Developmental changes in rat liver branched-chain 2-oxo acid dehydrogenase.

Branched-chain 2-oxo acid dehydrogenase catalyses the first irreversible step in the degradation of the branched-chain amino acids leucine, isoleucine and valine. With specifically labelled 4-methyl-2-oxo[1-14C]pentanoate as substrate, the enzyme's activity was measured in rat liver homogenates. Activity (per g wet wL of liver or per mg of protein) increased most rapidly during the perinatal period (2 days before to 1 day after birth), reaching approximately adult values by the time of weaning. The apparent Vmax, of the enzyme increased with age, but its Km appeared unchanged. The data suggest that hepatic branched-chain 2-oxo acid dehydrogenase is induced or activated during the perinatal period. The enzyme's activity at birth was unaffected by maternal diabetes, or by treating the mother with pharmacological doses of corticosterone or 3,3',5-tri-iodothyronine, during the last 5 days of pregnancy.     

Role of branched-chain 2-oxo acid dehydrogenase and pyruvate dehydrogenase in 2-oxobutyrate metabolism.

Purified branched-chain 2-oxo acid dehydrogenase (BCODH) and pyruvate dehydrogenase (PDH) had apparent Km values (microM) for 2-oxobutyrate of 26 and 114, with a relative Vmax. (% of Vmax. for 3-methyl-2-oxobutyrate and pyruvate) of 38 and 45% respectively. The phosphorylation state of both complexes in extracts of mitochondria from rat liver, kidney, heart and skeletal muscle was shown to influence oxidative decarboxylation of 2-oxobutyrate. Inhibitory antibodies to BCODH and an inhibitor of PDH (3-fluoropyruvate) were used with mitochondrial extracts to determine the relative contribution of both complexes to oxidative decarboxylation of 2-oxobutyrate. Calculated rates of 2-oxobutyrate decarboxylation in mitochondrial extracts, based on the kinetic constants given above and the activities of both complexes, were the same as the measured rates. Hydroxyapatite chromatography of extracts of mitochondria from rat liver revealed only two peaks of oxidative decarboxylation of 2-oxobutyrate, with one peak associated with PDH and the other with BCODH. Competition studies with various 2-oxo acids revealed a different inhibition pattern with mitochondrial extracts from liver compared with those from heart or skeletal muscle. We conclude that both intramitochondrial complexes are responsible for oxidative decarboxylation of 2-oxobutyrate. However, the BCODH is probably the more important complex, particularly in liver, on the basis of kinetic analyses, activity or phosphorylation state of both complexes, competition studies, and the apparent physiological concentration of pyruvate, 2-oxobutyrate and the branched-chain 2-oxo acids.     

Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated adipocytes. Effects of 2-oxo acids.

Isolated adipocytes from rat epididymal fat-pads were incubated with [32P]Pi, and intracellular phosphoproteins were then analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. A phosphorylated polypeptide of apparent Mr 46,000 was identified as the alpha-subunit of branched-chain 2-oxo acid dehydrogenase complex by immunoprecipitation using antiserum raised against the homogeneous E1 component of branched-chain 2-oxo acid dehydrogenase complex. Immunoprecipitation of this phosphoprotein is blocked in a competitive manner by purified branched-chain 2-oxo acid dehydrogenase complex. Peptide mapping of the isolated phosphoprotein indicates that two sites on the polypeptide are phosphorylated in the intact cells. Addition of branched-chain 2-oxo acids to the incubation medium causes diminution in the extent of labelling of both phosphorylation sites on the alpha-subunit, an effect presumably mediated via their known inhibitory action on branched-chain 2-oxo acid dehydrogenase kinase. These observations provide direct evidence for phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in intact cells.     

Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated hepatocytes

Hepatocytes, isolated from rats fed a low-protein diet, were incubated with [32P]Pi and the phosphoproteins analysed. Immunoprecipitation using antibody against El of branched-chain 2-oxo acid dehydrogenase complex demonstrated phosphorylation of the alpha-subunit of El. Analysis of the tryptic phosphopeptides from the alpha-subunit indicated that two sites were phosphorylated. 4-methyl 2-oxopentanoate and DL-2-chloro 4-methylpentanoate decreased labelling of both sites. No major direct effects of several hormones on phosphorylation of branched-chain 2-oxo acid dehydrogenase was observed.     

Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex.

Branched-chain 2-oxo acid dehydrogenase complex was resolved into component E1 and E2-kinase subcomplex by gel filtration in the presence of 1 M-NaC1. Essentially all the original activity of the complex can be regained after reconstitution of the component enzymes, reassociation being a rapid process. The specific activities of E1 and E2 were 25.1 and 19.0 units/mg respectively. Non-phosphorylated active E1 has an approx. 6-fold higher affinity for E2 than does phosphorylated E1. The components of the branched-chain 2-oxo acid dehydrogenase complex do not crossreact with the respective components from the pyruvate dehydrogenase complex. The significance of these results and of the tight association of the kinase with E2 are discussed.     

Oxidative decarboxylation of 4-methylthio-2-oxobutyrate by branched-chain 2-oxo acid dehydrogenase complex.

Highly purified branched-chain 2-oxo acid dehydrogenase complex (BCOADC) oxidizes 4-methylthio-2-oxobutyrate and 2-oxobutyrate, with Km values of 67 microM and 18 microM respectively. The Vmax. for oxidation of these substrates is 27% and 53% respectively of that for 3-methyl-2-oxobutyrate. Highly purified pyruvate dehydrogenase complex (PDC) oxidizes 2-oxobutyrate (Km 100 microM; Vmax. 49% of that for pyruvate) but not 4-methylthio-2-oxobutyrate, whereas 2-oxoglutarate dehydrogenase complex will not utilize either 2-oxo acid as substrate. BCOADC kinase is inhibited by both 4-methylthio-2-oxobutyrate and 2-oxobutyrate, with half-maximal inhibition by 45 microM and 50 microM respectively. Phosphorylation of BCOADC in isolated adipocytes is inhibited by both 4-methylthio-2-oxobutyrate and 2-oxobutyrate, consistent with their inhibitory action of BCOADC kinase. Phosphorylation of PDC is decreased by 2-oxobutyrate, but not by 4-methylthio-2-oxobutyrate.     

Exercise-induced activation of the branched-chain 2-oxo acid dehydrogenase in human muscle

The present study was conducted to investigate the metabolic regulation of the oxidation of branched-chain amino acids (BCAA) by exercise in human skeletal muscle. Five trained male volunteers were exercised on a cycle ergometer at 70% +/- 10% (mean +/- SD) of their maximal oxygen consumption (VO2max). Percutaneous quadriceps muscle biopsies were obtained under local anaesthesia at rest and after 30 and 120 min of exercise. In the muscle samples the active and total amount of the branched-chain 2-oxo acid dehydrogenase complex (BC-complex), the regulatory enzyme in the oxidative pathway of the BCAA, were measured. Glycogen content and activity of mitochondrial marker enzymes were also measured. Blood samples were obtained every 20 min for the measurement of metabolites. Heart rate and rated perceived exertion on the Borg scale were recorded every 10 min. At rest 4.0% +/- 2.5% of the BC complex was active, after 30 min of exercise 9.9% +/- 9.0% and after 120 min 17.5% +/- 8.5% (mean +/- SD). Exercise did not change the total activity. The largest activation was seen in two of the subjects who developed higher blood lactates early on during exercise and decreased their muscle glycogen more (indications of anaerobic metabolism). These data demonstrate that in trained individuals significant increases in the activity of the BC-complex occur only after prolonged intense exercise. In spite of the 4-fold activation, the data support the classical view that amino acids and protein do not contribute substantially as an energy source during exercise, since VO2 increased more than 20-fold.     

Regulation by induction of branched-chain 2-oxo acid dehydrogenase complex in clofibrate-fed rat liver.

The activity of branched-chain 2-oxo acid dehydrogenase complex increased 3.0-fold in liver of rats fed on 0.1%(w/w) clofibrate. Immunotitration experiments with antibodies against the constituent enzymes of the complex revealed that this increase resulted mainly from the increased amounts of only two(a decarboxylase and a lipoate acyltransferase) of three components of the complex and that the other component(dihydrolipoamide dehydrogenase) remained unchanged in its content, irrespective of clofibrate administration. The increases of both enzyme components were associated with increases in their mRNA levels which were estimated by in vitro translation with poly(A)+ RNA.     

Quantitative control analysis of branched-chain 2-oxo acid dehydrogenase complex activity by feedback inhibition.

The potential for branched-chain 2-oxo acid dehydrogenase complex (BCOADC) activity to be controlled by feedback inhibition was investigated by calculating the Elasticity Coefficients for several feedback inhibitors. We suggest that feedback inhibition is a quantitatively important regulatory mechanism by which branched-chain 2-oxo acid dehydrogenase activity is regulated. The potential for control of enzyme activity is greater for NADH than for the acyl-CoA products, and suggests that factors that alter the redox potential may physiologically regulate BCOADC activity through a feedback inhibitory mechanism in vivo. Local pH may also be an important regulatory control factor.     

Adrenergic inhibition of branched-chain 2-oxo acid dehydrogenase in rat diaphragm muscle in vitro.

In theory, the complete oxidation to CO2 of amino acids that are metabolized by conversion into tricarboxylic acid-cycle intermediates may proceed via their conversion into acetyl-CoA. The possible adrenergic modulation of this oxidative pathway was investigated in isolated hemidiaphragms from 40 h-starved rats. Adrenaline (5.5 microM), phenylephrine (0.49 mM) and dibutyryl cyclic AMP (10 microM) inhibited 14CO2 production from 3 mM-[U-14C]valine by 35%, 28% and 19% respectively. At the same time, these agents stimulated glycogen mobilization (measured as a decrease in glycogen content) and glycolysis (measured as lactate release). Adrenaline, phenylephrine and dibutyryl cyclic AMP did not inhibit 14CO2 production from 3 mM-[U-14C]aspartate or 3 mM-[U-14C]glutamate, although, as in the presence of valine, the agents stimulated glycogen mobilization and glycolysis. The rate of proteolysis (measured as tyrosine release in the presence of cycloheximide) was not changed by adrenaline. The data indicate that the adrenergic inhibition of 14CO2 production from [U-14C]valine was not a consequence of radiolabel dilution. Inhibition was apparently specific for branched-chain amino acid metabolism in that the adrenergic agonists also inhibited 14CO2 production from [1-14C]valine, [1-14C]leucine and [U-14C]isoleucine. Since 14CO2 production from the 1-14C-labelled substrates is a specific measure of decarboxylation in the reaction catalysed by the branched-chain 2-oxo acid dehydrogenase complex, it is at this site that the adrenergic agents are concluded to act.     

Activation of rat liver branched-chain 2-oxo acid dehydrogenase in vivo by glucagon and adrenaline.

The activity of liver branched-chain 2-oxo acid dehydrogenase complex was measured in rats fed on low-protein diets and given adrenaline, glucagon, insulin or dibutyryl cyclic AMP in vivo. Administration of glucagon or adrenaline (200 micrograms/100 g body wt.) resulted in a 4-fold increase in the percentage of active complex. As with glucagon and adrenaline, treatment of rats with cyclic AMP (5 mg/100 g body wt.) resulted in marked activation of branched-chain 2-oxo acid dehydrogenase. Insulin administration (1 unit/100 g body wt.) also resulted in activation of enzyme; however, these effects were less than those observed with glucagon and adrenaline. In contrast with the results obtained with low-protein-fed rats, administration of adrenaline (200 micrograms/100 g body wt.) to rats fed with an adequate amount of protein resulted in only a modest (14%) increase in the activity of the complex. The extent to which these hormones activate branched-chain 2-oxo acid dehydrogenase appears to be correlated with their ability to stimulate amino acid uptake into liver.     

Rat tissue concentrations of branched-chain 2-oxo acid dehydrogenase complex. Re-evaluation by immunoassay and bioassay.

We point out that human low-Mr kininogen contains three cystatin-like sequences, rather than two, as had previously been thought. The protein was purified by affinity chromatography on carboxymethyl-papain-Sepharose, and subjected to limited proteolysis by trypsin and chymotrypsin. Fragments were isolated, and three corresponding to the individual cystatin-like domains were identified. By comparison with the known amino acid sequence of the protein they were numbered 1 to 3 from the N-terminus. Domain 1 was not found to have any inhibitory activity for cysteine proteinases, which is consistent with the absence of residues that are highly conserved in inhibitors of the cystatin superfamily, and have previously been suggested to be essential for activity. Domain 2 was a good inhibitor of chicken calpain, and also papain and cathepsin L. Domain 3 showed negligible inhibition of calpain, but inhibited papain and cathepsin L strongly. The probable arrangement of disulphide bonds in the heavy chain of low-Mr kininogen is deduced from the homology with the cystatins and other evidence contained in the present paper.     

The activity state of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues

An assay is described to define the proportion of the branched-chain 2-oxo acid dehydrogenase complex that is present in the active state in rat tissues. Activities are measured in homogenates in two ways: actual activities, present in tissues, by blocking both the kinase and phosphatase of the enzyme complex during homogenization, preincubation, and incubation with 1-14C-labelled branched-chain 2-oxo acid, and total activities by blocking only the kinase during the 5 min preincubation (necessary for activation). The kinase is blocked by 5 mM-ADP and absence of Mg2+ and the phosphatase by the simultaneous presence of 50 mM-NaF. About 6% of the enzyme is active in skeletal muscle of fed rats, 7% in heart, 20% in diaphragm, 47% in kidney, 60% in brain and 98% in liver. An entirely different assay, which measures activities in crude tissue extracts before and after treatment with a broad-specificity protein phosphatase, gave similar results for heart, liver and kidney. Advantages of our assay with homogenates are the presence of intact mitochondria, the simplicity, the short duration and the high sensitivity. The actual activities measured indicate that the degradation of branched-chain 2-oxo acids predominantly occurs in liver and kidney and is limited in skeletal muscle in the fed state.     

Activity of branched-chain 2-oxo acid dehydrogenase complex in rat liver mitochondria and in rat liver.

Four mitochondrial marker enzymes were used to show that: (1) high-protein (24%) diet increased the rat liver concentration and content of total branched-chain 2-oxo acid dehydrogenase complex (BCDC) by 31% by increasing mitochondrial specific activity of BCDC; (2) starvation increased the liver concentration of BCDC by 25% by decreasing liver weight; the liver content of mitochondria and the mitochondrial specific activity of BCDC were unchanged; (3) protein-free diet decreased rat liver BCDC concentration and content by 20%, by decreasing the liver concentration and content of mitochondria. Protein-free diet increased liver mitochondrial specific activities of L-glutamate, 2-oxoglutarate and NAD-isocitrate dehydrogenases. The validity of a mitochondrial method for the determination of the liver concentration of BCDC and the percentage in the active form in vivo is confirmed, and improvements are described. The experimental basis of criticisms of its use in this regard by Zhang, Paxton, Goodwin, Shimomura & Harris [(1987) Biochem. J. 246, 625-631] was not confirmed. The finding by Harris, Powell, Paxton, Gillim & Nagae [(1985) Arch. Biochem. Biophys. 243, 542-555], that starvation has no effect on the percentage of BCDC in the active form in rat liver, is confirmed.     

The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation.

1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies.     

Insulin regulation of the activity and phosphorylation of branched-chain 2-oxo acid dehydrogenase in adipose tissue.

The activity of the intramitochondrial branched-chain 2-oxo acid dehydrogenase (BCDH), like that of pyruvate dehydrogenase, is regulated, at least in part, by interconversion between the active dephosphorylated enzyme and its inactive phosphorylated form. The stimulatory effect of insulin on BCDH activity was compared with its effect on phosphorylation of the enzyme. Intact tissues were incubated in the presence or the absence of insulin, and then mitochondria were isolated and disrupted before assaying for enzyme activity or estimating the extent of enzyme phosphorylation. Tissues were incubated in either the presence or the absence of leucine, which also stimulated BCDH activity up to 10-fold. Insulin (1 munit/ml) doubled the activity of BCDH in the absence and in the presence of leucine. Together, 1 mM-leucine and insulin appeared to stimulate BCDH activity fully. Phosphorylation of BCDH was estimated indirectly by measuring the incorporation of 32P into phosphorylation sites that remained unesterified after preparing mitochondrial extracts under conditions that preserved the effect of insulin on BCDH activity. Increased incorporation of 32P in these experiments implies decreased phosphorylation in situ when tissues were incubated with insulin and leucine. In the absence of leucine, little incorporation of 32P into BCDH was detected. In the presence of leucine, however, incorporation of 32P into BCDH was markedly increased, and insulin increased 32P incorporation still further. The results support the hypothesis that leucine and insulin both stimulate the activity of BCDH by promoting its dephosphorylation.     

Inhibition of the bovine branched-chain 2-oxo acid dehydrogenase complex and its kinase by arylidenepyruvates

A novel class of inhibitors for the branched-chain 2-oxo acid dehydrogenase (BCOAD) complex has been synthesized and studied. The sodium salts of arylidenepyruvates: e.g., furfurylidenepyruvate (compound I), 4-(3-thienyl)-2-oxo-3-butenoate (compound II), cinnamalpyruvate (compound III) and 4-(2-thienyl)-2-oxo-3-butenoate (compound IV) inhibit the overall and kinase reactions of the BCOAD complex from bovine liver. Inhibitions of the overall reaction occur at the decarboxylase (E1) step as determined by a spectrophotometric assay with 2,6-dichlorophenolindophenol as an electron acceptor. Inhibition of the E1 reaction by compound I (Ki = 0.5 microM) is competitive, whereas inhibitions by compounds II (Ki = 150 microM) and III (Ki = 500 microM) are non-competitive with respect to the substrate 2-oxoisovalerate. The Km value for 2-oxoisovalerate is 6.7 microM as measured by the E1 assay. Inhibition of the E1 step by compounds I, II and III are reversible at low inhibitor concentrations based on the Michaelis-Menten kinetics observed. By comparison, compound I does not significantly inhibit pyruvate and 2-oxoglutarate dehydrogenase complexes. The arylidenepyruvates (compounds I, II and IV) inhibit the BCOAD kinase reaction in a manner similar to the substrate 2-oxo acids. The inhibition of the kinase reaction by compound I is non-competitive with respect to ATP, with an apparent Ki value of 4.5 mM. The results suggest that arylidenepyruvates may be useful probes for elucidating the reaction mechanisms of the BCOAD complex and its kinase.     

Preservation of the activity state of hepatic branched-chain 2-oxo acid dehydrogenase during the isolation of mitochondria.

Monoclonal antibody PH7 has specificity for the phosphorylated form of the human liver phenylalanine hydroxylase and negligible reactivity towards the dephosphorylated form of the native enzyme by enzyme-linked immunoassay. PH7 binds specifically to the phosphorylated form of the liver enzyme after SDS/polyacrylamide-gel electrophoresis and transfer to nitrocellulose. Competitive blocking assays have been applied in conjunction with reversed-phase h.p.l.c. of purified tryptic fragments of human liver phenylalanine hydroxylase to localize the epitope. The major immunoreactive tryptic peptide cross-reacting with PH7 had an amino acid analysis corresponding to the first 41 amino acids of the human liver phenylalanine hydroxylase sequence and included the serine residue that is thought to be the phosphorylation site. The monoclonal antibody recognized the phosphorylated form of the synthetic decapeptide corresponding to the local phosphorylation-site sequence Gly-Leu-Gly-Arg-Lys-Leu-Ser(P)-Asp-Phe-Gly, but not the dephosphodecapeptide. Thermolysin digestion of the peptide demonstrated the monoclonal antibody bound to the pentapeptide Leu-Ser(P)-Asp-Phe-Gly. Monoclonal antibody PH7 recognized the phosphodecapeptide at concentrations 10(3)-fold higher than with phenylalanine hydroxylase, compared with 10(4)-10(7)-fold higher for other phosphopeptides and phosphoproteins. The results demonstrate that monoclonal antibody PH7 has specificity for the phosphorylated form of phenylalanine hydroxylase at the phosphorylation site.     

Calcium plays an important role in the regulation of hepatic branched-chain 2-oxo acid dehydrogenase activity

A fetal haemoglobin variant was noted in a healthy Jamaican infant of mixed African/Chinese extraction. A two-dimensional chromatogram of the soluble tryptic peptides (Tp) showed 2 ‘new’ ones/ One was composed of the last 4 residues of the usually insoluble Tpγ41–59. To permit a tryptic split this required a change of residue γ55 Met to Lys or Arg. The other new Tp contained arginine and was in the position expected for a Tpγ41–55 (55 Arg). As the material was limited it could not be analysed. When after more than 6 years no example of Hb F Kingston had become available it was decided to describe the variant on the basis of the present evidence.     

Activities of branched-chain 2-oxo acid dehydrogenase and its components in skin fibroblasts from normal and classical-maple-syrup-urine-disease subjects.

1. Comparisons of the activity and kinetics of the branched-chain 2-oxo acid dehydrogenase in cultured skin fibroblasts from normal and classical maple-syrup-urine-disease (MSUD) subjects provide a kinetic explanation for the enzyme defect. 2. In the intact cell assays, normal fibroblasts demonstrated hyperbolic kinetics with 3-methyl-2-oxo[1-14C]butyrate as a substrate. Intact fibroblasts from four classical MSUD patients showed no decarboxylation over a substrate concentration range of 0.25 to 5.0 mM, and thiamin (4 mM) was without effect. 3. The overall reaction of the multienzyme complex was efficiently reconstituted by using a disrupted-cell system. Normals again showed typical hyperbolic kinetics at the 2-oxo acid concentrations of 0.1 to 5 mM. The Vmax. and apparent Km values were 0.10 +/- 0.02 m-unit/mg of protein and 0.05-0.1 mM respectively, with 3-methyl-2-oxobutyrate. In contrast, classical MSUD patients exhibited sigmoidal kinetics (Hill coefficient, 2.5) with activity approaching 40-60% of the normal value at 5 mM substrate. The K0.5 values from the Hill plots for MSUD patients were 4-7 mM. 4. The E1 (branched-chain 2-oxo acid decarboxylase) component of the multienzyme complex was measured in disrupted-particulate preparations. Normals again showed hyperbolic kinetics with the 2-oxo acid, whereas MSUD preparations exhibited sigmoidal kinetics with the activity of E1 strictly dependent on substrate concentration. Apparent Km or K0.5 were 0.1 and 1.0 mM for normal and MSUD subjects respectively. 5. Measurements of E2 (dihydrolipoyl transacylase) and E3 (dihydrolipoyl dehydrogenase) in MSUD preparations showed them to be in the normal range. 6. The above data suggest a defect in the E1 step of branched-chain 2-oxo acid dehydrogenase in classical MSUD patients.