title: Molecular evolution of alanine/glyoxylate aminotransferase 1 intracellular targeting. Analysis of the feline gene.
abstract: The subcellular distribution of hepatic alanine:glyoxylate aminotransferase 1 (AGT) has changed, under the influence of dietary selection pressure, on several o occasions during the evolution of mammals. In some species (e.g. human and rabbit) AGT is entirely peroxisomal; in other species (e.g. marmoset and rat) this enzyme is found in similar amounts in peroxisomes and mitochondria; in yet other species (e.g. cat) it is mainly mitochondrial. The molecular basis of the species-specific dual intracellular targeting of AGT has been partially elucidated in the human and rabbit (as examples of the first group), and in the rat and marmoset (as examples of the second group). As part of a wider study on the molecular evolution of AGT intracellular targeting, we report in the present paper the results of an investigation into the molecular basis of the subcellular distribution of AGT in the cat (as an example of the third group). Cat liver AGT cDNA has been cloned and sequenced, and shown to have a high degree of similarity to AGT from human, rabbit, marmoset and rat. Southern-blotting analysis showed that AGT in the cat is probably encoded by a single gene, as it is in other species. Transcript analysis by RNase protection indicated that almost all of the AGT mRNA would possess an open reading frame encoding a polypeptide of 414 amino acids and a molecular mass of 45,508 Da. The N-terminal 22 amino acids comprised the putative mitochondrial-targeting sequence (by analogy with the equivalent sequence in marmoset and rat pre-mitochondrial AGT). The very low level of peroxisomal AGT in cat liver is compatible with the absence of any RNase-protected transcripts initiating downstream of the first putative translation initiation codon (i.e. absence of any transcripts in which the mitochondrial-targeting sequence is excluded from the open reading frame). In vitro studies showed that the 45 kDa polypeptide was imported into rat liver mitochondria and processed to a mature protein of approximately 43 kDa, compatible with the cleavage of the N-terminal 22 amino acids, as is also the case in rat and marmoset. A polypeptide in which the N-terminal 22 amino acids was absent could not be imported into mitochondria in vitro.
authors.affiliation: Biochemical Genetics Research Group, MRC Clinical Research Centre, Harrow, England.
authors.forename: M J
authors.initials: MJ
authors.lastname: Lumb
authors.forename: P E
authors.initials: PE
authors.lastname: Purdue
authors.forename: C J
authors.initials: CJ
authors.lastname: Danpure
mesh_terms: D000410:Alanine Transaminase / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D015152:Blotting, Northern; D015139:Blotting, Southern; D002144:Callithrix; D002415:Cats / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008099:Liver / Q000201:enzymology; D008830:Microbodies / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011817:Rabbits; D051381:Rats; D017385:Sequence Homology; D013045:Species Specificity; D013347:Subcellular Fractions / Q000201:enzymology*; D000637:Transaminases; D014158:Transcription, Genetic
chemical_list: D018076:DNA, Complementary; D000637:Transaminases; D000410:Alanine Transaminase; C019635:Alanine-glyoxylate transaminase