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R W Moreadith

Publications and source records attributed to R W Moreadith.

25 records · Page 2Linked to original sources
PubMed1984

Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria.

The purification and kinetic characterization of an NAD(P)+-malic enzyme from 22aH mouse hepatoma mitochondria are described. The enzyme was purified 328-fold with a final yield of 51% and specific activity of 38.1 units/mg of protein by employing DEAE-cellulose chromatography and an ATP affinity column. Sephadex G-200 chromatography yielded a native Mr = 240,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major subunit with Mr = 61,000, suggesting a tetrameric structure, and also showed that the preparation contained less than 10% polypeptide impurities. Use of the ATP affinity column required the presence of MnCl2 and fumarate (an allosteric activator) in the elution buffers. In the absence of fumarate, the Michaelis constants for malate, NAD+, and NADP+ were 3.6 mM, 55 microM, and 72 microM, respectively; in the presence of fumarate (2 mM), the constants were 0.34 mM, 9 microM, and 13 microM, respectively. ATP was shown to be an allosteric inhibitor, competitive with malate. However, the inhibition by ATP displayed hyperbolic competitive kinetics with a KI (ATP) of 80 microM (minus fumarate) and 0.5 mM (plus 2 mM fumarate). The allosteric properties of the enzyme are integrated into a rationale for its specific role in the pathways of malate and glutamate oxidation in tumor mitochondria.

Animals↗
PubMed1984

Isolation of mitochondria from ascites tumor cells permeabilized with digitonin.

A new, improved procedure for isolating mitochondria from ascites tumor cells is described. The unique feature of this technique is the use of digitonin to make the cells susceptible to disruption by Teflon pestle/glass vessel homogenization. The yield and respiratory control ratios of mitochondria isolated by this method from murine Ehrlich ascites tumor cells and rat AS30-D ascites hepatoma cells are significantly better than those obtained for mitochondria isolated by the commonly employed Nagarse method, which involves the use of proteolytic enzymes. Moreover, mitochondria isolated by this new procedure from three different lines of tumors exhibit respiratory control ratios with both adenosine diphosphate and a respiratory uncoupler comparable to those obtained with mitochondria present in situ within digitonin-permeabilized tumor cells.

Animals↗
PubMed1984

Deficiency of the iron-sulfur clusters of mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase (complex I) in an infant with congenital lactic acidosis.

We report the case of an infant with hypoglycemia, progressive lactic acidosis, an increased serum lactate/pyruvate ratio, and elevated plasma alanine, who had a moderate to profound decrease in the ability of mitochondria from four organs to oxidize pyruvate, malate plus glutamate, citrate, and other NAD+-linked respiratory substrates. The capacity to oxidize the flavin adenine dinucleotide-linked substrate, succinate, was normal. The most pronounced deficiency was in skeletal muscle, the least in kidney mitochondria. Enzymatic assays on isolated mitochondria ruled out defects in complexes II, III, and IV of the respiratory chain. Further studies showed that the defect was localized in the inner membrane mitochondrial NADH-ubiquinone oxidoreductase (complex I). When ferricyanide was used as an artificial electron acceptor, complex I activity was normal, indicating that electrons from NADH could reduce the flavin mononucleotide cofactor. However, electron paramagnetic resonance spectroscopy performed on liver submitochondrial particles showed an almost total loss of the iron-sulfur clusters characteristic of complex I, whereas normal signals were noted for other mitochondrial iron-sulfur clusters. This infant is presented as the first reported case of congenital lactic acidosis caused by a deficiency of the iron-sulfur clusters of complex I of the mitochondrial electron transport chain.

Acidosis↗
PubMed1983

Control of heart oxidative phosphorylation by creatine kinase in mitochondrial membranes.

Three important points must be emphasized in summary. First is the idea that a cellular microcompartment need not be limited by a semi-permeable membrane. We recognize microcompartments in multi-enzyme complexes where substrates are covalently transported from subunit to subunit. An example of this is the lipoic acid moiety of the pyruvate dehydrogenase complex. However, to act as a kinetic microcompartment, covalent transfer is not an obligatory requirement. Proximity effects may be sufficient for substantial rate enhancement. Our data clearly show that the kinetics of ADP translocation are influenced by the site of ADP formation. We contend that this represents a newly recognized and important form of cellular microcompartmentation. The second point is that we do not want our results misinterpreted as an overextension of the known data concerning tissue respiration. We believe that the primary parameter controlling heart mitochondrial oxygen consumption is the availability of ADP at the adenine nucleotide translocase. Our data show, however, that this is not a simple process. Secondary control is exerted by the localization of ADP formation, i.e. microcompartmentation. As a result of the kinetic data (Table 3), we conclude that the forward rate of mitochondrial creatine kinase is the preferential reaction controlling ADP delivery to the translocase. We are left, nonetheless, with questions concerning the secondary regulation of this enzyme in vivo by substrate (ATP and creatine) and inhibition by product (phosphocreatine). The nature of this control awaits further experimental data. Finally, the results are consistent with the creatine kinase energy transport hypothesis. Overall, the rate of tissue oxygen consumption reflects the metabolic activity of the organ, determined by the rate of ATP utilization (see right side of Figure 1). This results in the cytoplasmic production of ADP. In heart, this is coupled via the bound cytoplasmic isozymes of creatine kinase to the local rephosphorylation of ADP to ATP and the simultaneous production of creatine.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1982

Mitochondrial respiratory control. Evidence against the regulation of respiration by extramitochondrial phosphorylation potentials or by [ATP]/[ADP] ratios.

To explore how mitochondria can respire at high physiological, extramitochondrial phosphorylation potentials, two series of experiments were conducted. In the first, intact rat liver mitochondria were incubated in oxygraph medium containing 5 mM succinate (+rotenone), 1.0 mM ATP, 20 mM glucose, pH 7.2, at 37 degrees C. Yeast hexokinase (0.02 to 1.0 IU) was added to establish steady state rates of respiration. Samples were removed, assayed for ATP, ADP, and Pi content, and ratios were calculated. As previously reported, low rates of respiration were observed at high phosphorylation potential ([ATP]/[ADP] x [Pi]) or [ATP]/[ADP] ratio values, and the rates of respiration increased as these values declined. In a second series of experiments, only sufficient hexokinase was added to potentially stimulate respiration to 90% of the ADP State 3 rate. At constant hexokinase, 0.35 IU, ATP (5 microM to 10.0 mM) was titrated into the medium to establish steady state rates of oxygen consumption. Under these conditions, low rates of respiration correlated with low [ATP]/[ADP] ratios and extramitochondrial phosphorylation potentials, while maximum rates of respiration were observed at high values of these ratios, the opposite of the previous experimental case. Therefore, it may be concluded that these extramitochondrial parameters per se exert little or no regulatory influence on the rates of respiration, and thus matrix ATP synthesis. In both cases, the concentrations of ADP correlated with respiratory rates. Double reciprocal plots were used to estimate the apparent KmADP for respiratory stimulation. The values are 56 microM for constant [ATP] and 15 microM at constant hexokinase. The value calculated from direct ADP pulses was 25 microM. Together, these results suggest that the most plausible explanation of respiratory control is the availability of ADP and the kinetics of its transport by the adenine nucleotide translocase, a hypothesis first proposed by Chance and Williams more than 25 years ago (Chance, B., and Williams, G. R. (1955) J. Biol. Chem. 217, 385-393).

Adenosine Diphosphate↗