Branched-chain Aminotransferase Activity Research Articles

Reduced branched-chain aminotransferase activity alleviates metabolic vulnerability caused by dim light exposure at night in Drosophila

The rhythmic pattern of biological processes controlled by light over 24 h is termed the circadian rhythm. Disturbance of circadian rhythm due to exposure to light at night (LAN) disrupts the sleep-wake cycle and can promote cardiovascular disease, diabetes, cancer, and metabolic disorders in humans. We studied how dim LAN affects the circadian rhythm and metabolism using male Drosophila. Wild-type flies exposed to the dim light of 10 lux at night displayed altered 24 h sleep-wake behavior and expression patterns of circadian rhythm genes. In addition, the flies became more vulnerable to metabolic stress, such as starvation. Whole-body metabolite analysis revealed decreased amounts of branched-chain amino acids (BCAAs), such as isoleucine and valine. The dim light exposure also increased the expression of branched-chain amino acid aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase (BCKDC) enzyme complexes that regulate the metabolism of BCAAs. Flies with the Bcat heterozygous mutation were not vulnerable to starvation stress, even when exposed to dim LAN, and hemolymph BCAA levels did not decrease in these flies. Furthermore, the vulnerability to starvation stress was also suppressed when the Bcat expression level was reduced in the whole body, neurons, or fat body during adulthood using conditional GAL4 and RNA interference. Finally, the metabolic vulnerability was reversed when BCAAs were fed to wild-type flies exposed to LAN. Thus, short-term dim light exposure at night affects the expression of circadian genes and BCAA metabolism in Drosophila, implying a novel function of BCAAs in suppressing metabolic stress caused by disrupted circadian rhythm.

MiR-125a-3p-KLF15-BCAA Regulates the Skeletal Muscle Branched-Chain Amino Acid Metabolism in Nile Tilapia (Oreochromis niloticus) During Starvation.

The branched-chain amino acids (BCAAs) play a key role in the energy metabolism of the muscle tissue and the Krüppel-like factor 15 (KLF15) as a transcription factor, which is a key regulator of BCAA metabolism in the skeletal muscle. This study assessed the effect of starvation for 0, 3, 7, and 15 days on BCAA metabolism in the skeletal muscle of Nile tilapia. The results showed that the expression of KLF15 showed a trend of increasing first and then decreasing during starvation, as well as the expression and activity of branched-chain aminotransferase 2 (BCAT2) and alanine aminotransferase (ALT). On the other hand, the content of BCAA was at first decreased and then upregulated, and it reached the lowest level after starvation for 3 days. In addition, through dual-luciferase reporter assay and injection experiments, it was found that KLF15 is the target gene of miR-125a-3p, which further verified that miR-125a-3p can regulate the BCAA metabolism by targeting KLF15 in the skeletal muscle. Thus, our work investigated the possible mechanisms of BCAA metabolism adapting to nutritional deficiency in the skeletal muscle of Nile tilapia and illustrated the regulation of BCAA metabolism through the miR-125a-3p-KLF15-BCAA pathway in the skeletal muscle.

Emerging Moonlighting Functions of the Branched-Chain Aminotransferase Proteins

Significance: Unique to the branched-chain aminotransferase (BCAT) proteins is their redox-active CXXC motif. Subjected to post-translational modification by reactive oxygen species and reactive nitrogen species, these proteins have the potential to adopt numerous cellular roles, which may be fundamental to their role in oncogenesis and neurodegenerative diseases. An understanding of the interplay of the redox regulation of BCAT with important cell signaling mechanisms will identify new targets for future therapeutics. Recent Advances: The BCAT proteins have been assigned novel thiol oxidoreductase activity that can accelerate the refolding of proteins, in particular when S-glutathionylated, supporting a chaperone role for BCAT in protein folding. Other metabolic proteins were also shown to have peroxide-mediated redox associations with BCAT, indicating that the cellular function of BCAT is more diverse. Critical Issues: While the role of branched-chain amino acid metabolism and its metabolites has dominated aspects of cancer research, less is known about the role of BCAT. The importance of the CXXC motif in regulating the BCAT activity under hypoxic conditions, a characteristic of tumors, has not been addressed. Understanding how these proteins operate under various cellular redox conditions will become important, in particular with respect to their moonlighting roles. Future Directions: Advances in the quantification of thiols, their measurement, and the manipulation of metabolons that rely on redox-based interactions should accelerate the investigation of the cellular role of moonlighting proteins such as BCAT. Given the importance of cross talk between signaling pathways, research should focus more on these "housekeeping" proteins paying attention to their wider application. Antioxid. Redox Signal. 34, 1048-1067.

Branched-chain amino acid interactions in growing pig diets.

The branched-chain amino acids (BCAA) Leu, Ile, and Val share the first steps of their catabolism due to similarities in their structure. The BCAA are reversibly transaminated in skeletal muscle through the activity of branched-chain aminotransferase and then transported to the liver. They undergo an irreversible decarboxylation catalyzed by the branched-chain α-keto acid dehydrogenase complex. Both enzymes are common to Leu, Ile, and Val and increased enzymatic activity stimulated by an excess of one of them will increase the catabolism of all BCAA, which can result in antagonisms. Leucine and its keto acid are the most potent stimulators of BCAA catabolic enzymes. Moreover, BCAA and large neutral amino acids (LNAA) share common brain transporters. Research has shown that high concentrations of BCAA, especially Leu, can decrease the absorption of LNAA, such as Trp, which is a precursor of serotonin and can have a significant impact in feed intake regulation. Finally, high Leu concentrations have the ability to overstimulate the mTOR signaling pathway, resulting in an inhibitory effect on feed intake. Most of the research conducted to evaluate the impact of BCAA on growth performance of pigs seems to agree that high levels of Leu decrease weight gain, mostly due to a reduction in feed intake. However, some studies, mostly with finishing pigs, observed no evidence for an impact on growth performance even with extremely high levels of Leu. It could be hypothesized that these inconsistencies are driven by the entire dietary amino acid profile as opposed to only considering the level of Leu. Grow-finish diets typically contain high levels of Leu, but the other BCAA are also well above the requirement and could potentially mitigate the negative impact of Leu on BCAA catabolism. Indeed, some studies suggest that when diets contain high levels of Leu, more Ile and Val are needed to optimize growth performance. However, the precise relationship between BCAA and their balance in swine diets is not fully understood. More research is needed to understand and quantify the relationship between LNAA and BCAA.

PPARγ is a major regulator of branched-chain amino acid blood levels and catabolism in white and brown adipose tissues

ObjectiveWe investigated whether PPARγ modulates adipose tissue BCAA metabolism, and whether this mediates the attenuation of obesity-associated insulin resistance induced by pharmacological PPARγ activation. MethodsMice with adipocyte deletion of one or two PPARγ copies fed a chow diet and rats fed either chow, or high fat (HF) or HF supplemented with BCAA (HF/BCAA) diets treated with rosiglitazone (30 or 15 mg/kg/day, 14 days) were evaluated for glucose and BCAA homeostasis. ResultsAdipocyte deletion of one PPARγ copy increased mice serum BCAA and reduced inguinal white (iWAT) and brown (BAT) adipose tissue BCAA incorporation into triacylglycerol, as well as mRNA levels of branched-chain aminotransferase (BCAT)2 and branched-chain α-ketoacid dehydrogenase (BCKDH) complex subunits. Adipocyte deletion of two PPARγ copies induced lipodystrophy, severe glucose intolerance and markedly increased serum BCAA. Rosiglitazone abolished the increase in serum BCAA induced by adipocyte PPARγ deletion. In rats, HF increased serum BCAA, such levels being further increased by BCAA supplementation. Rosiglitazone, independently of diet, lowered serum BCAA and upregulated iWAT and BAT BCAT and BCKDH activities. This was associated with a reduction in mTORC1-dependent inhibitory serine phosphorylation of IRS1 in skeletal muscle and whole-body insulin resistance evaluated by HOMA-IR. ConclusionsPPARγ, through the regulation of both BAT and iWAT BCAA catabolism in lipoeutrophic mice and muscle insulin responsiveness and proteolysis in lipodystrophic mice, is a major determinant of circulating BCAA levels. PPARγ agonism, therefore, may improve whole-body and muscle insulin sensitivity by reducing blood BCAA, alleviating mTORC1-mediated inhibitory IRS1 phosphorylation.

Functional roles of a predicted branched chain aminotransferase encoded by the LkBAT1 gene of the yeast Lachancea kluyveri

Branched chain amino acid aminotransferases (BCATs) catalyze the last step of the biosynthesis and the first step of the catabolism of branched chain amino acids. In Saccharomyces cerevisiae, BCATs are encoded by the ScBAT1 and ScBAT2 paralogous genes. Analysis of Lachancea kluyveri genome sequence, allowed the identification of the LkBAT1 locus, which could presumably encode a BCAT. A second unlinked locus (LkBAT1bis), exhibiting sequence similarity to LkBAT1 was also identified. To determine the function of these putative BCATs, L. kluyveri mutant strains lacking LkBAT1, LkBAT1bis or both genes were generated and tested for VIL metabolism. LkBat1 displayed branched chain aminotransferase activity and is required for VIL biosynthesis and catabolism. However, Lkbat1Δ mutant is a valine and isoleucine auxotroph and a leucine bradytroph indicating that L. kluyveri harbors an alternative enzyme(s) involved in leucine biosynthesis. Additionally, heterologous reciprocal gene complementation between S. cerevisiae and L. kluyveri orthologous LkBAT1, ScBAT1 and ScBAT2 genes, confirmed that the mitochondrial LkBat1 functions as BCAT in S. cerevisiae, restoring wild type phenotype to the ScBAT1 null mutant. Conversely, LkBAT1bis did not display a role in BCAAs metabolism. However, when ethanol was used as carbon source, deletion of LkBAT1bis in an Lkbat1Δ null strain resulted in an extended ’lag’ growth phase, pointing to a potential function of LkBAT1 and LkBAT1bis in the aerobic metabolism of L. kluyveri. These results confirm the BCAT function of LkBAT1 in L. kluyveri, and further support the proposition that the BCAT function in ancestral-type yeasts has been distributed in the two paralogous genes present in S. cerevisiae.

The feasibility of assessing branched-chain amino acid metabolism in cellular models of prostate cancer with hyperpolarized [1-13C]-ketoisocaproate

Recent advancements in the field of hyperpolarized 13C magnetic resonance spectroscopy (MRS) have yielded powerful techniques capable of real-time analysis of metabolic pathways. These non-invasive methods have increasingly shown application in impacting disease diagnosis and have further been employed in mechanistic studies of disease onset and progression. Our goals were to investigate branched-chain aminotransferase (BCAT) activity in prostate cancer with a novel molecular probe, hyperpolarized [1-13C]-2-ketoisocaproate ([1-13C]-KIC), and explore the potential of branched-chain amino acid (BCAA) metabolism to serve as a biomarker. Using traditional spectrophotometric assays, BCAT enzymatic activities were determined in vitro for various sources of prostate cancer (human, transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse and human cell lines). These preliminary studies indicated that low levels of BCAT activity were present in all models of prostate cancer but enzymatic levels are altered significantly in prostate cancer relative to healthy tissue. The MR spectroscopic studies were conducted with two cellular models (PC-3 and DU-145) that exhibited levels of BCAA metabolism comparable to the human disease state. Hyperpolarized [1-13C]-KIC was administered to prostate cancer cell lines, and the conversion of [1-13C]-KIC to the metabolic product, [1-13C]-leucine ([1-13C]-Leu), could be monitored via hyperpolarized 13C MRS.

Three Different Classes of Aminotransferases Evolved Prephenate Aminotransferase Functionality in Arogenate-competent Microorganisms

The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1β in tested α- and β-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the β-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.

Widespread neuronal expression of branched‐chain aminotransferase in the CNS: implications for leucine/glutamate metabolism and for signaling by amino acids

Transamination of the branched-chain amino acids produces glutamate and branched-chain alpha-ketoacids. The reaction is catalyzed by branched-chain aminotransferase (BCAT), of which there are cytosolic and mitochondrial isoforms (BCATc and BCATm). BCATc accounts for 70% of brain BCAT activity, and contributes at least 30% of the nitrogen required for glutamate synthesis. In previous work, we showed that BCATc is present in the processes of glutamatergic neurons and in cell bodies of GABAergic neurons in hippocampus and cerebellum. Here we show that this metabolic enzyme is expressed throughout the brain and spinal cord, with distinct differences in regional and intracellular patterns of expression. In the cerebral cortex, BCATc is present in GABAergic interneurons and in pyramidal cell axons and proximal dendrites. Axonal labeling for BCATc continues into the corpus callosum and internal capsule. BCATc is expressed by GABAergic neurons in the basal ganglia and by glutamatergic neurons in the hypothalamus, midbrain, brainstem, and dorsal root ganglia. BCATc is also expressed in hypothalamic peptidergic neurons, brainstem serotoninergic neurons, and spinal cord motor neurons. The results indicate that BCATc accumulates in neuronal cell bodies in some regions, while elsewhere it is exported to axons and nerve terminals. The enzyme is in a position to influence pools of glutamate in a variety of neuronal types. BCATc may also provide neurons with sensitivity to nutrient-derived BCAAs, which may be important in regions that control feeding behavior, such as the arcuate nucleus of the hypothalamus, where neurons express high levels of BCATc.

Mitochondrial branched chain aminotransferase gene expression in AS-30D hepatoma rat cells and during liver regeneration after partial hepatectomy in rat

Branched chain aminotransferase (BCAT) is the first enzyme in the catabolism of branched chain amino acids (BCAA). Unlike other amino acid degrading enzymes present in liver, BCAT is only expressed in extrahepatic tissues, and is not regulated by dietary protein, glucagon or glucocorticoids. However, the mitochondrial (m) isoform of BCAT is highly expressed in the fetal liver and rapidly decays after birth. The purpose of the present work was to establish if liver cells under conditions of rapid cell proliferation such as in hepatoma AS30D cells or during liver regeneration after partial hepatectomy were associated with an increase in the activity and expression of BCATm. BCAT activity in mitochondria of AS30D cells was 18.6 mU/mg protein. Western, Northern blot, and immunohistochemical analysis revealed that AS30D hepatoma cells expressed only BCATm. The apparent Km of BCATm in isolated AS30D cells mitochondria for leucine, isoleucine and valine was 1.0 ± 0.02, 1.3 ± 0.1 and 2.1 ± 0.1 mM, respectively. The regenerated liver showed BCAT activity from day 3 to day 6, and the maximal BCAT activity (7.0 mU/mg protein) was on day 5. By day 14 after partial hepatectomy BCAT activity and expression was almost undetectable. Interestingly, there was a relationship between BCAT activity and the Mr. of the immunoreactive band of BCATm. The presence of a 41 kDa band was associated with BCAT activity, whereas the 43 kDa band with undetectable activity. The results of this study indicate that BCATm activity is required in liver cells under conditions of rapid cell proliferation.

Cooperation between wild lactococcal strains for cheese aroma formation

Several wild lactococcal strains were tested for their ability to produce aroma compounds during growth in milk. Strains were incubated alone and in combination with Lactococcus lactis IFPL730, which is characterized by showing α-keto acid decarboxylase activity. Volatile compounds from incubated milks were analyzed by means of solid-phase microextraction (SPME) and evaluated by gas chromatography–mass spectrometry (GC–MS). Incubated milks were also sniffed for sensory analysis to describe aroma attributes. The combination of L. lactis IFPL326 that showed the highest branched chain aminotransferase activity with IFPL730 contributed to the highest formation of leucine-derived volatile compounds, such as 3-methylbutanal, 3-methyl-1-butanol and 2-hydroxy-4-methyl pentanoic acid methyl ester. In addition, the milk incubated with this combination of strains was awarded, by the test panellists, the highest scores for “ripened cheese” attribute and aroma intensity. The results indicate that combination of L. lactis strains harbouring complementary catabolic routes can contribute to improved cheese aroma formation, the combined cultures with L. lactis IFPL730 resulting in higher volatile compound formation than isolate strains.

Localization and expression of BCAT during pregnancy and lactation in the rat mammary gland.

During lactation, branched-chain aminotransferase (BCAT) gene expression increases in the mammary gland. To determine the cell type and whether this induction is present only during lactation, female rats were randomly assigned to one of three experimental groups: pregnancy, lactation, or postweaning. Mammary gland BCAT activity during the first days of pregnancy was similar to that of virgin rats, increasing significantly from day 16 to the last day of pregnancy. Maximal BCAT activity occurred on day 12 of lactation. During postweaning, BCAT activity decreased rapidly to values close to those observed in virgin rats. Analyses by Western and Northern blot revealed that changes in enzyme activity were accompanied by parallel changes in the amount of enzyme and its mRNA. Immunohistochemical studies of the mammary gland showed a progressive increase in mitochondrial BCAT (mBCAT)-specific staining of the epithelial acinar cells during lactation, reaching high levels by day 12. Immunoreactivity decreased rapidly after weaning. There was a significant correlation between total BCAT activity and milk production. These results indicate that the pattern of mBCAT gene expression follows lactogenesis stages I and II and is restricted to the milk-producing epithelial acinar cells. Furthermore, BCAT activity is associated with milk production in the mammary gland during lactation.

Dietary Protein Level Regulates Expression of the Mitochondrial Branched-Chain Aminotransferase in Rats

The first step in the degradation of branched-chain amino acids (BCAA) is transamination catalyzed by the branched-chain aminotransferase (BCAT), which is located in extrahepatic tissues. Studies of the effect of dietary protein on BCAT activity have given contradictory results. Therefore, we established the levels of BCAT activity and mitochondrial BCAT (BCATm) mRNA expression in different organs and tissues of rats. We then determined the effect of different levels of dietary protein in well-nourished rats, the effect of feeding a 0.5% casein diet for 5 wk (protein-malnourished rats) and nutritional rehabilitation of these rats with different levels of dietary protein on BCAT activity and BCATm mRNA expression in selected tissues. Finally, the response of tissue BCAT activity and BCATm mRNA levels in rats fed a 10% casein diet and injected with glucagon (4 d) or hydrocortisone (7 d) was determined. The highest concentration of BCATm mRNA was found in stomach, followed by kidney, heart, muscle, brain, skin and lung. Low levels were found in intestine, and no BCATm mRNA was detectable in liver. Although BCAT activity was significantly higher in muscle, kidney and brain from rats adapted to consume a 50% casein diet for 7 h/d for 10 d than in rats fed 6, 18 or 35% casein diets, only muscle had significantly higher levels of BCATm mRNA. In protein-malnourished rats, BCAT activity and BCATm mRNA expression in kidney, muscle and heart were not different from those of rats with free access to an 18% casein diet. Nutritional rehabilitation of the protein-malnourished rats with 50% casein for 21 d significantly increased the BCAT activity and BCATm mRNA expression in muscle. Neither hydrocortisone nor glucagon injection affected BCAT activity or BCATm mRNA concentrations in rat kidney, muscle or heart. We conclude that the nutritional regulation of BCATm is extrahepatic, tissue specific and may involve transcriptional and post-translational mechanisms.

The effect of tyrosine-deficient total parenteral nutrition on the synthesis of dihydroxyphenylalanine in neural tissue and the activities of tyrosine and branched-chain aminotransferases

The poor solubility of tyrosine (Tyr) limits the amount of this amino acid in total parenteral nutrition (TPN). In rats maintained on a standard pediatric TPN mixture, plasma and brain concentrations of Tyr are reduced to about 25% of the levels in chow-fed controls. To determine whether these low concentrations of Tyr affect the synthesis of catecholamines in neural tissue, the rate-limiting step (conversion of Tyr to dihydroxyphenylalanine [DOPA]) is studied by administering NSD-1015 to block the pyridoxal phosphate (PLP)-dependent decarboxylation of DOPA. However, in TPN rats, plasma concentrations of Tyr are increased by drug treatment. Because brain Tyr is also increased, these and other experiments using NSD-1015 clearly overestimate the rate of DOPA synthesis for drug-free rats on TPN. Nevertheless, in TPN rats, there is less DOPA in the brain in one experiment and less DOPA in the olfactory bulbs in another, versus control rats. Further examination of the metabolic effects of NSD-1015 reveals that the drug also elevates the concentration of branched-chain amino acids (BCAAs) in the plasma of TPN rats. These findings result from inhibition by NSD-1015 of the PLP-dependent aminotransferases that initiate catabolism of Tyr in the liver and BCAAs in the muscle. Despite the pronounced reduction in plasma Tyr, TPN rats showed a marked increase in the activity of hepatic Tyr aminotransferase compared with chow-fed controls. Conversely, although TPN elevates BCAA concentrations in plasma, the activity of branched-chain aminotransferase (BCAT) in the heart muscle of TPN rats is not different from control values. Different values but the same relationships are seen in drug-free rats.

Overexpression and Characterization of the Human Mitochondrial and Cytosolic Branched-chain Aminotransferases

We have developed overexpression systems for the human branched-chain aminotransferase isoenzymes. The enzymes function as dimers and have substrate specificity comparable with the rat enzymes. The human cytosolic enzyme appears to turn over 2-5 times faster than the mitochondrial enzyme, and there may be anion and cation effects on the kinetics of both enzymes. The two proteins demonstrate similar absorption profiles, and the far UV circular dichroism spectra show that no global structural changes occur when the proteins are converted from the pyridoxal to pyridoxamine form. On the other hand, the near UV circular dichroism spectra suggest differences in the local environment surrounding tyrosines within these proteins. Both enzymes require a reducing environment for maximal activity, but the mitochondrial enzyme can be inhibited by nickel ions in the presence of reducing agents, while the cytosolic enzyme is unaffected. Chemical denaturation profiles of the proteins show that there are differences in structural stability. Titration of -SH groups with 5,5'-dithiobis(2-nitrobenzoic acid) suggests that no disulfide bonds are present in the mitochondrial enzyme and that at least two disulfide bonds are present in the cytosolic enzyme. Two -SH groups are titrated in the native form of the mitochondrial enzyme, leading to complete inhibition of activity, while only one -SH group is titrated in the cytosolic enzyme with no effect on activity. Although these proteins share 58% identity in primary amino acid sequence, the local environment surrounding the active site appears unique for each isoenzyme.

Reduced kidney branched chain aminotransferase expression in puromycin aminonucleoside-induced nephrotic syndrome

Injection of puromycin aminonucleoside to rats induces nephrotic syndrome characterized by hypoalbuminemia, proteinuria and hypercholesterolemia. In these rats, a low protein diet (6% casein diet) increased serum albumin by 26.3%, decreased proteinuria by 39 % and reduced total cholesterol by 32%. Branched chain aminotransferase activity in kidney mitochondria of nephrotic rats fed 20 or 6% casein diet decreased by 30 and 24 % with respect to their pair-fed groups and it was not modified by the protein content of the diet. Mitochondrial branched chain aminotransferase mRNA expression decreased by 67.3 and 72.5% in nephrotic rats fed 20 and 6% casein diet in comparison to their pair-fed groups. Total serum branched chain amino acids concentration (leucine, isoleucine, valine) in nephrotic rats was 30% higher than their pair-fed groups and it was associated with a decrease in the branched chain aminotransferase activity and mRNA expression suggesting that the catabolism of branched chain amino acid is reduced to conserve body nitrogen.

Regulation of valine catabolism in canine tissues: tissue distributions of branched-chain aminotransferase and 2-oxo acid dehydrogenase complex, methacrylyl-CoA hydratase and 3-hydroxyisobutyryl-CoA hydrolase

To clarify the valine catabolism, the activities of principal enzymes in its catabolic pathway, branched-chain aminotransferase, branched-chain 2-oxo acid dehydrogenase complex, methacrylyl-CoA hydratase and 3-hydroxyisobutyryl-CoA hydrolase, were measured using canine tissues. After killing of beagle dogs, tissues (liver, pancreas, kidney, heart, skeletal muscle and mucosae of digestive organs such as stomach, small intestine and colon) were removed and immediately frozen. Branched-chain aminotransferase activity in liver was the lowest among the tissues measured. In contrast, the activities of branched-chain 2-oxo acid dehydrogenase complex in liver as well as in kidney were relatively high and the enzyme complex activities were markedly low in small intestine and skeletal muscle. The activities of methacrylyl-CoA hydratase and 3-hydroxyisobutyryl-CoA hydrolase were relatively high in all tissues, suggesting that a cytotoxic intermediate, methacrylyl-CoA, is immediately degraded to non-toxic compounds, 3-hydroxyisobutyrate and free CoA. These findings suggest that the consumption of branched-chain amino acids in the absorption site (small intestine) is suppressed in order to supply them to the whole body, in particular to skeletal muscle and that skeletal muscle might act as a storage of gluconeogenic amino acids. The high capacity to dispose methacrylyl-CoA produced in the valine catabolism is suggested to play an important role in protecting cells against the toxic effects of methacrylyl-CoA.

Identification of the mitochondrial branched chain aminotransferase as a branched chain alpha-keto acid transport protein.

Conditions were developed which optimized reconstitution of branched chain alpha-keto acid transport activity, which was measured as alpha-ketoisocaproate (KIC) transport, and pyruvate transport activity. Reconstitutable KIC transport activity was about 40-fold higher in heart than in liver mitochondrial extracts and 40-fold higher than heart pyruvate transport activity but only about 7-fold higher than liver pyruvate transport activity. A purification procedure was developed for the branched chain alpha-keto acid and pyruvate transport proteins which resulted in partial purification of the proteins from rat heart mitochondria. Pyruvate transport activity appeared to be associated with a 32.5-kDa protein, whereas KIC transport activity appeared to be associated with two proteins around 41 kDa. As shown by immunoblotting and immunoaffinity chromatography, these proteins were recognized by an antiserum raised against purified rat heart mitochondrial branched chain aminotransferase (BCATm). Procedures used to extract BCATm from mitochondria effectively solubilized KIC transport activity, and both transaminase and transport activities in the sonicate supernatant were immunoprecipitated by BCATm antiserum. The tissue distribution of reconstitutable KIC transport activity was identical with the tissue distribution of BCATm. On the other hand, the distribution of reconstitutable pyruvate transport activity was distinct from that of KIC transport and branched chain aminotransferase activities, and pyruvate transport activity could not be immunoprecipitated by BCATm antiserum. When incorporated into phospholipid vesicles, purified BCATm exhibited branched chain alpha-keto acid transport activity but did not transport pyruvate. Transport was saturable, and Km values for KIC and alpha-ketoisovalerate uptake were 10 and 25 microM, respectively. Substrate competition experiments indicated that KIC transport could be inhibited substantially by branched chain alpha-keto acids and their derivatives but not by substrates for the pyruvate transporter. Transport was also inhibited by several aromatic carboxylic acid derivatives and alpha-ketoglutarate, but not by N-butylmalonate and succinate. Studies with covalent protein-modifying reagents indicated that transport was inhibited by sulfhydryl reagents, the histidine reagent diethyl pyrocarbonate, and the tyrosine reagent N-acetylimidazole. When BCATm was incorporated into phospholipid vesicles, pyridoxal 5-phosphate was an inhibitor of transport (75% inhibition at 10 mM) but had little effect on aminotransferase activity. The data indicate BCATm is a bifunctional protein catalyzing branched chain amino acid transamination and branched chain alpha-keto acid transport. The transport properties of BCATm suggest that this protein may be the branched chain alpha-keto acid transporter that was originally identified and characterized kinetically in isolated rat heart mitochondria (Hutson, S.M., and Rannels, S.L. (1985) J. Biol. Chem. 260, 14189-14193).

Branched chain aminotransferase isoenzymes. Purification and characterization of the rat brain isoenzyme.

This paper presents the first complete purification of the branched chain aminotransferase (EC 2.6.1.42) from rat brain cytosol (BCATc). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme appeared as a single band with a molecular mass of 47 kDa; however, gel exclusion chromatography suggested that BCATc is a dimer. Comparison of tryptic peptide maps of BCATc and the mitochondrial form of the enzyme (BCATm) indicated that they are different proteins. Experiments with protein labeling reagents, in particular sulfhydryl reagents, also suggested that there may be some distinct structural differences in BCATc and BCATm. Nevertheless, BCATc and BCATm showed similar specificities for amino acid and alpha-keto acid substrates. Both enzymes transaminated branched chain amino acids, their straight chain analogs, L-alloisoleucine and glutamate. A broader range of alpha-keto acids than amino acids was accepted as substrate including alpha-ketobutyrate and the alpha-keto acid of methionine. Both enzymes exhibited ping-pong kinetics with apparent Km values for leucine and isoleucine of about 1 and 5 mM for valine, respectively. Km values for alpha-ketoglutarate ranged from about 0.6 to 3 mM depending on the amino acid substrate. Polyclonal antibodies were raised in rabbits against purified BCATc. BCATc antiserum neutralized branched chain aminotransferase activity in rat brain cytosol but did not affect the activity in a heart mitochondrial extract. However, immunoblotting showed that BCATc and BCATm do share common epitopes since BCATm antiserum recognized BCATc on the immunoblots. The tissue distribution of BCATc was examined using BCATc and BCATm antisera. These data showed that BCATc was found in adult and fetal rat brain, cultured cells from fetal rat brain cortex, ovary, and placenta. Brain had the highest activity followed by ovary, fetal brain, and placenta. BCATc was not found in fetal liver, adult rat liver, or a rat hepatoma cell line. These data provide clear evidence that BCATc, unlike BCATm, is restricted to several highly specialized tissues.

Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues.

The tissue distribution and subcellular location of branched chain aminotransferase was analyzed using polyclonal antibodies against the enzyme purified from rat heart mitochondria (BCATm). Immunoreactive proteins were visualized by immunoblotting. The antiserum recognized a 41-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate. The 41-kDa protein was always present in mitochondria which contained branched chain aminotransferase activity, skeletal muscle, kidney, stomach, and brain, but not in cytosolic fractions. In liver mitochondria, which have very low levels of branched chain aminotransferase activity, the 41-kDa protein was not present. However, two immunoreactive proteins of slightly higher molecular masses were identified. These proteins were located in hepatocytes. The 41-kDa protein was present in fetal liver mitochondria but not in liver mitochondria from 5-day neonates. Thus disappearance of the 41-kDa protein coincided with the developmental decline in liver branched chain aminotransferase activity. Two-dimensional immunoblots of isolated BCATm immunocomplexes showed that the liver immunoreactive proteins were clearly different from the heart and kidney proteins which exhibited identical immunoblots. Investigation of BCATm in subcellular fractions prepared from different skeletal muscle fiber types revealed that branched chain aminotransferase is exclusively a mitochondrial enzyme in skeletal muscles. Although total detergent-extractable branched chain aminotransferase activity was largely independent of fiber type, branched chain aminotransferase activity and BCATm protein concentration were highest in mitochondria prepared from white gastrocnemius followed by mixed skeletal muscles with lowest activity and protein concentration found in soleus mitochondria. These quantitative differences in mitochondrial branched chain aminotransferase activity and enzyme protein content suggest there may be differential expression of BCATm in different muscle fiber types.