Amphibian allantoinase. Molecular cloning, tissue distribution, and functional expression.
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 phylogenetic evolution. Previous studies have assigned the localization of both urate oxidase and allantinase to the peroxisome in the amphibian liver. This study reports the cloning of a cDNA encoding bullfrog (Rana catesbeiana) allantoinase, an enzyme that converts allantoin 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 suggested two potential transmembrane segments and the presence of a putative mitochondrial localization sequence in the amino terminus. Immunocytochemical analysis revealed that allantoinase is localized to mitochondria and not to peroxisomes. On Northern blotting, a single mRNA species was detected in the liver and kidney of frog but not in other tissues; this distribution was confirmed by immunoblotting. The hepatic- and renal-specific expression of allantoinase coincides with the distribution of urate oxidase in these tissues in the frog. The allantoinase expressed in Saccharomyces cerevisiae and in Spodoptera frugiperda (Sf9) insect cells exhibits catalytic activity and is antigenically identical to the native frog enzyme.
Hayashi
S
S
Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611.
Jain
S
S
Chu
R
R
Alvares
K
K
Xu
B
B
Erfurth
F
F
Usuda
N
N
Rao
M S
MS
Reddy
S K
SK
Noguchi
T
T
D000581:Amidohydrolases / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D007150:Immunohistochemistry; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008830:Microbodies / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009928:Organ Specificity; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011892:Rana catesbeiana; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D012333:RNA, Messenger; D000581:Amidohydrolases; C019659:allantoinase