Activation of branched-chain alpha-ketoacid dehydrogenase in isolated hepatocytes by branched-chain alpha-ketoacids

The effects of branched-chain alpha-ketoacids on flux through and activity state of the branched-chain alpha-ketoacid dehydrogenase complex were studied in hepatocytes prepared from chow-fed, starved, and low-protein-diet-fed rats. Very low concentrations of alpha-ketoisocaproate caused a dramatic stimulation (50% activation at 20 microM) of alpha-ketoisovalerate decarboxylation in hepatocytes from low-protein-fed rats. alpha-Keto-beta-methylvalerate was also effective, but less so than alpha-ketoisocaproate. alpha-Ketoisocaproate did not stimulate alpha-ketoisovalerate decarboxylation by hepatocytes from chow-fed or starved rats. To a smaller degree, alpha-keto-beta-methylvalerate and alpha-ketoisovalerate stimulated alpha-ketoisocaproate decarboxylation by hepatocytes from low-protein-fed rats. The implied order of potency of stimulation of flux through branched-chain alpha-ketoacid dehydrogenase was alpha-ketoisocaproate greater than alpha-keto-beta-methylvalerate greater than alpha-ketoisovalerate, i.e., the same order of potency of these compounds as branched-chain alpha-ketoacid dehydrogenase kinase inhibitors. Fluoride, known to inhibit branched-chain alpha-ketoacid dehydrogenase phosphatase, largely prevented alpha-ketoisocaproate and alpha-chloroisocaproate activation of flux through the branched-chain alpha-ketoacid dehydrogenase. Assay of the branched-chain alpha-ketoacid complex in cell-free extracts of hepatocytes isolated from low-protein-diet-fed rats confirmed that alpha-ketoacids affected the activity state of the complex. Branched-chain alpha-ketoacids failed to activate flux in hepatocytes prepared from chow-fed and starved rats because essentially all of the complex was already in the dephosphorylated, active state. These findings indicate that inhibition of branched-chain alpha-ketoacid dehydrogenase kinase activity by branched-chain alpha-ketoacids is important for regulation of the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase.     

Multivalent regulation of isoleucine-valine transaminase in an Escherichia coli K-12 ilvA deletion strain.

In a strain of Escherichia coli K-12 lacking threonine deaminase, the enzyme converting alpha-ketoisovalerate and alpha-keto-beta-methylvalerate to valine and isoleucine, respectively, was multivalently repressed by valine, isoleucine, and leucine. This activity was due to transaminase B, specified by the ilvE structural gene.     

Quantitation of the efflux of acylcarnitines from rat heart, brain, and liver mitochondria

The efflux of individual short-chain and medium-chain acylcarnitines from rat liver, heart, and brain mitochondria metabolizing several substrates has been measured. The acylcarnitine efflux profiles depend on the substrate, the source of mitochondria, and the incubation conditions. The largest amount of any acylcarnitine effluxing per mg of protein was acetylcarnitine produced by heart mitochondria from pyruvate. This efflux of acetylcarnitine from heart mitochondria is almost 5 times greater with 1 mM than 0.2 mM carnitine. Apparently the acetyl-CoA generated from pyruvate by pyruvate dehydrogenase is very accessible to carnitine acetyltransferase. Very little acetylcarnitine effluxes from heart mitochondria when octanoate is the substrate except in the presence of malonate. Acetylcarnitine production from some substrates peaks and then declines, indicating uptake and utilization. The unequivocal demonstration that considerable amounts of propionylcarnitine or isobutyrylcarnitine efflux from heart mitochondria metabolizing alpha-ketoisovalerate and alpha-keto-beta-methylvalerate provides evidence for a role (via removal of non-metabolizable propionyl-CoA or slowly metabolizable acyl-CoAs) for carnitine in tissues which have limited capacity to metabolize propionyl-CoA. These results also show propionyl-CoA must be formed during the metabolism of alpha-ketoisovalerate and that extra-mitochondrial free carnitine rapidly interacts with matrix short-chain aliphatic acyl-CoA generated from alpha-keto acids of branched-chain amino acids and pyruvate in the presence and absence of malate.     

Isolation of a branched-chain alpha-keto acid decarboxylase from rat liver.

An enzyme which catalyses oxidative decarboxylation of branched-chain alpha-keto acids was extracted from rat liver mitochondria with the aid of NaClO4. Purification yielded a product which appeared homogenous upon electrophoresis. Some kinetic data are reported; however, the enzyme is inactive with alpha-ketoisovalerate. The tenacity of binding to mitochondria, specificity, and other features, suggest that the decarboxylase may be a component of an enzyme complex named alpha-ketoisocaproate: alpha-keto-beta-methylvalerate dehydrogenase.     

Transient and long-term differential modulations of branched-chain alpha-keto acid decarboxylase activity in hypophysectomized rats

Hypophysectomy caused a marked but transient increase in branched-chain alpha-keto acid decarboxylase activities in rat liver mitochondria, peaking at about nine days post-surgery. The magnitude of increase is different for each of the three branched-chain alpha-keto acids. The activities then fall to a new steady state in three weeks with alpha-ketoisovalerate decarboxylase activity within the normal range, alpha-keto-beta-methylvalerate decarboxylase activity at twice normal, and alpha-ketoisocaproate decarboxylase activity decreased to a level too low for accurate measurements.     

Hypobaric hypoxia affects endogenous levels of alpha-keto acids in murine heart ventricles

Alpha-keto acids have recently been identified as potent regulators of cellular adaptations to hypoxia. Their actual intracellular concentrations under such conditions are unknown. Here, we determined concentrations of alpha-ketobutyrate, alpha-ketoglutarate, alpha-ketoisocaproate, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, phenylpyruvate, and pyruvate by a recently developed ultra-sensitive fluorescence HPLC method in ventricular myocardium of mice exposed to hypobaric hypoxia for up to 3 weeks. We observed characteristic alterations of cardiac alpha-keto acid concentrations that are specific for individual alpha-keto acids, show significant side differences (right versus left ventricles), and are suited to trigger some of the cardiac metabolic and structural adaptations to chronic hypoxia.     

An investigation of the metabolism of isoleucine to active Amyl alcohol in Saccharomyces cerevisiae

The metabolism of isoleucine to active amyl alcohol (2-methylbutanol) in yeast was examined by the use of (13)C nuclear magnetic resonance spectroscopy, combined gas chromatography-mass spectrometry, and a variety of mutants. From the identified metabolites a number of routes between isoleucine and active amyl alcohol seemed possible. All involved the initial decarboxylation of isoleucine to alpha-keto-beta-methylvalerate. The first, via branched chain alpha-ketoacid dehydrogenase to alpha-methylbutyryl-CoA, was eliminated because abolition of branched-chain alpha-ketoacid dehydrogenase in an lpd1 disruption mutant did not prevent the formation of active amyl alcohol. However, the lpd1 mutant still produced large amounts of alpha-methylbutyrate which initially seemed contradictory because it had been assumed that alpha-methylbutyrate was derived from alpha-methylbutyryl-CoA via acyl-CoA hydrolase. Subsequently it was observed that alpha-methylbutyrate arises from the non-enzymic oxidation of alpha-methylbutyraldehyde (the immediate decarboxylation product of alpha-keto-beta-methylvalerate). Mutant studies showed that one of the decarboxylases encoded by PDC1, PDC5, PDC6, YDL080c, or YDR380w must be present to allow yeast to utilize alpha-keto-beta-methylvalerate. Apparently, any one of this family of decarboxylases is sufficient to allow the catabolism of isoleucine to active amyl alcohol. This is the first demonstration of a role for the gene product of YDR380w, and it also shows that the decarboxylation steps for each alpha-keto acid in the catabolic pathways of leucine, valine, and isoleucine are accomplished in subtly different ways. In leucine catabolism, the enzyme encoded by YDL080c is solely responsible for the decarboxylation of alpha-ketoisocaproate, whereas in valine catabolism any one of the isozymes of pyruvate decarboxylase will decarboxylate alpha-ketoisovalerate.     

Purification, resolution, and reconstitution of rat liver branched-chain alpha-keto acid dehydrogenase complex

Branched-chain alpha-keto acid dehydrogenase (BCKADH) was solubilized as an enzyme complex from rat liver mitochondria by sonic treatment. Dehydrogenase (E1) and dihydrolipoyltransacylase (E2) components of the complex were purified in an associated form and resolved into individual components in the presence of 1 M NaCl, while lipoamide dehydrogenase (E3) component was dissociated from the complex during purification. Analysis by gel electrophoresis in dodecyl sulfate revealed the E1 comprised two different subunits with apparent molecular weights of 36,000 and 45,500, presumably in an equal molar ratio, while E2 consisted of a single subunit with an apparent molecular weight of 51,000. The BCKADH complex was reconstituted by combining E1, E2, and E3, and the formation of the complex was confirmed by analysis by sucrose density gradient centrifugation. The reconstituted enzyme complex oxidized not only alpha-ketoisovalerate (KIV), alpha-ketoisocaproate (KIC), and alpha-keto-beta-methylvalerate (KMV), but also pyruvate and alpha-ketoglutarate. Apparent Km values were 10-12 microM for the branched-chain alpha-keto acids, 2.2 mM for pyruvate, and 2.5 mM for alpha-ketoglutarate.     

Repression and inhibition of transport systems for branched-chain amino acids in Salmonella typhimurium.

Kinetics of the transport systems common for entry of L-isoleucine, L-leucine, and L-valine in Salmonella typhimurium LT2 have been analyzed as a function of substrateconcentration in the range of 0.5 to 45 muM. The systems of transport mutants, KA203 (ilvT3) and KA204 (ilvT4), are composed of two components; apparent Km values for uptake of isoleucine, leucine, and valine by the low Km component are 2 muM, 2 to 3 muM, and 1 muM, respectively, and by the high Km component 30 muM, 20 to 40 muM, and 0.1 mM, respectively. The transport system(s) of the wild type has not been separated into components but rather displays single Km values of 9 muM for isoleucine, 10 muM for leucine, and 30 muM for valine. The transport activity of the wild type was repressed by L-leucine, alpha ketoisocaproate, glycyl-L-isoleucine, glycyl-L-leucine, and glycyl-L-methionine. That for the transport mutants was repressed by L-alanine, L-isoleucine, L-methionine, L-valine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, glycyl-L-alanine, glycyl-L-threonine, and glycyl-L-valine, in addition to the compounds described above. Repression of the mutant transport systems resulted in disappearance of the low Km component for valine uptake, together with a decrease in Vmax of the high Km component; the kinetic analysis with isoleucine and leucine as substrates was not possible because of poor uptake. The maximum reduction of the transport activity for isoleucine was obtained after growing cells for two to three generations in a medium supplemented with repressor, and for the depression, protein synthesis was essential after removal of the repressor. The transport activity for labeled isoleucine in the transport mutant and wild-type strains was inhibited by unlabeled L-alanine, L-cysteine, L-isoleucine, L-leucine, L-methionine, L-threonine, and L-valine. D-Amino acids neither repressed nor inhibited the transport activity of cells for entry of isoleucine.     

Purification and characterization of branched chain alpha-ketoacid dehydrogenase from bovine liver mitochondria.

Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.3(4)) was solubilized and purified from bovine liver mitochondria for the first time. Decarboxylation of alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and alpha-ketoisocaproate was catalyzed by this multienzyme complex and this activity was co-purified for each substrate. Three enzymatic functions were contained in the complex including decarboxylation of the above ketoacids, transacylation of their simple acid derivatives, and reduction of NAD+ as an overall reaction. Product stoichiometry of these three reactions was 1 CO2:1 acyl-CoA:1 NADH. Activity depended upon the addition of thiamin pyrophosphate, CoASH, and NAD+ which were dissociable cofactors. Physically, two active forms of the enzyme complex were found: a 275,000-dalton unit and a 2 x 10(6)-dalton component. Both showed a characteristic flavin spectra and catalyzed all functions of the complex, implying that 10 small units aggregated into the larger unit. The soluble complex as visualized by electron microscopy had a diameter ranging from 12 to 24 nm corresponding to a molecular weight of 2 x 10(6). The size of the native membrane-bound component remains to be determined.     

Bovine heart pyruvate dehydrogenase kinase stimulation by alpha-ketoisovalerate

Purified bovine heart pyruvate dehydrogenase complex was used to investigate the effects of monovalent cations and alpha-ketoisovalerate on pyruvate dehydrogenase (PDH) kinase inhibition by thiamin pyrophosphate. Initial velocity patterns for thiamin pyrophosphate inhibition were consistent with hyperbolic non-competitive or hyperbolic uncompetitive inhibition at various K+ concentrations between 0 and 120 mM. The Kis, Kid, and Kin for thiamin pyrophosphate were in the range of 0.009 to 5.1 microM over the range of K+ concentrations tested. In the absence of K+, 1 mM alpha-ketoisovalerate had no effect on PDH kinase inhibition by thiamin pyrophosphate, whereas in the presence of 20 mM K+, alpha-ketoisovalerate stimulated PDH kinase activity almost 2-fold over the range of 0-80 microM thiamin pyrophosphate. Half-maximal stimulation by alpha-ketoisovalerate occurred at about 200 microM in the presence of 100 microM thiamin pyrophosphate and 20 mM K+. Similar but less extensive changes occurred in the presence of 100 microM thiamin pyrophosphate and 1 mM NH4+. Initial velocity patterns for PDH kinase inhibition by thiamin pyrophosphate in the presence of 2 mM alpha-ketoisovalerate were mixed noncompetitive, but alpha-ketoisovalerate increased the Vm and Km for adenosine 5'-triphosphate in the presence of inhibitor. In the presence of thiamin pyrophosphate, PDH kinase remained stimulated after chromatography on Sephadex G-25 to remove alpha-ketoisovalerate. The results indicate that acylation of pyruvate dehydrogenase complex by alpha-ketoisovalerate results in PDH kinase stimulation but only in the presence of monovalent cations and thiamin pyrophosphate.     

The regulation of hepatic gluconeogenesis using alpha-ketoisovalerate and propionate as precursors

The regulation of gluconeogenesis from alpha-ketoisovalerate and propionate was investigated in perfused livers from fasted rats. With alpha-ketoisovalerate as the gluconeogenic precursor, infusion of beta-hydroxybutyrate and acetate stimulated the rate of alpha-ketoisovalerate decarboxylation, but inhibited the rate of glucose production. Oleate, on the other hand, inhibited both alpha-ketoisovalerate decarboxylation and glucose production. When propionate was the primary gluconeogenic substrate, oleate, beta-hydroxybutyrate, and acetate infusion did not significantly alter hepatic glucose production. The present studies suggest that gluconeogenesis from alpha-ketoisovalerate is regulated at the level of various dehydrogenases prior to formation of propionyl-CoA, but subsequent to the branched-chain alpha-keto acid dehydrogenase reaction.     

Ketopantoate hydroxymethyltransferase. II. Physical, catalytic, and regulatory properties.

Some physical, catalytic, and regulatory properties of ketopantoate hydroxymethyltransferase (5,10-methylenetetrahydrofolate: alpha-ketoisovalerate hydroxymethyltranferase) from Escherichia coli are described. This enzyme catalyzes the reversible synthesis of ketopantoate (Reaction 1), an essential precursor of pantothenic acid. (1) HC(CH3)2COCOO- + 5,10-methylene tetrahydrofolate f in equilibrium r HOCH2C(CH3)2COCOO- + tetrahydrofolate It has a molecular weight by sedimentation equilibrium of 255,000, a sedimentation coefficient (S20,w) of 11 S, a partial specific volume of 0.74 ml/g, an isoelectric point of 4.4, and an absorbance, (see article), of 0.85. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and amino acid analyses give a subunit molecular weight of 27,000 and 25,700, respectively; both procedures indicate the presence of 10 identical subunits. The NH2-terminal sequence is Met-Tyr---. The enzyme is stable and active over a broad pH range, with an optimum from 7.0 to 7.6. It requires Mg2+ for activity; Mn2+, Co2+, Zn2+ are progressively less active. The enzyme is not inactivated by borohydride reduction in the presence of excess substrates, i.e. it is a Class II aldolase. Reaction 1f is partially inhibited by concentrations of formaldehyde (0.8 mM) and tetrahydrofolate (0.38 mM) below or near the Km values, apparent Km values are 0.18, 1.1 and 5.9 mM for tetrahydrofolate, alpha-ketoisovalerate, and formaldehyde, respectively. For Reaction 1r, apparent Km values are 0.16 and 0.18 mM, respectively, for ketopantoate and tetrahydrofolate, and the saturation curves for both substrates show positive cooperativity. Forward and reverse reactions occur at similar maximum velocities (Vmax approximately equal to 8 mumol of ketopantoate formed or decomposed per min per mg of enzyme at 37 degrees). Only 1-tetrahydrofolate is active in Reaction 1; d-tetrahydrofolate, folate, and methotrexate were neither active nor inhibitory. However, 1-tetrahydrofolate was effectively replaced with conjugates containing 1 to 6 additional glutamate residues; of these, tetrahydropterolpenta-, tetra-, and triglutamate were effective at lower concentrations than tetrahydrofolate itself; they were also the predominant conjugates of tetrahydrofolate present in E. coli. Alpha-Ketobutyrate, alpha-ketovalerate, and alpha-keto-beta-methylvalerate replaced alpha-ketoisovalerate as substrates; pyruvate was inactive as a substrate, but like isovalerate, 3-methyl-2-butanone and D- or L-valine, inhibited Reaction 1. the transferase has regulatory properties expected of an enzyme catalyzing the first committed step in a biosynthetic pathway. Pantoate (greater than or equal to 500 muM) and coenzyme A (above 1 mM) all inhibit; the Vmax is decreased, Km is increased, and the cooperativity for substrate (ketopantoate) is enhanced. Catalytic activity of the transferase is thus regulated by the products of the reaction path of which it is one component; transferase synthesis is not repressed by growth in the presence of pantothenate.     

Analysis of an avtA::Mu d1(Ap lac) mutant: metabolic role of transaminase C

Escherichia coli can synthesize alpha-ketoisovalerate, the precursor of valine, leucine, and pantothenate, by three routes: anabolically via dihydroxyacid dehydrase and catabolically via both the branched-chain amino acid transaminase (transaminase B) and the alanine-valine transaminase (transaminase C). An E. coli K-12 mutant devoid of transaminase C (avtA) was isolated by mutagenizing an isoleucine-requiring strain devoid of transaminase B (ilvE::Tn5) with Mu d1(Ap lac) and selecting for valine-requiring derivatives which were ampicillin resistant, Lac+, able to crossfeed an ilvD mutant, and unable to grow on alpha-ketoisovalerate in place of valine. Strains defective in one, two, or all three alpha-ketoisovalerate metabolic enzymes were constructed, and their properties were analyzed. The data indicated that avtA is the structural gene for transaminase C, that transaminase C is a single enzyme species, and that the sole pathway for pantothenate biosynthesis is from alpha-ketoisovalerate. The data further showed that isoelectric inhibits the transaminase B-catalyzed deamination of valine in vivo.     

Effects of branched chain alpha-ketoacids on the metabolism of isolated rat liver cells. I. Regulation of branched chain alpha-ketoacid metabolism.

alpha-Ketoisocaproate (ketoleucine) is shown to be metabolized to ketone bodies rapidly by isolated rat liver cells. Acetoacetate is the major end product and maximum rates were observed with 2 mM substrate. Studies with 2-tetradecylglycidic acid (an inhibitor of long chain fatty acid oxidation) showed that ketogenesis from alpha-ketoisocaproate and from endogenous fatty acids were additive. With alpha-ketoisocaproate present as soole substrate at 2 mM, leucine production was less than 10% of alpha-ketoisocaproate uptake and only 30% of the acetyl coenzyme A generated was oxidized in the citric acid cycle. Metabolism of alpha-ketoisocaproate was inhibited by fatty acids, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and pyruvate. Oxidation of acetyl-CoA generated from alpha-ketoisocaproate was suppressed by oleate and by pyruvate, but was enhanced by lactate. Metabolism between the different branched chain alpha-ketoacids was mutually competitive. When alpha-ketoisocaproate (2 mM) was added in the presence of high pyruvate concentrations (4.4 mM), flux through pyruvate dehydrogenase was decreased, and the proportion of total pyruvate dehydrogenase in the active form (PDHa) also fell. With lactate as substrate, PDHa was only 25% of total activity and was little affected by addition of alpha-ketoisocaproate. These data suggest that enhanced oxidation of acetyl-CoA from alpha-ketoisocaproate by lactate addition is caused by a low activity of pyruvate dehydrogenase combined with increased flux through the citric acid cycle in response to the energy requirements for gluconeogenesis. However, acetyl-CoA generation from pyruvate is apparently insufficiently inhibited by alpha-ketoisocaproate to cause a diversion of acetyl-CoA formed during alpha-ketoisocaproate metabolism from ketone body formation to oxidation in the citric acid cycle. Measurements of the cell contents of CoASH, acetyl-CoA, acid-soluble acyl-CoA, and acid-insoluble fatty acyl-CoA indicated that when the branched chain alpha-ketoacids were added as sole substrate, their oxidation was limited at a step distal to the branched chain alpha-ketoacid dehydrogenase. Acid-soluble acyl-CoA derivatives were depleted after oleate addition in the presence of alpha-ketoisocaproate, suggesting an inhibition of the branched chain alpha-ketoacid dehydrogenase by the elevation of the mitochondrial NADH/NAD+ ratio observed during fatty acid oxidation. This effect was not observed in the presence of oleate and 2-tetradecylglycidic acid.     

Ethanol and oleate inhibition of alpha-ketoisovalerate and 3-hydroxyisobutyrate metabolism by isolated hepatocytes.

Ethanol inhibited glucose synthesis from alpha-ketoisovalerate by isolated rat hepatocytes without significant inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex. Accumulation of 3-hydroxyisobutyrate, an intermediate in the catabolism of alpha-ketoisovalerate, was increased by ethanol, indicating inhibition of flux at the level of 3-hydroxyisobutyrate dehydrogenase. 3-Hydroxybutyrate caused the same effects as ethanol, suggesting inhibition was a consequence of an increase in the mitochondrial NADH/NAD+ ratio. Flux through the 3-hydroxyisobutyrate dehydrogenase was more sensitive to regulation by the mitochondrial NADH/NAD+ ratio than flux through the branched-chain alpha-ketoacid dehydrogenase. Oleate also inhibited glucose synthesis from alpha-ketoisovalerate, but marked inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex was caused by this substrate.     

Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase

The regulatory effects of alpha-ketoisovalerate on purified bovine heart pyruvate dehydrogenase complex and endogenous pyruvate dehydrogenase kinase were investigated. Incubation of pyruvate dehydrogenase complex with 0.125 to 10 mM alpha-ketoisovalerate caused an initial lag in enzymatic activity, followed by a more linear but inhibited rate of NADH production. Incubation with 0.0125 or 0.05 mM alpha-ketoisovalerate caused pyruvate dehydrogenase inhibition, but did not cause the initial lag in pyruvate dehydrogenase activity. Gel electrophoresis and fluorography demonstrated the incorporation of acyl groups from alpha-keto[2-14C]isovalerate into the dihydrolipoyl transacetylase component of the enzyme complex. Acylation was prevented by pyruvate and by arsenite plus NADH. Endogenous pyruvate dehydrogenase kinase activity was stimulated specifically by K+, in contrast to previous reports, and kinase stimulation by K+ correlated with pyruvate dehydrogenase inactivation. Maximum kinase activity in the presence of K+ was inhibited 62% by 0.1 mM thiamin pyrophosphate, but was inhibited only 27% in the presence of 0.1 mM thiamin pyrophosphate and 0.1 mM alpha-ketoisovalerate. Pyruvate did not affect kinase inhibition by thiamin pyrophosphate at either 0.05 or 2 mM. The present study demonstrates that alpha-ketoisovalerate acylates heart pyruvate dehydrogenase complex and suggests that acylation prevents thiamin pyrophosphate-mediated kinase inhibition.     

The effect of octanoate and palmitate on the metabolism of valine in perfused hindquarter of rat.

The effect of octanoate and palmitate on the oxidation of 14C-labelled valine has been studied in perfused hindquarter of rat. 1. The oxidation rate of valine increases 30 times when the concentration of valine is increased from 0.1 mM to 5 mM. 2. Octanoate at a 5 mM concentration effected a 10-fold increase in the flux through the alpha-ketoisovalerate dehydrogenase step and a 5-fold increase at 0.5 mM concentration. 3. Palmitate (1 mM) effects only a moderate increase in the valine oxidation. 4. With no octanoate there was a great accumulation of alpha-ketoisovalerate in both the muscle and the perfusion medium. 5. With octanoate little alpha-ketoisovalerate accumulated whereas 3-hydroxyisobutyrate was found in high concentration both in the muscle and in the medium. 6. Octanoate stimulated the production of citric-acid-cycle intermediates and lactate. 7. The results are discussed in relation to valine metabolism in the body.     

Biosynthesis of branched-chain fatty acid in bacilli: FabD (malonyl-CoA:ACP transacylase) is not essential for in vitro biosynthesis of branched-chain fatty acids

It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.     

Interactions between alpha-ketoisovalerate metabolism and the pathways of gluconeogenesis and urea synthesis in isolated hepatocytes

The mechanism of inhibition of pyruvate carboxylase, pyruvate dehydrogenase, and carbamyl phosphate synthetase induced by alpha-ketoisovalerate metabolism has been investigated in isolated rat hepatocytes incubated with lactate, pyruvate, ammonia, and ornithine as substrates. Half-maximum inhibitions of flux through each of these enzyme steps were obtained with 0.3 mM alpha-ketoisovalerate. The inhibition of pyruvate carboxylase flux by alpha-ketoisovalerate was largely reversed by oleate addition, but pyruvate dehydrogenase flux was inhibited further. Inhibition of flux through pyruvate carboxylase could be attributed mainly to the fall of its allosteric activator, acetyl-CoA, with some additional effect due to inhibition by methylmalonyl-CoA. Tissue acetyl-CoA levels decrease as a result of an inhibition of the active form of pyruvate dehydrogenase. Kinetic studies with the purified pig heart pyruvate dehydrogenase complex showed that methyl-malonyl-CoA, propionyl-CoA, and isobutyryl-CoA were inhibitory, the latter noncompetitive with CoASH with an apparent Ki of 90 microM. The observed inhibition of pyruvate dehydrogenase flux correlated with increases of the acetyl-CoA/CoASH and propionyl-CoA/CoASH ratios and isobutyryl-CoA levels, while increases of the mitochondrial NADH/NAD+ ratio explained differences between the effects of alpha-ketoisovalerate and propionate. Carbamyl phosphate synthetase I purified from rat liver was shown to be inhibited directly by methylmalonyl-CoA (apparent Ki of 5 mM). Inhibition of flux through carbamyl phosphate synthetase during alpha-ketoisovalerate metabolism could be attributed both to a direct inhibitory effect of methyl-malonyl-CoA and to a diminished activation by N-acetylglutamate. Direct effects of various acyl-CoA metabolites on these key enzymes may explain symptoms of hypoglycemia and hyperammonemia observed in patients with inherited disorders of organic acid metabolism.