(enviado para su publicacion) Metabolic Control Analysis to Introduce to the Study of Systems in Biochemistry: the red blood cell Glycolytic Pathway

ANGELANI C.R; CARABIAS P; CRUZ KM; DELFINO J.M; DE SAUTU M; ESPELT V; FERREIRA GOMES M; GÓMEZ G.E (CORRESPONDING AUTHORS); INCICCO J; MANGIALAVORI IC (CORRESPONDING AUTHORS); MANZI M; PIGNATARO MF; SAFFIOTI N; SALVATIERRA D; SANTOS J (CORRESPONDING AUTHORS); SCHWARZBAUM P

Biochemistry and Molecular Biology Education

John Wiley & Sons, Inc.

Año: 2018

1539-3429

Metabolic Control Analysis (MCA) is a relatively new field in biochemistry where the contribution of enzymes and transporters to the control of a pathway flux and metabolites concentrations is determined and expressed quantitatively by means of coefficients. Metabolic flux can be influenced by a wide variety of modulators acting on one or more metabolic steps along the pathway.In this context, we describe a teaching module to study metabolic regulation of human erythrocytes (RBCs). Within the framework of MCA, students use these cells to determine the sensitivity of glycolytic flux to two inhibitors (iodoacetic, and iodoacetamide) of the enzyme glyceraldehyde-3-phosphate-deshydrogenase (GAPDH). Flux was estimated by determining the concentration of extracellular lactate, the end product of RBC glycolysis, at different time points. For this purpose, a low-cost colorimetric assay was used, followed by quantification of the signal using a custom-built CCD-device. Subsequent colorimetric analysis was carried out using the ImageJ program and a plug-in developed ad hoc.Students estimated flux response coefficients for the inhibitors by fitting an empirical function to experimental data, followed by analytical derivation of this function. Accordingly, continuous curves showing the values of response coefficient as a function of inhibitors concentrations could be built, where IA and IAA exhibited qualitatively different patterns, which were thoroughly analyzed in terms of the properties of the inhibitors and the target enzymes. In general, a lower IA concentration was required to produce the highest glycolytic flux inhibition. This work opens the possibility to use the MCA approach to study key variables of a simple metabolic system such as the RBC, in a context of an upper level biochemistry course.