3] Aconitases: A class of metalloproteins highly sensitive to nitric oxide synthesis

Abstract

This chapter describes the methods for measuring the effects of nitric oxide (NO) production of enzymatic activity of m-aconitase/c-aconitase and on iron-responsive element (IRE) binding to c-aconitase. Aconitases are monorneric [Fe-S] proteins that catalyze the stereospecific interconversion of citrate and isocitrate by the intermediate cis-aconitate. The reaction of NO with the [Fe-S] centers of m-aconitase and c-aconitase has important functional consequences. Mitochondrial aconitase is measured after treating cells with digitonin. This technique is based on the fact that digitonin selectively permeabilizes the plasma membrane and leaves inner mitochondrial membrane intact. Inhibition of m-aconitase interrupts the flow of citrate through the Krebs cycle. Inhibition of c-aconitase results in suppression of catalytic activity for cytosolic citrate and in modulation of translation of messenger RNA (mRNA) of key proteins involved in intracellular iron homeostasis. Measurement of enzymatic activity of c-aconitase requires removal of mitochondria to avoid contribution of m-aconitase. These modifications of m-aconitase and c-aconitase activity appear to be part of the pattern of changes in cellular metabolism that occur when the immune/inflammatory NO synthase is induced by cytokines.