Bovine 5-oxo-L-prolinase: simple assay method, purification, cDNA cloning, and detection of mRNA in the coronary artery.
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Watanabe T, Abe K, Ishikawa H, Iijima Y
Bovine 5-oxo-L-prolinase: simple assay method, purification, cDNA cloning, and detection of mRNA in the coronary artery.
Biol Pharm Bull. 2004 Mar;27(3):288-94.
- PubMed ID
- 14993790 [ View in PubMed]
- Abstract
RS-7897 is a novel antianginal nitrate containing an L-2-oxothiazolidine-4-carboxylic acid (OTCA) and unlike other organic nitrates does not induce nitrate tolerance. OTCA is known to be converted to L-cysteine (L-Cys) by rat 5-oxo-prolinase (5-OPase) and was detected in the plasma of RS-7897-treated dogs. Nitrate tolerance is considered to develop mainly through sulfhydryl depletion. We hypothesized that RS-7897-derived OTCA was converted into L-Cys by 5-OPase and supplied sulfhydryl groups in vascular smooth muscle cells, the targets of the nitrate. As the initial step for clarifying the presumed role of 5-OPase in RS-7897 metabolism, we established a non-radiochemical assay method highly specific for 5-OPase. Using this assay method, we purified 5-OPase from bovine kidney cytosol until homogeneous results were obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The catalytic activity of bovine 5-OPase to convert OTCA to L-Cys was confirmed, as was the case for the rat enzyme. Then the cDNA encoding bovine 5-OPase was cloned. The deduced amino acid sequence revealed that bovine 5-OPase was highly homologous to rat 5-OPase. Based on the bovine cDNA sequence, oligonucleotide primers were synthesized and used for RT-PCR. 5-OPase mRNA was significantly detected in the RT-PCR product of the bovine coronary artery, a major target organ of RS-7897. These results suggest that OTCA may be converted to L-Cys by 5-OPase in the mammalian coronary artery when treated with RS-7897, and thus generated L-Cys may reduce sulfhydryl depletion and contribute to the prevention of the development of nitrate tolerance.