ATP-dependent degradation of a mutant serine: pyruvate/alanine:glyoxylate aminotransferase in a primary hyperoxaluria type 1 case

Primary hyperoxaluria type 1 (PH 1), an inborn error of glyoxylate metabolism characterized by excessive synthesis of oxalate and glycolate, is caused by a defect in serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT). This enzyme is peroxisomal in human liver. Recently, we cloned SPT/AGT-cDNA from a PH 1 case, and demonstrated a point mutation of T to C in the coding region of the SPT/AGT gene encoding a Ser to Pro substitution at residue 205 (Nishiyama, K., T. Funai, R. Katafuchi, F. Hattori, K. Onoyama, and A. Ichiyama. 1991. Biochem. Biophys. Res. Commun. 176:1093-1099). In the liver of this patient, SPT/AGT was very low with respect to not only activity but also protein detectable on Western blot and immunoprecipitation analyses. Immunocytochemically detectable SPT/AGT labeling was also low, although it was detected predominantly in peroxisomes. On the other hand, the level of translatable SPT/AGT-mRNA was higher than normal, indicating that SPT/AGT had been synthesized in the patient's liver at least as effectively as in normal liver. Rapid degradation of the mutant SPT/AGT was then demonstrated in transfected COS cells and transformed Escherichia coli, accounting for the low level of immunodetectable mutant SPT/AGT in the patient's liver. The mutant SPT/AGT was also degraded much faster than normal in an in vitro system with a rabbit reticulocyte extract, and the degradation in vitro was ATP dependent. These results indicate that a single amino acid substitution in SPT/AGT found in the PH1 case leads to a reduced half- life of this protein. It appears that the mutant SPT/AGT is recognized in cells as an abnormal protein to be eliminated by degradation.     

Peroxisomal and mitochondrial targeting of serine: Pyruvate/alanine: Glyoxylate aminotransferase in rat liver

Serine: pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide. In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, STPp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.     

Primary hyperoxaluria type 1: AGT mistargeting highlights the fundamental differences between the peroxisomal and mitochondrial protein import pathways

Primary hyperoxaluria type 1 (PH1) is an atypical peroxisomal disorder, as befits a deficiency of alanine:glyoxylate aminotransferase (AGT), which is itself an atypical peroxisomal enzyme. PH1 is characterized by excessive synthesis and excretion of the metabolic end-product oxalate and the progressive accumulation of insoluble calcium oxalate in the kidney and urinary tract. Disease in many patients is caused by a unique protein trafficking defect in which AGT is mistargeted from peroxisomes to mitochondria, where it is metabolically ineffectual, despite remaining catalytically active. Although the peroxisomal import of human AGT is dependent upon the PTS1 import receptor PEX5p, its PTS1 is exquisitely specific for mammalian AGT, suggesting the presence of additional peroxisomal targeting information elsewhere in the AGT molecule. This and many other functional peculiarities of AGT are probably a consequence of its rather chequered evolutionary history, during which much of its time has been spent being a mitochondrial, rather than a peroxisomal, enzyme. Analysis of the molecular basis of AGT mistargeting in PH1 has thrown into sharp relief some of the fundamental differences between the requirements of the peroxisomal and mitochondrial protein import pathways, particularly the properties of peroxisomal and mitochondrial matrix targeting sequences and the different conformational limitations placed upon importable cargos.     

TAT-Mediated Delivery of Human Alanine:Glyoxylate Aminotransferase in a Cellular Model of Primary Hyperoxaluria Type I

Defects in liver peroxisomal alanine:glyoxylate aminotransferase (AGT), as a consequence of inherited mutations on the AGXT gene, lead to primary hyperoxaluria type I (PH1), a rare metabolic disorder characterized by the formation of calcium oxalate stones at first in the urinary tract and then in the whole body. The curative treatments currently available for PH1 are pyridoxine therapy, effective in only 10–30 % of the patients, and liver transplantation, an invasive procedure with potentially serious complications. A valid therapeutic option for PH1 patients would be the development of an enzyme administration therapy. However, the exogenous administration of the missing AGT would require the crossing of the plasma membrane to deliver the protein to liver peroxisomes. In this study, we constructed, purified and characterized the fusion protein of AGT with the membrane-penetrating Tat peptide (Tat-AGT). Although Tat-AGT shows subtle active site conformational changes as compared with untagged AGT, it retains a significant transaminase activity. Western-blot analyses, enzymatic assays and immunofluorescence studies show that active Tat-AGT can be successfully delivered to a mammalian cellular model of PH1 consisting of chinese hamster ovary cells expressing glycolate oxidase (CHO-GO), whereas untagged AGT cannot. Moreover, the intracellular transduced Tat-AGT makes CHO-GO cells able to detoxify endogenously produced glyoxylate to an extent similar to that of CHO-GO cells stably expressing AGT. Altogether, these results show that the Tat peptide is capable of delivering a functional AGT to mammalian cells, thus paving the way for the possibility to use Tat-AGT as an enzyme replacement therapy to counteract PH1.     

Differential enzyme targeting as an evolutionary adaptation to herbivory in carnivora

Not all members of the order Carnivora are carnivorous. Some are omnivorous, and a few, such as the giant panda, Ailuropoda melanoleuca, are almost exclusively herbivorous. Although a number of adaptations to increased plant-eating are recognized within Carnivora, few have been studied at the molecular level. One molecular adaptation to diet that is spread widely across Mammalia is the differential intracellular targeting of the intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT), which tends to be mitochondrial in carnivores, peroxisomal in herbivores, and both mitochondrial and peroxisomal in omnivores. In the present study, we have analyzed the targeting of AGT in Carnivora in relation to species' natural diets. We show not only that there has been an adaptive shift in AGT targeting from the mitochondrion toward the peroxisome as diets have shifted from being mainly carnivorous to ones that are more omnivorous and herbivorous but also that in one lineage, namely that of the giant panda, there is evidence for positive selection pressure at the molecular level on the AGT mitochondrial targeting sequence to decrease its efficiency, thereby allowing more AGT to be targeted to the peroxisomes.     

Immunocytochemical localization of human hepatic alanine: glyoxylate aminotransferase in control subjects and patients with primary hyperoxaluria type 1

Primary hyperoxaluria type 1 (PH1) is an inherited disorder of glyoxylate metabolism caused by a deficiency of the hepatic peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT; EC 2.6.1.44) [FEBS Lett (1986) 201:20]. The aim of the present study was to investigate the intracellular distribution of immunoreactive AGT protein, using protein A-gold immunocytochemistry, in normal human liver and in livers of PH1 patients with (CRM+) or without (CRM-) immunologically crossreacting enzyme protein. In all CRM+ individuals, which included three controls, a PH1 heterozygote and a PH1 homozygote immunoreactive AGT protein was confined to peroxisomes, where it was randomly dispersed throughout the peroxisomal matrix with no obvious association with the peroxisomal membrane. No AGT protein could be detected in the peroxisomes or other cytoplasmic compartments in the livers of CRM- PH1 patients (homozygotes). The peroxisomal labeling density in the CRM+ PH1 patient, who was completely deficient in AGT enzyme activity, was similar to that of the controls. In addition, in the PH1 heterozygote, who had one third normal AGT enzyme activity, peroxisomal labeling density was reduced to 50% of normal.     

Glycolate and glyoxylate metabolism in HepG2 cells

Oxalate synthesis in human hepatocytes is not well defined despite the clinical significance of its overproduction in diseases such as the primary hyperoxalurias. To further define these steps, the metabolism to oxalate of the oxalate precursors glycolate and glyoxylate and the possible pathways involved were examined in HepG2 cells. These cells were found to contain oxalate, glyoxylate, and glycolate as intracellular metabolites and to excrete oxalate and glycolate into the medium. Glycolate was taken up more effectively by cells than glyoxylate, but glyoxylate was more efficiently converted to oxalate. Oxalate was formed from exogenous glycolate only when cells were exposed to high concentrations. Peroxisomes in HepG2 cells, in contrast to those in human hepatocytes, were not involved in glycolate metabolism. Incubations with purified lactate dehydrogenase suggested that this enzyme was responsible for the metabolism of glycolate to oxalate in HepG2 cells. The formation of ¹⁴C-labeled glycine from ¹⁴C-labeled glycolate was observed only when cell membranes were permeabilized with Triton X-100. These results imply that peroxisome permeability to glycolate is restricted in these cells. Mitochondria, which produce glyoxylate from hydroxyproline metabolism, contained both alanine:glyoxylate aminotransferase (AGT)2 and glyoxylate reductase activities, which can convert glyoxylate to glycine and glycolate, respectively. Expression of AGT2 mRNA in HepG2 cells was confirmed by RT-PCR. These results indicate that HepG2 cells will be useful in clarifying the nonperoxisomal metabolism associated with oxalate synthesis in human hepatocytes. liver; peroxisomes; hepatocytes; hyperoxaluria; alanine:glyoxylate aminotransferase; glyoxylate reductase     

Crystal structure of alanine:glyoxylate aminotransferase and the relationship between genotype and enzymatic phenotype in primary hyperoxaluria type 1

A deficiency of the liver-specific enzyme alanine:glyoxylate aminotransferase (AGT) is responsible for the potentially lethal hereditary kidney stone disease primary hyperoxaluria type 1 (PH1). Many of the mutations in the gene encoding AGT are associated with specific enzymatic phenotypes such as accelerated proteolysis (Ser205Pro), intra-peroxisomal aggregation (Gly41Arg), inhibition of pyridoxal phosphate binding and loss of catalytic activity (Gly82Glu), and peroxisome-to-mitochondrion mistargeting (Gly170Arg). Several mutations, including that responsible for AGT mistargeting, co-segregate and interact synergistically with a Pro11Leu polymorphism found at high frequency in the normal population. In order to gain further insights into the mechanistic link between genotype and enzymatic phenotype in PH1, we have determined the crystal structure of normal human AGT complexed to the competitive inhibitor amino-oxyacetic acid to 2.5A. Analysis of this structure allows the effects of these mutations and polymorphism to be rationalised in terms of AGT tertiary and quaternary conformation, and in particular it provides a possible explanation for the Pro11Leu-Gly170Arg synergism that leads to AGT mistargeting.     

Flux of the L-serine metabolism in rabbit, human, and dog livers. Substantial contributions of both mitochondrial and peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase.

L-Serine metabolism in rabbit, dog, and human livers was investigated, focusing on the relative contributions of the three pathways, one initiated by serine dehydratase, another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Under quasi-physiological in vitro conditions (1 mM L-serine and 0.25 mM pyruvate), flux through serine dehydratase accounted for only traces, and that through SPT/AGT substantially contributed no matter whether the enzyme was located in peroxisomes (rabbit and human) or largely in mitochondria (dog). As for flux through serine hydroxymethyltransferase and GCS, the conversion of serine to glycine occurred fairly rapidly, followed by GCS-mediated slow decarboxylation of the accumulated glycine. The flux through GCS was relatively high in the dog and low in the rabbit, and only in the dog was it comparable with that through SPT/AGT. An in vivo experiment with L-[3-3H,14C]serine as the substrate indicated that in rabbit liver, gluconeogenesis from L-serine proceeds mainly via hydroxypyruvate. Because an important role in the conversion of glyoxylate to glycine has been assigned to peroxisomal SPT/AGT from the studies on primary hyperoxaluria type 1, these results suggest that SPT/AGT in this organelle plays dual roles in the metabolism of glyoxylate and serine.     

Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1

Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway. Although the COOH-terminal KKL of human AGT was shown to be necessary for its peroxisomal import, this tripeptide was unable to direct the peroxisomal import of the bona fide peroxisomal protein firefly luciferase or the reporter protein bacterial chloramphenicol acetyltransferase. An ill-defined region immediately upstream of the COOH-terminal KKL was also found to be necessary for the peroxisomal import of AGT, but again this region was found to be insufficient to direct the peroxisomal import of chloramphenicol acetyltransferase. Substitution of the COOH-terminal KKL of human AGT by the COOH-terminal tripeptides found in the AGTs of other mammalian species (SQL, NKL), the prototypical PTS1 (SKL), or the glycosomal PTS1 (SSL) also allowed peroxisomal targeting, showing that the allowable PTS1 motif in AGT is considerably more degenerate than, or at least very different from, that acceptable in luciferase. AGT possessing the two amino acid substitutions responsible for its mistargeting in PH1 (i.e., Pro11-- >Leu and Gly170-->Arg) was targeted mainly to the mitochondria. However, AGTs possessing each amino acid substitution on its own were targeted normally to the peroxisomes. This suggests that Gly170-->Arg- mediated increased functional efficiency of the otherwise weak mitochondrial targeting sequence (generated by the Pro11-->Leu polymorphism) is not due to interference with the peroxisomal targeting or import of AGT.     

Identification and characterization of HAOX1, HAOX2, and HAOX3, three human peroxisomal 2-hydroxy acid oxidases

Computer-based approaches identified three distinct human 2-hydroxy acid oxidase genes, HAOX1, HAOX2, and HAOX3, that encode proteins with significant sequence similarity to plant glycolate oxidase, a prototypical 2-hydroxy acid oxidase. The products of these genes are targeted to peroxisomes and have 2-hydroxy acid oxidase activities. Each gene displays a distinct tissue-specific pattern of expression, and each enzyme exhibits distinct substrate preferences. HAOX1 is expressed primarily in liver and pancreas and is most active on the two-carbon substrate, glycolate, but is also active on 2-hydroxy fatty acids. HAOX2 is expressed predominantly in liver and kidney and displays highest activity toward 2-hydroxypalmitate. HAOX3 expression was detected only in pancreas, and this enzyme displayed a preference for the medium chain substrate 2-hydroxyoctanoate. These results indicate that all three human 2-hydroxy acid oxidases are involved in the oxidation of 2-hydroxy fatty acids and may also contribute to the general pathway of fatty acid alpha-oxidation. Primary hyperoxaluria type 1 (PH1) is caused by defects in peroxisomal alanine-glyoxylate aminotransferase, the enzyme that normally eliminates intraperoxisomal glyoxylate. The presence of HAOX1 in liver and kidney peroxisomes and the ability of HAOX1 to oxidize glyoxylate to oxalate implicate HAOX1 as a mediator of PH1 pathophysiology.     

Primary hyperoxaluria in Coton de Tulear

Primary hyperoxaluria (PH) is a rare autosomal recessive disorder of glyoxylate metabolism in humans. It is characterized by the accumulation of oxalate and subsequent precipitation of calcium oxalate crystals, primarily in the kidneys. Deficiencies in glyoxylate-metabolizing enzymes alanine-glyoxylate aminotransferase (AGXT) or glyoxylate reductase/hydroxypyruvate reductase (GRHPR) occur in 95% of PH cases. Seven Coton de Tulear puppies from four apparently unrelated litters were examined owing to sudden illness at the age of 3-4 weeks. A complete necropsy was performed. The typical finding was tubular necrosis with extensive oxalate crystal deposition. Based on history and necropsy findings, PH was suspected. Eight microsatellite loci flanking AGXT and GRHPR were analysed, and based on segregation results, AGXT was suspected as to be the candidate gene. AGXT exon sequencing revealed a single base change (c.996G>A) that changed one conserved residue (p.Gly102Ser). The mutation was tested in of 118 Finnish Coton de Tulear dogs, ten (8.5%) of which were revealed as carriers. This preliminary study reports PH as a cause of neonatal death in Finnish Coton de Tulear and suggests that genetic testing of dogs be carried out before breeding to prevent the birth of affected offspring.     

An enzyme trafficking defect in two patients with primary hyperoxaluria type 1: peroxisomal alanine/glyoxylate aminotransferase rerouted to mitochondria

Most patients with the autosomal recessive disease primary hyperoxaluria type 1 (PH1) have a complete deficiency of alanine/glyoxylate aminotransferase (AGT) enzyme activity and immunoreactive protein. However a few possess significant residual activity and protein. In normal human liver, AGT is entirely peroxisomal, whereas it is entirely mitochondrial in carnivores, and both peroxisomal and mitochondrial in rodents. Using the techniques of isopycnic sucrose and Percoll density gradient centrifugation and quantitative protein A-gold immunoelectron microscopy, we have found that in two PH1 patients, possessing 9 and 27% residual AGT activity, both the enzyme activity and immunoreactive protein were largely mitochondrial and not peroxisomal. In addition, these individuals were more severely affected than expected from the levels of their residual AGT activity. In these patients, the PH1 appears to be due, at least in part, to a unique trafficking defect, in which peroxisomal AGT is diverted to the mitochondria. To our knowledge, this is the first example of a genetic disease caused by such interorganellar rerouting.     

Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease

The pyridoxal-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria in patients with the hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) due to the synergistic interaction between a common Pro(11)Leu polymorphism and a PH1-specific Gly(170)Arg mutation. The kinetic partitioning of newly synthesised AGT between peroxisomes and mitochondria is determined by the combined effects of (1) the generation of cryptic mitochondrial targeting information, and (2) the inhibition of AGT dimerization. The crystal structure of AGT has recently been solved, allowing the effects of the various polymorphisms and mutations to be rationalised in terms of AGT's three-dimensional conformation. Procedures that increase dimer stability and/or increase the rate of dimer formation have potential in the formulation of novel strategies to treat this otherwise intractable life-threatening disease.     

Inhibition of alanine:glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1

Peroxisome-to-mitochondrion mistargeting of the homodimeric enzyme alanine:glyoxylate aminotransferase 1 (AGT) in the autosomal recessive disease primary hyperoxaluria type 1 (PH1) is associated with the combined presence of a normally occurring Pro(11)Leu polymorphism and a PH1-specific Gly170Arg mutation. The former leads to the formation of a novel NH2-terminal mitochondrial targeting sequence (MTS), which although sufficient to direct the import of in vitro-translated AGT into isolated mitochondria, requires the additional presence of the Gly170Arg mutation to function efficiently in whole cells. The role of this mutation in the mistargeting phenomenon has remained elusive. It does not interfere with the peroxisomal targeting or import of AGT. In the present study, we have investigated the role of the Gly170Arg mutation in AGT mistargeting. In addition, our studies have led us to examine the relationship between the oligomeric status of AGT and the peroxisomal and mitochondrial import processes. The results obtained show that in vitro-translated AGT rapidly forms dimers that do not readily exchange subunits. Although the presence of the Pro(11)Leu or Gly170Arg substitutions alone had no effect on dimerization, their combined presence abolished homodimerization in vitro. However, AGT containing both substitutions was still able to form heterodimers in vitro with either normal AGT or AGT containing either substitution alone. Expression of various combinations of normal and mutant, as well as epitope-tagged and untagged forms of AGT in whole cells showed that normal AGT rapidly dimerizes in the cytosol and is imported into peroxisomes as a dimer. This dimerization prevents mitochondrial import, even when the AGT possesses an MTS generated by the Pro(11)Leu substitution. The additional presence of the Gly170Arg substitution impairs dimerization sufficiently to allow mitochondrial import. Pharmacological inhibition of mitochondrial import allows AGT containing both substitutions to be imported into peroxisomes efficiently, showing that AGT dimerization is not a prerequisite for peroxisomal import.     

Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana

Leaf peroxisomes are present in greening cotyledons and contain enzymes of the glycolate pathway that functions in photorespiration. However, only a few leaf peroxisomal proteins, that is hydroxypyruvate reductase (HPR), glycolate oxidase (GO) and alanine:glyoxylate aminotransferase 1 (AGT1), have been characterized, and other functions in leaf peroxisomes have not been solved. To better understand the functions of leaf peroxisomes, we established a method to isolate leaf peroxisomes of greening cotyledons. We analyzed 53 proteins by MALDI-TOF MS and then identified 29 proteins. Among them, five proteins are related to the glycolate pathway, four proteins function in scavenging of hydrogen peroxide and additionally 20 novel leaf peroxisomal proteins were identified. In particular, protein kinases and protein phosphatase were first identified as peroxisomal proteins suggesting that protein phosphorylation is one of the regulatory mechanisms in leaf peroxisomes. Novel leaf peroxisomal proteins contained five PTS1-like proteins that have sequences where one amino acid is substituted with another one in PTS1 sequences. The PTS1 motif was suggested to have novel PTS1 sequences.     

Primary hyperoxalurias: Disorders of glyoxylate detoxification

Glyoxylate detoxification is an important function of human peroxisomes. Glyoxylate is a highly reactive molecule, generated in the intermediary metabolism of glycine, hydroxyproline and glycolate mainly. Glyoxylate accumulation in the cytosol is readily transformed by lactate dehydrogenase into oxalate, a dicarboxylic acid that cannot be metabolized by mammals and forms tissue-damaging calcium oxalate crystals. Alanine-glyoxylate aminotransferase, a peroxisomal enzyme in humans, converts glyoxylate into glycine, playing a central role in glyoxylate detoxification. Cytosolic and mitochondrial glyoxylate reductase also contributes to limit oxalate production from glyoxylate. Mitochondrial hydroxyoxoglutarate aldolase is an important enzyme of hydroxyproline metabolism. Genetic defect of any of these enzymes of glyoxylate metabolism results in primary hyperoxalurias, severe human diseases in which toxic levels of oxalate are produced by the liver, resulting in progressive renal damage. Significant advances in the pathophysiology of primary hyperoxalurias have led to better diagnosis and treatment of these patients, but current treatment relies mainly on organ transplantation. It is reasonable to expect that recent advances in the understanding of the molecular mechanisms of disease will result into better targeted therapeutic options in the future. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease.     

Enxymological characterization of a putative canine analogue of primary hyperoxaluria type 1

This paper concerns an enzymological investigation into a putative canine canalogue of the human autosomal recesive disease primary hyperoxaluria type 1 (alanine:glyoxylate / serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and seribe:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephripathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including d-glycerate dehydrogenese / glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate / serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal humanb liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is anlogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.