In order to facilitate the purification of isocitrate lyase from
Aspergillus nidulans, the isocitrate lyase overexpressing strain
JCB4a was
derived. Isocitrate lyase was purified to homogeneity by the criterion
of
polyacrylamide gel electrophoresis and anti-isocitrate lyase
polyclonal antibodies were raised. Stabilization of purified enzyme, when
stored at
−20°C, required the addition of 1 mM
dithiothreitol (DTT) plus 1 mM ethylenediaminetetraacetate (EDTA).
Aspergillus nidulans isocitrate lyase is a multimeric enzyme with
a native molecular weight of 240 kDa and composed of four monomers of 59
kDa.
The enzyme required 5 mM Mg2+ and
1 mM DTT or cysteine for full activity. EDTA at 1 mM
replaced
the requirement of a thiol compound for activity. The Km
for threo-DS-isocitrate was 0·050 mM,
and the enzyme activity was inhibited by succinate, itaconate and structural
analogs of glyoxylate as well as by fructose-1,6-bisphosphate.