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 p roducts, urea and glyoxylic acid in vertebrates, is truncated through the successive loss of allantoicase, allantoinase and urate oxidase du ring evolution. In the rats, and most other animals that have urate ox idase activity, this enzyme is associated with crystalloid inclusion p resent within the peroxisomes in hepatocytes. We have previously shown that the absence of urate oxidase activity in man and hominoid primat es is due to mutations in the gene encoding this protein. The presence of all three enzymes of uric acid catabolism in fish and amphibian li ver enabled us to undertake their subcellular localization. Using spec ific antibodies against frog urate oxidase and allantoinase we have lo calized these two proteins by Immunohistochemical methods to frog live r and kidney. We demonstrated that urate oxidase is present in peroxis omes, allantoinase is localized to mitochondria and allantoicase in cy tosol 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-ba se pairs in length containing a 1449-bp open reading frame which corre spond to a 483-residue protein. Structural analysis of the deduced pro tein suggested two potential transmembrane segments and also showed th at it lacks a typical peroxisomal targeting signal. The protein sequen ce indicates the presence of a putative mitochondrial localization seq uence in the amino terminus. Northern blotting revealed a single mRNA species in the liver and kidney of frog, which was confirmed by immuno blotting. The hepatic and renal specific expression of allantoinase co incides with the distribution of urate oxidase in the frog tissues. Th ese studies clearly established that urate oxidase, allantoinase, and allantoicase the three enzymes involved in uric acid degradation are l ocalized in different subcellular organelles in the frog liver and kid ney.