A METABOLIC CONTROL ANALYSIS APPROACH TO STUDY OF SYSTEMS: THE RED BLOOD CELL GLYCOLYTIC FLUX UNDER INHIBITION BY ALKYLATING REAGENTS

M. FLORENCIA PIGNATARO; ALLIERA CARLOS; CARABIAS PABLO; CRUZ KAREN; DELFINO JOSE MARIA; DE SAUTU, MARILINA; FERREIRA GOMES, MARIELA; GOMEZ GABRIELA; INCICCO JEREMIAS; MANZI MALENA; MANGIALAVORI IRENE; SAFFIOTI NICOLÁS; SALVATIERRA DAMIANA; SANTOS JAVIER

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

Sociedades de Biociencias

Metabolic Control Analysis (MCA) is a system-level methodologyto the study of metabolism in a quantitative fashion. Red blood cells(RBCs) are interesting systems to develop non-reductionist biologicalstudies of metabolic pathways. Moreover, they are relativelysimple in structure and do not contain organelles, thus becominga simple model without internal compartments. This work providesa simple experimental setup to measure the glycolytic flux and theflux response (sensitivity) to external modulators. Here we assayedtwo different inhibitors of the enzyme glyceraldehyde-3-phosphate-deshydrogenase (GAPDH) often used as text book examplefor an irreversible enzyme inhibition: iodoacetic acid (IA) and iodoacetamide(IAA). These compounds are widely used as alkylatingreagents to modify thiol groups in proteins by S-carboxymethylationor S-cabamidomethylation. IAA and IA inactivate to GAPDH byblocking an essential Cys residue in the active center of the enzymeand, consequently, glycolysis flux is inhibited.Glucose was the source and lactate was the sink of the metabolicpathway. The glycolytic flux was determined by measuring theextracellular lactate concentration through time, using a colorimetriclow-cost setup based on two coupled enzymatic reactions andphotographic analysis using the ImageJ program and a plugin developedto this end. The system reached steady state and, underthese conditions, MCA tools were applied to evaluate the sensitivityof the system?s flux to the enzyme inhibitors. The response coefficientwas calculated by fitting a derivable empirical equation to theexperimental data. A lower IA concentration was required to producethe highest flux inhibition compared with IAA. Moreover, the systemsshowed to be very robust and of easy implementation opening thepossibility of RBCs ageing or viability measurements. Besides, testingresponse to hormones like insulin, among other modulators isalso feasible.All authors contributed equally