Pyruvate kinase activity enzyme assay

A continuous, enzyme-coupled assay, which uses lactate dehydrogenase (LDH) and measures the depletion of NADH via absorbance at 340 nm was utilized to determine the pyruvate kinase activity. For AC₅₀ measurements (concentration of activator necessary to achieve half-maximal activation) with ML-265, assays were performed in 96-well format using 200 μL/well assay volume with final concentrations of 20 nM human recombinant PKM2 (Sigma, SAE0021), differing concentrations of ML-265, 0.5 mM PEP, 1 mM ADP, 0.2 mM NADH, and 8 U of lactate dehydrogenase (LDH) in an assay buffer of 50 mM Tris-HCl (pH 7.4), 100 mM KCl, and 5 mM MgCl₂ as previously described^15,17. The decrease in absorbance at 340 nm was monitored using a SPECTROstar Omega plate reader (BMG LABTECH Inc., Cary, NC, USA). Initial velocities were calculated with the MARS software. Data were normalized to DMSO (dimethyl sulfoxide)-treated PKM2 activity.

For 661 W cell line experiments, media was replaced prior to the start of treatment with DMSO or ML-265. Cells were incubated with DMSO or different concentrations of ML-265 for 2 hours. Cells were lysed and homogenized in RIPA Lysis and Extraction Buffer (Catalog number: 89900, Life Technologies Corporation, Grand Island, NY) with protease inhibitors (Complete-Mini, Roche Diagnostics, Indianapolis, IN) and cellular debris was removed by centrifugation at 10,000 rpm for 10 minutes. Ten microliters of the supernatant was used to assess pyruvate kinase activity, and the activity was normalized to total protein content as previously described²².

Intravitreal injections of ML-265 or vehicle (DMSO) were performed in Brown-Norway, adult rats. Rodents were anesthetized with a mix of ketamine (100 mg/mL) and xylazine (20 mg/mL), and pupils were dilated with topical phenylephrine (2.5%) and tropicamide (1%). A 25-gauge microvitreoretinal blade (Walcott Rx Products, Ocean View, NJ) was used to create a sclerotomy located 1–2 mm posterior to the limbus with care taken to avoid lens damage. A blunt, 34-gauge cannula was introduced through the sclerotomy into the vitreous cavity. Two microliters of different concentrations of ML-265 or DMSO was slowly injected into the vitreous cavity. This volume was utilized as it has been shown to produce minimal reflux, and lower volumes may not produce adequate reproducibility^45,46. An average vitreous volume in rats of 15 μL was assumed in order to estimate intravitreal concentrations of ML-265 after injection throughout this study^45,46. Left eyes were injected with ML-265, and right eyes were injected with DMSO, which served as the control that pyruvate kinase activation was based upon. Four hours after intravitreal injection, retinas from experimental rat eyes were dissected from the RPE-choroid, homogenized, and lysed in RIPA Lysis and Extraction Buffer with protease inhibitors. The assay was carried out using 4 microliters of rat retinal lysate in an enzyme buffer mixture (50 mM Tris-HCl, pH 7.4, 100 mM KCl, 5 mM MgCl₂, 1 mM ADP, 0.5 mM PEP, and 0.2 mM NADH) and 8 units of LDH similar to what has been previously described^15,17. Pyruvate kinase activity was normalized to total protein in each retinal lysate.