Further studies on cis-aconitic decarboxylase

Summary

• 1. Localization studies indicated that cis-aconitic decarboxylase occurs in the soluble fraction resulting on centrifugation at 15,000xg.

• 2. A high degree of purification of the enzyme was possible by initial fractionation of mycelial extracts with ammonium sulfate, followed by treatment with tricalcium phosphate gel, or chromatography on DEAE cellulose column.

• 3. Some of the purified fractions were devoid of aconitase contamination, or its concentration was considerably reduced.

• 4. Purified cis-aconitic decarboxylase was highly sensitive to storage in the frozen condition and to lyophilization.

• 5. The enzyme was employed on a spectrophotometric estimation of cis-aconitic acid, based on the fact that the transformation to itaconic acid is attended with a sharp drop in the absorption at 240 mμ.

• 6. Spectrophotometric assay of cis-aconitic decarboxylase in crude and purified preparations was possible, based on the differential absorption of cis-aconitic acid and itaconic acid in the ultraviolet and on the reaction of itaconic acid with bromine.