Bovine 5-oxo-L-prolinase: simple assay method, purification, cDNA cloning, and detection of mRNA in the coronary artery.
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.
Watanabe
Tohru
T
Pharmacology and Molecular Biology Research Laboratories, Sankyo Co., Ltd., Hiromachi, Tokyo, Japan. tohruw@sankyo.co.jp
Abe
Koji
K
Ishikawa
Hirokazu
H
Iijima
Yasuteru
Y
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D003331:Coronary Vessels / Q000187:drug effects* / Q000201:enzymology; D003545:Cysteine / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009566:Nitrates / Q000494:pharmacology*; D011761:Pyrrolidonecarboxylic Acid; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013844:Thiazoles / Q000378:metabolism / Q000494:pharmacology*; D053778:Thiazolidines; D014665:Vasodilator Agents / Q000494:pharmacology*
C494609:4-N-(N-2-nitroxyethyl)-2-oxothiazolidine-4-carboxamide; D018076:DNA, Complementary; D009566:Nitrates; D012333:RNA, Messenger; D013844:Thiazoles; D053778:Thiazolidines; D014665:Vasodilator Agents; D000581:Amidohydrolases; C118889:5-oxoprolinase (ATP-hydrolyzing); D003545:Cysteine; D011761:Pyrrolidonecarboxylic Acid; C029536:2-oxothiazolidine-4-carboxylic acid