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Biomedical subjects

R M Denton

Publications and source records attributed to R M Denton.

At least 91 records · Page 5Linked to original sources
PubMed1987

Use of rapid gel-permeation chromatography to explore the inter-relationships between polymerization, phosphorylation and activity of acetyl-CoA carboxylase. Effects of insulin and phosphorylation by cyclic AMP-dependent protein kinase.

Superose 6 chromatography was used to separate rapidly the polymeric and dimeric forms of acetyl-CoA carboxylase. With preparations of acetyl-CoA carboxylase purified by Sepharose-avidin chromatography, it is shown that citrate promotes polymerization and that the extent of polymerization is diminished, but not eliminated, after phosphorylation by cyclic-AMP-dependent protein kinase. After exposure of rat epididymal adipose tissue to insulin, evidence was obtained for a marked increase in polymerization. The polymeric form, which was active in the absence of citrate, exhibited increased phosphorylation, particularly on a tryptic peptide designated the I-peptide in an earlier study [Brownsey & Denton (1982) Biochem. J. 202, 77-86]. In contrast, in tissue exposed to the beta-agonist isoprenaline, most of the phosphorylated acetyl-CoA carboxylase appeared to be in the dimeric form if chromatography was carried out in the absence of citrate, whereas in the presence of citrate the degree of polymerization was diminished.

Acetyl-CoA Carboxylase↗
PubMed1987

Hypoglycaemia due to an insulin-receptor antibody in Hodgkin's disease.

Severe fasting hypoglycaemia developed in a patient with Hodgkin's disease after many courses of chemotherapy. Her serum contained a factor which stimulated glucose uptake by rat adipocytes, and this factor was found in the immunoglobulin fraction. The serum also displaced insulin bound to human erythrocytes and both precipitated and phosphorylated insulin receptors extracted from human placenta. The insulin-like substance is probably an antibody to the insulin receptor.

Adipose Tissue↗
PubMed1987

Effects of Ca2+ and Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within toluene-permeabilized mitochondria.

Mitochondria from rat epididymal white adipose tissue were made permeable to small molecules by toluene treatment and were used to investigate the effects of Mg2+ and Ca2+ on the re-activation of pyruvate dehydrogenase phosphate by endogenous phosphatase. Re-activation of fully phosphorylated enzyme after addition of 0.18 mM-Mg2+ showed a marked lag of 5-10 min before a maximum rate of reactivation was achieved. Increasing the Mg2+ concentration to 1.8 mM (near saturating) or the addition of 100 microM-Ca2+ resulted in loss of the lag phase, which was also greatly diminished if pyruvate dehydrogenase was not fully phosphorylated. It is concluded that, within intact mitochondria, phosphatase activity is highly sensitive to the degree of phosphorylation of pyruvate dehydrogenase and that the major effect of Ca2+ may be to overcome the inhibitory effects of sites 2 and 3 on the dephosphorylation of site 1. Apparent K0.5 values for Mg2+ and Ca2+ were determined from the increases in pyruvate dehydrogenase activity observed after 5 min. The K0.5 for Mg2+ was diminished from 0.60 mM at less than 1 nM-Ca2+ to 0.32 mM at 100 microM-Ca2+; at 0.18 mM-Mg2+, the K0.5 for Ca2+ was 0.40 microM. Ca2+ had little or no effect at saturating Mg2+ concentrations. Since effects of Ca2+ are readily observed in intact coupled mitochondria, it follows that Mg2+ concentrations within mitochondria are sub-saturating for pyruvate dehydrogenase phosphate phosphatase and hence less than 0.5 mM.

Animals↗
PubMed1987

The role of Ca2+ in the regulation of intramitochondrial energy production in heart.

In the heart and other mammalian tissues, there are three exclusively intramitochondrial dehydrogenases that occupy key sites in oxidative metabolism which can be activated by increases in Ca2+ in the range 0.1-10 microM. They are the pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases. Activation of these enzymes can be demonstrated within intact mitochondria (incubated with Na+ and Mg2+) when extramitochondrial Ca2+ is raised within the expected physiological range (0.05-5 microM). Evidence is presented to suggest that the increase in cytoplasmic [Ca2+] caused by positive inotropic agents in the heart result in increases in intramitochondrial [Ca2+] and activation of the dehydrogenases and hence oxidative energy metabolism. Therefore it is proposed that the main function of the mitochondrial Ca2+-transport system is to regulate matrix rather than cytoplasmic Ca2+. In this way the stimulation of energy utilisation as contraction is enhanced is balanced by a parallel stimulation of ATP producing reactions.

Animals↗
PubMed1987

Hormonal regulation of fluxes through pyruvate dehydrogenase and the citric acid cycle in mammalian tissues.

Three key dehydrogenases in the mitochondria of higher animals have been found to be activated by Ca2+ ions; these are pyruvate dehydrogenase and two enzymes in the citric acid cycle, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. Activation can also be demonstrated within permeabilized and intact mitochondria. Evidence is summarized that when hormones and other extracellular stimuli increase the cytoplasmic concentration of Ca2+, then this results in an increase in the intramitochondrial concentration of Ca2+. In this way, rates of pyruvate oxidation and citric acid cycle flux are increased, and hence there is an increase in NADH supply for the respiratory chain under conditions where there is an enhanced demand for ATP. In contrast, the activation of pyruvate dehydrogenase which is observed in adipose and other tissues exposed to insulin is brought about by a Ca2+-independent mechanism.

Animals↗
PubMed1986

Sensitivity of pyruvate dehydrogenase phosphate phosphatase to magnesium ions. Similar effects of spermine and insulin.

The effects of Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within intact mitochondria prepared from control and insulin-treated rat epididymal adipose tissue was explored by incubating the mitochondria in medium containing the ionophore A23187. The apparent Ka for Mg2+ was approximately halved in the mitochondria derived from insulin-treated tissue in both the absence and the presence of Ca2+. In this system, the major effect of Ca2+ was also to decrease the apparent Ka for Mg2+, rather than to change the Vmax. of the phosphatase. Damuni, Humphreys & Reed [(1984) Biochem. Biophys. Res. Commun. 124, 95-99] have reported that spermine activates ox kidney pyruvate dehydrogenase phosphate phosphatase. Studies were carried out on phosphatase from pig heart and rat epididymal adipose tissue which confirm and extend this observation. The major effect of spermine is shown to be a decrease in the Ka for Mg2+, which is apparent in both the presence and the absence of Ca2+. Spermine did not affect the sensitivity of the phosphatase to Ca2+ at saturating concentrations of Mg2+. Other polyamines tested were not as effective as spermine. No alteration in the maximum activity or Mg2+-sensitivity of pyruvate dehydrogenase phosphate phosphatase was apparent in extracts of mitochondria from insulin-treated tissue. The close similarity of the effects of spermine and the changes in kinetic properties of pyruvate dehydrogenase phosphate phosphatase within mitochondria from insulin-treated adipose tissue suggests that insulin may activate pyruvate dehydrogenase by increasing the concentration of spermine within the mitochondria. However, it is concluded that insulin is more likely to alter the interaction of the pyruvate dehydrogenase system with some other polybasic intramitochondrial component whose action can be mimicked by spermine.

Adipose Tissue↗
PubMed1986

Use of toluene-permeabilized mitochondria to study the regulation of adipose tissue pyruvate dehydrogenase in situ. Further evidence that insulin acts through stimulation of pyruvate dehydrogenase phosphate phosphatase.

Rat epididymal-adipose-tissue mitochondria were made selectively permeable to small molecules without the loss of matrix enzymes by treating the mitochondria with toluene under controlled conditions. With this preparation the entire pyruvate dehydrogenase system was shown to be retained within the mitochondrial matrix and to retain its normal catalytic activity. By using dilute suspensions of these permeabilized mitochondria maintained in the cuvette of a spectrophotometer, it was possible to monitor changes of pyruvate dehydrogenase activity continuously while the activities of the interconverting kinase and phosphatase could be independently manipulated. Permeabilized mitochondria were prepared from control and insulin-treated adipose tissue, and the properties of both the pyruvate dehydrogenase kinase and the phosphatase were compared in situ. No difference in kinase activity was detected, but increases in phosphatase activity were observed in permeabilized mitochondria from insulin-treated tissue. Further studies showed that the main effect of insulin treatment was a decrease in the apparent Ka of the phosphatase for Mg2+, in agreement with earlier studies with mitochondria made permeable to Mg2+ by using the ionophore A23187 [Thomas, Diggle & Denton (1986) Biochem. J. 238, 83-91]. No effects of spermine were detected, although spermine diminishes the Ka of purified phosphatase preparations for Mg2+. Since effects of insulin on pyruvate dehydrogenase phosphatase activity are not evident in mitochondrial extracts, it is concluded that insulin may act by altering some high-Mr component which interacts with the pyruvate dehydrogenase system within intact or permeabilized mitochondria, but not when the mitochondrial membranes are disrupted.

Adipose Tissue↗
PubMed1986

Studies on the specific activity of [gamma-32P]ATP in adipose and other tissue preparations incubated with medium containing [32P]phosphate.

The specific activity of the gamma-32P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717-720). The HPLC method also allowed the incorporation of 32P into the (alpha + beta)-positions of ATP to be determined. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [gamma-32P]ATP attained a steady-state value after 1-2 h incubation in medium containing 0.2 mM [32P]phosphate. Addition of insulin or the beta-agonist isoprenaline increased this value by 5-10% within 15 min. Under these conditions the steady-state specific activity of [gamma-32P]ATP was 30-40% of the initial specific activity of the medium [32P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the gamma-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [32P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components.

Adenosine Diphosphate↗
PubMed1986

The calcium sensitive dehydrogenases of vertebrate mitochondria.

Three important dehydrogenases in vertebrate mitochondria are activated by Ca2+ ions with half-maximal effects at about 1 microM. These are pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase. Activation of these enzymes can also be demonstrated within intact mitochondria when extramitochondrial Ca2+ is increased within the range of concentrations generally considered to occur in the cytoplasm of vertebrate cells. It is argued that the main role of the calcium transport system in the inner membrane of vertebrate mitochondria is to relay changes in the cytoplasmic concentration of Ca2+ into the mitochondrial matrix. In this way, hormones and other extracellular stimuli which stimulate ATP-requiring processes such as contraction and secretion through increases in the cytoplasmic concentration of Ca2+ may also increase intramitochondrial oxidative metabolism and hence the replenishment of ATP.

Adenosine Triphosphate↗
PubMed1985

Adipose-tissue phosphofructokinase. Rapid purification and regulation by phosphorylation in vitro.

A new procedure for the purification of phosphofructokinase using Blue Dextran-Sepharose is described. This allowed an approx. 1000-fold purification of phosphofructokinase from rat white and brown adipose tissue to be achieved in essentially a single step. The purified enzymes from both tissues were found to exhibit hyperbolic kinetics with fructose 6-phosphate, to be inhibited by ATP and citrate, and to be activated by 5'-AMP, phosphate and fructose 2,6-bisphosphate. The enzymes were phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, and phosphorylation was found to be associated with increases in activity when the enzymes were assayed under appropriate sub-optimal conditions. In particular, the phosphorylated enzymes exhibited less inhibition by ATP and the white-adipose-tissue enzyme was more sensitive to activation by fructose 2,6-bisphosphate. It is suggested that an increase in the cytoplasmic concentration of cyclic AMP in tissues other than liver may result in an increase in glycolysis through the phosphorylation of phosphofructokinase by cyclic AMP-dependent protein kinase.

Adenosine Triphosphate↗
PubMed1985

Beta-adrenergic agents increase the phosphorylation of phosphofructokinase in isolated rat epididymal white adipose tissue.

Pieces of rat epididymal adipose tissue were incubated in medium containing [32P]phosphate for 2 h to achieve steady-state labelling of intracellular phosphoproteins and then with or without hormones for a further 15 min. Phosphofructokinase was rapidly isolated from the tissue by use of either Blue Dextran-Sepharose chromatography or immunoprecipitation with antisera raised against phosphofructokinase purified from rat interscapular brown adipose tissue. Similar extents of incorporation of 32P into phosphofructokinase were measured by both techniques. Exposure of the tissue to adrenaline or the beta-agonist isoprenaline increased phosphorylation by about 5-fold (to about 1.4 mol of phosphate/mol of enzyme tetramer). No change in phosphorylation was detected with the alpha-agonist phenylephrine, but exposure to insulin resulted in an approx. 2-fold increase. The increased phosphorylation observed with isoprenaline was found to be associated with a decrease in the apparent Ka for fructose 2,6-bisphosphate similar to that observed on phosphorylation of phosphofructokinase purified from rat epididymal white adipose tissue with the catalytic subunit of cyclic AMP-dependent protein kinase. These results support the view [Sale & Denton (1985) Biochem. J. 232, 897-904] that an increase in cyclic AMP in adipose tissue may result in an increase in glycolysis through the phosphorylation of phosphofructokinase by cyclic AMP-dependent protein kinase.

Adipose Tissue↗
PubMed1985

Evidence that noradrenaline increases pyruvate dehydrogenase activity and decreases acetyl-CoA carboxylase activity in rat interscapular brown adipose tissue in vivo.

The rate of fatty acid synthesis in interscapular brown adipose tissue of female cold-adapted rats, as measured by the incorporation of 3H from 3H2O into tissue lipid, was decreased by about 70% after injection of noradrenaline. There was a similar decrease in the activity of acetyl-CoA carboxylase. In contrast, the proportion of pyruvate dehydrogenase in its active non-phosphorylated form was greatly increased after injection of noradrenaline. This finding suggests that the oxidation of glucose may be important in noradrenaline-induced thermogenesis in rat brown adipose tissue.

Acetyl-CoA Carboxylase↗
PubMed1985

Ca2+ transport by mammalian mitochondria and its role in hormone action.

Three key dehydrogenases in mammalian mitochondria have been found to be activated by Ca2+ with a half-maximal effect at approximately 1 microM. These are pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase, and oxoglutarate dehydrogenase. Activation of these enzymes can also be demonstrated in intact coupled mitochondria when extra mitochondrial Ca2+ is increased in the range of concentrations (0.1 to 2 microM) generally considered to occur in the cytoplasm of normal cells. It is argued that the main role of the calcium transport system in mammalian mitochondria is to relay changes in cytoplasmic Ca2+ into the mitochondrial matrix. Hormones and other extracellular messengers which stimulate ATP-requiring processes such as secretion or muscle contraction through increasing the cytoplasmic concentration of Ca2+ could in this way also increase intramitochondrial oxidative metabolism and hence promote the replenishment of ATP. Recent evidence obtained with heart and liver preparations in support of this view is reviewed.

Adenosine Triphosphate↗