Branched-chain Alpha-keto Acids Research Articles

Chemical conversion of alpha-keto acids in relation to flavor formation in fermented foods.

Formation of flavor compounds from branched-chain alpha-keto acids in fermented foods such as cheese is believed to be mainly an enzymatic process, while the conversion of phenyl pyruvic acid, which is derived from phenylalanine, also proceeds chemically. In this research, the chemical conversion of alpha-keto acids to aldehydes with strong flavor characteristics was studied, with the main focus on the conversion of alpha-ketoisocaproic acid to the aldehyde 2-methylpropanal, and a manganese-catalyzed reaction mechanism is proposed for this conversion. The mechanism involves keto-enol tautomerism, enabling molecular oxygen to react with the beta-carbon atom of the alpha-keto acid, resulting in a peroxide. This peroxide can react in several ways, leading to unstable dioxylactone or noncyclic intermediates. These intermediates will break down into an aldehyde and oxalate or carbon oxides (CO and CO(2)). All the alpha-keto acids tested were converted at pH 5.5 and in the presence of manganese, although their conversion rates were rather diverse. This chemical reaction might provide new ways for controlling cheese flavor formation with the aim of acceleration of the ripening process or diversification of the flavor characteristics.

Inhibition of brain energy metabolism by the alpha-keto acids accumulating in maple syrup urine disease.

Neurological dysfunction is a common finding in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly known. In the present study, we investigated the effect of the in vitro effect of the branched chain alpha-keto acids (BCKA) accumulating in MSUD on some parameters of energy metabolism in cerebral cortex of rats. [14CO(2)] production from [14C] acetate, glucose uptake and lactate release from glucose were evaluated by incubating cortical prisms from 30-day-old rats in Krebs-Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of 1-5 mM of alpha-ketoisocaproic acid (KIC), alpha-keto-beta-methylvaleric acid (KMV) or alpha-ketoisovaleric acid (KIV). All keto acids significantly reduced 14CO(2) production by around 40%, in contrast to lactate release and glucose utilization, which were significantly increased by the metabolites by around 42% in cortical prisms. Furthermore, the activity of the respiratory chain complex I-III was significantly inhibited by 60%, whereas the other activities of the electron transport chain, namely complexes II, II-III, III and IV, as well as succinate dehydrogenase were not affected by the keto acids. The results indicate that the major metabolites accumulating in MSUD compromise brain energy metabolism by blocking the respiratory chain. We presume that these findings may be of relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients.

Effect of the branched-chain alpha-ketoacids accumulating in maple syrup urine disease on the high molecular weight neurofilament subunit (NF-H) in rat cerebral cortex.

In this study we investigated the effects of the branched chain alpha-ketoacids accumulating in maple syrup urine disease (MSUD) on the concentrations of the high molecular weight neurofilament subunit (NF-H) associated with the cytoskeletal fraction of the cerebral cortex of 12-day-old rats. Cortical slices were incubated with alpha-ketoisocaproic acid (KIC), alpha-keto beta-methylvaleric acid (KMV) and alpha-ketoisovaleric acid (KIV) at concentrations ranging from 0.5 to 1.0 mM. The cytoskeletal fraction was extracted and the immunoreactivity for phosphorylated and total NF-H was analyzed by immunoblotting. The in vitro 32P incorporation into NF-H was also determined. Results showed that treatment of tissue slices induced with KMV increased Triton-insoluble phosphorylated NF-H immunoreactivity, with no alteration in total NF-H immunoreactivity. Furthermore, KIC treatment drastically increased the total amount of NF-H, whereas KIV did not change either phosphorylated or total NF-H immunoreactivity. KMV also increased the in vitro 32P incorporation into NF-H, confirming the highly phosphorylated NF-H levels detected in the immunoblot. These findings demonstrate that KIC and KMV alter the dynamic regulation of NF-H assembly in the cytoskeletal fraction. Therefore we may suggest that cytoskeletal disorganization may be one of the factors associated with the neurodegeneration characteristic of MSUD disease.

Function of Leucine in Excitatory Neurotransmitter Metabolism in the Central Nervous System

A novel hypothesis for the role of branched-chain amino acids (BCAA) in regulating levels of the major excitatory neurotransmitter glutamate in the central nervous system is described. It is postulated that the branched-chain aminotransferase (BCAT) isoenzymes (mitochondrial BCATm and cytosolic BCATc) are localized in different cell types and operate in series to provide nitrogen for optimal rates of de novo glutamate synthesis. BCAA enter the astrocyte where transamination is catalyzed by BCATm, producing glutamate and branched-chain alpha-keto acids (BCKA). BCKA, which are poorly oxidized in astrocytes, exit and are taken up by neurons. Neuronal BCATc catalyzes transamination of the BCKA with glutamate. The products, BCAA, exit the neuron and return to the astrocyte. The alpha-ketoglutarate product in the neurons may undergo reductive amination to glutamate via neuronal glutamate dehydrogenase. Operation of the shuttle in the proposed direction provides a mechanism for efficient nitrogen transfer between astrocytes and neurons and synthesis of glutamate from astrocyte alpha-ketoglutarate. Evidence in favor of the hypothesis is: 1) The two BCAT isoenzymes appear to be localized separately in the neurons (BCATc) or in the astroglia (BCATm). 2) Inhibition of the shuttle in the direction of glutamate synthesis can be achieved by inhibiting BCATc using the neuroactive drug gabapentin. Although gabapentin does not inhibit BCATm, it does block de novo glutamate synthesis from alpha-ketoglutarate. 3) Conversely, gabapentin stimulates oxidation of glutamate. Inhibition of BCATc may allow BCKA to accumulate in the astroglia, thus facilitating conversion of glutamate to alpha-ketoglutarate.

Regulation of Branched-Chain α-Keto Acid Dehydrogenase Kinase Expression in Rat Liver

Branched-chain amino acids are toxic in excess but have to be conserved for protein synthesis. This is accomplished in large part by control of the activity of the branched-chain alpha-keto acid dehydrogenase complex by phosphorylation/dephosphorylation. Regulation of the activity of the hepatic enzyme appears particularly important, at least in rats, since an exceptional high activity of the complex in this tissue makes the liver the primary clearing house for excess branched-chain alpha-keto acids released by other tissues. The degree to which the branched-chain alpha-keto acid dehydrogenase complex is inactivated by phosphorylation is determined by the activity of the branched-chain alpha-keto acid dehydrogenase kinase, which is itself regulated by allosteric effectors as well as factors that affect its level of expression. Well established among these are the alpha-keto acid produced by leucine transamination, which is a potent inhibitor of the kinase, and starvation for dietary protein, which causes increased expression of the branched-chain alpha-keto acid dehydrogenase kinase. The latter finding resulted in the working hypothesis that nutrients and hormones regulate expression of the branched-chain alpha-keto acid dehydrogenase kinase. Evidence has been obtained for the involvement of thyroid hormone, glucocorticoids and ligands for peroxisome proliferator-activated receptor alpha. Thyroid hormone induces, whereas glucocorticoids and peroxisome proliferator-activated receptor alpha ligands repress, expression of the kinase. Increased blood levels of thyroid hormone are proposed to be responsible for increased expression of branched-chain alpha-keto acid dehydrogenase kinase in animals starved for protein.

Catabolism of branched-chain alpha-keto acids in Enterococcus faecalis: the bkd gene cluster, enzymes, and metabolic route.

Genes encoding a branched-chain alpha-keto acid dehydrogenase from Enterococcus faecalis 10C1, E1alpha (bkdA), E1beta (bkdB), E2 (bkdC), and E3 (bkdD), were found to reside in the gene cluster ptb-buk-bkdDABC. The predicted products of ptb and buk exhibited significant homology to the phosphotransbutyrylase and butyrate kinase, respectively, from Clostridium acetobutylicum. Activity and redox properties of the purified recombinant enzyme encoded by bkdD indicate that E. faecalis has a lipoamide dehydrogenase that is distinct from the lipoamide dehydrogenase associated with the pyruvate dehydrogenase complex. Specific activity of the ptb gene product expressed in Escherichia coli was highest with the substrates valeryl-coenzyme A (CoA), isovaleryl-CoA, and isobutyryl-CoA. In cultures, a stoichiometric conversion of alpha-ketoisocaproate to isovalerate was observed, with a concomitant increase in biomass. We propose that alpha-ketoisocaproate is converted via the BKDH complex to isovaleryl-CoA and subsequently converted into isovalerate via the combined actions of the ptb and buk gene products with the concomitant phosphorylation of ADP. In contrast, an E. faecalis bkd mutant constructed by disruption of the bkdA gene did not benefit from having alpha-ketoisocaproate in the growth medium, and conversion to isovalerate was less than 2% of the wild-type conversion. It is concluded that the bkd gene cluster encodes the enzymes that constitute a catabolic pathway for branched-chain alpha-keto acids that was previously unidentified in E. faecalis.

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.

Effects of leucine on whole body leucine, valine, and threonine metabolism in humans.

We tested whether expansion of the plasma leucine pool distorts leucine or valine tracer kinetics, causing errors in the derived values of whole body proteolysis. Seven normal adults received a 10-h primed-continuous tracer infusion of L-[5,5,5-2H3]leucine, L-[(1-13)C]valine, and L-[(1-13)C]threonine, during the final 7 h of which L-leucine was infused at a rate that more than tripled the plasma leucine concentration. Leucine, valine, and threonine rates of appearance were converted to a common value of whole body proteolysis on the basis of their concentrations in body proteins. The conversion of labeled leucine and valine to their corresponding branched-chain alpha-keto and alpha-hydroxy acids was also monitored. Before the unlabeled leucine infusion, postabsorptive whole body proteolysis was estimated similarly by the three tracers (approximately 180 mg protein.kg-1.h-1. The leucine infusion reduced proteolysis by an average of 21% (P < 0.006), as estimated by use of valine or threonine kinetics, and by 10% by use of leucine kinetics (P < 0.02). No delay in the conversion of valine to alpha-ketoisovalerate occurred during the leucine infusion. Thus all three tracers indicated similar postabsorptive rates of whole body proteolysis and a reduction of proteolysis during leucine administration, although the magnitude of the effect was underestimated with use of the leucine tracer.

The valine catabolic pathway in human liver: Effect of cirrhosis on enzyme activities

The activities of key enzymes in the valine catabolic pathway–branched-chain aminotransferase, branched-chain alpha-keto acid dehydrogenase complex, methacrylyl (MC)-coenzyme A (CoA) hydratase (crotonase), and 3- hydroxyisobutyryl-CoA (HIB-CoA) hydrolase–were measured in normal and cirrhotic human livers. Unlike rat liver, which does not contain branched-chain aminotransferase, the aminotransferase activity in the normal liver was measurable and is increased somewhat in cirrhosis of the human liver. The total activity of branched-chain alpha-keto acid dehydrogenase complex in the normal human liver was approximately 1% of that in rat liver, and 20% to 30% of the complex was in the active form in both normal and cirrhotic livers. Only the actual activity of the enzyme was significantly decreased by cirrhosis. These results suggest that human liver is less active than rat liver in the catabolism of branched-chain amino and alpha-keto acids. Activities of MC-CoA hydratase and HIB-CoA hydrolase in human liver were very high compared with that of branched-chain alpha-keto acid dehydrogenase complex, suggesting an important role for these enzymes in catabolism of a potentially toxic compound, MC-CoA, formed as an intermediate in the catabolism of valine and isobutyrate. Cirrhosis resulted in a significant decrease in HIB-CoA hydrolase activity but had no effect on the citrate synthase activity, suggesting that the decrease in HIB-CoA hydrolase activity does not reflect a general decrease in mitochondria but that it may contribute to cellular damage that culminates in liver failure. (Hepatology 1996 Dec;24(6):1395-8)

The valine catabolic pathway in human liver: effect of cirrhosis on enzyme activities.

The activities of key enzymes in the valine catabolic pathway--branched-chain aminotransferase, branched-chain alpha-keto acid dehydrogenase complex, methacrylyl (MC)-coenzyme A (CoA) hydratase (crotonase), and 3-hydroxyisobutyryl-CoA (HIB-CoA) hydrolase--were measured in normal and cirrhotic human livers. Unlike rat liver, which does not contain branched-chain aminotransferase, the aminotransferase activity in the normal liver was measurable and is increased somewhat in cirrhosis of the human liver. The total activity of branched-chain alpha-keto acid dehydrogenase complex in the normal human liver was approximately 1% of that in rat liver, and 20% to 30% of the complex was in the active form in both normal and cirrhotic livers. Only the actual activity of the enzyme was significantly decreased by cirrhosis. These results suggest that human liver is less active than rat liver in the catabolism of branched-chain amino and alpha-keto acids. Activities of MC-CoA hydratase and HIB-CoA hydrolase in human liver were very high compared with that of branched-chain alpha-keto acid dehydrogenase complex, suggesting an important role for these enzymes in catabolism of a potentially toxic compound, MC-CoA, formed as an intermediate in the catabolism of valine and isobutyrate. Cirrhosis resulted in a significant decrease in HIB-CoA hydrolase activity but had no effect on the citrate synthase activity, suggesting that the decrease in HIB-CoA hydrolase activity does not reflect a general decrease in mitochondria but that it may contribute to cellular damage that culminates in liver failure.

Molecular basis of maple syrup urine disease and stable correction by retroviral gene transfer.

Maple syrup urine disease (MSUD) or branched-chain ketoaciduria is caused by a deficiency of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex. This results in the accumulation of the branched-chain amino acids (BCAA) and branched-chain alpha-keto acids (BCKA), which often produce severe neurological damage and mental retardation. The present studies focus on mutations in the E1 alpha gene of the BCKAD complex and their effects on the assembly of the E1 decarboxylase component of the enzyme complex. We have developed an efficient histidine-tagged bacterial expression system that allows the folding and assembly of E1 alpha and E1 beta subunits into the E1 heterotetramer (alpha 2 beta 2) in the presence of overexpressed chaperonins GroEL and GroES. The results of pulse-chase experiments with this bacterial expression system showed that a majority of the 15 known E1 alpha mutations, including the prevalent Y393N of Mennonite MSUD patients, decrease the rate of association of normal E1 beta with mutant E1 alpha. This results in limited or no assembly of mutant E1. It is concluded that the carboxy-terminal region of the E1 alpha subunit encoded by exons 7-9 is important for subunit interaction. To stably correct MSUD, we have developed a retroviral vector that contains a normal E1 alpha precursor complementary DNA. Transduction of cultured lymphoblasts from a Mennonite MSUD patient with this recombinant retroviral vector completely restored the rate of decarboxylation of BCKA. The normal decarboxylation activity in transduced MSUD cells remained stable without antibiotic selection during the 14-week study.(ABSTRACT TRUNCATED AT 250 WORDS)

Branched-chain alpha-keto acid dehydrogenase complex in rat skeletal muscle: regulation of the activity and gene expression by nutrition and physical exercise.

Branched-chain alpha-keto acid dehydrogenase complex is the rate-limiting enzyme in the catabolism of branched-chain amino acids in skeletal muscle. It is suggested that activation of this enzyme in the muscle during exercise plays an important role in the increased oxidation of branched-chain amino acids in the muscle. Evidence suggests that branched-chain alpha-keto acids, the substrates for the enzyme, regulate the activity state of the enzyme in the muscle during exercise through phosphorylation/dephosphorylation cycle of the enzyme protein. We propose a model for the mechanism of enzyme activation by exercise. In addition to this acute effect of exercise, we present evidence suggesting that exercise training modulates the enzyme activity and gene expression for the enzyme. Increases in the total activity as well as enzyme proteins by exercise training are suggested to be associated with mitochondrial biogenesis in the muscle.

α-Keto and α-Hydroxy Branched-Chain Acid Interrelationships in Normal Humans

Plasma concentrations of the branched-chain amino acids leucine, isoleucine and valine, and those of leucine's and isoleucine's transamination products alpha-ketoisocaproic acid (KICA) and alpha-keto-beta-methylvaleric acid (KMVA), respectively, are known to increase after a protein meal or during extended fasting, but little or no increase in the concentration of valine's transamination product, alpha-ketoisovaleric acid (KIVA), has been observed under these conditions. To determine whether this could be explained by the conversion of KIVA to its alpha-hydroxy analogue, we measured the plasma concentrations of KICA, KMVA and KIVA, as well as their alpha-hydroxy analogues [alpha-hydroxyisocaproic acid (HICA), alpha-hydroxy-beta-methylvaleric acid (HMVA) and alpha-hydroxyisovaleric acid (HIVA)], in normal volunteers immediately after a protein meal or during a 60-h fast. We also determined the oxidoreduction equilibrium constants for HIVA/KIVA and HICA/KICA and their extent of plasma protein binding. In subjects in the postabsorptive state, the plasma concentrations of KICA and KMVA were 100 times those of HICA and HMVA, whereas that of KIVA was only twice that of HIVA. Shortly after a protein meal, KICA and KMVA concentrations increased significantly by 30 and 60%, respectively, whereas that of KIVA decreased by 25% (P < 0.05). HICA, HMVA and HIVA concentrations did not change. During prolonged fasting the plasma concentrations of all six metabolites increased gradually. The high plasma keto/hydroxy acid ratios were not related to their K(eq), which favored alpha-hydroxy analogue formation. The reduction of the branched-chain alpha-keto acids to their alpha-hydroxy analogues seems to take place too slowly to attain thermodynamic equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)

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).

EFFECT OF ALCOHOL ON BLOOD LEVELS OF BRANCHED-CHAIN ALPHA-KETO ACIDS IN MALE WISTAR RATS

The effect of oral alcohol administration on branched-chain alpha-keto acids was studied in Wistar rats. Rats were divided into alcohol loading groups 1 (ethanol single daily dose: 2.5 g/kg body wt for 14 days) and 2 (ethanol dose: 5 g/kg body wt), and a pair-fed control group (isocaloric glucose and food equal to the amount taken by group 1 the previous day). Branched-chain alpha-keto acids, other alpha-keto acids and amino acids were analysed using high-performance liquid chromatography. Results showed a significant body weight increase in the alcohol loading groups. Concentrations of branched-chain amino acids and keto acids were significantly increased in the alcohol-treated groups. The blood levels of methionine, threonine, alanine and glutamic acid were also increased by alcohol administration. Blood levels of taurine and ornithine, however, showed a significant decrease in the alcohol-treated groups. These results show that alcohol increases the blood levels of branched-chain keto acids and branched-chain amino acids.

High branched-chain alpha-keto acid intake, branched-chain alpha-keto acid dehydrogenase activity, and plasma and brain amino acid and plasma keto acid concentrations in rats

Diets containing high quantities of individual branched-chain alpha-keto acids (BCKAs) or a combination of BCKAs as used for treatment of renal disease were fed to rats. When the diet contained a single BCKA, its concentration was high in plasma and the concentration of its corresponding amino acid was high in plasma and brain. Liver BCKA dehydrogenase (BCKD) was 42% active in control rats. Consumption of diets containing 0.38 mol/kg diet of alpha-ketoisocaproate (KIC), alpha-keto-beta-methylvalerate (KMV), or alpha-ketoisovalerate (KIV) resulted in complete activation of liver BCKD. Consumption of the diet containing the combination of BCKAs increased basal BCKD activity of liver twofold. Muscle BCKD was activated after feeding the KIV diet (2-fold), the KIC diet (3-fold), and the KMV diet (15-fold). Total BCKD activity of liver and muscle was unaffected by dietary treatments. Activation of liver and muscle BCKD by dietary BCKA is consistent with their ability to inhibit BCKD kinase in vitro.

Solubilization and functional reconstitution of the branched-chain alpha-keto acid transporter from rat heart mitochondria.

The mitochondrial branched-chain alpha-keto acid transporter was solubilized from rat heart mitochondria and its function was reconstituted in phospholipid vesicles. The transporter was extracted from mitoplasts with Triton X-114 in the presence of exogenous cardiolipin and alpha-ketoisocaproate. Upon incorporation of this extract into asolectin vesicles by the freeze-thaw-sonication technique, a p-chloromercuribenzoate-sensitive, protein-dependent transport of alpha-ketoisocaproate into the proteoliposomes was observed. Significant inhibition of alpha-ketoisocaproate transport was observed in the reconstituted system with branched-chain alpha-keto acids (64-83%) and the related carboxylates alpha-ketocaproate (58%) and alpha-ketovalerate (49%), but not with substrates for the pyruvate carrier (less than 5%). The reconstituted carrier was substantially inhibited by sulfhydryl reagents, by the histidine-specific reagent diethyl pyrocarbonate, and by the tyrosine-specific reagent N-acetylimidazole. The extraction and functional reconstitution of the branched-chain alpha-keto acid transporter represents an important first step towards purification and molecular characterization of this anion carrier.

Nutritional and hormonal regulation of the activity state of hepatic branched-chain alpha-keto acid dehydrogenase complex.

The hepatic branched-chain alpha-keto acid dehydrogenase complex plays an important role in regulating branched-chain amino acid levels. These compounds are essential for protein synthesis but are toxic if present in excess. When dietary protein is deficient, the hepatic enzyme is present in the inactive, phosphorylated state to allow conservation of branched-chain amino acids for protein synthesis. When dietary protein is excessive, the enzyme is in the active, dephosphorylated state to commit the excess branched-chain amino acids to degradation. Inhibition of protein synthesis by cycloheximide, even when the animal is starving for protein, results in activation of the hepatic branched-chain alpha-keto acid dehydrogenase complex to prevent accumulation of branched-chain amino acids. Likewise, the increase in branched-chain amino acids caused by body wasting during starvation and uncontrolled diabetes is blunted by activation of the hepatic branched-chain alpha-keto acid dehydrogenase complex. The activity state of the hepatic branched-chain alpha-keto acid dehydrogenase complex is regulated in the short term by the concentration of branched-chain alpha-keto acids (inhibitors of branched-chain alpha-keto acid dehydrogenase kinase) and in the long term by alteration in the total branched chain alpha-keto acid dehydrogenase kinase activity.

Reversion of the Maple Syrup Urine Disease Phenotype of Impaired Branched Chain α-Ketoacid Dehydrogenase Complex Activity in Fibroblasts from an Affected Child

Branched chain alpha-ketoacid dehydrogenase is a heteroprotein complex of mitochondria and commits the branched chain alpha-ketoacids to their catabolic fate. Inherited nuclear mutations in humans decrease the activity of this complex and result in maple syrup urine disease. Here we demonstrate the restoration of branched chain alpha-ketoacid dehydrogenase activity to fibroblasts from a child with this disorder by transfection with a cDNA for the prebranched chain acyltransferase. Prior to transfection these fibroblasts contained the prebranched chain acyltransferase gene but failed to transcribe the gene and thus lacked the protein. Regulation of the restored complex by phosphorylation mechanisms resembles that of wild-type cells. These results describe a human cell modeling system for testing engineered proteins and support the possibility of gene replacement therapy for this human disorder.

Branched-chain alpha-ketoacids and related acids in thiamin-deprived rats.

We showed elevated plasma levels of branched-chain amino acids, alpha-ketoacids, alpha-hydroxyacids in thiamin-deprived rats, which had decreased liver thiamin levels (2.4% of control) after 4 weeks of feeding of thiamin-deficient diet. The ratios of mean levels of these acids in thiamin-deficient rats to those in control rats were as follows: total branched-chain amino acids 1.6; total branched-chain alpha-ketoacids 2.7; total branched-chain alpha-hydroxyacids 3.6; pyruvic acid 1.6. The plasma level of total branched-chain alpha-hydroxyacids was correlated to liver thiamin concentration (r = -0.67, p less than 0.01), but those of the other two acids were not correlated. Although the increased levels of branched-chain acids in thiamin-deprived rats were less remarkable than those of ill patients with maple syrup urine disease, they reflect the reportedly decreased branched-chain alpha-ketoacid dehydrogenase activities (about 40% of control), and we discussed whether the toxic effects of individual branched-chain acid could be demonstrated by its administration to the thiamin-deprived rats.