Detection and partial characterization of the three isoforms of rat liver uroporphyrinogen decarboxylase.

CORVI M. M.,; GUIDI S.,; CHAUFAN G.,; SAN MARTÍN DE VIALE L. C.,; RÍOS DE MOLINA M.

San Francisco, California

Congreso; 17th International Congress of Biochemestry and Molecular Biology; 1997

American Society for Biochemistry and Molecualr Biology

With the aim to characterize the drastic decrease of Uroporphyrinogen decarboxylase (UroD) activity produced in experimental polyhalogenated aromatic hydrocarbons porphyria we begain to study normal rat liver UroD. In order to purify the enzyme a liver homogenate was subjected to the following purification schem: centrifugation 20 min at 11,000 xg, supernatant ammoniun sulphate precipitation (35-70 %), phosphate gel absortion, affinity chromatography and gel filtration. We found a complex mixture of proteins exhibiting UroD activity. At first we detected two isoforms with MW of 54,000 and 30,000 (IfA and IfB respectively). Trying to further purify IfA by re-cromatography on G-75 column, we detected a third form of MW 17,000 which appeared in a molecular ratio 1:1 respect to IfB. We started the characterization of IfA and IfB, obtaining a Km of 0.27 mM and 0.55 mM for IfA and 5.0 mM and 0.5 mM for IfB. Using Uroporphyrinogen III or pentacarbocyporphyrinogen III as substrate respectively. Besides, we assayed 2 carboxylic group reagents which showed to have an inhibiting effect on both proteins meaning that in the enzymes a carboxyl group would be involved in their active center. This is a first report about 3 hepatic that show UroD activity.