AMPHIBIAN ALLANTOINASE - MOLECULAR-CLONING, TISSUE DISTRIBUTION, AND FUNCTIONAL EXPRESSION

The chain of enzymes necessary to convert uric acid to its metabolic p roducts urea and glyoxylic acid in vertebrates is truncated through th e successive loss of allantoicase, allantoinase, and urate oxidase dur ing phylogenetic evolution. Previous studies have assigned the localiz ation of both mate oxidase and allantoinase to the peroxisome in the a mphibian liver. This study reports the cloning of a cDNA encoding bull frog (Rana catesbeiana) allantoinase, an enzyme that converts allantoi n to allantoic acid. The cDNA is 2112 base pairs in length containing a 1449-base pair open reading frame which corresponds to a 483-residue protein (53,296 Da). Structural analysis of the deduced protein sugge sted two potential transmembrane segments and the presence of a putati ve mitochondrial localization sequence in the amino terminus. Immunocy tochemical analysis revealed that allantoinase is localized to mitocho ndria and not to peroxisomes. On Northern blotting, a single mRNA spec ies was detected in the liver and kidney of frog but not in other tiss ues; this distribution was confirmed by immunoblotting. The hepatic- a nd renal- specific expression of allantoinase coincides with the distr ibution of urate oxidase in these tissues in the frog. The allantoinas e expressed in Saccharomyces cerevisiae and in Spodoptera frugiperda ( Sf9) insect cells exhibits catalytic activity and is antigenically ide ntical to the native frog enzyme.