Search PubMed⌕ Search

Biomedical subjects

E J Davis

Publications and source records attributed to E J Davis.

At least 37 records · Page 2Linked to original sources
PubMed1986

Carnitine palmitoyltransferase: activation and inactivation in liver mitochondria from fed, fasted, hypo- and hyperthyroid rats.

The sensitivity of carnitine palmitoyltransferase to malonyl-CoA is lost when liver mitochondria are preincubated in a KCl-containing medium. This loss of sensitivity is slowed down in mitochondria from hypothyroid rats and accelerated in mitochondria from fasted and hyperthyroid rats. Glucagon seems to enhance the effect of fasting. The loss of sensitivity is significantly slowed down by 50-500 nM malonyl-CoA and accelerated by small amounts of palmitoyl-CoA in the preincubation medium.

Acyltransferases↗
PubMed1986

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.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1986

Amino acid catabolism by perfused rat hindquarter. The metabolic fates of valine.

Hindquarters from starved rats were perfused with plasma concentrations of amino acids, but without other added substrates. Release of amino acids was similar to that previously reported, but, if total amino acid changes were recorded, alanine and glutamine were not formed in excess of their occurrence in muscle proteins. In protein balance (excess insulin) there was no net formation of either alanine or glutamine, even though the branched-chain amino acids and methionine were consumed. If [U-14C]valine was present, radiolabelled 3-hydroxyisobutyrate and, to a lesser extent, 2-oxo-3-methylbutyrate accumulated and radiolabel was incorporated into citrate-cycle intermediates and metabolites closely associated with the citrate cycle (glutamine and glutamate, and, to a smaller extent, lactate and alanine). If a 2-chloro-4-methylvalerate was present to stimulate the branched-chain oxo acid dehydrogenase, flux through this step was accelerated, resulting in increased accumulation of 3-hydroxyisobutyrate, decreased accumulation of 2-oxo-3-methylbutyrate, and markedly increased incorporation of radiolabel (specific and total) into all measured metabolites formed after 3-hydroxyisobutyrate. It is concluded that: amino acid catabolism by skeletal muscle is confined to degradation of the branched-chain amino acids, methionine and those that are interconvertible with the citrate cycle; amino acid catabolism is relatively minor in supplying carbon for net synthesis of alanine and glutamine; and partial degradation products of the branched-chain amino acids are quantitatively significant substrates released from muscle for hepatic gluconeogenesis. For valine, 3-hydroxyisobutyrate appears to be quantitatively the most important intermediate released from muscle. A side path for inter-organ disposition of the branched-chain amino acids is proposed.

Amino Acids↗
PubMed1986

A sensitive spectrophotometric assay of pyruvate dehydrogenase activity.

The recently reported highly sensitive method for assay of acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) [H. H. Andres, A. J. Klein, S. M. Szabo, and W. W. Weber (1985) 145, 367-375] has been adapted for determination of pyruvate dehydrogenase activity. This method provides an improvement in sensitivity over extant spectrophotometric methods and circumvents limitations of assays using radioactive pyruvate. In addition, the assay is simple and inexpensive and can be readily adapted for measurement of enzyme activity in crude tissue extracts or homogenates.

Acetyl Coenzyme A↗
PubMed1986

Amino acid catabolism by perfused rat hindquarters: degradation of threonine and isoleucine.

Rat hindquarters were perfused without added substrate other than trace amounts of [U-14C]threonine or [U-14C]isoleucine. Comparison of incorporation of radiolabel into some nonessential amino acids, citrate cycle intermediates, and lactate is presented. Activities of three enzymes for the initial reactions in threonine degradation are reported. It is concluded that skeletal muscle catabolizes threonine, and that the latter is a potential source of carbon for glucogenic precursors for the liver. In contrast, label from isoleucine was incorporated into glutamate, glutamine, and alanine much more than was that from threonine. Large amounts of organic acids accumulated, and more than 60% of total radioactivity was lost as CO2 during a 2-h perfusion period.

Amino Acids↗
PubMed1985

Serine synthesis by an isolated perfused rat kidney preparation.

The isolated perfused rat kidney was shown to synthesize serine from aspartate or glutamate, both of which are also precursors of glucose. The major products of aspartate metabolism were ammonia, serine, glutamate, glucose, glutamine and CO2. Perfusion of kidneys with aspartate in the presence of amino-oxyacetate resulted in a near-complete inhibition of aspartate metabolism, illustrating the essential role of aspartate aminotransferase in the metabolism of this substrate. Radioactivity from 14C-labelled aspartate and from 14C-labelled glycerol was incorporated into serine and glucose. Production of both glucose and serine from aspartate was suppressed in the presence of 3-mercaptopicolinic acid. These data provide evidence for the operation of the phosphorylated and/or non-phosphorylated pathway for serine production to the presence of 3-mercaptopicolinic acid. This is explained by simultaneous glycolysis. The rate of glucose production, but not that of serine, was greater in kidneys perfused with glutamate or with aspartate plus glycerol than the rates obtained by perfusion with aspartate alone. These data are taken to suggest that serine synthesis occurred at a near-maximal rate, and that the capacity of the kidney for serine synthesis from glucose precursors is lower than that for glucose synthesis.

Amino Acids↗
PubMed1985

Amino acid metabolism by perfused rat hindquarter. Effects of insulin, leucine and 2-chloro-4-methylvalerate.

Hindquarters from starved rats were perfused without substrates but in the presence of an O2- and CO2-carrying perfluorocarbon emulsion to evaluate principally the metabolism of individual endogenous and protein-derived amino acids by this muscle preparation. This experimental model was shown, by a battery of metabolite measurements, to maintain cellular homoeostasis for at least 2h. The net appearance of most amino acids closely approximated their frequency of occurrence in muscle proteins, showing that they are not significantly metabolized. Exceptions were the branched-chain amino acids, methionine and those amino acids that are interconvertible with intermediates of the citrate cycle and pyruvate through coupled transaminations. The evidence indicates that only valine, isoleucine, aspartate and probably methionine can be catabolized by skeletal muscle to provide carbon precursors for glutamate/glutamine and alanine that are formed de novo by protein-catabolic muscle. The protein-sparing effects of insulin and leucine were confirmed. Although each decreased proteolysis and the net appearance of free amino acids, they were generally without effect on the ratios of amino acids formed. 2-Chloro-4-methylvalerate selectively stimulated the removal rate for the branched-chain amino acids, confirming the idea that the branched-chain oxo acid dehydrogenase normally limits the rate of their oxidation by muscle. It is also concluded that, since alanine was not formed in excess of that found in muscle proteins when no glucose was added as substrate, the excess of alanine (carbon) released from muscles in other studies is derived to a large extent, but not exclusively, from preformed carbohydrate.

Amino Acids↗
PubMed1985

Interference by ethanol of coupling between gluconeogenesis and ureagenesis from proline in isolated hepatocytes.

Proline stimulated equally the production of glucose and urea by isolated hepatocytes. Ethanol suppressed glucose production much more strongly than urea synthesis. The proline-derived carbon not reaching glucose was found as lactate. Inhibition of phosphoenolpyruvate synthesis with 3-mercaptopicolinate blocked gluconeogenesis, but was without effect on lactate production. Acetate was formed from endogenous sources, as well as from ethanol. Its accumulation from ethanol was enhanced both by proline and lactate. The differential effect of ethanol on gluconeogenesis and ureagenesis appears to be related to its effect on the redox state of the cell.

Animals↗
PubMed1984

Control of cellular redox potential as measured in a steady-state, cell-free system.

A cell-free system consisting of rat liver mitochondria, liver cytosol, lactate, and the substrates intrinsic to the malate-aspartate shuttle was reconstituted for studies of steady-state substrate fluxes and, more specifically, to evaluate further the mechanism of control of the intra- and extramitochondrial steady states of the free NAD+/NADH ratios. Soluble (F1) ATPase or 2,4-dinitrophenol (DNP) were added in varying amounts to alter substrate fluxes and the constant energy state of this 'open' metabolizing system. The steady-state redox segregation (1.36 log NAD+/NADH ratio out vs NAD+/NADH in the mitochondrial matrix) was maximally about 3 kcal, and declined together with the membrane potential (delta psi) and log ATP/ADP, which obtain on imposing an increasing energy load on the system. It is concluded that transmembrane movement of reducing equivalents is coupled to electron transfer through delta psi, mediated by the electrogenic exchange of glutamate and aspartate. When delta psi was high (near State 4), delta G redox was approximately the same as that generated without flux of reducing equivalents [E. J. Davis, J. Bremer, and K. E. Akerman (1980) J. Biol. Chem. 255, 2277-2283], suggesting that delta Gredox is in near thermodynamic equilibrium with delta psi. If the steady-state ATP/ADP ratio was altered with an energy load (F1-ATPase), delta Gredox decreased more steeply than delta psi (tetraphenyl phosphonium-sensitive electrode used to measure delta psi). At comparable ranges of ATP/ADP, both delta Gredox and delta psi decreased more steeply with uncoupler than with an external ADP-regenerating system.

2,4-Dinitrophenol↗
PubMed1984

Rate control of phosphorylation-coupled respiration by rat liver mitochondria.

Liver mitochondria provided with an oxidizable substrate, ATP, oxygen, and an ADP-generating system (soluble F1-ATPase) were used to reevaluate the rate-controlling step(s) intrinsic to all of the processes of mitochondrial oxidative phosphorylation. The quantity termed "control strength" (C), previously defined as the fractional change in flux through a (system) induced by a fractional change in the concentration of an individual enzyme in the system, has been used to evaluate rate-influencing steps in this overall process by carefully defining the dimensions of the "system" under analysis. If the system is defined by a suspension of mitochondria provided with substrates, plus an extrinsic ADP-generating process (ATPase), the value of C of the latter for the overall process of phosphorylation-linked respiration is near 1.0 until the capacity of the mitochondria to phosphorylate ADP is approached, after which C for the soluble ATPase becomes zero as the maximum capacity for phosphorylation is attained. Carboxyatractyloside was found only marginally to inhibit respiration stimulated by ATPase, even when a large percentage of adenine nucleotide translocase molecules were immobilized. The relative lack of effect of carboxyatractyloside on phosphorylating respiration is explained by the readjustment of the concentration of one of the substrates (ADP) and an inhibitor (ATP), which results from inhibition of adenine nucleotide translocase. The residual blunted inhibition of respiration is explained by product inhibition of the ADP-regenerating ATPase, and not necessarily to any intrinsically mitochondrial intermediate process. The system being evaluated can be redefined to include only the processes intrinsic to mitochondria. This can be achieved by providing exactly comparable substrate concentrations to the mitochondria under comparable incubation conditions. Under these conditions, the adenine nucleotide translocase is the principal, if not the only, rate-controlling step in the overall process of oxidative phosphorylation until a new rate-limitation is attained (ATP synthesis). These data are consistent with the conclusion that, at intermediate rates of phosphorylation-coupled respiration, the extramitochondrial ATP/ADP ratio regulates this process through its kinetic effects on the catalytic properties of the adenine nucleotide translocase.

Adenosine Diphosphate↗
PubMed1984

The effect of glucagon on the carbon flux from palmitate into glucose, lactate and ketone bodies, studied with isolated hepatocytes.

Glucagon induced a rapid (within 3 min) increase in glucose radioactivity and a decrease in the labeling of ketone bodies when isolated hepatocytes were incubated in the presence of [1-14C]palmitate. Simultaneously, the hormone induced a decrease in the levels of pyruvate and Krebs cycle intermediates and an increase in the level of phosphoenolpyruvate (PEP). The glucagon-induced increase in glucose radioactivity was much larger than the simultaneous decrease in lactate labeling. A comparison of the incorporation of labeled carbon from [1-14C]palmitate and [U-14C]palmitate into glucose and CO2 indicates a selective stimulatory action of glucagon on the flux through the phosphoenolpyruvate carboxykinase (PEPCK) reaction.

Animals↗
PubMed1983

Reconstruction of steady state in cell-free systems. Interactions between glycolysis and mitochondrial metabolism: regulation of the redox and phosphorylation states.

A reconstituted "open" system comprising respiring mitochondria and actively glycolyzing muscle extract was devised for studies of vectorially mediated interactions. Glycogen particles were the substrate for the glycolyzing enzymes. Purified soluble (F1) ATPase was added in varying quantities to establish a range of energetic steady states. The data generally confirm our recent conclusions (Wu and Davis, (1981) Arch. Biochem. Biophys. 208, 85-89) on the relative efficacy of the adenine nucleotides and their ratios, and of inorganic phosphate on flux through rate-controlling steps of glycolysis. When mitochondrial ATP synthesis was blocked, glycolytic flux was relatively rapid, and the lactate/pyruvate ratio increased with time to values up to greater than 300. If functional mitochondria were present, glycolytic flux was very strongly suppressed, provided the energy state (ATP/ADP) was high, and the phosphate concentration[Pi] was low. Adenine nucleotide control of glycolysis was to a large extent lost when the steady-state ATP/ADP was below about 10, or if [Pi] was elevated. In the two-phase system containing respiring mitochondria and components of the malate-aspartate shuttle, the ATP/ADP and both extra- and intramitochondrial NAD+/NADH ratios were maintained constant, and to various perturbable levels with varying energy load (ATPase). The gradient in reduction potentials attained values (delta Gredox) of up to about 2.5 kcal. The extramitochondrial redox state was not positively correlated with the external phosphorylation potential ([ATP]/[ADP] X [Pi]). The following conclusions are drawn on the basis of the present data, together with other reports (Davis, Bremer, and Akerman (1980) J. Biol. Chem. 255, 2277-2283) and (Klingenberg and Rottenberg (1977) Eur. J. Biochem. 73, 125-130): (a) the gradient in reduction potential is driven by the membrane potential (delta psi), mediated by the electrogenic glutamate-aspartate exchange, and the poise or set point of this gradient is a function of delta psi; and (b) the gradient of ATP/ADP ratios across the membrane is also driven principally by delta psi, mediated by the electrogenic ATP-ADP exchange. Hence, segregation of phosphorylation and reduction potentials is linked through a mutually shared electrical driving force.

Adenosine Diphosphate↗
PubMed1981

The disposition of citric acid cycle intermediates by isolated rat heart mitochondria.

The mechanism of depletion of tricarboxylic acid cycle intermediates by isolated rat heart mitochondria was studied using hydroxymalonate (an inhibitor of malic enzymes) and mercaptopicolinate (an inhibitor of phosphoenolpyruvate carboxykinase) as tools. Hydroxymalonate inhibited the respiration rate of isolated mitochondria in state 3 by 40% when 2 mM malate was the only external substrate, but no inhibition was found with 2 mM malate plus 0.5 mM pyruvate as substrates. In the presence of bicarbonate, arsenite and ATP propionate was converted to pyruvate and malate at the rates of 14.0 +/- 2.9 and 2.8 +/- 1.8 nmol/mg protein in 5 min, respectively. Under these conditions, 0.1 mM mercaptopicolinate did not affect this conversion, but 2 mM hydroxymalonate inhibited pyruvate formation completely and resulted in an accumulation of malate up to 13.2 +/- 2.9 nmol/mg protein. No accumulation of phosphenolpyruvate was found under any condition tested. It is concluded that malic enzymes but not phosphoenolpyruvate carboxykinase, are involved in conversion of propionate to pyruvate in isolated rat heart mitochondria.

Animals↗
PubMed1981

Metabolism of pent-4-enoate in rat heart. Reduction of the double bond.

1. Soluble extracts from rat heart and liver mitochondria were used to evaluate the early steps in the conversion of pent-4-enoyl-CoA into tricarboxylic acid-cycle intermediates. Hitherto the unresolved problem was the reduction of the double bond of pent-4-enoate. 2. Soluble extracts from heart mitochondria reduced pent-4-enoyl-CoA and penta-2,4-dienoyl-CoA in the presence of NADPH at rates (nmol/min per mg of protein) of 0.9 +/- 0.1 and 132 +/- 8 and from the liver mitochondria at the rates of 1.9 +/- 0.2 and 52 +/- 6 respectively. No reduction of acryloyl-CoA was found. 3. We show that primarily the double bond in position 4, not in position 2, of penta-2,4-dienoyl-CoA is reduced. 4. It is concluded that the principal metabolic pathway of penta-4-enoate is reduction of the double bond in position 4 after an initial oxidation of penta-2,4-dienoyl-CoA. The pent-2-enoyl-CoA thus formed can be further metabolized by the usual enzymes of beta-oxidation, and by the further metabolism of propionyl-CoA to tricarboxylic acid-cycle intermediates.

Acyl Coenzyme A↗
PubMed1981

Three-factor reciprocal cross mapping of a gene that causes expression of feedback-resistant acetohydroxy acid synthase in Escherichia coli K-12.

The ilv-662 allele was previously identified as a mutation that caused acetohydroxy acid synthase activity to be resistant to feedback inhibition by valine (Davis et al. 1977). This allele was mapped between thr and leu by cotransduction analysis and labeled ilvJ. This report describes the mapping of ilvJ relative to genes that lie between thr and leu (ara, carA and pdxA) by three factor reciprocal cross analyses. We find that the probable gene order is thr-carA-pdxA-ilvJ-ara-leu. Although the phenotypic properties of ilvJ662 appear to be quite distinct from brnS, a gene reported to involve branched chain amino acid transport (Guardiola et al. 1974), we do not rule out possible allelism because of the uncertainty of the map position of brnS.

Acetolactate Synthase↗
PubMed1980

Thermodynamic aspects of translocation of reducing equivalents by mitochondria.

The "poise" in which there is no net transport of reducing equivalents mediated by glutamate and aspartate movements across the mitochondrial membrane has been studied using nonrespiring liver mitochondria, supplied with a given extramitochondrial ATP/ADP ratio. Absorbance shifts in safranine have been used as a probe to estimate the membrane potential generated under these conditions. Measurements of the asymmetric distribution of substrate anions and of reducing potential across the limiting membrane gave estimated differences of between 2.2 and 3.3 kcal when the ATP/ADP ratio in the suspending medium was about 30. The estimated membrane potential under these conditions was about 140 mV. All of the above parameters were decreased in a corresponding manner when the ATP/ADP ratio imposed from outside was decreased. A strong inflection in the slope of correlated parameters was obtained when the ATP/ADP ratio was below about 0.5. On the other hand, there was a linear relationship between the membrane potential and the asymmetric poise of reducing equivalents and of carbon metabolites. It is concluded that the poise of reducing equivalents mediated by the malate-aspartate shuttle is determined principally by the membrane potential through its effect on the electrogenic exchange of glutamate and aspartate.

Adenosine Diphosphate↗