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Purification and properties of isocitrate lyase from Aspergillus nidulans, a model enzyme to study catabolite inactivation in filamentous fungi

  • J. R. DE LUCAS (a1), C. AMOR (a1), M. DÍAZ (a1), G. TURNER (a2) and F. LABORDA (a1)...

Abstract

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.

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Address for correspondence: Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Alcalá de Henares, Campus Universitario, E-28871 Alcalá de Henares, Madrid, Spain.

Purification and properties of isocitrate lyase from Aspergillus nidulans, a model enzyme to study catabolite inactivation in filamentous fungi

  • J. R. DE LUCAS (a1), C. AMOR (a1), M. DÍAZ (a1), G. TURNER (a2) and F. LABORDA (a1)...

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