MOLECULAR ANALYSIS, SUBCELLULAR-LOCALIZATION AND TISSUE DISTRIBUTION OF ENZYMES INVOLVED IN URIC-ACID DEGRADATION

The chain of enzymes necessary to convert uric acid to its metabolic products, urea and glyoxylic acid in vertebrates, is truncated through the successive loss of allantoicase, allantoinase and urate oxidase during evolution. In the rats, and most other animals that have urate oxidase activity, this enzyme is associated with crystalloid inclusion present within the peroxisomes in hepatocytes. We have previously shown that the absence of urate oxidase activity in man and hominoid primates is due to mutations in the gene encoding this protein. The presence of all three enzymes of uric acid catabolism in fish and amphibian liver enabled us to undertake their subcellular localization. Using specific antibodies against frog urate oxidase and allantoinase we have localized these two proteins by Immunohistochemical methods to frog liver and kidney. We demonstrated that urate oxidase is present in peroxisomes, allantoinase is localized to mitochondria and allantoicase in cytosol by protein A-gold immunocytochemical method. We have cloned the cDNA encoding allantoinase by immunoscreening a lambda gt11 bull-frog liver cDNA library and polymerase chain reactions. The cDNA is 2112-base pairs in length containing a 1449-bp open reading frame which correspond to a 483-residue protein. Structural analysis of the deduced protein suggested two potential transmembrane segments and also showed that it lacks a typical peroxisomal targeting signal. The protein sequence indicates the presence of a putative mitochondrial localization sequence in the amino terminus. Northern blotting revealed a single mRNA species in the liver and kidney of frog, which was confirmed by immunoblotting. The hepatic and renal specific expression of allantoinase coincides with the distribution of urate oxidase in the frog tissues. These studies clearly established that urate oxidase, allantoinase, and allantoicase the three enzymes involved in uric acid degradation are localized in different subcellular organelles in the frog liver and kidney.