Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 3 de 3
Filter
Add more filters










Database
Language
Publication year range
1.
J Biol Chem ; 276(1): 215-24, 2001 Jan 05.
Article in English | MEDLINE | ID: mdl-11013238

ABSTRACT

Although the pseudohalide thiocyanate (SCN(-)) is the preferred substrate for eosinophil peroxidase (EPO) in fluids of physiologic halide composition, the product(s) of this reaction have not been directly identified, and mechanisms underlying their cytotoxic potential are poorly characterized. We used nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry, and quantitative chemical analysis to identify the principal reaction products of both the EPO/SCN(-)/H(2)O(2) system and activated eosinophils as roughly equimolar amounts of OSCN(-) (hypothiocyanite) and OCN(-) (cyanate). Red blood cells exposed to increasing concentrations of OSCN(-)/OCN(-) are first depleted of glutathione, after which glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase then ATPases undergo sulfhydryl (SH) reductant-reversible inactivation before lysing. OSCN(-)/OCN(-) inactivates red blood cell membrane ATPases 10-1000 times more potently than do HOCl, HOBr, and H(2)O(2). Exposure of glutathione S-transferase to [(14)C]OSCN(-)/OCN(-) causes SH reductant-reversible disulfide bonding and covalent isotope labeling. We propose that EPO/SCN(-)/H(2)O(2) reaction products comprise a potential SH-targeted cytotoxic system that functions in striking contrast to HOCl, the highly but relatively indiscriminantly reactive product of the neutrophil myeloperoxidase system.


Subject(s)
Eosinophils/enzymology , Erythrocytes/drug effects , Oxidants/metabolism , Oxidants/toxicity , Peroxidases/metabolism , Thiocyanates/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/metabolism , Cyanates/metabolism , Eosinophil Peroxidase , Eosinophils/drug effects , Eosinophils/metabolism , Erythrocytes/enzymology , Erythrocytes/metabolism , Glutathione/metabolism , Glutathione Transferase/antagonists & inhibitors , Glutathione Transferase/metabolism , Hemolysis/drug effects , Humans , Hydrogen Peroxide/metabolism , Hydrogen-Ion Concentration , Kinetics , Magnetic Resonance Spectroscopy , Neutrophils/enzymology , Neutrophils/metabolism , Oxidants/chemistry , Peroxidase/metabolism , Spectrometry, Mass, Electrospray Ionization , Temperature , Tetradecanoylphorbol Acetate/pharmacology , Thiocyanates/chemistry , Thiocyanates/toxicity
2.
Drug Metab Dispos ; 29(1): 60-9, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11124231

ABSTRACT

Omapatrilat, a potent vasopeptidase inhibitor, is currently under development for the treatment of hypertension and congestive heart failure. This study describes the plasma profile along with isolation and identification of urinary metabolites of omapatrilat from subjects dosed orally with 50 mg of [(14)C]omapatrilat. Only a portion of the radioactivity in plasma was unextractable (40-43%). Prominent metabolites identified in plasma were S-methyl omapatrilat, acyl glucuronide of S-methyl omapatrilat, and S-methyl (S)-2-thio-3-phenylpropionic acid. Omapatrilat accounted for less than 3% of the radioactivity. However, after dithiothreitol reduction all of the radioactivity was extractable and was characterized to be omapatrilat and its hydrolysis product (S)-2-thio-3-phenylpropionic acid, both apparently bound to proteins via reversible disulfide bonds. Urinary profile of radioactivity showed no parent compound but the presence of several metabolites that can be grouped into three categories. 1) Three metabolites, accounting for 56% of the urinary radioactivity, resulted from the hydrolysis of the exocyclic amide bond of omapatrilat. Two metabolites were diastereomers of S-methyl sulfoxide of (S)-2-thio-3-phenylpropionic acid, and the third was the acyl glucuronide of S-methyl (S)-2-thio-3-phenylpropionic acid. 2) One disulfide, identified as the L-cysteine mixed disulfide of omapatrilat, accounted for 8% of the radioactivity in the urine. 3) Five metabolites, derived from omapatrilat, accounted for 30% of the radioactivity in the urine. Two of these metabolites were mixtures of diastereomers of S-methyl sulfoxide of omapatrilat and the third was the S-methyl omapatrilat ring sulfoxide. The other two metabolites were S-methyl omapatrilat and its acyl glucuronide. These results indicate that omapatrilat undergoes extensive metabolism in humans.


Subject(s)
Enzyme Inhibitors/pharmacokinetics , Pyridines/pharmacokinetics , Thiazepines/pharmacokinetics , Angiotensin-Converting Enzyme Inhibitors/blood , Angiotensin-Converting Enzyme Inhibitors/pharmacokinetics , Angiotensin-Converting Enzyme Inhibitors/urine , Biotransformation , Carbon Radioisotopes , Chromatography, High Pressure Liquid , Enzyme Inhibitors/blood , Enzyme Inhibitors/urine , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Pyridines/blood , Pyridines/urine , Thiazepines/blood , Thiazepines/urine
3.
Biochemistry ; 29(22): 5245-58, 1990 Jun 05.
Article in English | MEDLINE | ID: mdl-2383544

ABSTRACT

We have previously suggested that variations in the 31P chemical shifts of individual phosphates in duplex oligonucleotides are attributable to torsional angle changes in the deoxyribose phosphate backbone. This hypothesis is not directly supported by analysis of the 1H/31P two-dimensional J-resolved spectra of a number of mismatch dodecamer oligonucleotide duplexes including the following sequences: d-(CGTGAATTCGCG), d(CGUGAATTCGCG), d(CGGGAATTCGCG), d(CGAGAATTCGCG), and d(CGCGAATTCACG). The 31P NMR signals of the dodecamer mismatch duplexes were assigned by 2D 1H/31P pure absorption phase constant time (PAC) heteronuclear correlation spectra. From the assigned H3' and H4' signals, the 31P signals of the base-pair mismatch dodecamers were identified. JH3'-P coupling constants for each of the phosphates of the dodecamers were obtained from 1H/31P J-resolved selective proton flip 2D spectra. By use of a modified Karplus relationship, the C4'-C3'-O3'-P torsional angles (epsilon) were obtained. JH3'-P coupling constants were measured for many of the oligonucleotides as a function of temperature. There exists a good linear correlation between 31P chemical shifts and the epsilon torsional angle. This correlation can be further extended to the C3'-O3'-P-O5' torsional angle (zeta) by using a linear relationship between epsilon and zeta obtained from crystal structure studies. The 31P chemical shifts follow the general observation that the more internally the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. In addition, 31P chemical shifts show sequence- and site-specific variations. Analysis of the backbone torsional angle variations from the coupling constant analysis has provided additional information regarding the origin of these variations in 31P chemical shifts.


Subject(s)
DNA , Oligodeoxyribonucleotides , Base Sequence , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemical synthesis , Phosphorus , Temperature
SELECTION OF CITATIONS
SEARCH DETAIL
...