Modulation of branched-chain 2-oxoacid dehydrogenase activity by adenine nucleotides in isolated rat liver mitochondria

Feeding a 17.5% amino acid diet to rats results in inactivation of the hepatic branched-chain 2-oxoacid dehydrogenase complex. Reactivation occurs when preincubating mitochondria in the presence of 0.3 mM ATP, ADP, and AMP. The effect of AMP is assumed to be due to de novo formation of ADP. NaF (25 mM) blocks reactivation suggesting the involvement of a protein phosphatase in the activation process. At high nucleotide concentrations (3 mM) the enzyme is inactive. In the presence of Mg2+ ions nucleotide induced activation is further increased. Mg2+ ions themselves influence the equilibrium state of the enzyme complex. Low concentrations (1 mM) favor inactivation while high concentrations (10 mM) stimulate activation of the enzyme suggesting that Mg2+ ions may act by regulating the associated kinase and phosphatase.     

Mitochondrial 2-oxoacid dehydrogenase complexes of animal tissues

The pyruvate dehydrogenase and branched-chain 2-oxoacid dehydrogenase complexes of animal mitochondria are inactivated by phosphorylation of serine residues, and reactivated by dephosphorylation. In addition, phosphorylated branched-chain complex is reactivated, apparently without dephosphorylation, by a protein or protein-associated factor present in liver and kidney mitochondria but not in heart or skeletal muscle mitochondria. Interconversion of the branched-chain complex may adjust the degradation of branched-chain amino acids in different tissues in response to supply. Phosphorylation is inhibited by branched-chain ketoacids, ADP and TPP. The pyruvate dehydrogenase complex is almost totally inactivated (99%) by starvation or diabetes, the kinase reactions being accelerated by products of fatty acid oxidation and by a protein or protein-associated factor induced by starvation or diabetes. There are three sites of phosphorylation, but only sites 1 and 2 are inactivating. Site 1 phosphorylation accounts for 98% of inactivation except during dephosphorylation when its contribution falls to 93%. Sites 2 and 3 are only fully phosphorylated when the complex is fully inactivated (starvation, diabetes). Phosphorylation of sites 2 and 3 inhibits reactivation by phosphatase. The phosphatase reaction is activated by Ca2+ (which may mediate effects of muscle work) and possibly by uncharacterized factors mediating insulin action in adipocytes.     

The catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses 2-oxoacid dehydrogenase (EC 1.2.4.4) similar to that found in mammalian cells. The activity is readily detected in cells which have been cultured in a minimal medium containing a branched-chain amino acid. Mutants defective in lipoamide dehydrogenase also lack 2-oxoacid dehydrogenase and are thus unable to catabolize branched-chain amino acids: 2-oxoacids accumulate in the cultures of these cells. The 2-oxoacid dehydrogenase activity is distinct from both 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase, because it could not be detected in assay conditions which permitted the measurement of 2-oxoglutarate dehydrogenase and vice versa. In addition, a strain lacking 2-oxoglutarate dehydrogenase (kgd1::URA3) retained 2-oxoacid dehydrogenase as did a mutant specifically lacking pyruvate dehydrogenase (pda1::Tn5ble). In complex media the specific activity of this enzyme is highest in YEP (yeast extract-peptone)-glycerol and lowest in YEP-acetate and YEP-fructose. 2-Oxoacid dehydrogenase could not be detected in cells which had been transferred to sporulation medium. These results suggest that in S. cerevisiae the catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase, not via the 'Ehrlich Pathway'.     

Branched-chain 2-oxoacid dehydrogenase kinase activity is regulated by alteration of protein thiol groups.

Effects of sulfhydryl reagents (5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide) and potassium ferricyanide on the activities of branched-chain 2-oxoacid dehydrogenase complex and its kinase were studied. The dehydrogenase activity was inhibited by the sulfhydryl reagents, but not by potassium ferricyanide. The kinase activity of branched-chain 2-oxoacid dehydrogenase-kinase complex was inhibited with an increase in concentration of all three compounds. However, direct treatment of the purified kinase with N-ethylmaleimide prior to reconstitution with kinase-depleted branched-chain 2-oxoacid dehydrogenase resulted in no loss of kinase activity. These results suggest that protein thiol groups of the E2 component of the dehydrogenase complex are involved in the interaction between the dehydrogenase and its kinase.     

Calpain inhibition by peptide epoxides.

The protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex was purified greater than 1000-fold from extracts of rat liver mitochondria; the specific activity was greater than 1000 units/mg of protein (1 unit gives half-maximum re-activation of 10 munits of phosphorylated complex). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave two bands (Mr 47700 and 35300) indistinguishable from the alpha- and beta-subunits of the branched-chain dehydrogenase component of the complex. On gel filtration (Sephacryl S-300), apparent Mr was 190000. This and other evidence suggests that activator protein is free branched-chain dehydrogenase; this conclusion is provisional until identical amino acid composition of the subunits has been demonstrated. Activator protein (i.e. free branched-chain dehydrogenase) was inhibited (up to 30%) by NaF, whereas branched-chain complex was not inhibited. There was no convincing evidence for interconvertible active and inactive forms of activator protein in rat liver mitochondria. Activator protein was detected in mitochondria from liver (ox, rabbit and rat) and kidney (ox and rat), but not in rat heart or skeletal-muscle mitochondria. In rat liver mitochondrial extracts, branched-chain complex sedimented with the mitochondrial membranes, whereas activator protein remained in the supernatant. Activator protein re-activated phosphorylated (inactive) particulate complex from rat liver mitochondria, but it did not activate dephosphorylated complex. Liver and kidney, but not muscle, mitochondria apparently contain surplus free branched-chain dehydrogenase, which is bound by the complex with lower affinity than is the branched-chain dehydrogenase intrinsic to the complex. It is suggested that this functions as a buffering mechanism to maintain branched-chain complex activity in liver and kidney mitochondria.     

Nature of the Cytoplasmic Factor(s) Modulating Adenylate Cyclase in the Developing Rat Brain

Abstract: Adenylate cyclase activity in the particulate fraction from rat brain was markedly enhanced by the cytoplasmic fraction, which itself contained negligible enzyme activity, indicating the presence of some stimulatory factor(s) in the supernatant. Activation of adenylate cyclase was dependent on the supernatant concentration up to 1 mgiml, but higher concentration of the supernatant did not produce further activation of the enzyme. The supernatant retained its stimulatory activity after boiling for 5 min, extensive dialysis, and phospholipase A and DNAase treatments, but was completely inactivated by digestion with trypsin. Ability of the supernatant to activate adenylate cyclase was low during fetal life, increased severalfold neonatally, and declined somewhat thereafter to an adult level. Adenylate cyclase in the particulate fraction from 2-day-old rat brain was also activated by GTP, calcium-dependent regulator (CDR) of cyclic AMP phosphodiesterase in the presence of 100 pM-Ca¹, and by NaF. The supernatant produced additive activation of the enzyme with NaF but not with GTP or CDR, suggesting a common site of action of the supernatant factor(s) and the latter two agents. DEAE-cellulose chromatography of the boiled supernatant resolved the heat-stable proteins into several peaks. Adenylate cyclase activator eluted in two distinct peaks, one of which also contained CDR activity. It is concluded that rat brain supernatant contains some factor in addition to CDR which activates particulate adenylate cyclase.     

The 2-oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization

The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (M(r) = 5 x 10(6)) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.     

Discovery of a putative acetoin dehydrogenase complex in the hyperthermophilic archaeon Sulfolobus solfataricus

Like many other aerobic archaea, the hyperthermophile Sulfolobus solfataricus possesses a gene cluster encoding components of a putative 2-oxoacid dehydrogenase complex. In the current paper, we have cloned and expressed the first two genes of this cluster and demonstrate that the protein products form an α₂β₂ hetero-tetramer possessing the catalytic activity characteristic of the first component enzyme of an acetoin dehydrogenase multienzyme complex. This represents the first report of an acetoin multienzyme complex in archaea, and contrasts with the branched-chain 2-oxoacid dehydrogenase complex activities characterised in two other archaea, Thermoplasma acidophilum and Haloferax volcanii.     

Discovery of the catalytic function of a putative 2-oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon Thermoplasma acidophilum

Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an alpha2beta2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.     

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.     

Physiological covalent regulation of rat liver branched-chain alpha-ketoacid dehydrogenase

A radiochemical assay was developed for measuring branched-chain alpha-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain alpha-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain alpha-ketoacids, established inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently starved, inhibition of branched-chain alpha-ketoacid dehydrogenase kinase by branched-chain alpha-ketoacids generated as a consequence of endogenous proteolysis most likely promotes the greater branched-chain alpha-ketoacid dehydrogenase activity state.     

Dephosphorylation and reactivation of phosphorylated purified ox-kidney branched-chain dehydrogenase complex by co-purified phosphatase

The branched-chain 2 oxoacid dehydrogenase complex has been purified from well-washed ox-kidney mitochondria together with branched-chain dehydrogenase kinase. The complex was inactivated and phosphorylated by ATP in about 5 min at 30 degrees C. After hydrolysis of ATP by a contaminating ATPase (5-10 min) the complex was dephosphorylated and reactivated. Dephosphorylation and reactivation were linearly correlated. Reactivation was dependent upon Mg2+ (K0.5 greater than 1 mM) and inhibited completely by 50 mM fluoride. Reactivation and dephosphorylation are attributed to a mitochondrial branched-chain dehydrogenase phosphatase.     

A novel branched-chain amino acid metabolon. Protein-protein interactions in a supramolecular complex

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain alpha-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.     

Pyruvate dehydrogenase phosphate (PDHb) phosphatase in brain: activity, properties, and subcellular localization

The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg . ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho-PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K-phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K-phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A:cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA:cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and 'reactivation' of enzyme activity. Cytosol caused a 78% increase in acyl-CoA:cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl2 and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A:cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA:cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500 X g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl2, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA:cholesterol acyltransferase activity is observed.     

Engineering Escherichia coli to produce branched-chain fatty acids in high percentages

Branched-chain fatty acids (BCFAs) are key precursors of branched-chain fuels, which have cold-flow properties superior to straight chain fuels. BCFA production in Gram-negative bacterial hosts is inherently challenging because it competes directly with essential and efficient straight-chain fatty acid (SCFA) biosynthesis. Previously, Escherichia coli strains engineered for BCFA production also co-produced a large percentage of SCFA, complicating efficient isolation of BCFA. Here, we identified a key bottleneck in BCFA production: incomplete lipoylation of 2-oxoacid dehydrogenases. We engineered two protein lipoylation pathways that not only restored 2-oxoacid dehydrogenase lipoylation, but also increased BCFA production dramatically. E. coli expressing an optimized lipoylation pathway produced 276 mg/L BCFA, comprising 85% of the total free fatty acids (FFAs). Furthermore, we fine-tuned BCFA branch positions, yielding strains specifically producing ante-iso or odd-chain iso BCFA as 77% of total FFA, separately. When coupled with an engineered branched-chain amino acid pathway to enrich the branched-chain α-ketoacid pool, BCFA can be produced from glucose at 181 mg/L and 72% of total FFA. While E. coli can metabolize BCFAs, we demonstrated that they are not incorporated into the cell membrane, allowing our system to produce a high percentage of BCFA without affecting membrane fluidity. Overall, this work establishes a platform for high percentage BCFA production, providing the basis for efficient and specific production of a variety of branched-chain hydrocarbons in engineered bacterial hosts.     

Immunology, biosynthesis and in vivo assembly of the branched-chain 2-oxoacid dehydrogenase complex from bovine kidney

Specific, polyclonal antisera have been raised to the native branched-chain 2-oxoacid dehydrogenase complex (BCOADC) from bovine kidney and each of its three constituent enzymes: E1, the substrate-specific 2-oxoacid dehydrogenase; E2, the multimeric dihydrolipoamide acyltransferase 'core' enzyme and E3, dihydrolipoamide dehydrogenase. Purified BCOADC, isolated by selective poly(ethyleneglycol) precipitation and hydroxyapatite chromatography, contains only traces of endogenous E3 as detected by a requirement for this enzyme in assaying overall complex activity and by immunoblotting criteria. A weak antibody response was elicited by the E1 beta subunit relative to the E2 and E1 alpha polypeptides employing either purified E1 or BCOADC as antigens. Anti-BCOADC serum showed no cross-reaction with high levels of pig heart E3 indicating the absence of antibody directed against this component. However, immunoprecipitates of mature BCOADC from detergent extracts of NBL-1 (bovine kidney) or PK-15 (porcine kidney) cell lines incubated for 3-4 h in the presence of [35S]methionine contained an additional 55,000-Mr species which was identified as E3 on the basis of immunocompetition studies. Accumulation of newly synthesised [35S]methionine-labelled precursors for E2, E1 alpha and E3 was achieved by incubation of PK-15 cells for 4 h in the presence of uncouplers of oxidative phosphorylation. Pre-E2 exhibited an apparent Mr value of 56,500, pre-E1 alpha, 49,000 and pre-E3, 57,000 compared to subunit Mr values of 50,000, 46,000 and 55,000, respectively, for the mature polypeptides. Thus, like the equivalent lipoate acyltransferases of the mammalian pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) complexes, pre-E2 of BCOADC characteristically contains an extended presequence. In NBL-1 cells, pre-E2 was found to be unstable since no cytoplasmic pool of this precursor could be detected; moreover, processed E1 alpha was not assembled into intact BCOADC as evidenced by the absence of E2 or E3 in immunoprecipitates with anti-(BCOADC) serum after a 45-min 'chase' period in the absence of uncoupler. Dihydrolipoamide dehydrogenase (E3), in its precursor state, was not present in immune complexes with anti-(BCOADC) serum, indicating that its co-precipitation with mature complex is by virtue of its high affinity for assembled complex in vivo whereas no equivalent interaction of pre-E3 with its companion precursors occurs prior to mitochondrial import.     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme a reductase and acylcoenzyme a: Cholesterol acyltransferase activities by phoshorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA: cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and ‘reactivation’ of enzyme activity. Cytosol caused a 78% increase in acyl-CoA: cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl₂ and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A: cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA: cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500×g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl₂, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA: cholesterol acyltransferase activity is observed.     

Interaction of thioredoxins with target proteins: role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2-oxoacid dehydrogenase complexes

The thioredoxin action upon the 2-oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild-type and mutated. The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxin-dependent activation of the complexes. Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide. Site-directed mutagenesis of D26, W31, F/W12, and Y/A70 (the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied) indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2-oxoacid dehydrogenase complexes. Sequences of 11 thioredoxin species tested biochemically are aligned. The thioredoxin residues at the contact between the alpha3/3(10) and alpha1 helices, the length of the alpha1 helix and the charges in the alpha2-beta3 and beta4-beta5 linkers are found to correlate with the protein influence on the 2-oxoacid dehydrogenase complexes (the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11:13-28). The distribution of the charges on the surface of the thioredoxin molecules is analyzed. The analysis reveals the species specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2-oxoacid dehydrogenase systems. The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule. Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action. The dipole direction is found to be significantly influenced by the charges of the residues 13/14, 51, and 83/85, which distinguish the activating and inhibiting thioredoxin disulfides.     

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.