Purification and cloning of the mitochondrial branched-chain amino acid aminotransferase from sheep placenta

This paper presents the first purification of the mitochondrial branched-chain amino acid aminotransferase (BCATm) from sheep placenta. It is a homodimer with an apparent subunit molecular mass of 41 kDa. The enzyme differs from those of the rat and human as it appears to form at least one intermolecular disulfide bond. The sheep BCATm cDNA (1.4 kb) encodes a mature polypeptide of 366 amino acids with a calculated molecular mass of 41 329 Da and a partial mitochondrial targeting sequence of seven amino acids. The sheep BCATm sequence shares higher identity with other mammalian BCATm isoenzymes (82-85%) than with the cytosolic isoenzymes (60%). By Northern blot analysis, a message of 1.7 kb was detected in sheep placenta and skeletal muscle. Measurements of BCAT activity, mRNA and BCATm protein in sheep placenta and skeletal muscle revealed that BCATm is the sole BCAT isoenzyme expressed in placenta, whereas it contributes 57 and 71% of the BCAT activity in tensor fascia latae and masseter muscles from weaned lambs respectively. Skeletal muscle, the main site of branched-chain amino acid transamination, exhibits significantly lower BCAT activity in sheep than in rat. Our results suggest that the low BCATm mRNA level probably accounts for the low BCAT activity in sheep skeletal muscle, and that the metabolic scheme for branched-chain amino acid catabolism is specific to each species.     

Regulation of branched-chain amino acid metabolism in the lactating rat

There is evidence that during lactation, uptake of the essential branched-chain amino acids (BCAA) by mammary glands exceeds their output in milk protein. In this study, we have measured the potential of lactating rats to catabolize BCAA. The activity, relative protein and specific mRNA levels of the first two enzymes in the BCAA catabolic pathway, branched-chain aminotransferase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD), were measured in mammary gland, liver and skeletal muscle obtained from rat dams at peak lactation (12 d), from rat dams 24 h after weaning at peak lactation and from age-matched virgin controls. Western analysis showed that the mitochondrial BCATm isoenzyme was found in mammary gland. Comparison of lactating and control rats revealed that tissue BCATm activity, protein and mRNA were at least 10-fold higher in mammary tissue during lactation. Values were 1.3- to 1. 9-fold higher after 24 h of weaning. In mammary gland of lactating rats, the BCKD complex was fully active. In virgin controls and weaning dams, only about 20% of the complex was in the active state. Hypertrophy of the liver and mammary gland during lactation resulted in a 73% increase in total oxidative capacity in lactating rats. The results are consistent with increased expression of the BCATm gene in the mammary gland during lactation, whereas oxidation appears to be regulated primarily by changes in activity state (phosphorylation state) of BCKD.     

Acidosis and glucocorticoids induce branched-chain amino acid catabolism

Chronic renal failure (CRF) is frequently complicated by malnutrition and wasting. The loss of lean body mass in CRF is the result of accelerated protein and amino acid degradation. Both appear to occur via acidosis-induced, glucocorticoid-dependent processes. In skeletal muscle, acidosis stimulates the activity of the rate-limiting enzyme in branched-chain amino acid metabolism, branched-brain ketoacid dehydrogenase (BCKAD). The activation of BCKAD in acidosis is likely to be glucocorticoid-dependent.     

Tissue-specific responses of branched-chain alpha-ketoacid dehydrogenase activity in metabolic acidosis

In adrenalectomized rats, acidosis does not increase whole-body leucine oxidation unless a physiologic amount of glucocorticoids (dexamethasone) is also provided; an equivalent dose of dexamethasone without acidosis does not change leucine catabolism. Because the influences of acidification and glucocorticoids on branched-chain amino acid metabolism in specific organs are unknown, the function of branched-chain alpha-ketoacid dehydrogenase (BCKAD), the rate-limiting enzyme in branched-chain amino acid catabolism, in adrenalectomized rat skeletal muscle and liver, the two major tissues that degrade branched-chain amino acid was measured. In muscle of acidotic adrenalectomized rats receiving dexamethasone, basal and total BCKAD activities were increased 2.6- (P < 0.05) and 2.8-fold (P < 0.05), respectively. Neither acidosis nor dexamethasone alone increased these activities. BCKAD E1alpha subunit mRNA in muscle of acidotic rats given dexamethasone was increased 1.89-fold (P < 0.05) in parallel with the change in BCKAD activity; BCKAD E2 subunit mRNA was increased by acidosis, dexamethasone, or a combination of both stimuli. In contrast, basal BCKAD activity in liver of rats with acidosis or dexamethasone was nearly threefold lower (P < 0.05) and changes in enzyme activity reflected reduced subunit mRNA. Thus, there are reciprocal, tissue-specific changes in BCKAD function in response to acidosis.     

Mitochondrial and cytosolic branched-chain amino acid transaminases from yeast, homologs of the myc oncogene-regulated Eca39 protein

We have isolated a high copy suppressor of a temperature-sensitive mutation in ATM1, which codes for an ABC transporter of Saccharomyces cerevisiae mitochondria. The suppressor, termed BAT1, encodes a protein of 393 amino acid residues with an NH2-terminal extension that directs Bat1p to the mitochondrial matrix. A highly homologous protein, Bat2p, of 376 amino acid residues was found in the cytosol. Both Bat proteins show striking similarity to the mammalian protein Eca39, which is one of the few known targets of the myc oncogene. Deletion of a single BAT gene did not impair growth of yeast cells. In contrast, deletion of both genes resulted in an auxotrophy for branched-chain amino acids (Ile, Leu, and Val) and in a severe growth reduction on glucose-containing media, even after supply of these amino acids. Mitochondria and cytosol isolated from bat1 and bat2 deletion mutants, respectively, contained largely reduced activities for the conversion of branched-chain 2-ketoacids to their corresponding amino acids. Thus, the Bat proteins represent the first known isoforms of yeast branched-chain amino acid transaminases. The severe growth defect of the double deletion mutant observed even in the presence of branched-chain amino acids suggests that the Bat proteins, in addition to the supply of these amino acids, perform another important function in the cell.     

Alterations in the excess synthesis of riboflavin in Pichia guilliermondii under the influence of branched-chain amino acids

The effect of the branched-chain amino acids: L-valine, L-isoleucine and L-leucine on riboflavin overproduction was studied in the Pichia (Candida) guilliermondii (Cast.) Lang. et G. yeast, L-Val, L-Ile and L-Leu were found to inhibit riboflavin overproduction only under iron-deficient growth conditions. Other amino acids used did not show this effect. In crude extracts of P. guilliermondii the specific activity of the alpha-acetolactate forming enzyme, pH 8.0, is inhibited by L-Val. It is revealed that the activity of alpha-acetolactate synthetase in iron-deficient riboflavin-overproduction cells was exceedingly higher than in the valine-inhibited cells. Under iron deficiency alpha-acetolactate synthetase shows maximal activity after 48 h of growth. It was possible to detect diacetyl (and aceton) in the culture fluid.     

Role of branched-chain aminotransferase isoenzymes and gabapentin in neurotransmitter metabolism

Because it is well known that excess branched-chain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with alpha-ketoglutarate to form the branched-chain alpha-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BCAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic (BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-14C]glutamate we found that BCKAs, but not BCAAs, stimulate glutamate transamination to alpha-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an alpha-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAA/BCKA shuttle, where BCATs and BCAAs provide the amino group for glutamate synthesis from alpha-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.     

Maple syrup urine disease: branched-chain amino acid concentrations and metabolism in cultured human lymphoblasts

The intracellular concentration of free leucine, isoleucine, and valine and their metabolism were studied in lymphoblast cultures established from peripheral blood of an individual with maple syrup urine disease (MSUD) and a control subject. Branched-chain alpha-keto acid decarboxylase activity in the MSUD cells was 10% or less of the control value as measured by the ability of the cells to release 14CO2 from the corresponding [1-14C]labeled branched-chain amino acid. The intracellular concentrations of free leucine and isoleucine were increased three-fold in MSUD lymphoblasts as compared to control cells. Free valine was present in only trace amounts of less than 0.1 mM in both cell lines. Exposure of normal and mutant cells to a 10 mM load of leucine, isoleucine, and valine revealed in a comparable concentration within cells after 24 hr. Concentrations returned to base values in normal cells 12 hr after removal of load, but leucine remained elevated in MSUD cells after 3 days. Leucine and its keto acid, alpha-ketoisocaproic acid, added to the culture medium gave significant growth inhibition of MSUD lymphoblasts but not of normal cells, in the millimolar range. Isoleucine, valine, and their keto acids had no effect.     

Pre-exercise branched-chain amino acid administration increases endurance performance in rats

This study investigated the effects of pre-exercise branched-chain amino acid (BCAA) administration on blood ammonia levels and on time to exhaustion during treadmill exercise in rats. Adult female Wistar rats were trained on a motor driven treadmill. After a 24-h fast, rats were injected intraperitoneally (i.p.) with 1 mL of placebo or BCAA (30 mg), 5 min before performing 30 min of submaximal exercise (N = 18) or running to exhaustion (N = 12). In both cases, rats were sacrificed immediately following exercise, and blood was collected for the measurement of glucose, nonesterified fatty acid (NEFA), lactic acid, BCAA, ammonia, and free-tryptophan (free-TRP) levels. Control values were obtained from sedentary rats that were subjected to identical treatments and procedures (N = 30). Plasma BCAA levels increased threefold within 5 min after BCAA administration. Mean run time to exhaustion was significantly longer (P < 0.01) after BCAA administration (99 +/- 9 min) compared with placebo (76 +/- 4 min). During exercise, blood ammonia levels were significantly higher (P < 0.01) in the BCAA treated compared with those in the placebo treated rats both in the 30-min exercise bout (113 +/- 25 mumol.L-1 (BCAA) vs 89 +/- 16 mumol.L-1) and following exercise to exhaustion (186 +/- 44 mumol.L-1 (BCAA) vs 123 +/- 19 mumol.L-1). These data demonstrate that BCAA administration in rats results in enhanced endurance performance and an increase in blood ammonia during exercise.     

An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport

The azlB locus of Bacillus subtilis was defined previously by a mutation conferring resistance to a leucine analog, 4-azaleucine (J. B. Ward, Jr., and S. A. Zahler, J. Bacteriol. 116:727-735, 1973). In this report, azlB is shown to be the first gene of an operon apparently involved in branched-chain amino acid transport. The product of the azlB gene is an Lrp-like protein that negatively regulates expression of the azlBCDEF operon. Resistance to 4-azaleucine in azlB mutants is due to overproduction of AzlC and AzlD, two novel hydrophobic proteins.     

Oral branched-chain amino acids do not improve exercise capacity in McArdle disease

To determine whether oral branched-chain amino acids (BCAAs) improve exercise capacity, six fasting patients with McArdle's disease were given a solution of BCAA (77 mg/kg) or a control noncaloric beverage 30 minutes before exercise. The BCAA meal tripled plasma BCAA levels, increased BCAA catabolism as indicated by greater exercise increases in plasma glutamine and alanine, but lowered mean peak free fatty acid levels and reduced exercise capacity in five of six patients. Lower work capacity may be attributed to a net reduction in muscle fuel availability after BCAA administration.     

Branched-chain amino acid oxidation by isolated rat tissue preparations

Branched-chain amino acid transaminase activity, branced-chain alpha-keto acid dehydrogenase activity, and leucine oxidation were measured in homogenates and slices of several rat tissues. Transaminase activity was highest in heart, while dehydrogenase activity was highest in liver. Leucine oxidation in isolated tissues may be limited by either transaminase or dehydrogenase activity depending upon the relative activities of these two enzymes in the tissue. The results suggest that, as the load of branched-chain amino acids increases, the liver may become an increasingly important site for the degradation of branched-chain alpha-keto acids.     

Pancreatic cancer in rats and hamsters does not induce IAPP-related hyperglycaemia

The X-ray crystallographic structure of the branched-chain amino acid aminotransferase from Escherichia coli was determined by means of isomorphous replacement using the selenomethionyl enzyme as one of the heavy atom derivatives. The enzyme is a homo hexamer with D3 symmetry, and the polypeptide chain of the subunit is folded into two domains (small and large domains). The coenzyme, pyridoxal 5'-phosphate, resides at the domain interface, its re-face facing toward the protein. The active site structure shows that the following sites can recognize branched-chain amino acids and glutamate as substrates: (1) a hydrophobic core formed by Phe36, Tyr164, Tyr31*, and Val109* for a branched-chain; (2) Arg97 for an acidic side chain of glutamate; and (3) Tyr95 and two main chain NH groups of Thr257 and Ala258 for the alpha-carboxylate of substrates. Although the main chain conformation of the active site is homologous to that of D-amino acid aminotransferase, many of the active site residues are different between them.     

Stimulation of albumin synthesis by keto analogues of amino acids

(1) The effect of ketoanalogues of branched-chain amino acids on albumin synthesis was examined in two biological systems using the [14C]carbonate technique. (2)alpha-Ketocaproic acid, the ketoanalogue of leucine, was able to reverse the reduced synthesis rate observed when isolated livers, from well-nourished animals were perfused with blood from rats deprived of dietary protein for 48 h. (3) A mixture of ketoanalogues of the three branched-chain amino acids, leucine, isoleucine and valine, was able to increase albumin synthesis per unit dry liver weight to above normal levels when administered intragastrically to rats 16 h after partial hepatectomy.     

The antinociceptive effects of branched-chain amino acids: evidence for their ability to potentiate morphine analgesia

The effect of branched-chain amino acids (BCAA) on pain threshold was studied in rats. Nociception was induced by the hot-plate analgesia meter, a method measuring supraspinally organized pain responses. After a single intravenous injection of BCAA (320 mg/kg), the percent change in latency time to the pain response significantly increased by 19% in 60 min, and by 22% in 75 min (p < 0.005), as compared to an injection of an equal volume of a standard concentration of an amino acid solution or physiological saline. Subsequently, we studied the interaction of BCAA with opioid-type analgesia. In combination with intravenously injected morphine (3 mg/kg), BCAA significantly potentiated and prolonged the action of morphine using the hot-plate test. From 5 min after morphine injection, the latencies to a pain response were markedly higher with the combination of BCAA and morphine (+80% and +89% at 5 min after morphine injection, if BCAA was administered 45 or 60 min prior to morphine injection, respectively) when compared with the effect of morphine alone (+13% at 5 min; p < 0.005). BCAA demonstrated analgesic effects, which, in combination with morphine, potentiated and prolonged the antinociceptive action of morphine. BCAA may represent a new adjunct treatment modality for acute and chronic pain, and give us further insight into the mechanisms of pain control.     

Time after feeding and dietary arginine deficiency alter splanchnic and hepatic amino acid flux in rats

The effect of an arginine-deficient diet containing 3.4% glutamate on net flux of amino acids across the portal-drained viscera and liver was studied in rats at 0, 1 or 2 h after a meal and compared with that in arginine-fed controls. Net portal-drained viscera flux for most amino acids was greater in the fed state compared with the postabsorptive state except for glycine and cystine, which did not change, and methionine, which declined. Net amino acid recovery in portal blood 2 h after feeding compared with amounts consumed was highest for alanine (17.3%); recovery of other amino acids ranged from 5.6 to 15.3%. No net portal-drained viscera recovery of consumed cystine was observed. For the branched-chain and aromatic amino acids, methionine, threonine, histidine and lysine, net hepatic uptake was nearly equal to net portal-drained viscera absorption (range 77-127% of portal-drained viscera flux). Correlation coefficients between net hepatic and portal-drained viscera fluxes for leucine, valine, isoleucine, methionine and phenylalanine were 0.84 to 0.93. Postabsorptive hepatic extraction for most amino acids was zero, but after a meal, ranged from 13.3 to 22.9% for the branched-chain and aromatic amino acids. Net hepatic production of ornithine and proline occurred in arginine-fed control rats. This value was near zero for ornithine in rats fed the arginine-deficient diet. Models of interorgan amino acid metabolism in the food-deprived and fed state are presented.     

Amino acid metabolism in the piglet. 2. Influence of fasting on plasma free amino acid concentration and in vivo oxidation of methionine, isoleucine and threonine

1. The influence of a 24 h fast on the concentrations of free amino acids in the plasma, and upon the oxidation rates of methionine, isoleucine and threonine was studied (using early weaned, 4-week-old piglets which were receiving a semi-purified diet. 2. There was no change in the total concentration of the essential amino acids as a result of the 24 h fast: the concentration of the branched-chain amino acids increased, but the effect of this was offset by decreases in the concentrations of arginine, histidine, lysine, methionine and phenylalanine. There was a reduction in the concentration of the non-essential amino acids. 3. The piglets received infusions of L-[I-14C]methionine, L-[U-14C]isoleucine and L-[U-14C]-threonine, and the recovery of the label in carbon dioxide was determined. Less than 5% of the activity from methionine was recovered in the CO2 from the fed piglets, whereas 12% was recovered from the fasted piglets. The corresponding values with threonine were 11 and 19% but there was no effect of fasting on the recovery of the label from isoleucine in CO2. 4. The initial dilution of a single dose of a labelled amino acid infused into the bloodstream depends on the plasma concentration of the amino acid. Nutritional regimens may effect the free amino acid concentration in the plasma. Thus comparisons based upon direct determination of activity recovered in CO2 from the labelled dose of an amino acid with animals on different nutritional regimens could not misleading, unless the differences in the concentrations of the amino acid in the plasma are considered.     

Diurnal changes in plasma amino acids in maple syrup urine disease

Protein turnover is a cyclic process with a net loss of protein in the (catabolic) fasted state and a net gain in the (anabolic) fed state. In maple syrup urine disease (MSUD) the early block of degradation of the branched-chain amino acids (BCAA) brings about the opportunity for evaluation of the diurnal variation in net protein anabolism and catabolism by studying cyclic changes in the plasma concentrations of BCAA. The alterations in plasma BCAA in a 3-y-old boy with classical MSUD were studied in the fed and fasted state over a period of 19 months. For each amino acid a total of 34 data pairs was calculated. The plasma concentrations of the BCAA leucine, valine and isoleucine were constantly higher in the fasted than in the fed state. Plasma concentrations of alloisoleucine, being a non-protein amino acid, did not participate in cyclic changes. In contrast, the essential amino acid pair tyrosine and phenylalanine increased after meals. The fasting concentration of alanine increased after feeding, while glycine did not change significantly. Healthy subjects show a decrease in all amino acids in the fasted (mild catabolic) state and an increase in the fed state. These findings in MSUD suggest a net decrease in non-BCAA as result of a greater rate of amino acid oxidation rate than of protein breakdown and a net entry of BCAA into plasma in the fasted state due to the specific metabolic block. Such changes in amino acid plasma pools have to be taken into account during monitoring of treatment and especially when in vivo leucine oxidation is assessed.