Isocitrate dehydrogenase

alpha-PBP and alpha-PEP were provided by the Bavarian State Criminal Police Office (Munich, Germany). NADP-Na₂, acetonitrile (LC-MS grade), and methanol (LC-MS grade) were obtained from VWR (Darmstadt, Germany), MgCl₂, K₂HPO₄, K₃PO₄, superoxide dismutase, isocitrate dehydrogenase, isocitrate, ammonium formate, ammonium acetate, and formic acid from Sigma (Taufkirchen, Germany). Water was purified with a Millipore filtration unit (18.2 Ω × cm water resistance). pHLM (pool of 25 donors, 20 mg microsomal protein/mL) were obtained from Corning (Amsterdam, The Netherlands). After delivery, the pHLM were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.

Aconitase is an [4Fe-4S] cluster-containing TCA cycle protein that catalyzes the isomerization of citrate to isocitrate via cis-aconitate [58 (link)]. HEK293T cells were grown in T75 culture flasks until the cells were 90% confluent. The cells were harvested and lysed in nondenaturing lysis buffer [50 mM Tris-HCl and 1% NP-40 (pH 8)]. The protein concentration was determined via a Bradford assay. The aconitase activity was measured using 50 μL of cell lysate mixed with 250 μL of reaction buffer [50 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, 0.5 mM NADP+ and 0.05 unit of isocitrate-dehydrogenase (Sigma) (pH 8)]. This was incubated for 5 min at 37 °C before the addition of 200 μL of starting buffer (50 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl₂ and 2.5 mM cis-aconitate). The reaction was followed at 340 nm by the reduction of NADPH as ICDH converted the product of aconitase. The specific activity was calculated using the extinction coefficient of NADPH (ε340 = 6220 mM^–1) [59 (link)].

In-gel aconitase activities were measured as described previously (Holmes-Hampton et al., 2018 (link), Tong and Rouault, 2006 (link)). Aconitase activity gels are composed of a separating gel containing 8% acrylamide (132 mM Tris base, 132 mM borate, 3.6mM citrate) and a 4% acrylamide (67 mM Tris base, 67 mM borate, 3.6 mM citrate) stacking gel. The running buffer contains 25 mM Tris pH 8.3, 192 mM glycine, and 3.6 mM citrate. Protein concentration of macrophage lysates was measured by using the Pierce BCA Protein Assay Kit and the sample buffer contained 25 mM Tris-Cl, pH 8.0, 10% glycerol, and 0.025% bromophenol blue. Electrophoresis was carried out at 180 V, 4°C, for 2 hours. Aconitase activities were assayed by incubating the gel in the dark at 37°C in 100 mM Tris (pH 8.0), 1 mM NADP, 2.5 mM cis-aconitic acid, 5 mM MgCl₂, 1.2 mM MTT, 0.3 mM phenazine methosulfate, and 5 U/ml isocitrate dehydrogenase (Sigma).

Methoxyacetylfentanyl hydrochloride (purity ≥ 98%), U-51754 hydrochloride (purity 98%), and U-47931E were purchased from LGC Standards (Wesel, Germany). Isocitrate, Isocitrate dehydrogenase, superoxide dismutase, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), S-(5′-adenosyl)-L-methionine (SAM), dithiothreitol (DTT), reduced glutathione (GSH), acetyl carnitine, and acetyl coenzyme A (AcCoA) were all purchased from Sigma (Taufkirchen, Germany), NADP⁺ from Biomol (Hamburg, Germany), and acetonitrile (LC-MS grade), ammonium formate (analytical grade), formic acid (LC-MS grade), methanol (LC-MS grade), glucuronidase (EC No. 3.2.1.32)/arylsulfatase (EC No. 3.1.6.1) from Helix pomatia L, and all other chemicals and reagents (analytical grade) from VWR (Darmstadt, Germany). phS9 (20 mg protein/mL, from 30 individual donors), UGT reaction mix solution A (25 mM UDP-glucuronic acid), and UGT reaction mix solution B (250 mM Tris–HCl, 40 mM MgCl₂, and 0.125 mg/mL alamethicin) were obtained from Corning (Amsterdam, The Netherlands). After delivery, the enzyme preparations were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.

Ten micrograms of isolated mitochondria were resuspended in 170 µl of 100 mM triethanolamine buffer, pH 8.0, containing 1.2 mM NADP⁺ and 10 µL isocitrate dehydrogenase (Sigma-Aldrich, I2002) in a clear bottom 96-well plate (Falcon). Twenty microliters of cis-aconitate was added (final concentration 240 µM) and reduction of NADP⁺ was monitored at 340 nm for a total of 10 min. The rate of reaction was calculated from the slope of the linear part of the kinetic curve. As a control, activity was also measured in pure buffer or mitochondrial samples before and after the addition of cis-aconitate^{54 (link)}. Aconitase activity was normalized to citrate synthase activity and displayed as a percentage of WT activity.
For citrate synthase activity, 20 µg of isolated mitochondria were resuspended in 100 µl of 10 mM Tris-HCl buffer, pH 7.5, 0.2% Triton X-100 (v/v) and 200 µM of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), in a clear bottom 96-well plate (Falcon). Fifty microliters of acetyl-CoA (2 mM) was added to this solution. After 5 min of incubation, reaction was started by adding 50 µl of oxaloacetate (2 mM) and turn-over of acetyl-CoA was monitored by observing the absorbance at 412 nm for 10 min. The initial rate of reaction was calculated from the slope of the linear part of the kinetic curve^{55 (link)}.

A-CHMINACA was provided by the EU project ADEBAR/State Bureau of Criminal Investigation Schleswig-Holstein (Kiel, Germany) for research purpose. The chemical purity and identity of the compound were verified by MS and nuclear magnetic resonance analysis. Ammonium formate, ammonium acetate, formic acid, isocitrate dehydrogenase, isocitrate, dipotassium phosphate, tripotassium phosphate, magnesium chloride, and superoxide dismutase were obtained from Sigma (Taufkirchen, Germany). Acetonitrile (LC-MS grade), methanol (LC-MS grade), and NADP-Na₂ were from VWR (Darmstadt, Germany). pHLM (20 mg microsomal protein mL⁻¹) was obtained from Corning (Amsterdam, The Netherlands). After delivery, pHLM were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.