L lactate dehydrogenase ldh

Racemic HMG and CHA were synthesized and purified as described previously [21 (link)]. L-lactate dehydrogenase (LDH, rabbit muscle), L-malate dehydrogenase (MDH, porcine heart), and oxaloacetic acid were from Sigma-Aldrich (Oakville, ON). Pfu polymerase was from Invitrogen (Burlington, ON). All other chemicals were analytical grade and were obtained from either Sigma-Aldrich or Fisher Scientific unless otherwise stated.

An MPST activity was assayed according to the method of Valentine and Frankenfeld [44 (link)]. The incubation mixture contained: 250 μL of 0.12 M sodium phosphate buffer, pH 8.0, 50 μL of 0.5 M sodium sulfate (Sigma-Aldrich, Poznan, Poland), 50 μL of 0.15 M D, L-dithiothreitol (DTT, Sigma-Aldrich, Poznan, Poland), 50 μL of distilled water, 50 μL of supernatant and 50 μL of 0.1 M 3-mercaptopyruvate acid sodium salt (Sigma-Aldrich, Poznan, Poland) of the final volume of 500 μL. The mixture was incubated for 15 min, then 250 μL of 1.2 M PCA was added to stop the reaction. Samples were centrifuged at 1600× g for 5 min, and then 100 μL of supernatant was transferred to a 1350 μL solution containing: 1200 μL of 0.12 M sodium phosphate buffer, pH 8.0, 100 μL of 0.1 M N-ethylmaleimide (NEM, Sigma-Aldrich, Poznan, Poland) and 50 μL of β-Nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH, Sigma-Aldrich, Poznan, Poland) (5 mg/mL). After equilibration at 37 °C, 2.5 μL (7 IU) of L-lactate dehydrogenase (LDH, Sigma-Aldrich, Poznan, Poland) was added, and the decrease in absorbance was measured at 340 nm (Genesys 10UV Scanning UV/Visible Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). The MPST activity was expressed as nmoles of pyruvate produced during one minute-incubation at 37 °C per 1 mg of protein.