Dl isocitrate

Phenacetin, acetaminophen, 2-acetamidophenol, coumarin, 7-hydroxy-comuarin (umbelliferone), flurbiprofen, dextromethorphan, dextrorphan, trazodone, thymol, bupropion, 6-hydroxy-bupropion, alprazolam, 1′-hydroxy-alprazolam, NADP⁺, isocitrate dehydrogenase, and DL-isocitrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pronethalol was from Tocris (Minneapolis, MN, USA). Propofol was provided by Zeneca Pharmaceuticals (Wilmington, DE, USA). 4′-hydroxy-flurbiprofen, 2-fluoro-4-biphenyl-acetic acid, 4-hydroxy-propofol, chlorzoxazone, and 6-hydroxy-chlorzoxazone were purchased from Toronto Research Chemicals (Toronto, ON, Canada). S-Mephenytoin and 4-hydroxymephenytoin were purchased from Gentest (Corning, Corning, NY, USA). GW340416A, a chemical analogue of bupropion, was kindly provided by GlaxoSmithKline (Research Triangle Park, NC, USA). Omeprazole was purchased from BeanTown Chemical, Inc. (Hudson, NH, USA) and omeprazole sulfone was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Sodium hydroxide, potassium phosphate monobasic and potassium phosphate dibasic and EDTA were purchased from J.T. Baker (Center Valley, PA, USA). HPLC-grade acetonitrile and methanol were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ultra-pure water was obtained using a Milli-Q^® Q-POD Millipore System (EMD Millipore, Burlington, MA, USA).

MDH and ICDH activities were determined in a 1-mL reaction buffer containing 32.5 mM Tris/HCl, pH 7.5, at indicated molar concentrations of NaCl or 2.5 M GB (pH adjusted with concentrated HCl). For MDH, 0.3 mM NADH was used with oxaloacetate at indicated concentrations. For ICDH, 1 mM MgCl₂, 0.8 mM NADP⁺ (Sigma-Aldrich) was used with DL-isocitrate at indicated concentrations (Sigma-Aldrich). Assays were started by addition of (freshly diluted) purified enzyme and followed by the decrease (MDH) or increase (ICDH) of the absorbance at 340 nm, resulting from the oxidation of NADH to NAD⁺ or reduction of NADP⁺ to NADPH, respectively. The protein concentrations were determined from the absorbance at 280 nm using the calculated extinction coefficient from the amino acid sequence and were confirmed by the microbiuret method using BSA as protein standard.