How does hexacjlorobenzene treatment affect liver uroporphyrinogen decarboxylase?

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

Institut Pasteur, Paris, France.

Congreso; Millennium muting on Porphyrins and Porhyrias 2000; 2000

Centre Francais des Porphyries. Hopital Louis Mourier. Universite Paris.

The aim of this work was to investigate how does hexachlorobenzene affect uroporphyrinogen decarboxylase (UroD). We evaluated if the strong decrease of liver UroD observed in experimental porphyria cutanea tarda (PCT) is due to a protein alteration, and improved, in parallel, the knowledge about the normal liver enzyme. With these purposes several physicochemical studies for enzymatic characterization were carried out comparatively on the enzyme of both normal and hexachlorobenzene (HCB) porphyric rat liver. The enzyme was purified from their homogenates through differential centrifugation, ammonium sulphate fractionation and calcium phosphate gel adsorption. The porphyric preparation showed higher activation energy, lower reactivity index and lower optimum pH when compared with the normal enzyme. In addition, it did not reach the Vmax at any of the substrate concentrations assayed (up to 28 µM uroporphyrinogen III) while the normal enzyme reached the plateau around 14 µM. The porphyric enzyme appears to be more protected than the normal against the inhibitory action of several metals, particularly Cu+2 and Pb+2, and against thermal inactivation. Zn+2 did not affect enzymatic activity, whereas Cu+2, Hg+2, Fe+2, Pb+2, and Cd+2 lowered the activities of both normal and porphyric enzyme in a dose-related way. It was also observed that the bigger the atomic radium in its hydrated state, the lower the effect of the metal. Neither glutathione nor dithiothreitol significantly altered enzymatic activity in the range of concentrations assayed. b-Mercaptoethanol had diverse effects, as both regards the concentration assayed and the enzymatic sample used. Assays with cystine showed a dual behaviour of the enzymatic activity both in normal and porphyric enzymes. Western blots for both preparations revealed a single band (65 kD) with similar intensity. These study show that HCB modifies the physicochemical properties of the enzyme leading to a sharp decrease of its activity, without affecting its antigenic reactivity probably as consequence of changes at the conformational level promoted by the inhibitor-enzyme binding. The molecular changes of the UroD protein, involving a modification of its aminoacidic structure can not be discarded and could be additive to the conformational ones. Since the presence of an UroD inhibitor is being reported in human porphyria, on the basis of an analogous enzyme-inhibitor binding, it could be possible to assume that the human enzyme would behave similarly to the here described for its experimental model.