Alanine In Rat Liver Research Articles

Metabolism of D‐ and L‐[13C]alanine in rat liver detected by 1H and 13C NMR spectroscopy in vivo and in vitro

Alanine is the major amino acid utilized by the liver for gluconeogenesis under normal conditions. The metabolism of alanine in rat liver was investigated by means of 1H and 13C NMR spectroscopic studies in vivo and in vitro after infusion of L- and D-alanine labelled with 13C at the carboxyl and methyl group into normal, fasted rats. Valuable information about different metabolic pathways of alanine in rat liver and their regulation under the conditions of gluconeogenesis were obtained. The enrichment of the alanine pool by the infusate was estimated to be 11% for L-alanine and 70% for D-alanine. After infusion of labelled D-alanines, the metabolic pathway via D-amino acid oxidase was observed. The labelled alanines entered the tricarboxylic acid cycle mainly via pyruvate carboxylase; the ratio of pyruvate dehydrogenase activity to that of pyruvate carboxylase is about 28%. The ratio of flux from phosphoenolpyruvate (PEP) through phosphoenolpyruvate kinase as compared with the flux from PEP to glucose was approximately 42%. From the labelling pattern of glucose it was concluded that the pentose phosphate cycle was active under the experimental conditions. © 2000 John Wiley & Sons, Ltd.

Metabolism ofD- andL-[13C]alanine in rat liver detected by1H and13C NMR spectroscopyin vivo andin vitro

Alanine is the major amino acid utilized by the liver for gluconeogenesis under normal conditions. The metabolism of alanine in rat liver was investigated by means of (1)H and (13)C NMR spectroscopic studies in vivo and in vitro after infusion of L- and D-alanine labelled with (13)C at the carboxyl and methyl group into normal, fasted rats. Valuable information about different metabolic pathways of alanine in rat liver and their regulation under the conditions of gluconeogenesis were obtained. The enrichment of the alanine pool by the infusate was estimated to be 11% for L-alanine and 70% for D-alanine. After infusion of labelled D-alanines, the metabolic pathway via D-amino acid oxidase was observed. The labelled alanines entered the tricarboxylic acid cycle mainly via pyruvate carboxylase; the ratio of pyruvate dehydrogenase activity to that of pyruvate carboxylase is about 28%. The ratio of flux from phosphoenolpyruvate (PEP) through phosphoenolpyruvate kinase as compared with the flux from PEP to glucose was approximately 42%. From the labelling pattern of glucose it was concluded that the pentose phosphate cycle was active under the experimental conditions.

13C DEPT spectroscopy in vivo of [3- 13C]Alanine in rat liver using a 13C surface coil

13C DEPT spectroscopy in vivo of [3- 13C]Alanine in rat liver using a 13C surface coil

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.

Identification of mammalian aminotransferases utilizing glyoxylate or pyruvate as amino acceptor. Peroxisomal and mitochondrial asparagine aminotransferase.

The subcellular distribution of asparagine:oxo-acid aminotransferase (EC 2.6.1.14) in rat liver was examined by centrifugation in a sucrose density gradient. About 30% of the homogenate activity after the removal of the nuclear fraction was recovered in the peroxisomes, about 56% in the mitochondria, and the remainder in the soluble fraction from broken peroxisomes. The mitochondrial asparagine aminotransferase had identical immunological properties with the peroxisomal one. Glucagon injection to rats resulted in the increase of its activity in the mitochondria but not in the peroxisomes. Immunological evidence was obtained that the enzyme was identical with alanine:glyoxylate aminotransferase 1 (EC 2.6.1.44) which had been reported to be identical with serine:pyruvate aminotransferase (EC 2.6.1.51) (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The same results as described above were obtained with mouse liver. All of alanine:glyoxylate aminotransferase 1 in livers of mammals other than rodents, which cross-react with the antibody against rat liver alanine:glyoxylate aminotransferase 1, had no asparagine aminotransferase activity.

The adaptive behaviour of isoenzyme forms of rat liver alanine amino transferases during development

1. The activities of the mitochondrial and cytosol isoenzyme forms of l-alanine-glyoxylate and l-alanine-2-oxoglutarate aminotransferases were determined in rat liver during foetal and neonatal development. 2. The mitochondrial glyoxylate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. The cytosol glyoxylate aminotransferase and the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities first appear prenatally, increase further after birth and then rise to the adult values during weaning. 4. In foetal liver the mitochondrial glyoxylate aminotransferase and the cytosol 2-oxoglutarate aminotransferase activities are increased after injection in utero of glucagon, dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) or thyroxine. The cytosol glyoxylate aminotransferase and the mitochondrial 2-oxoglutarate aminotransferase activities are increased after injection in utero of cortisol or thyroxine. 5. After birth the further normal increases in the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities can be hastened by cortisol injection, whereas the increase in cytosol glyoxylate aminotransferase activity requires cortisol treatment together with the intragastric administration of casein. 6. The results are discussed with reference to the metabolic patterns and the changes in regulatory stimuli (hormonal and dietary) that occur during the period of development.

Rat Liver Alanine Aminotransferase: Crystallization, composition, and role of sulfhydryl groups

Abstract Rat liver alanine aminotransferase has been crystallized and has a specific activity of 500 to 550 units (micromoles per min) per mg. The enzyme contains two pyridoxal phosphate groups and 23 to 30 titratable sulfhydryl equivalents per molecule (114,000 daltons). The amino acid composition has been determined and includes 34 half-cystine residues per molecule. Reaction of the first seven or eight sulfhydryl groups with p-mercuribenzoate or 5,5'-dithiobis-(2-nitrobenzoic acid) led to no loss of activity, after which there was a loss of activity proportional to the extent of reaction of the next 16 equivalents with 5,5'-dithiobis-(2-nitrobenzoic acid). Resolution and partial reconstitution of the apoenzyme has been achieved. The spectrum of the pyridoxal form of the enzyme is pH dependent, with an acid peak at 430 mµ, a basic peak at 335 mµ, and an isosbestic point at 380 mµ.

Interaction of rat liver alanine aminotransferase with L-proline

A recent report from this laboratory ( Segal, Abraham,and Schatz, 1968) described some in vitro stability studies with rat liver alanine aminotransferase in which it was found that L-proline protected the enzyme against heat denaturation better than any other amino acid tested. It was of interest to us to pursue this observation in order to decide whether the interaction was at the active site, and if so whether a complex was formed with the pyridoxal moiety. The results allow us to conclude that L-proline does interact with the prosthetic group and to speculate about the nature of the complex and its relationship to normal intermediates in the transamination reaction.

Further Characterization of Alanine Aminotransferase of Rat Liver

Abstract Rat liver alanine aminotransferase has been obtained in a homogeneous state according to ultracentrifugal, electrophoretic, and immunological criteria. The molecular weight of the enzyme is 114,000. No physical differences were detected in the enzyme prepared from normal and glucocorticoid-treated animals. The enzyme exhibited an absorption maximum at 430 mµ at pH 5.0 and at 335 mµ at pH 8.3. After conversion to the pyridoxamine form, a maximum at 325 mµ appeared. Reaction of the enzyme with amino-oxyacetate shifted the 430 mµ peak to 370 mµ. The enzyme appeared to exist in several closely related forms of slightly different mobility on acrylamide gel. In the absence of the reducing agent, β-mercaptoethanol, a series of aggregated forms of the enzyme arose.