Identity of alanine:glyoxylate aminotransferase with alanine:2-oxoglutarate aminotransferase in rat liver cytosol

Rat liver soluble fraction contained 3 forms of alanine: glyoxylate aminotransferase. One with a pI of 5.2 and an Mr of approx. 110,000 was found to be identical with cytosolic alanine:2-oxoglutarate aminotransferase. The pI 6.0 enzyme with an Mr of approx. 220,000 was suggested to be from broken mitochondrial alanine:glyoxylate aminotransferase 2 and the pI 8.0 enzyme with an Mr of approx. 80,000 enzyme from broken peroxisomal and mitochondrial alanine:glyoxylate aminotransferase 1. These results suggest that the cytosolic alanine: glyoxylate aminotransferase activity is due to cytosolic alanine: 2-oxoglutarate aminotransferase.     

Developmental profiles and properties of hepatic peroxisomal apo- and mitochondrial holoalanine:glyoxylate aminotransferase during chick embryogenesis

Alanine:glyoxylate aminotransferase was present as the apoenzyme in the peroxisomes and as the holoenzyme in the mitochondria in chick embryos. The peroxisomal enzyme predominated in the early stage and gradually decreased during embryonic development and disappeared after hatching. In contrast, the mitochondrial enzyme gradually increased and predominated in the later stage of chick embryos. Peroxisomal alanine:glyoxylate aminotransferase in chick embryos was a single peptide with a molecular weight of about 40,000. The enzyme differed from the mitochondrial enzyme in the embryos, and mammalian alanine:glyoxylate aminotransferases 1 (with a molecular weight of about 80,000 with two identical subunits) and 2 (with a molecular weight of about 200,000 with four identical subunits) in molecular weights and immunological properties. Mitochondrial alanine:glyoxylate aminotransferase in chick embryos had an identical molecular weight and immunologically cross-reacted with mammalian mitochondrial alanine:glyoxylate aminotransferase 2. Pyridoxal 5'-phosphate dissociated easily from the peroxisomal enzyme saturated with pyridoxal 5'-phosphate. Hepatic aspartate:2-oxoglutarate aminotransferase and alanine:2-oxoglutarate aminotransferase in chick embryos, and hepatic alanine:glyoxylate aminotransferases in different animal species were all present as the holoenzyme.     

Purification and characterization of peroxisomal apo and holo alanine:glyoxylate aminotransferase from bird liver.

Alanine:glyoxylate aminotransferase has been reported to be present as the apo enzyme in the peroxisomes and as the holo enzyme in the mitochondria in chick (white leghorn) embryonic liver. However, surprisingly, birds were found to be classified into two groups on the basis of intraperoxisomal forms of liver alanine:glyoxylate aminotransferase. In the peroxisomes, the enzyme was present as the holo form in group 1 (pigeon, sparrow, Java sparrow, Australian budgerigar, canary, goose, and duck), and as the apo form in group 2 (white leghorn, bantam, pheasant, and Japanese mannikin). In the mitochondria, the enzyme was present as the holo form in both groups. The peroxisomal holo enzyme was purified from pigeon liver, and the peroxisomal apo enzyme from chicken (white leghorn) liver. The pigeon holo enzyme was composed of two identical subunits with a molecular weight of about 45,000, whereas the chicken apo enzyme was a single peptide with the same molecular weight as the subunit of the pigeon enzyme. The peroxisomal holo enzyme of pigeon liver was not immunologically cross-reactive with the peroxisomal apo enzyme of chicken liver, the mitochondrial holo enzymes from pigeon and chicken liver, and mammalian alanine:glyoxylate aminotransferases 1 and 2. The mitochondrial holo enzymes from both pigeon and chicken liver had molecular weights of about 200,000 with four identical subunits and were cross-reactive with mammalian alanine:glyoxylate aminotransferase 2 but not with mammalian alanine:glyoxylate aminotransferase 1.     

A novel hyperthermophilic archaeal glyoxylate reductase from Thermococcus litoralis. Characterization, gene cloning, nucleotide sequence and expression in Escherichia coli.

A novel NADH-dependent glyoxylate reductase has been found in a hyperthermophilic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophilic archaea. NADH-dependent glyoxylate reductase was purified approximately 560-fold from a crude extract of the hyperthermophile by five successive column chromatographies and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodimer with a subunit molecular mass of approximately 37 kDa. The optimum pH and temperature for enzyme activity were approximately 6.5 and 90 degrees C, respectively. The enzyme was extremely thermostable; the activity was stable up to 90 degrees C. The glyoxylate reductase catalyzed the reduction of glyoxylate and hydroxypyruvate, and the relative activity for hydroxypyruvate was approximately one-quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron donor compared with NADH. The Km values for glyoxylate, hydroxypyruvate, and NADH were determined to be 0.73, 1.3 and 0.067 mM, respectively. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the glyoxylate reductase gene was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular mass of 36,807. The amino-acid sequence of the T. litoralis enzyme showed high similarity with those of probable dehydrogenases in Pyrococcus horikoshii and P. abyssi. The purification of the enzyme from recombinant E. coli was much simpler compared with that from T. litoralis; only two steps of heat treatment and dye-affinity chromatography were needed.     

Dimethylarginine:pyruvate aminotransferase in rats. Purification, properties, and identity with alanine:glyoxylate aminotransferase 2

Dimethylarginine:pyruvate aminotransferase, which plays a role in the metabolism of dimethylarginines, has been purified to homogeneity from rat kidney. The enzyme has a molecular weight of approximately 200,000 and an isoelectric point at about pH 6.3. The enzyme consists of four similar subunits having a molecular weight of about 50,000. The enzyme catalyzes the effective transaminations of guanidino-N methylated L-arginines (e.g. NG,NG-dimethyl-L-arginine, NG,N'G-dimethyl-L-arginine and NG-monomethyl-L-arginine) and the alpha-amino group of L-ornithine to pyruvate or glyoxylate. The enzyme was always accompanied by the known alanine:glyoxylate amino-transferase activity with the ratios of their specific activities remaining constant during the purification steps. The physicochemical and immunological properties of the purified enzyme were shown to be identical with those of the isozyme of alanine:glyoxylate aminotransferase (EC 2.6.1.44), designated as alanine:glyoxylate aminotransferase 2 (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The distribution profiles in tissues and the negative response to glucagon treatment further supported the identity of the two enzymes. The present data show that alanine:glyoxilate aminotransferase 2 functions in dimethylarginine metabolism in vivo in rats.     

Alanine: glyoxylate aminotransferase 1 is present in the peroxisomes of guinea pig kidney

The subcellular distribution of alanine: glyoxylate aminotransferase 1 in guinea pig and rabbit kidneys was examined by centrifugation in a sucrose density gradient. The enzyme was located in the peroxisomes of guinea pig kidney and cross-reactive with the antibody against rat liver alanine: glyoxylate aminotransferase 1. This is the first report on the presence of the enzyme in the peroxisomes of mammalian kidney. The enzyme was found to be located in the mitochondria but not in the peroxisomes in rabbit kidney.     

Enzymological characterization of a putative canine analogue of primary hyperoxaluria type 1.

This paper concerns an enzymological investigation into a putative canine analogue of the human autosomal recessive disease primary hyperoxaluria type 1 (alanine:glyoxylate/serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and serine:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephropathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including D-glycerate dehydrogenase/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 human 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 analogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.     

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.     

The evolution of peroxisomal and mitochondrial alanine: glyoxylate aminotransferase 1 in mammalian liver

Immunological distances of alanine: glyoxylate aminotransferase 1 (serine:pyruvate aminotransferase) in mitochondria or peroxisomes from eight different mammalian liver were determined with rabbit anti-serum against the mitochondrial enzyme of rat liver by microcomplement fixation. Results suggest that heterotopic alanine:glyoxylate aminotransferase 1 are orthologous proteins and their subcellular localization and substrate specificity changed during rapid molecular evolution.     

Peroxisomal alanine:glyoxylate aminotransferase deficiency in primary hyperoxaluria type I

Activities of alanine:glyoxylate aminotransferase in the livers of two patients with primary hyperoxaluria type I were substantially lower than those found in five control human livers. Detailed subcellular fractionation of one of the hyperoxaluric livers, compared with a control liver, showed that there was a complete absence of peroxisomal alanine:glyoxylate aminotransferase. This enzyme deficiency explains most of the biochemical characteristics of the disease and means that primary hyperoxaluria type I should be added to the rather select list of peroxisomal disorders.     

Alanine:glyoxylate aminotransferase is present as the apoenzyme in the peroxisomes of chicken kidney

The subcellular distribution of alanine:glyoxylate aminotransferase in chicken kidney was examined by centrifugation in a sucrose density gradient. The enzyme was found to be present as the apoform in the peroxisomes and as the holoform in the mitochondria. Alanine:glyoxylate aminotransferase in different mammalian kidneys were all present as the holoenzyme in the mitochondrial and soluble fractions.     

Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase.

The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes.     

Intraperoxisomal form of alanine:glyoxylate aminotransferase in the peroxisomes of bird kidney

Alanine:glyoxylate aminotransferase has been reported to be present as the apo form in the peroxisomes and as the holo form in the mitochondria in chicken kidney. In contrast, the enzyme was found to be present as the holo form both in the peroxisomes and in the mitochondria in pigeon kidney, suggesting that birds are classified into two groups on the basis of intraperoxisomal form of kidney alanine:glyoxylate aminotransferase. In the kidney, the pigeon peroxisomal holo enzyme did not cross-react immunologically with the chicken peroxisomal apo enzyme.     

Purification and properties of an asparagine aminotransferase from Pisum sativum leaves

The enzyme responsible for the transamination of L-asparagine in pea leaves has been partially purified. It appears to be the same protein as the serine-glyoxylate aminotransferase. It is able to use serine or asparagine as amino donors and pyruvate or glyoxylate as amino acceptors. The reaction is reversible but the equilibrium is toward glycine or alanine production. The favored substrates are serine and glyoxylate: serine shows competitive inhibition toward asparagine, as does pyruvate toward glyoxylate. Substrate interaction and product inhibition patterns are consistent with a ping-pong mechanism. The enzyme has a pH optimum at 8.1. Gel filtration indicates a Mr of 105,000. Inhibition was caused by aminoxyacetate and hydroxylamine, but the enzyme was unaffected by isonicotinic acid hydrazide. The apoenzyme was resolved and was inactive: addition of pyridoxal 5'-phosphate restored 85% of the original activity.     

Glyoxylate metabolism and fatty acid oxidation in mango fruit during development and ripening

Throughout the development (maturation) of mango fruit the contents of citric and glyoxylic acids increased steadily. As the fruit matured the levels of isocitrate lyase, malate lyase and alanine: glyoxylate aminotransferase increased and reached maximum values prior to the time of harvesting. At and after harvest the levels of malate lyase and alanine : glyoxylate aminotransferase began to decrease but that of isocitrate lyase remained high until after the harvest when it decreased. The level of glyoxylate reductase was highest in the early developmental stage but declined as the fruit matured and ripened. As the fruit ripened, after harvest, the amounts of citric and glyoxylic acids decreased concomitant with a considerable increase in the levels of isocitrate dehydrogenase, malic dehydrogenase, malic enzyme and glyoxylate dehydrogenase.Fatty acid oxidizing capacity of mitochondria isolated from immature (developing) and postclimacteric fruit pulps was much less than that observed with mitochondria from preclimacteric and climacteric fruit. Glyoxylate stimulated the oxidation of caprylic, lauric, myristic and palmitic acids and inhibited the activity of isocitrate dehydrogenase in vitro.     

Characteristics of alanine: glyoxylate aminotransferase from Saccharomyces cerevisiae, a regulatory enzyme in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates.

Alanine: glyoxylate aminotransferase (EC 2.6.1.44), which is involved in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates in Saccharomyces cerevisiae, was highly purified and characterized. The enzyme had Mr about 80 000, with two identical subunits. It was highly specific for L-alanine and glyoxylate and contained pyridoxal 5'-phosphate as cofactor. The apparent Km values were 2.1 mM and 0.7 mM for L-alanine and glyoxylate respectively. The activity was low (10 nmol/min per mg of protein) with glucose as sole carbon source, but was remarkably high with ethanol or acetate as carbon source (930 and 430 nmol/min per mg respectively). The transamination of glyoxylate is mainly catalysed by this enzyme in ethanol-grown cells. When glucose-grown cells were incubated in medium containing ethanol as sole carbon source, the activity markedly increased, and the increase was completely blocked by cycloheximide, suggesting that the enzyme is synthesized de novo during the incubation period. Similarity in the amino acid composition was observed, but immunological cross-reactivity was not observed among alanine: glyoxylate aminotransferases from yeast and vertebrate liver.     

Response of hepatic alanine:glyoxylate aminotransferase 1 to hormone differs among mammalia

The subcellular distribution and substrate specificity of hepatic alanine:glyoxylate aminotransferase 1 have been reported to differ among mammalia. In the present study, the response of this enzyme to hormone (glucagon) was found to differ among mammalia.     

Purification and Characterization of L-Alanine: 4,5-Dioxovalerate (Glyoxylate) Aminotransferase from Radish (Raphanus sativus L.) Seedlings

L-Alanine:4,5-dioxovalerate (DOVA) aminotransferase was purified131-fold and characterized from greening seedlings of radish(Raphanus sativus L.). The enzyme was shown to be identicalwith alanine:glyoxylate aminotransferase. The rate of activityof DOVA aminotransferase was 15 times less than that of glyoxylateaminotransferase. Its molecular weight was estimated to be approximately123,000 with two identical subunits, and exhibited a single,broad pH optimum at 8.0. DOVA aminotransferase activity wascompetitively inhibited by glyoxylate. A kinetic study of theenzyme at different alanine concentrations suggested a pingpong reaction mechanism. The K_m values for DOVA and L-alaninewere 0.71 and 1.7 mM, respectively. The activity ratio of transamination under various conditions,the cellular localization of the enzyme and the lack of correlationbetween the activity of this enzyme and chlorophyll synthesis,indicate that DOVA aminotransferase in radish is not involvedin 5-aminolevulinate synthesis. (Received July 7, 1984; Accepted September 11, 1984)