relatively stable H2G-triphosphate, which is a potent inhibitor of the viral DNA polymerases.

Antiviral Agents↗
PubMed1992

Designer dyes: 'biomimetic' ligands for the purification of pharmaceutical proteins by affinity chromatography.

Affinity chromatography has been extensively refined over the past few years to meet the more stringent criteria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in process-scale protein purification. Exploitation of sophisticated molecular modelling techniques in conjunction with binding and crystallographic studies has permitted the design of new, highly selective 'biomimetic' ligands for the target proteins.

Binding Sites↗