A highly purified cytosol aconitase [citrate (isocitrate) hydrolyase, EC 18.104.22.168] was isolated from pig liver. Whereas purified aconitase preparations previously reported in the literature require cysteine and Fe2+ for maximal catalytic activity, the purified cytosol enzyme and the partially purified mitochondrial aconitase described here need no added activator. The molecular weight of the purified cytosol enzyme as determined by sucrose density gradient centrifugation was between 107,000 and 111,000. Inhibitory effects of (-)-erythro-fluorocitrate on both aconitase isoenzymes were studied in the absence and presence of Mg2+ and Mn2+. Fluorocitrate was a competitive reversible inhibitor of both aconitases, as deduced from steady-state kinetic analyses (average Ki = 22-45 µM). The bivalent cations Mg2+ and especially Mn2+, when incubated with either aconitase isoenzyme, inhibited enzymatic activity, an effect which increased with time. A high concentration (30 mM) of citrate reversed the inhibition and protected the enzyme against inhibition by Mg2+ or Mn2+. When (-)-erythro-fluorocitrate and Mg2+ (or Mn2+) were present simultaneously, as in the isocitrate dehydrogenase coupled assay system, the time-dependent inhibition which ensued was a result of competitive inhibition by fluorocitrate and the more complex inhibition of the enzyme by Mg2+ or Mn2+
ACKNOWLEDGMENT It is a pleasure to acknowledge the contribution of Miss Barbara Lancaster to the writing of the manuscript.
- Copyright ©, 1974, by Academic Press, Inc.