Porphyrinogen carboxylyase. Studies on existence of isoenzymes

RESUMO

Porphyrinogen carboxylyase from normal rat liver was subjected to purification methods. Two different purification protocols were used. In both cases, the inital steps consisted in obtaining a liver homogenate, followed by centrifugation, salt precipitation and phosphate gel absorption. Scheme I consisted in then submiting the preparation to DEAE-cellulose, followed by Sephacryl S-200 and Phenyl-sepharose sequential column chromatographies. Scheme II involved an affinity column followed by a Sephadex G-75 gel filtration column. In both cases, the enzyme was stored at-20 degrees Celsius until its assay. The addition of 2mM dithiotreytol to the incubation media or to the enzyme extract before storage, did not help improve the activity nor the stability of the enzyme. Those fractions containing the maximal enzyme activity, detected using Uroporphyrinogen III or Pentacarboxy-porphyrinogen III as substrate, were not alawys present in the same tubes for the different columns employed. In addition, the degree of purification obtained in some steps was different according to the substrate employed. The results suggest the existence of at least two isoenzymes for rat liver porphyrinogen carboxy-lyase.