Branched-chain Amino Acid Aminotransferase Activity Research Articles

An electricalchemical method to detect the branch-chain aminotransferases activity in lactic acid bacteria

In this study, an electrochemical system was established to detect the branched-chain amino acid aminotransferase (BCAT) activity in lactic acid bacteria (LAB). A nanocomposite of chitosan (CS) with multi-walled carbon nanotubes (MWCNTs) was synthesized, and the composite solution were uniformly spread over the glassy carbon electrode (GCE) surface by drop-casting to fabricate an electrochemical biosensor. The composite was characterized by scanning electron microscopy (SEM) and cyclic voltammetry (TEM). Results indicated that the MWCNTs–CS/GCE electrode exhibited higher stability and sensitivity, compared with the GCE electrode. The linear response for nicotinamide adenine dinucleotide (NADH) was 1.0–9.0 μM and the response limit was 0.12 µM. The system effectively and sensitively detected the BCAT activity by NADH concentration in the LAB culture, comparing with the optical method. The culture condition of LAB was optimized by using this system, evidencing that established method was available to detect the BCAT activity of LAB.

Physicochemical, taste, and functional changes during the enhanced fermentation of low-salt Sufu paste, a Chinese fermented soybean food

ABSTRACTIn this study, the physicochemical, taste, and functional changes in low-salt Sufu paste were investigated during fermentation as enhanced by a mixed starter (Pichia fermentans, Kodamaea ohmeri, and Lactococcus lactis subsp. lactis) and α-ketoglutarate. The total free amino acids increased from 0.02 mg/g dry matter to 12.63 mg/g dry matter, and monosodium glutamate-like was the major free amino acid group (3.89 mg/g dry matter), followed by bitter according to the taste characteristics. Functional components such as γ-aminobutyric acid, riboflavin (VB2), and puerarin significantly increased to 30.96, 4.97, and 12.03 mg/g dry matter, respectively, at the end of post-ripening (p < 0.05). Protease, lipase, peptidase, α-amylase, aromatic amino acid aminotransferase, and branched-chain amino acid aminotransferase activities increased and were significantly correlated (p < 0.05) with most physicochemical and functional components, indicating that enzymes may play an important role in the fermentation process of Sufu paste. Additionally, the color of the Sufu paste changed from pale yellow to yellowish brown, whereas the Sufu paste fermented without the mixed starter and α-ketoglutarate changed to gray, the unique color of gray Sufu. The preliminary results indicated that the mixed starter and α-ketoglutarate are beneficial for taste quality and color and that fermentation may be an effective method to enhance the functional components in Sufu paste. This information would be useful for future improvements in the manufacturing process and quality of Sufu paste.

Identification of (R)-selective ω-aminotransferases by exploring evolutionary sequence space.

Several (R)-selective ω-aminotransferases (R-ωATs) have been reported. The existence of additional R-ωATs having different sequence characteristics from previous ones is highly expected. In addition, it is generally accepted that R-ωATs are variants of aminotransferase group III. Based on these backgrounds, sequences in RefSeq database were scored using family profiles of branched-chain amino acid aminotransferase (BCAT) and d-alanine aminotransferase (DAT) to predict and identify putative R-ωATs. Sequences with two profile analysis scores were plotted on two-dimensional score space. Candidates with relatively similar scores in both BCAT and DAT profiles (i.e., profile analysis score using BCAT profile was similar to profile analysis score using DAT profile) were selected. Experimental results for selected candidates showed that putative R-ωATs from Saccharopolyspora erythraea (R-ωAT_Sery), Bacillus cellulosilyticus (R-ωAT_Bcel), and Bacillus thuringiensis (R-ωAT_Bthu) had R-ωAT activity. Additional experiments revealed that R-ωAT_Sery also possessed DAT activity while R-ωAT_Bcel and R-ωAT_Bthu had BCAT activity. Selecting putative R-ωATs from regions with similar profile analysis scores identified potential R-ωATs. Therefore, R-ωATs could be efficiently identified by using simple family profile analysis and exploring evolutionary sequence space.

Ontogeny and subcellular localization of rat liver mitochondrial branched chain amino-acid aminotransferase.

Branched chain amino-acid aminotransferase (BCAT) activity is present in fetal liver but the developmental pattern of mitochondrial BCAT (BCATm) expression in rat liver has not been studied. The aim of this study was to determine the activity, protein and mRNA concentration of BCATm in fetal and postnatal rat liver, and to localize this enzyme at the cellular and subcellular levels at both developmental stages. Maximal BCAT activity and BCATm mRNA expression occurred at 17 days' gestation in fetal rat liver and then declined significantly immediately after birth. This pattern was observed only in liver; rat heart showed a different developmental pattern. Fetal liver showed intense immunostaining to BCATm in the nuclei and mitochondria of hepatic cells and blood cell precursors; in contrast, adult liver showed mild immunoreactivity located only in the mitochondria of hepatocytes. BCAT activity in isolated fetal liver nuclei was 0.64 mU x mg(-1) protein whereas it was undetectable in adult liver nuclei. By Western blot analysis the BCATm antibody recognized a 41-kDa protein in fetal liver nuclei, and proteins of 41 and 43 kDa in fetal liver supernatant. In adult rat liver supernatant, the BCATm antibody recognized only a 43-kDa protein; however, neither protein was detected in adult rat liver nuclei. The appearance of the 41-kDa protein was associated with the presence of the highly active form of BCATm. These results suggest the existence of active and inactive forms of BCAT in rat liver.

Branched-Chain Amino Acid Aminotransferase Activity Decreases during Development in Skeletal Muscles of Sheep

The catabolism of branched-chain amino acid (BCAA) differs between sheep and monogastric animals. The transamination of BCAA seems to be affected by development of the sheep. We studied the developmental changes in the activity and expression of the BCAA aminotransferase (BCAT) isoenzymes in skeletal muscle of sheep. Five muscles were taken from fetus, newborn, preruminant and ruminant lambs. BCAT specific activity and the contribution of each BCAT isoenzyme [mitochondrial and cytosolic (BCATm and BCATc, respectively)] were quantified using radioenzymatic and immunoprecipitation assays. BCATm and BCATc mRNAs were assessed by real-time reverse transcription-polymerase chain reaction. BCAT specific activities were 62% (diaphragma) to 83% (longissimus dorsi) lower in the ruminant lamb than in the fetal sheep. BCATm and BCATc were both expressed in sheep skeletal muscle at all developmental stages. BCATc was mainly responsible for the developmental decrease in BCAT specific activity. BCATc specific activities were 77% (diaphragma) to 92% (longissimus dorsi) lower in the ruminant lamb than in the fetal sheep, whereas BCATm specific activities were only 36% (semimembranosus) to 56% (longissimus dorsi) lower. BCATc and BCATm mRNAs in the longissimus dorsi were not affected by development of the sheep. The developmental decrease in BCATc activity, and to a lesser extent in BCATm activity, probably involves posttranscriptional mechanisms in sheep. The present results are consistent with lower in vivo metabolism of BCAA in ruminant than in the fetal sheep.

Enzymatic Method for Determination of Branched-Chain Amino Acid Aminotransferase Activity

A spectrophotometric assay for the determination of branched-chain L-amino acid aminotransferase activity is described. It is based on the transamination of L-leucine in the presence of 2-oxoglutarate yielding 4-methyl-2-oxopentanoate. The rate of formation of the branched-chain 2-oxo acid is specifically monitored in a coupled enzymatic reaction using NAD +-dependent D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei ssp. pseudoplantarum as coupling enzyme by measuring the decrease in NADH absorbance at 334 nm. Optimized assay conditions are provided as evaluated for the (iso)enzyme from rat heart.

Changes in activity levels and isozyme patterns of isoleucine aminotransferase in response to experimentally induced hepatic lesion

Branched-chain amino acid aminotransferase activities were measured in rats in which hepatic lesions were induced experimentally, that is, fatty liver produced by an orotic acid-containing diet and acute hepatic lesion induced by d-galactosamine injection into the abdominal cavity. The levels of the enzyme activities, using l-isoleucine as substrate, were elevated in both cases. Among the isozymes (enzymes I, II and III) of the enzyme, the activity of enzyme I was elevated by orotic acid treatment. However, with d-galactosamine treatment, another isoleucine aminotransferase activity was chromatographically separated from that of enzyme I and is considered to be enzyme III, and not enzyme II, based on its substrate specificity.

Regulation of valine and alpha-ketoisocaproate metabolism in rat kidney mitochondria.

Activities of branched-chain amino acid (BCAA) aminotransferase (BCAT) and alpha-keto acid dehydrogenase (BCKD) were assayed in mitochondria isolated from kidneys of rats. Rates of transamination of valine and oxidation of keto acids alpha-ketoisocaproate (KIC) or alpha-ketoisovalerate (KIV) were estimated using radioactive tracers of the appropriate substrate from amounts of 14C-labeled products formed (14CO2 or [1-14C]-keto acid). Because of the high mitochondrial BCAT activity, an amino acceptor for BCAT, alpha-ketoglutarate (alpha-KG) or KIC, was added to the assay medium when valine was the substrate. Rates of valine transamination and subsequent oxidation of the KIV formed were determined with 0.5 mM alpha-KG as the amino acceptor; these rates were 5- to 50-fold those without added alpha-KG. Rates of CO2 evolution from valine also increased when KIC (0.01-0.10 mM) was present; however, with KIC concentrations above 0.2 mM, rates of CO2 evolution from valine declined although rates of transamination continued to rise. When 0.05 mM KIC was added to the assay medium, oxidation of KIC was suppressed by inclusion of valine or glutamate in the medium. When valine was present KIC was not oxidized preferentially, presumably because it was also serving as an amino acceptor for BCAT. These results indicate that as the supply of amino acceptor, alpha-KG or KIC, is increased in mitochondria not only is the rate of valine transamination stimulated but also the rate of oxidation of the KIV formed from valine.(ABSTRACT TRUNCATED AT 250 WORDS)

Activities of branched-chain amino acid aminotransferase and branched-chain 2-oxo acid dehydrogenase complex in tissues of maternal and fetal sheep.

Branched-chain amino acid aminotransferase (BCAAT) and branched-chain 2-oxo acid dehydrogenase (BCODH) activities were determined in sheep maternal and fetal liver, kidney, skeletal muscle, adipose and placenta in the fed and 5-day-starved states at 125 days gestation (term is 147 days). BCAAT activities were quite low in maternal skeletal muscle, low in fetal and maternal liver, high in fetal muscle, and very high in placenta. No significant changes occurred with maternal starvation. The high BCAAT activity of the placenta may provide the essential branched-chain 2-oxo acids for the fetus. BCODH activity was highest in liver and kidney of both the mother and fetus; it was largely in the active (dephosphorylated) state in maternal liver and kidney, and in fetal kidney, and about 50% active in fetal liver. The enzyme was largely inactive in fetal muscle, and about 50% active in maternal muscle. Although starvation had little effect on the activity of BCODH in fetal tissues, a significant decrease in activity was observed in maternal tissues, thereby potentially sparing branched-chain amino acids or the corresponding 2-oxo acids for maintenance of the fetus during compromised maternal nutrition.

Activation of Liver Branched-Chain α-Keto Acid Dehydrogenase in Rats by Excesses of Dietary Amino Acids

As part of an effort to explain the leucine-induced depressions of plasma isoleucine and valine concentrations, and the concomitant stimulation of valine oxidation in vivo, branched-chain α-keto acid dehydrogenase (BCKAD) activity was measured in livers from rats that were fed for only 6 h/d large quantities of individual amino acids in a low protein diet. Preincubation of homogenates with buffer containing Mg2+ and Ca2+ allowed estimation of fully active complex. Cytosolic and mitochondrial branched-chain-amino-acid aminotransferase (BCAAT) activities were also measured in livers of rats fed an excess of leucine. The percentage of BCKAD in the active form in livers of rats fed the low protein diet containing an excess of leucine, isoleucine, valine or phenylalanine for 2 d was double that of rats fed the low protein control diet (control, leucine, isoleucine, valine and phenylalanine groups having, respectively, 45 ± 2, 85 ± 7, 85 ± 3, 95 ± 5, and 81 ± 4% of hepatic BCKAD in the active form). Consumption of a low protein diet containing an excess of leucine had no significant effect on either cytosolic or mitochondrial BCAAT activities of liver. The response of BCKAD in liver can contribute to the leucine-induced stimulation of valine oxidation in vivo but analysis of the results of this study leads to the conclusion that other mechanisms, probably in nonhepatic tissues, must also be involved.

A method for branched-chain amino acid aminotransferase activity in microgram and nanogram tissue samples

A method for branched-chain amino acid aminotransferase is described which is based on running the reaction in the reverse of the usual direction with glutamate and α-ketoisocaproate as substrates.The α-ketoglutarate generated is reduced with glutamate dehydrogenase and NADH. For sensitivity in the nanomole range, the NAD + generated is measured directly by converting to the highly fluorescent strong alkali product. For smaller samples, down to the 0.2- to 2-pmol range, the NAD + is amplified by enzymatic cycling.

Metabolism of 2-oxo-acid analogues of leucine and valine in isolated rat hepatocytes.

Isolated hepatocytes were incubated with 1-14C-labelled 2-oxo-acid analogues of leucine and valine. Decarboxylation and transamination rates were determined by measuring 14CO2 release and the appearance of the corresponding 14C-labelled amino acid. Decarboxylation exceeded transamination with both substrates, the ratio of decarboxylation/transamination being 3.4 for 4-methyl-2- oxovalerate and 78 for 3-methyl-2-oxobutyrate. Urea synthesis and ammonia utilization were not significantly decreased by the 2-oxo-acids even under conditions of stimulated urea production. To see if dietary treatment can alter the decarboxylation/ transamination ratio in favour of transamination rats were fed diets low in nitrogen content. These diets caused a reduction of the branched-chain 2-oxo-acid dehydrogenase activity by 80% but did not alter the branched-chain amino-acid aminotransferase activity in liver cells. This change in enzyme activities resulted in a marked decrease of the uptake and decarboxylation rates of 4-methyl-2- oxovalerate but did not enhance the conversion to the corresponding amino acid. Reducing the uptake of branched-chain 2-oxo acids by liver might increase the amount of orally administered 2-oxo acids reaching the muscle for transamination thus improving the nitrogen-sparing effect of these compounds.

Branched-chain amino acid aminotransferase activity in the tissues of lake trout

1. 1. The enzyme branched-chain amino acid aminotransferase (BCAT) was found in five tissues of fingerling lake trout, Salvelinus namaycush, (listed in order of decreasing tissue specific activity): posterior kidney, skeletal muscle, gill, liver, and anterior kidney. 2. 2. This pattern is consistent with that found in other animals. 3. 3. The results of this study seem to indicate that BCAT in the liver of lake trout has a higher specific activity than that of the rat and that the specific activity is higher in both the liver and skeletal muscle than it is in these organs of the chick.