A metabolic control analysis approach to study of systems: the red blood cell glycolytic flux under inhibition by alkylating reagents

PIGNATARO MF; CARABIAS P; DE SAUTU M; INCICCO J; SAFFIOTI N; ANGELANI C.R; DELFINO J.M; GÓMEZ G.E; MANGIALAVORI IC; SANTOS J; ALLIERA C; CRUZ KM; FERREIRA GOMES M; MANZI M; SALVATIERRA D

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedades de Biociencias

Metabolic Control Analysis (MCA) is a system-level methodology to the study of metabolism in a quantitative fashion. Red blood cells (RBCs) are interesting systems to develop non-reductionist biological studies of metabolic pathways. Moreover, they are relatively simple in structure and do not contain organelles, thus becoming a simple model without internal compartments. This work provides a simple experimental setup to measure the glycolytic flux and the flux response (sensitivity) to external modulators. Here we assayed two different inhibitors of the enzyme glyceraldehyde-3-phosphate- deshydrogenase (GAPDH) often used as text book example for an irreversible enzyme inhibition: iodoacetic acid (IA) and iodoacetamide (IAA). These compounds are widely used as alkylating reagents to modify thiol groups in proteins by S-carboxymethylation or S-cabamidomethylation. IAA and IA inactivate to GAPDH by blocking an essential Cys residue in the active center of the enzyme and, consequently, glycolysis flux is inhibited. Glucose was the source and lactate was the sink of the metabolic pathway. The glycolytic flux was determined by measuring the extracellular lactate concentration through time, using a colorimetric low-cost setup based on two coupled enzymatic reactions and photographic analysis using the ImageJ program and a plugin developed to this end. The system reached steady state and, under these conditions, MCA tools were applied to evaluate the sensitivity of the system?s flux to the enzyme inhibitors. The response coefficient was calculated by fitting a derivable empirical equation to the experimental data. A lower IA concentration was required to produce the highest flux inhibition compared with IAA. Moreover, the systems showed to be very robust and of easy implementation opening the possibility of RBCs ageing or viability measurements. Besides, testing response to hormones like insulin, among other modulators is also feasible.Keywords: Metabolic control analysis, red blood cells, glycolyticflux