title: An application of RP-HPLC for determination of the activity of cystathionine β-synthase and γ-cystathionase in tissue homogenates.
abstract: The RP-HPLC-based method of determination of the activity of cystathionine β-synthase and γ-cystathionase was undertaken in mouse liver, kidney and brain. Products of the reactions, such as cystathionine, α-ketobutyrate, cysteine and glutathione, were measured using the RP-HPLC method. A difference in the cystathionine level between homogenates with totally CTH-inhibiting concentrations of DL-propargylglycine and without the inhibitor was employed to evaluate the activity of cystathionine β-synthase. Gamma-cystathionase activity was measured using DL-homoserine as a substrate and a sensitive HPLC-based assay to measure α-ketobutyrate. The results confirmed high cystathionine β-synthase activity and no γ-cystathionase activity in brain, and high γ-cystathionase activity in mouse liver. The method presented here allows for evaluating the relative contribution of CBS and CTH to generation of H2S in tissues. Additionally, it provides results, which reflect the redox status (GSH/GSSG) of a tissue.
authors.affiliation: Collegium Medicum, Jagiellonian University, Kopernika 7, 31-034 Kraków, Poland.
authors.forename: Patrycja
authors.initials: P
authors.lastname: Bronowicka-Adamska
authors.affiliation: Collegium Medicum, Jagiellonian University, Kopernika 7, 31-034 Kraków, Poland.
authors.forename: Jacek
authors.initials: J
authors.lastname: Zagajewski
authors.affiliation: Collegium Medicum, Jagiellonian University, Kopernika 7, 31-034 Kraków, Poland. Electronic address: mbwrobel@cyf-kr.edu.pl.
authors.forename: Maria
authors.initials: M
authors.lastname: Wróbel
mesh_terms: D000480:Alkynes / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D003541:Cystathionine beta-Synthase / Q000032:analysis* / Q000378:metabolism; D003542:Cystathionine gamma-Lyase / Q000032:analysis* / Q000378:metabolism; D003545:Cysteine / Q000032:analysis / Q000378:metabolism; D019803:Glutathione Disulfide / Q000032:analysis / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D006714:Homoserine; D051379:Mice; D009928:Organ Specificity
chemical_list: D000480:Alkynes; C009055:propargylglycine; D006714:Homoserine; D003541:Cystathionine beta-Synthase; D003542:Cystathionine gamma-Lyase; D003545:Cysteine; D005998:Glycine; D019803:Glutathione Disulfide
keywords: Cystathionine; Cystathionine-beta-synthase; Gamma-cystathionase; H(2)S; Mouse tissue