scholarly journals Equilibrium relations between the cytoplasmic adenine nucleotide system and nicotinamide–adenine nucleotide system in rat liver

1970 ◽  
Vol 117 (3) ◽  
pp. 499-503 ◽  
Author(s):  
R. L. Veech ◽  
L. Raijman ◽  
H. A. Krebs
Keyword(s):  
Lactate Dehydrogenase ◽  
Rat Liver ◽  
Redox State ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Phosphoglycerate Kinase ◽  
Direct Determination ◽  
Phosphorylation State ◽  
Nadh Ratio

1. The ratio [ATP]/[ADP][Pi], as measured by direct determination of the three components in rat liver, was found in various nutritional states to have approximately the same value as the ratio [ATP]/[ADP][Pi] calculated from the concentrations of lactate, pyruvate, glyceraldehyde phosphate and 3-phosphoglycerate on the assumption that lactate dehydrogenase, glyceraldehyde phosphate dehydrogenase and 3-phosphoglycerate kinase are at near-equilibrium in the liver. This implies that the redox state of the NAD couple in the cytoplasm is linked to, and partially controlled by, the phosphorylation state of the adenine nucleotides. 2. The combined equilibrium constant of the glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase reactions at 38°C and I0.25, was found to be 5.9×10−6. 3. The fall of the [NAD+]/[NADH] ratio in starvation and other situations is taken to be the consequence of a primary fall of the [ATP]/[ADP][HPO42−] ratio.

scholarly journals Control of the redox state of the nicotinamide–adenine dinucleotide couple in rat liver cytoplasm

1972 ◽  
Vol 126 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Marion Stubbs ◽  
R. L. Veech ◽  
H. A. Krebs
Keyword(s):  
Rat Liver ◽  
Redox State ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Phosphoglycerate Kinase ◽  
Phosphorylation State ◽  
Crotyl Alcohol ◽  
Order Of Magnitude ◽  
The Individual

1. A study has been made of the ability of rat liver in vivo to maintain equilibrium in the combined glyceraldehyde 3-phosphate dehydrogenase, 3-phosphoglycerate kinase and lactate dehydrogenase reactions, i.e. in the system: [Formula: see text] Attempts were made to upset equilibrium. The [lactate]/[pyruvate] ratio was rapidly changed by injection of ethanol or crotyl alcohol, and the value of [ATP]/[ADP][HPO42-] was rapidly changed by injection of ethionine or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone. 2. The concentrations of the metabolites occurring in the above equation were measured in freeze-clamped liver. 3. Although the injected agents caused large changes in the concentrations of the individual components, near-equilibrium in the system was maintained, as indicated by the fact that the value of [ATP]/[ADP][HPO42-], referred to as the phosphorylation state of the adenine nucleotides, measured directly agreed with the value calculated for equilibrium conditions from the above equation. 4. The results are discussed and taken to confirm that the order of magnitude of the value of the redox state of the cytoplasmic NAD couple in rat liver is controlled by the phosphorylation state of the adenine nucleotide system.

The Effect of Chronic Consumption of Ethanol on the Redox State of the Rat Liver

1972 ◽  
Vol 50 (9) ◽  
pp. 949-957 ◽  
Author(s):  
Ellen R. Gordon
Keyword(s):  
Lactate Dehydrogenase ◽  
Redox Potential ◽  
Rat Liver ◽  
Redox State ◽  
Fatty Infiltration ◽  
Caloric Intake ◽  
Treated Group ◽  
Control Group ◽  
Control Groups ◽  
Nadh Ratio

Isocaloric replacement of the carbohydrate content of the diet by ethanol (36% of the total caloric intake) produced fatty infiltration and a reduced redox potential of the liver. The hepatic ratio of NAD+/NADH was 1.2 in the ethanol-treated group compared to 8.6 in the pair-fed control group. This change was associated with the consumption of ethanol as, 24 h after the removal of ethanol from the liver, the ratio had returned to the level of the control group. The hepatic substrate levels of NAD+-linked lactate dehydrogenase and the NAD+-linked β-hydroxybutyrate dehydrogenase systems were measured and used to calculate the NAD+/NADH ratio of the cytoplasm and mitochondria, respectively. The chronic consumption of ethanol did not alter the ratio in the cytoplasm but did shift the ratio in the mitochondria to one-fourth that observed in the control group. Twenty-four hours after ethanol was removed from the diet, this reduced redox potential in the mitochondria shifted to a level higher than that observed in the control groups.

scholarly journals The effect of butacaine on adenine nucleotide binding and translocation in rat liver mitochondria

1975 ◽  
Vol 148 (3) ◽  
pp. 527-531 ◽  
Author(s):  
D R Fayle ◽  
G J Barritt ◽  
F L Bygrave
Keyword(s):  
Rat Liver ◽  
Local Anaesthetic ◽  
Binding Sites ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Nucleotide Binding ◽  
Local Anaesthetics ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Inner Membrane

The effect of the local anaesthetic, butacaine, on adenine nucleotide binding and translocation in rat liver mitochondria partially depleted of their adenine nucleotide content was investigated. The range of butacaine concentrations that inhibit adenine nucleotide translocation and the extent of the inhibition are similar to the values obtained for native mitochondria. Butacaine does not alter either the total number of atractyloside-sensitive binding sites of depleted mitochondria, or the affinity of these sites for ADP or ATP under conditions where a partial inhibition of the rate of adenine nucleotide translocation is observed. The data are consistent with an effect of butacaine on the process by which adenine nucleotides are transported across the mitochondrial inner membrane rather than on the binding of adenine nucleotides to sites on the adenine nucleotide carrier. The results are briefly discussed in relation to the use of local anaesthetics in investigations of the mechanism of adenine nucleotide translocation.

scholarly journals A quantitative study of the biospecific desorption of rat liver (M4) lactate dehydrogenase from 10-carboxydecylamino-Sepharose. Determination of the number of ligand-binding sites blocked on adsorption

1982 ◽  
Vol 207 (3) ◽  
pp. 549-556 ◽  
Author(s):  
P Kyprianou ◽  
R J Yon
Keyword(s):  
Lactate Dehydrogenase ◽  
Ligand Binding ◽  
Rat Liver ◽  
Binding Sites ◽  
Free Ligand ◽  
Factors Affecting ◽  
Adsorption Sites ◽  
Ligand Binding Sites ◽  
Chromatographic Parameters

1. The theory of Nichol, Ogston, Winzor & Sawyer [(1974) Biochem. J. 143, 435-443] for quantitative affinity chromatography, when adapted for use with a non-specific column from which a multi-site protein can be specifically desorbed by its free ligand, permits determination of the concentration of adsorption sites on the column, their adsorptive affinity (as an association constant) and either the intrinsic (site) constant for ligand-binding to the protein or an ‘occlusion coefficient’ (defined as the number of ligand-binding sites blocked on adsorption), one of which must be known. 2. The theory has been applied to the NADH-specific desorption of rat liver M4 lactate dehydrogenase from 10-carboxydecylamino-Sepharose. It suggests that most of the enzyme molecules are adsorbed with at least two NADH-binding sites blocked, indicating an extensive adsorption interface in relation to the protein surface. Other chromatographic parameters were also determined for the system. 3. Among topics discussed are (a) factors affecting the experimentally determined value for the number of blocked sites, (b) the nature of the adsorption sites on the column and (c) the similarity of the analysis to that for determining Hill coefficients, and other possible applications.

scholarly journals Stable enhancement of calcium retention in mitochondria isolated from rat liver after the administration of glucagon to the intact animal

1978 ◽  
Vol 176 (3) ◽  
pp. 705-714 ◽  
Author(s):  
Veronica Prpić ◽  
Terry L. Spencer ◽  
Fyfe L. Bygrave
Keyword(s):  
Rat Liver ◽  
Molecular Mechanisms ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Mitochondrial Protein ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Matrix Space ◽  
The Matrix

1. Mitochondria isolated from rat liver by centrifugation of the homogenate in buffered iso-osmotic sucrose at between 4000 and 8000g-min, 1h after the administration in vivo of 30μg of glucagon/100g body wt., retain Ca2+ for over 45min after its addition at 100nmol/mg of mitochondrial protein in the presence of 2mm-Pi. In similar experiments, but after the administration of saline (0.9% NaCl) in place of glucagon, Ca2+ is retained for 6–8min. The ability of glucagon to enhance Ca2+ retention is completely prevented by co-administration of 4.2mg of puromycin/100g body wt. 2. The resting rate of respiration after Ca2+ accumulation by mitochondria from glucagon-treated rats remains low by contrast with that from saline-treated rats. Respiration in the latter mitochondria increased markedly after the Ca2+ accumulation, reflecting the uncoupling action of the ion. 3. Concomitant with the enhanced retention of Ca2+ and low rates of resting respiration by mitochondria from glucagon-treated rats was an increased ability to retain endogenous adenine nucleotides. 4. An investigation of properties of mitochondria known to influence Ca2+ transport revealed a significantly higher concentration of adenine nucleotides but not of Pi in those from glucagon-treated rats. The membrane potential remained unchanged, but the transmembrane pH gradient increased by approx. 10mV, indicating increased alkalinity of the matrix space. 5. Depletion of endogenous adenine nucleotides by Pi treatment in mitochondria from both glucagon-treated and saline-treated rats led to a marked diminution in ability to retain Ca2+. The activity of the adenine nucleotide translocase was unaffected by glucagon treatment of rats in vivo. 6. Although the data are consistent with the argument that the Ca2+-translocation cycle in rat liver mitochondria is a target for glucagon action in vivo, they do not permit conclusions to be drawn about the molecular mechanisms involved in the glucagon-induced alteration to this cycle.

scholarly journals Dual-digitonin-pulse perfusion. Concurrent sampling of periportal and perivenous cytosol of rat liver for determination of metabolites and enzyme activities

1987 ◽  
Vol 243 (1) ◽  
pp. 87-95 ◽  
Author(s):  
B Quistorff ◽  
N Grunnet
Keyword(s):  
Lactate Dehydrogenase ◽  
Glutamine Synthetase ◽  
Rat Liver ◽  
High Purity ◽  
Alanine Aminotransferase ◽  
Specific Activity ◽  
Perfusion Technique ◽  
Short Intervals ◽  
Specific Activities

A previously described digitonin-perfusion technique [Quistorff, Grunnet & Cornell (1985) Biochem. J. 226, 289-297], by which intracellular material of rat liver could be liberated, has been refined, now allowing release of cytosol of high purity from both periportal and perivenous parts of the same liver. The cytosolic fractions are obtained by perfusing the liver for short intervals (10-20 s) with digitonin (4-5 mg/ml), first in the normal perfusion direction and then, after an interval of 1-2 min, in the retrograde direction, the eluate being collected during and after both intervals. The technique is termed ‘dual-digitonin-pulse perfusion’. The eluate fractions showed a peak specific activity of the cytosolic enzymes alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and pyruvate kinase (PK) of 3-5-fold higher than obtained in a biopsy from the same liver. For glutamine synthetase (GS) a 10-fold higher specific activity was obtained. Zonation, defined as the ratio of the specific activities in periportal and perivenous eluates, of ALAT, LDH and PK was 10, 1.7 and 0.70 respectively. Zonation of GS was less than 0.01. These factors may be modified by a slight zonation of cytosolic protein of 1.2-1.3. Peak concentrations in the eluate of ATP, ADP, Pi, NAD+ and glycerol 3-phosphate were 32.5 +/- 11.4, 19.9 +/- 4.3, 71.9 +/- 25.4, 2.41 +/- 0.83 and 6.84 +/- 2.74 nmol/mg of protein for periportal eluates. There was no difference between periportal and perivenous eluates except for glycerol 3-phosphate, which was significantly higher in perivenous eluates, 12.8 +/- 4.5 nmol/mg of protein.

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