A continuous spectrophotometric assay for human cystathionine beta-synthase
ABSTRACT We report a new continuous spectrophotometric assay for human cystathionine beta-synthase (hCBS). This assay relies upon the finding that hCBS will take cysteamine in place of L-homocysteine, thereby producing thialysine. Thialysine is, in turn, decarboxylated by lysine decarboxylase, releasing CO2 that is monitored by the sequential action of phosphoenolpyruvate carboxylase and L-malate dehydrogenase. The decrease in absorbance at 340 nm is monitored as reduced nicotinamide adenine dinucleotide is consumed. Using this four-enzyme couple, we find that Km(app) = 1.2+/-0.2 mM for L-serine and 5.6+/-2.2 mM for cysteamine, with kcat = 1.3+/-0.1s(-1) for the formation of thialysine by hCBS. For comparison purposes, the same hCBS reaction was monitored via a radioactive single time point assay using 14C-(C-1)-labeled L-serine and cysteamine as substrates, counting the thialysine product, following ion exchange chromatography. This assay yielded Km(app) = 2.2+/-0.5 mM for L-serine and 6.6+/-2.2 for cysteamine, with kcat = 2.5+/-0.4 s(-1). These numbers indicate that, although it possesses a shortened carbon chain and lacks a carboxyl group, cysteamine displays a catalytic efficiency (kcat/Km) with hCBS that is within an order of magnitude of that observed with its natural thiol cosubstrate, L-homocysteine.
Full-textDOI: · Available from: David B Berkowitz, May 27, 2015
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ABSTRACT: Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD; systematic name: 1,2-3,4-5,6-7,8-octahydro-1,8a-diaza-4,6-dithiafluoren-9(8aH)-one) is a previously described metabolite of cysteamine that has been reported to be present in mammalian brain, urine, plasma, and cells in culture and vegetables and to possess potent antioxidative properties. Here, we describe a stable isotope gas chromatography-tandem mass spectrometry (GC-MS/MS) method for specific and sensitive determination of AECK-DD in biological samples. (13)C(2)-labeled AECK-DD was synthesized and used as the internal standard. Derivatization was carried out by N-pentafluorobenzylation with pentafluorobenzyl bromide in acetonitrile. Quantification was performed by selected reaction monitoring of the mass transitions m/z 328 to 268 for AECK-DD and m/z 330 to 270 for [(13)C(2)]AECK-DD in the electron capture negative ion chemical ionization mode. The procedure was systematically validated for human plasma and urine samples. AECK-DD was not detectable in human plasma above approximately 4nM but was present in urine samples of healthy humans at a maximal concentration of 46nM. AECK-DD was detectable in rat brain at very low levels of approximately 8pmol/g wet weight. Higher levels of AECK-DD were detected in mouse brain (∼1nmol/g wet weight). Among nine dietary vegetables evaluated, only shallots were found to contain trace amounts of AECK-DD (∼6.8pmol/g fresh tissue).Analytical Biochemistry 07/2012; 430(1):4-15. DOI:10.1016/j.ab.2012.07.022 · 2.31 Impact Factor
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ABSTRACT: Background: In order to correctly assess the efficacy of therapy or diet in intervention studies on the activity of cystathionine β-synthase (CBS) a sensitive analytical method is necessary. Methods: An electrospray LC-MS/MS method preceded by a solid phase extraction step was developed for the measurement of CBS activity in cell extracts. Nonafluoropentanoic acid was used as an ionpair to provide the underivatized cystathionine the desired retention on a C18 column. Results: A detection limit of 50pmol cystathionine/h/mg protein was achieved. In fibroblasts, intra- and inter-assay CVs for the CBS activity were 5.2% and 14.7%, respectively. A K(m) value of 8μmol/L for homocysteine, and 2.5μmol/L for serine was calculated. In fibroblasts wildtype, heterozygous, and homozygous CBS activity ranges measured were 8.5-27.0, 4.2-13.4, 0.0-0.7nmol/h×mg protein, respectively. The method was applied to a study where rats were fed 2 diets. Increase of dietary methionine (7.7 versus 3.8mg/kg methionine) significantly increased the CBS activity in rat liver lysates from a median of 58.0 to a median of 71.5 (P=0.037)nmol/h×mg protein. In a lymphoblasts cell culture experiment, the addition of Hcy to the culture media increased the activity of CBS 3 fold. Conclusion: This LC-MS/MS is able to diagnose CBS deficiency at the enzyme level, and can accurately measure the effect diets or therapy might have on the CBS activity in a variety of cell types.Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 12/2012; 911C:186-191. DOI:10.1016/j.jchromb.2012.10.041 · 2.69 Impact Factor