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Biomedical subjects

P G Wang

Publications and source records attributed to P G Wang.

At least 19 recordsLinked to original sources
PubMed2001

Fluorophore-labeled S-nitrosothiols.

A series of fluorophore-labeled S-nitrosothiols were synthesized, and their fluorescence enhancements upon removal of the nitroso (NO) group were evaluated either by transnitrosation or by photolysis. It was shown that, with a suitable alkyl linker, the fluorescence intensity of dansyl-labeled S-nitrosothiols could be enhanced up to 30-fold. The observed fluorescence enhancement was attributed to the intramolecular energy transfer from fluorophore to the SNO moiety. Ab initio density functional theory (DFT) calculations indicated that the "overlap" between the SNO moiety and the dansyl ring is favored because of their stabilizing interaction, which was in turn affected by both the length of the alkyl linker and the rigidity of the sulfonamide unit. In addition, one of the dansyl-labeled S-nitrosothiols was used to explore the kinetics of S-nitrosothiol/thiol transnitrosation and was evaluated as a fluorescence probe of S-nitrosothiol-bound NO transfer in human umbilical vein endothelial cells.

Cell Membrane↗
PubMed2001

N-hydroxyl derivatives of guanidine based drugs as enzymatic NO donors.

Recent research suggests that NO may play a role in the physiological effects of some guanidine-containing drugs. In this report, three guanidine-containing drugs (guanadrel, guanoxan, and guanethidine) together with their N-hydroxyl derivatives were synthesized and their NO-releasing abilities catalyzed by nitric oxide synthases (NOSs) and horseradish peroxidase were evaluated. The guanidine containing compounds could not release NO in the presence of NOS or peroxidase. The corresponding N-hydroxyl compounds exhibited weak NO-releasing ability under the catalyzed of NOS and good NO-releasing ability under the oxidation by horseradish peroxidase in the presence of H(2)O(2). These compounds also displayed vasodilatory activity.

Animals↗
PubMed2001

Identification and characterization of GDP-d-mannose 4,6-dehydratase and GDP-l-fucose snthetase in a GDP-l-fucose biosynthetic gene cluster from Helicobacter pylori.

In this study two open reading frames, namely HP0044 and HP0045 from H. pylori, were cloned and overexpressed in E. coli. The two recombinant proteins were demonstrated to have GDP-d-mannose 4,6-dehydratase (GMD) and GDP-l-fucose synthetase (GFS) activities, respectively. The recombinant GMD was a tetramer and had an optimum pH of 6.5. Exogenous NADP(+) was essential for its activity. The K(m) and K(cat) for GDP-d-mannose were 117.3 microM and 0.27 s(-1), respectively. The recombinant GFS was a homodimer with an optimum pH of 8.0. The K(m) and K(cat) for GDP-4-keto-6-deoxy-d-mannose were 64.08 microM and 0.75 s(-1), respectively. It can use both NADPH and NADH, but less efficient with the latter. Amino acid sequence alignment and phylogenetic analysis showed that H. pylori GFS was highly homologous to the GFS of E. coli O111 and both of them were located on a separate phylogenetic branch from other GFS. The unique clustering and origin of the two genes were also discussed.

Amino Acid Sequence↗
PubMed2001

Equilibrium and kinetics studies of transnitrosation between S-nitrosothiols and thiols.

Using UV-vis spectrometrical measurements, equilibrium constants for NO transfer between S-nitroso-N-acetyl-penicillamine (SNAP) and different thiols as well as kinetic data for NO transfer from S-nitroso bovine serum albumin (BSANO) to thiols have been obtained. NO transfer from SNAP to other primary/secondary thiols are thermodynamically favorable, whereas other S-nitrosothiols exhibit similar NO transfer potential. The obtained Gibbs free energy, enthalpy and entropy data indicated that NO transfer reactions from SNAP to four thiols are exothermic with entropy loss. The kinetic behavior of BSANO/RSH transfer can be related to both the acidity of sulfhydryl group and the electronic structure in thiol.

Kinetics↗
PubMed2001

N-nitrosoanilines: a new class of caspase-3 inhibitors.

Caspases are a family of cysteine proteases activated during apoptosis. In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. Previous studies have demonstrated that NO inhibits the activity of caspase-3 by S-nitrosylation of the enzyme. In this study, the inhibitory effect of a new class of NO donors. N-nitrosoaniline derivatives, were examined against caspase-3. Initially eight small molecule inhibitors bearing N-nitroso moieties were assayed. It was found that the presence of an electron-donating group on the phenyl ring led to better inhibitory potency, a trend consistent with the results from the previous papain studies. Based on the analysis of the enzyme and substrates' structures, two peptidyl N-nitrosoaniline inhibitors [Ac-DVAD-NNO (1) and Ac-DV-AMO (2)] were designed and synthesized. Both compounds exhibited enhanced inhibitory potency against caspase-3.

Aniline Compounds↗
PubMed2001

Alpha 1,3 galactosyltransferase: new sequences and characterization of conserved cysteine residues.

Nucleotide sequences were determined for alpha1,3 galactosyltransferases (alpha1,3 GalTs) from several species (bat, mink, dog, sheep, and dolphin) and compared with those previously determined for this enzyme and members of the alpha1,3 galactosyl/N-acetylgalactosyltransferase (alpha1,3 Gal(NAc)Ts) family of enzymes. Sequence comparison of the newly characterized alpha1,3 GalT nucleotide and predicted amino acid sequences with those previously characterized for other alpha1,3GalT enzymes demonstrated a remarkable level of sequence identity at the nucleotide and amino acid level. The identity of each sequence as an alpha1,3 GalT was confirmed by expressing the encoded protein and characterizing the resulting enzyme. The alpha1,3 GalTs have a significant degree of sequence homology with A and B transferases, the alpha1,3 GalNAcT that catalyzes the synthesis of Forssman antigen, and the recently cloned iso-globotriaosylceramide synthase. Among the conserved residues, there are two Cys residues. To determine if these conserved residues are free or involved in the formation of a disulfide bond, bovine alpha1,3 GalT was characterized by chemical modification and mass spectrometry. Each peptide containing a Cys residue was chemically labeled with an alkylating reagent demonstrating that these enzymes do not contain disulfide bonds. Similar results have recently been reported for A and B transferases (Yen et al., 2000, J. Mass. Spectrom., 35, 990-1002). Thus, the highly conserved Cys residues found in these members of the alpha1,3 Gal(NAc)Ts family of enzymes are likely involved in other important aspects of enzyme structure/function within this enzyme family.

Amino Acid Sequence↗
PubMed2000

Specificity and mechanism of metal ion activation in UDP-galactose:beta -galactoside-alpha -1,3-galactosyltransferase.

UDP-galactose:beta-galactosyl-alpha1,3-galactosyltransferase (alpha3GT) catalyzes the synthesis of galactosyl-alpha-1,3-beta-galactosyl structures in mammalian glycoconjugates. In humans the gene for alpha3GT is inactivated, and its product, the alpha-Gal epitope, is the target of a large fraction of natural antibodies. alpha3GT is a member of a family of metal-dependent-retaining glycosyltransferases that includes the histo blood group A and B enzymes. Mn(2+) activates the catalytic domain of alpha3GT (alpha3GTcd), but the affinity reported for this ion is very low relative to physiological levels. Enzyme activity over a wide range of metal ion concentrations indicates a dependence on Mn(2+) binding to two sites. At physiological metal ion concentrations, Zn(2+) gives higher levels of activity and may be the natural cofactor. To determine the role of the cation, metal activation was perturbed by substituting Co(2+) and Zn(2+) for Mn(2+) and by mutagenesis of a conserved D(149)VD(151) sequence motif that is considered to act in cation binding in many glycosyltransferases. The aspartates of this motif were found to be essential for activity, and the kinetic properties of a Val(150) to Ala mutant with reduced activity were determined. The results indicate that the cofactor is involved in binding UDP-galactose and has a crucial influence on catalytic efficiency for galactose transfer and for the low endogenous UDP-galactose hydrolase activity. It may therefore interact with one or more phosphates of UDP-galactose in the Michaelis complex and in the transition state for cleavage of the UDP to galactose bond. The DXD motif conserved in many glycosyltransferases appears to have a key role in metal-mediated donor substrate binding and phosphate-sugar bond cleavage.

Animals↗
PubMed2000

Synthesis of alpha-Gal epitope derivatives with a galactosyltransferase-epimerase fusion enzyme.

Alpha-Gal epitopes are carbohydrate structures bearing an alpha-D-Galp-(1-->3)-beta-D-Galp terminus and are the main cause of antibody-mediated hyperacute rejection in xenotransplantation. Nine monosaccharides and ten disaccharides were evaluated as substrates for a fusion protein, which contains both alpha-(1-->3)-galactosyltransferase and uridine-5'-diphosphogalactose 4-epimerase. Four disaccharide and six trisaccharide alpha-Gal epitope derivatives were synthesized utilizing this novel fusion enzyme.

Animals↗
PubMed2000

Changing the donor cofactor of bovine alpha 1, 3-galactosyltransferase by fusion with UDP-galactose 4-epimerase. More efficient biocatalysis for synthesis of alpha-Gal epitopes.

Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC ) and alpha1, 3-galactosyltransferase (EC ) with an N-terminal His(6) tag and an intervening three-glycine linker were constructed by in-frame fusion of the Escherichia coli galE gene either to the 3' terminus (f1) or to the 5' terminus (f2) of a truncated bovine alpha1, 3-galactosyltransferase gene, respectively. Both fusion proteins were expressed in cell lysate as active, soluble forms as well as in inclusion bodies as improperly folded proteins. Both f1 and f2 were determined to be homodimers, based on a single band observed at about 67 kDa in SDS-polyacrylamide gel electrophoresis and on a single peak with a molecular mass around 140 kDa determined by gel filtration chromatography for each of the enzymes. Without altering the acceptor specificity of the transferase, the fusion with the epimerase changed the donor requirement of alpha1, 3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Galalpha1,3Gal-terminated oligosaccharides by more than 40-fold. For enzymatic synthesis of Galalpha1,3Galbeta1,4Glc from UDP-glucose and lactose, the genetically fused enzymes f1 and f2 exhibited kinetic advantages with overall reaction rates that were 300 and 50%, respectively, higher than that of the system containing equal amounts of epimerase and galactosyltransferase. These results indicated that the active sites of the epimerase and the transferase in fusion enzymes were in proximity. The kinetic parameters suggested a random mechanism for the substrate binding of the alpha1, 3-galactosyltransferase. This work demonstrated a general approach that fusion of a glycosyltransferase with an epimerase can change the required but expensive sugar nucleotide to a less expensive one.

Animals↗
PubMed2000

S-nitrosothiols as novel, reversible inhibitors of human rhinovirus 3C protease.

Human rhinovirus (HRV) 3C protease was inactivated by a series of S-nitrosothiols. These compounds exhibited different inhibitory activities in a time- and concentration-dependent manner with second-order rate constants (kinact/K(I)) ranging from 131 to 5360 M(-1) min(-1). The inactive enzyme could be re-activated by DTT, GSH and ascorbate, which indicated the inactivation mechanism was through an S-transnitrosylation process.

3C Viral Proteases↗
PubMed2000

O-Alkylation of cupferron: aiming at the design and synthesis of controlled nitric oxide releasing agents.

O-Alkylation of N-nitroso-N-phenylhydroxylamine ammonium salt (cupferron) was studied for the synthesis of novel nitric oxide (NO) releasing agents. The alkylation occurred regioselectively at the terminal oxygen, leading to a single product N-(alkyloxy)-N'-phenyldiimide N'-oxide as indicated by NMR and X-ray analysis. The O-alkyl derivatives exhibited significantly improved stability compared to their parent compound, cupferron. It was demonstrated that the cupferron O-alkyl derivatives could function as photoreleasing NO donor compounds. N-(N"-acetylphenylalanylmethylenyloxy)-N'-phenyldiimide N'-oxide), which linked the cupferron portion with an amino acid via an acetal moiety, was synthesized as an model NO prodrug where controlled NO release would occur either by increasing pH or by a protease-catalyzed hydrolysis.

Alkylation↗
PubMed2000

Inhibition of papain by S-nitrosothiols. Formation of mixed disulfides.

S-Nitrosylation of protein thiols is one of the cellular regulatory mechanisms induced by NO. The cysteine protease papain has a critical thiol residue (Cys(25)). It has been demonstrated that NO or NO donors such as sodium nitroprusside and N-nitrosoaniline derivatives can reversibly inhibit this enzyme by S-NO bond formation in its active site. In this study, a different regulated mechanism of inactivation was reported using S-nitrosothiols as the NO donor. Five S-nitroso compounds, S-nitroso-N-acetyl-dl-penicillamine, S-nitrosoglutathione, S-nitrosocaptopril, glucose-S-nitroso-N-acetyl-dl-penicillamine-2, and the S-nitroso tripeptide acetyl-Phe-Gly-S-nitrosopenicillamine, exhibited different inhibitory activities toward the enzyme in a time- and concentration-dependent manner with second-order rate constants (k(i)/K(I)) ranging from 8.9 to 17.2 m(-1) s(-1). The inhibition of papain by S-nitrosothiol was rapidly reversed by dithiothreitol, but not by ascorbate, which could reverse the inhibition of papain by NOBF(4). Incubation of the enzyme with a fluorescent S-nitroso probe (S-nitroso-5-dimethylaminonaphthalene-1-sulfonyl) resulted in the appearance of fluorescence of the protein, indicating the formation of a thiol adduct. Moreover, S-transnitrosylation in the incubation of S-nitroso inactivators with papain was excluded. These results suggest that inactivation of papain by S-nitrosothiols is due to a direct attack of the highly reactive thiolate (Cys(25)) in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between the inactivator and papain.

Binding Sites↗
PubMed2000

Inhibition of protein tyrosine phosphatases by low-molecular-weight S-nitrosothiols and S-nitrosylated human serum albumin.

The homogeneous recombinant mammalian protein tyrosine phosphatase 1B (PTP1B) and Yersinia protein tyrosine phosphatase (PTPase) are inactivated by a series of low-molecular-weight S-nitrosothiols. These compounds exhibited different inhibitory activities in a time- and concentration-dependent manner with second-order rate constants (k(inact)/K(I)) ranging from 37 to 113 M(-1) min(-1) against mammalian PTP1B and from 66 to 613 M(-1) min(-1) against Yersinia PTPase. Furthermore, the inactivation of Yersinia PTPase by S-nitrosylated protein:S-nitroso human serum albumin was investigated. Both single-S-nitrosylated and poly-S-nitrosylated human serum albumin show good inhibitory ability to Yersinia PTPase. The second-order rate constants are 472 and 1188 M(-1) min(-1), respectively. This result indicates a possibility that S-nitrosylated albumin in vivo may function as an inhibitor for a variety of cysteine-dependent enzymes.

Enzyme Inhibitors↗
PubMed2000

para-Substituted N-nitroso-N-oxybenzenamine ammonium salts: a new class of redox-sensitive nitric oxide releasing compounds.

N-Nitroso-N-oxybenzenamine ammonium salts with -OMe, -Me, -H, -F, -Cl, -CF3, and -SO2Me substituents at the para position of the phenyl ring constitute a new class of-redox sensitive nitric oxide (NO) releasing compounds. These compounds yield nitric oxide and the corresponding nitrosobenzene derivatives by a spontaneous dissociation mechanism after undergoing a one electron oxidation. Oxidation of these compounds can be achieved through chemical, electrochemical and enzymatic methods. It was observed electrochemically that the amount of NO generated was dependent on the substituent effect and the applied oxidation potential. Electron-withdrawing substituents increase the oxidation potential of the compound. A linear correlation was observed when the peak potentials for the oxidation were graphed versus the Hammett substituent constant. Density functional theory calculations were also performed on this series of compounds. The theoretical oxidation energies of the corresponding anions show a strong linear correlation with the experimental potentials. Furthermore, enzymatic oxidation using horseradish peroxidase showed a similar substituent effect. These results indicate that substitution at the para position of the phenyl ring has a profound effect on the stability, oxidation potential and enzymatic kinetic properties of the compounds. Thus para-substituted N-nitroso-N-oxybenzenamine salts comprise a new class of redox-sensitive nitric oxide releasing agents.

Electrochemistry↗