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     

Primary hyperoxaluria type 1 in Japan

Glyoxylate is an immediate precursor of oxalate, but in its metabolism the conversion into glycine catalyzed by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) appears to be the main route. When SPT/AGT is missing as in the case of primary hyperoxaluria type 1 (PH1) more glyoxylate is used for the oxalate production, resulting in calcium oxalate urolithiasis and finally systemic oxalosis. SPT/AGT is a unique enzyme of species-specific dual organelle localization; it is located largely in mitochondria in carnivores and entirely in peroxisomes in herbivores and man. For herbivores, the peroxisomal localization of SPT/AGT is indispensable to avoid massive production of oxalate, probably because liver peroxisomes are the main site of glyoxylate production from glycolate, and plants contain glycolate much more than animal tissues. Recently, we took charge of laboratory examination for 8 cases of primary hyperoxaluria in Japan, and felt that symptoms of some of the Japanese PH1 patients are apparently milder than those of Western patients. The reason of this is not clear, but from the above mentioned seemingly indispensable association of grass-eating with the peroxisomal localization of SPT/AGT it may be related, at least in part, to the food habit of Japanese, especially that of old generation, that they prefer boiled greens rather than frying or raw vegetables.     

Isolation and characterization of glycolic acid dehydrogenase from human liver.

Glycolic acid dehydrogenase has been purified over 800-fold from human liver by (NH4)2SO4 fractionation and column chromatography with DEAE-cellulose and hydroxyapatite. The enzyme catalyzes the direct oxidation of glycolate to oxalate without forming glyoxylate as a free intermediate. Activity is found only in the liver in the soluble fraction. The enzyme is specific for glycolate and inhibits no activity towards glycine or glyoxylate. Glyoxylate and DL-phenyllactate exhibit the enzyme. Optimum activity occurs sharply at pH 6.1 and the Michaelis constant for glycolate was 6.3.10(-5)M. Molecular oxygen does not appear to be the electron acceptor and no requirement for cofactors has been demonstrated, althoug flavin mononucleotide, ascorbate and cytochrome c stimulate activity. The isolation of this enzyme which may account for a significant part of the normal oxalate excretion in man, provides a more complete understanding of the pathways of oxalate biosynthesis and must be taken into account when considering possible methods for controlling disorders of oxalate metabolism.     

A central role for the peroxisomal membrane in glyoxylate cycle function

The glyoxylate cycle provides the means to convert C2-units to C4-precursors for biosynthesis, allowing growth on fatty acids and C2-compounds. The conventional view that the glyoxylate cycle is contained within peroxisomes in fungi and plants is no longer valid. Glyoxylate cycle enzymes are located both inside and outside the peroxisome. Thus, the operation of the glyoxylate cycle requires transport of several intermediates across the peroxisomal membrane. Glyoxylate cycle progression is also dependent upon mitochondrial metabolism. An understanding of the operation and regulation of the glyoxylate cycle, and its integration with cellular metabolism, will require further investigation of the participating metabolite transporters in the peroxisomal membrane.     

Metabolism of [13C5]hydroxyproline in vitro and in vivo: implications for primary hyperoxaluria

Hydroxyproline (Hyp) metabolism is a key source of glyoxylate production in the body and may be a major contributor to excessive oxalate production in the primary hyperoxalurias where glyoxylate metabolism is impaired. Important gaps in our knowledge include identification of the tissues with the capacity to degrade Hyp and the development of model systems to study this metabolism and how to suppress it. The expression of mRNA for enzymes in the pathway was examined in 15 different human tissues. Expression of the complete pathway was identified in liver, kidney, pancreas, and small intestine. HepG2 cells also expressed these mRNAs and enzymes and were shown to metabolize Hyp in the culture medium to glycolate, glycine, and oxalate. [(18)O]- and [(13)C(5)]Hyp were synthesized and evaluated for their use with in vitro and in vivo models. [(18)O]Hyp was not suitable because of an apparent tautomerism of [(18)O]glyoxylate between enol and hydrated forms, which resulted in a loss of isotope. [(13)C(5)]Hyp, however, was metabolized to [(13)C(2)]glycolate, [(13)C(2)]glycine, and [(13)C(2)]oxalate in vitro in HepG2 cells and in vivo in mice infused with [(13)C(5)]Hyp. These model systems should be valuable tools for exploring various aspects of Hyp metabolism and will be useful in determining whether blocking Hyp catabolism is an effective therapy in the treatment of primary hyperoxaluria.     

Inhibition and stimulation of ribulose-1,5-bisphosphate carboxylase/oxygenase by glyoxylate

Glyoxylate is a slowly reversible inhibitor of the CO2/Mg2+-activated form of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach leaves. Inactivation occurred with an apparent dissociation constant of 3.3 mM and a maximum pseudo-first-order rate constant of 7 X 10(-3) s-1. The rate constant for reactivation was 1.2 X 10(-2) s-1. Glyoxylate did not cause differential inhibition of ribulosebisphosphate carboxylase or oxygenase activities. 6-Phosphogluconate protected the enzyme from inactivation by glyoxylate. Glyoxylate was incorporated irreversibly into the large subunit of ribulosebisphosphate carboxylase after reduction with sodium borohydride. Activated enzyme incorporated 1.3 mol of glyoxylate per mole protomer, while enzyme treated with carboxyarabinitol 1,5-bisphosphate (CABP) to protect the active sites incorporated only 0.3 mol glyoxylate per mole protomer. The data suggest that glyoxylate forms a Schiff base with a lysyl residue in the region of the catalytic site. Glyoxylate stimulated the activity of the unactivated enzyme by about twofold. Pseudo-first-order inactivation also occurred with the unactivated enzyme after the initial stimulation by glyoxylate, although at a much slower rate than with the activated enzyme. Glyoxylate treatment of partially activated enzyme did not stimulate formation of the quaternary complex of enzyme X CO2 X Mg2+ X CABP.     

Glyoxylate Reductase Isoform 1 is Localized in the Cytosol and Not Peroxisomes in Plant Cells

Glyoxylate reductase (GLYR) is a key enzyme in plant metabolism which catalyzes the detoxification of both photorespiratory glyoxylate and succinic semialdehdye, an intermediate of the γ-aminobutyrate (GABA) pathway. Two isoforms of GLYR exist in plants, GLYR1 and GLYR2, and while GLYR2 is known to be localized in plastids, GLYR1 has been reported to be localized in either peroxisomes or the cytosol. Here, we reappraised the intracellular localization of GLYR1 in Arabidopsis thaliana L. Heynh (ecotype Lansberg erecta) using both transiently-transformed suspension cells and stably-transformed plants, in combination with fluorescence microscopy. The results indicate that GLYR1 is localized exclusively to the cytosol regardless of the species, tissue and/or cell type, or exposure of plants to environmental stresses that would increase flux through the GABA pathway. Moreover, the C-terminal tripeptide sequence of GLYR1, -SRE, despite its resemblance to a type 1 peroxisomal targeting signal, is not sufficient for targeting to peroxisomes. Collectively, these results define the cytosol as the intracellular location of GLYR1 and provide not only important insight to the metabolic roles of GLYR1 and the compartmentation of the GABA and photorespiratory pathways in plant cells, but also serve as a useful reference for future studies of proteins proposed to be localized to peroxisomes and/or the cytosol.     

Oxalate accumulation from citrate by Aspergillus niger. I. Biosynthesis of oxalate from its ultimate precursor.

Carbon-14 was incorporated from citrate-1,5-14C, glyoxylate-14C(U), or glyoxylate-1-14C into oxalate by cultures of Aspergillus niger pregrown on a medium with glucose as the sole source of carbon. Glyoxylate-14C(U) was superior to glyoxylate-1-14C and citrate-1,5-14C as a source of incorporation. By addition of a great amount of citrate the accumulation of oxalate was accelerated and its maximum yield increased. In a cell-free extract from mycelium forming oxalate from citrate the enzyme oxaloacetate hydrolase (EC3.7.1.1) was identified. Its in vitro activity per flask exceeded the rate of in vivo accumulation of oxalate. Glyoxylate oxidizing enzymes (glycolate oxidase, EC1.1.3.1; glyoxylate oxidase, EC1.2.3.5;NAD(P)-dependent glyoxylate dehydrogenase; glyoxylate dehydrogenase, CoA-oxalylating, EC1.2.1.7) could not be detected in cell-free extracts. It is concluded that in cultures accumulating oxalate from citrate after pregrowth on glucose, oxalate arises by hydrolytic cleavage of oxaloacetate but not by oxidation of glyoxylate.     

The enzymic reduction of glyoxylate and hydroxypyruvate in leaves of higher plants

Glyoxylate and hydroxypyruvate are metabolites involved in the pathway of carbon in photorespiration. The chief glyoxylate-reducing enzyme in leaves is now known to be a cytosolic glyoxylate reductase that uses NADPH as the preferred cofactor but can also use NADH. Glyoxylate reductase has been isolated from spinach leaves, purified to homogeneity, and characterized kinetically and structurally. Chloroplasts contain lower levels of glyoxylate reductase activity supported by both NADPH and NADH, but it is not yet known whether a single chloroplastic enzyme catalyzes glyoxylate reduction with both cofactors. The major hydroxypyruvate reductase activity of leaves has long been known to be a highly active enzyme located in peroxisomes; it uses NADH as the preferred cofactor. To a lesser extent, NADPH can also be used by the peroxisomal enzyme. A second hydroxypyruvate reductase enzyme is located in the cytosol; it preferentially uses NADPH but can also use NADH as cofactor. In a barley mutant deficient in peroxisomal hydroxypyruvate reductase, the NADPH-preferring cytosolic form of the enzyme permits sufficient rates of hydroxypyruvate reduction to support continued substrate flow through the terminal stages of the photosynthetic carbon oxidation (glycolate/glycerate) pathway. The properties and metabolic significance of the cytosolic and organelle-localized glyoxylate and hydroxypyruvate reductase enzymes are discussed.     

Oxalate accumulation from citrate by Aspergillus niger

Carbon-14 was incorporated from citrate-1,5-¹⁴C, glyoxylate-¹⁴C(U), or glyoxylate-1-¹⁴C into oxalate by cultures of Aspergillus niger pregrown on a medium with glucose as the sole source of carbon. Glyoxylate-¹⁴C(U) was superior to glyoxylate-1-¹⁴C and citrate-1,5-¹⁴C as a source of incorporation. By addition of a great amount of citrate the accumulation of oxalate was accelerated and its maximum yield increased. In a cell-free extract from mycelium forming oxalate from citrate the enzyme oxaloacetate hydrolase (EC 3.7.1.1) was identified. Its in vitro activity per flask exceeded the rate of in vivo accumulation of oxalate. Glyoxylate oxidizing enzymes (glycolate oxidase, EC 1.1.3.1; glyoxylate oxidase, EC 1.2.3.5; NAD(P)-dependent glyoxylate dehydrogenase; glyoxylate dehydrogenase, CoA-oxalylating, EC 1.2.1.17) could not be detected in cell-free extracts. It is concluded that in cultures accumulating oxalate from citrate after pregrowth on glucose, oxalate arises by hydrolytic cleavage of oxaloacetate but not by oxidation of glyoxylate.Abbreviations Used DCPIP 2,6-dichlorophenolindophenol     

The role of peroxisomes in the integration of metabolism and evolutionary diversity of photosynthetic organisms

The peroxisome is a metabolic compartment serving for the rapid oxidation of substrates, a process that is not coupled to energy conservation. In plants and algae, peroxisomes connect biosynthetic and oxidative metabolic routes and compartmentalize potentially lethal steps of metabolism such as the formation of reactive oxygen species and glyoxylate, thus preventing poisoning of the cell and futile recycling. Peroxisomes exhibit properties resembling inside-out vesicles and possess special systems for the import of specific proteins, which form multi-enzyme complexes (metabolons) linking numerous reactions to flavin-dependent oxidation, coupled to the decomposition of hydrogen peroxide by catalase. Hydrogen peroxide and superoxide originating in peroxisomes are important mediators in signal transduction pathways, particularly those involving salicylic acid. By contributing to the synthesis of oxalate, formate and other organic acids, peroxisomes regulate major fluxes of primary and secondary metabolism. The evolutionary diversity of algae has led to the presence of a wide range of enzymes in the peroxisomes that are only similar to higher plants in their direct predecessors, the Charophyceae. The appearance of seed plants was connected to the acquirement by storage tissues, of a peroxisomal fatty acid oxidation function linked to the glyoxylate cycle, which is induced during seed germination and maturation. Rearrangement of the peroxisomal photorespiratory function between different tissues of higher plants led to the appearance of different types of photosynthetic metabolism. The peroxisome may therefore have played a key role in the evolutionary formation of metabolic networks, via establishing interconnections between different metabolic compartments.     

Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.     

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.     

Metabolism of Glycolate in Isolated Spinach Leaf Peroxisomes : KINETICS OF GLYOXYLATE, OXALATE, CARBON DIOXIDE, AND GLYCINE FORMATION

The flow of glyoxylate derived from glycolate into various metabolic routes in the peroxisomes during photorespiration was assessed. Isolated spinach leaf peroxisomes were fed [¹⁴C] glycolate in the absence or presence of exogenous glutamate, and the formation of radioactive glyoxylate, CO₂, glycine, oxalate, and formate was monitored at time intervals. In the absence of glutamate, 80% of the glycolate was consumed within 2 hours and concomitantly glyoxylate accumulated; CO₂, oxalate, and formate each accounted for less than 5% of the consumed glycolate. In the presence of equal concentration of glutamate, glycolate was metabolized at a similar rate, and glycine together with some glyoxylate accumulated; CO₂, oxalate, and formate each accounted for an even lesser percentage of the consumed glycolate. CO₂ and oxalate were not produced in significant amounts even in the absence of glutamate, unless glycolate had been consumed completely and glyoxylate had accumulated for a prolonged period. These in vitro findings are discussed in relation to the extent of CO₂ and oxalate generated in leaf peroxisomes during photorespiration.     

Molecular aetiology of primary hyperoxaluria and its implications for clinical management

The primary hyperoxalurias type 1 (PH1) and type 2 (PH2) are autosomal recessive calcium oxalate kidney stone diseases caused by deficiencies of the metabolic enzymes alanine:glyoxylate aminotransferase (AGT) and glyoxylate/hydroxypyruvate reductase (GR/HPR), respectively. Over 50 mutations have been identified in the AGXT gene (encoding AGT) in PH1, associated with a wide variety of effects on AGT, including loss of catalytic activity, aggregation, accelerated degradation, and peroxisome-to-mitochondrion mistargeting. Some of these mutations segregate and interact synergistically with a common polymorphism. Over a dozen mutations have been found in the GRHPR gene (encoding GR/HPR) in PH2, all associated with complete loss of glyoxylate reductase enzyme activity and immunoreactive protein. The crystal structure of human AGT, but not human GR/HPR, has been solved, allowing the effects of many of the mutations in PH1 to be rationalised in structural terms. Detailed analysis of the molecular aetiology of PH1 and PH2 has led to significant improvements in all aspects of their clinical management. Enzyme replacement therapy by liver transplantation can provide a metabolic cure for PH1, but it has yet to be tried for PH2. New treatments that aim to counter the effects of specific mutations on the properties of the enzymes could be feasible in the not-too-distant future.     

A preliminary account of the properties of recombinant human Glyoxylate reductase (GRHPR), LDHA and LDHB with glyoxylate, and their potential roles in its metabolism

Human lactate dehydrogenase (LDH) is thought to contribute to the oxidation of glyoxylate to oxalate and thus to the pathogenesis of disorders of endogenous oxalate overproduction. Glyoxylate reductase (GRHPR) has a potentially protective role metabolising glyoxylate to the less reactive glycolate. In this paper, the kinetic parameters of recombinant human LDHA, LDHB and GR have been compared with respect to their affinity for glyoxylate and related substrates. The Km values and specificity constants (Kcat/K(M)) of purified recombinant human LDHA, LDHB and GRHPR were determined for the reduction of glyoxylate and hydroxypyruvate. K(M) values with glyoxylate were 29.3 mM for LDHA, 9.9 mM for LDHB and 1.0 mM for GRHPR. For the oxidation of glyoxylate, K(M) values were 0.18 mM and 0.26 mM for LDHA and LDHB respectively with NAD+ as cofactor. Overall, under the same reaction conditions, the specificity constants suggest there is a fine balance between the reduction and oxidation reactions of these substrates, suggesting that control is most likely dictated by the ambient concentrations of the respective intracellular cofactors. Neither LDHA nor LDHB utilised glycolate as substrate and NADPH was a poor cofactor with a relative activity less than 3% that of NADH. GRHPR had a higher affinity for NADPH than NADH (K(M) 0.011 mM vs. 2.42 mM). The potential roles of LDH isoforms and GRHPR in oxalate synthesis are discussed.     

Modification of the active site of isocitrate lyase from Pseudomonas indigofera

Kinetic analysis of inactivation of isocitrate lyase from Pseudomonas indigofera by 3-bromopyruvate established that enzyme binds this compound prior to alkylation and that substrate, D_s-isocitrate, competes for the same site on the enzyme. The rate of inactivation was increased by EDTA which is a promoter of catalysis in the presence of activated (reduced) enzyme and substrate. The combination of products, glyoxylate plus succinate, also protected against inactivation. Glyoxylate plus itaconate, phosphoenolpyruvate, or maleate also protected. However, each of the latter three compounds or glyoxylate or succinate alone provided little or no protection. Pyruvate, a competitive inhibitor with respect to glyoxylate in the condensation reaction, also failed to protect. However, two dicarboxylates, meso-tartrate and oxalate, that are also competitive inhibitors with respect to glyoxylate provide some protection against inactivation by BrP perhaps by bridging across cationic sites that facilitate glyoxylate and succinate binding. These and other results imply that alkylation by 3-bromopyruvate occurs at the succinate part of the active site. A mechanism which includes a catalytic role for the cysteine residue at the active site is presented and discussed.     

Distribution of peroxisomes and glycolate metabolism in relation to calcium oxalate formation in Lemna minor L

Calcium oxalate formation in Lemna minor L. occurs in structurally specialized cells called crystal idioblasts. Cytochemical and immunocytochemical protocols were employed to study the distribution of peroxisomes and the enzymes glycolate oxidase, glycine decarboxylase and ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) in relation to synthesis of oxalate used for Ca oxalate formation. These enzymes are necessary for photorespiratory glycolate synthesis and metabolism. Using catalase cytochemistry, microbodies were found to exist in crystal idioblasts but were smaller and fewer than those found in mesophyll cells. Glycolate oxidase, which can oxidize glycolate to oxalate via glyoxylate, could not be found in microbodies of crystal idioblasts at any stage of development. This enzyme increased in amount in microbodies of mesophyll cells as they matured and could even be found in dense amorphous inclusions of mature cell peroxisomes. Glycine decarboxylase and RuBisCO could also be detected in increasing amount in mesophyll cells as they matured but could not be detected in idioblasts or were just detectable. Thus, Lemna idioblasts lack the machinery for synthesis of oxalate from glycolate. Based on these results and other available information, two general models for the generation and accumulation of oxalate used for Ca oxalate formation in crystal idioblasts are proposed. The biochemical specialization of crystal idioblasts indicated by this study is also discussed with respect to differentiation of cellular structure and function.     

Control of the citric acid cycle by glyoxylate. The mechanism of inhibition of oxoglutarate dehydrogenase, isocitrate dehydrogenase and aconitate hydratase

1. The effects of glyoxylate on partially purified preparations of aconitate hydratase, isocitrate dehydrogenase and oxoglutarate dehydrogenase were compared with those of oxalomalate and hydroxyoxoglutarate (obtained by condensation of glyoxylate with oxaloacetate and pyruvate respectively). 2. Glyoxylate (1mm) did not affect aconitate hydratase and isocitrate dehydrogenase, whereas oxalomalate (1mm) inhibited the enzyme activities completely. 3. Glyoxylate (0.025mm) inhibited oxoglutarate dehydrogenase irreversibly, whereas the same concentrations of oxalomalate and hydroxyoxoglutarate were ineffective. This inhibitory effect was prevented if oxoglutarate, pyruvate or oxaloacetate was mixed with the enzyme before the glyoxylate. 4. Incubation of oxoglutarate dehydrogenase with radioactive glyoxylate produced radioactive carbon dioxide; radioactivity was also recovered in the portion of the enzyme identified with thiamin pyrophosphate. 5. The behaviour of glyoxylate in producing multiple inhibitions of the citric acid cycle, either by direct interaction with oxoglutarate dehydrogenase, or by means of its condensation compounds which inhibit aconitate hydratase and isocitrate dehydrogenase, is discussed.     

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.