Amphibian allantoinase. Molecular cloning, tissue distribution, and functional expression

Area of Science:

• Biochemistry
• Molecular Biology
• Evolutionary Biology

Background:

• Vertebrate uric acid metabolism involves enzymes like allantoicase, allantoinase, and urate oxidase.
• Phylogenetic studies indicate the loss of these enzymes during vertebrate evolution.
• Previous research suggested amphibian liver allantoinase and urate oxidase are peroxisomal.

Purpose of the Study:

• To clone the cDNA encoding bullfrog (Rana catesbeiana) allantoinase.
• To determine the subcellular localization of bullfrog allantoinase.
• To investigate the expression pattern and catalytic activity of bullfrog allantoinase.

Main Methods:

• cDNA cloning and sequencing of bullfrog allantoinase.
• Bioinformatic analysis of the deduced protein sequence.
• Immunocytochemistry and subcellular fractionation.
• Northern and immunoblotting analyses.
• Enzyme activity assays in yeast and insect cells.

Main Results:

• A cDNA encoding a 483-residue bullfrog allantoinase was successfully cloned.
• Structural analysis suggested potential transmembrane segments and a mitochondrial localization signal.
• Immunocytochemistry confirmed mitochondrial localization, refuting peroxisomal localization.
• Allantoinase mRNA and protein were specifically detected in frog liver and kidney.
• Recombinant bullfrog allantoinase exhibited catalytic activity and was antigenically similar to the native enzyme.

Conclusions:

• Bullfrog allantoinase is localized to mitochondria, not peroxisomes.
• Hepatic and renal expression of allantoinase in frogs correlates with urate oxidase distribution.
• The cloned bullfrog allantoinase is catalytically active and suitable for further functional studies.