scholarly journals Sub-mitochondrial location of Ruthenium Red-sensitive calcium-ion transport and evidence for its enrichment in a specific population of rat liver mitochondria

1978 ◽  
Vol 174 (3) ◽  
pp. 1011-1019 ◽  
Author(s):  
Fyfe L. Bygrave ◽  
Thomas P. Heaney ◽  
Chidambaram Ramachandran
Keyword(s):  
Rat Liver ◽  
Liver Homogenate ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Protonmotive Force ◽  
Glycerophosphate Dehydrogenase ◽  
Biogenesis Of Mitochondria ◽  
The Individual ◽  
Calcium Ion Transport

1. Seven fractions sedimenting at between 3000 and 120000g-min were prepared from a rat liver homogenate by differential centrifugation in buffered iso-osmotic sucrose. The following measurements were carried out on each of these fractions: Ruthenium Red-sensitive Ca2+ transport in the absence and in the presence of Pi as well as in the presence of N-ethylmaleimide to prevent Pi cycling, succinate-supported respiration in the absence and in the presence of ADP, the ΔE and −59 ΔpH components of the protonmotive force, cytochrome oxidase, uncoupler-stimulated adenosine triphosphatase, α-glycerophosphate dehydrogenase, Pi content and the effect on the ‘resting’ rate of respiration of repeated additions of a fixed Ca2+ concentration. 2. Ca2+ transport either in the presence or in the absence of added Pi and in the presence of N-ethylmaleimide exhibits significantly higher rates in the fraction sedimenting at 8000g-min. By contrast, respiration in the presence or in the absence of added ADP and the values for ΔE and −59 ΔpH were similar in those fractions sedimenting between 4000 and 20000g-min, indicating that the driving force for Ca2+ transport was similar in each of these fractions. 3. Experiments designed to determine the capacity of the individual fractions for Ca2+, as measured by the effect of repeated additions of Ca2+ on the resting rate of respiration, showed that fraction 2, i.e. that sedimenting at 8000g-min, also exhibited the greatest tolerance towards the uncoupling action of the ion. 4. Of the three enzyme activity profiles, only that of α-glycerophosphate dehydrogenase was similar to that of Ca2+ transport. Because previous workers have assigned this enzyme to loci in the inner peripheral membrane [Werner & Neupert (1972) Eur. J. Biochem.25, 379–396], it is concluded that the Ruthenium Red-sensitive Ca2+- transport system also is located in this domain of the inner membrane. The relation of these findings to the mechanisms of mitochondrial Ca2+ transport and the biogenesis of mitochondria is discussed.

scholarly journals Maturation in liver mitochondria of Ruthenium Red-sensitive calcium-ion-transport activity and the influence of glucagon administration in vivo and in utero

1981 ◽  
Vol 196 (1) ◽  
pp. 207-216
Author(s):  
Veronica Prpić ◽  
Fyfe L. Bygrave
Keyword(s):  
Rat Liver ◽  
Post Partum ◽  
Liver Mitochondria ◽  
In Utero ◽  
Ruthenium Red ◽  
Protonmotive Force ◽  
Period 2 ◽  
Calcium Ion Transport ◽  
Energy Dependent

The maturation of Ca2+ transport in mitochondria isolated from rat liver was examined, from 5 days before birth. The mitochondria used were isolated from liver homogenates by centrifugation at 22000g-min. Ca2+ transport by mitochondria isolated from foetal liver is energy-dependent and Ruthenium Red-sensitive. The transmembrane pH gradient in these mitochondria is higher by about 7mV and the membrane potential lower by about 20mV than in adult mitochondria. The inclusion of 2mm-Pi in the incubation medium enhances the protonmotive force by approx. 30mV. The rate of Ca2+ influx in foetal mitochondria measured in buffered KCl plus succinate is low until about 2–3h after birth, when it increases to about 60% of adult values; approx. 24h later it has reached near-adult values. Higher rates of Ca2+ influx are observed in the presence of 2mm-Pi; 3–5 days before birth the rates are about one-third of adult values and decline slightly as birth approaches. By 2–3h post partum they have reached adult values. The inclusion of 12.5μm-MgATP with the Pi stimulates further the initial rate of Ca2+ influx in foetal mitochondria. The rates observed are constant over the prenatal period examined and are 50–60% of those observed in adult mitochondria. Mitochondria isolated from foetal livers 4–5 days before birth retain the accumulated Ca2+ for about 50min in the presence of 2mm-Pi. In the period 2 days before birth to birth, this ability is largely lost, but by 2–3h after birth Ca2+ retention is similar to that of adult mitochondria. The presence of 12.5μm-MgATP progressively enhances the Ca2+ retention time as development proceeds until 2–3h after birth, when it becomes less sensitive to added MgATP. Glucagon administration to older foetuses in utero enhances both the rate of mitochondrial Ca2+ influx assayed in the presence of 2mm-Pi and the time for which mitochondria retain accumulated Ca2+ in the presence of 12.5μm-MgATP and 2mm-Pi. Its administration to neonatal animals leads to an increase in mitochondrial Ca2+ retention similar to that seen in adult mitochondria. The data provide evidence that the Ruthenium Red-sensitive Ca2+ transporter is potentially as active in foetal mitochondria 5 days before birth as it is in adult mitochondria. They also show that foetal mitochondria have an ability to retain accumulated Ca2+ reminiscent of mitochondria from tumour cells and from hormone-challenged rat liver.

Effects of 5-5'-Diphenyl-2-thiohydantoin (DPTH) and Thyroxine on the Ultrastructure and Chemical Composition of Rat Liver

1971 ◽  
Vol 29 ◽  
pp. 570-571
Author(s):  
E. A. Elfont ◽  
R. B. Tobin ◽  
D. G. Colton ◽  
M. A. Mehlman
Keyword(s):  
Chemical Composition ◽  
Rat Liver ◽  
Future Study ◽  
Mode Of Action ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Effective Inhibitor ◽  
Biochemical Effects ◽  
Glycerophosphate Dehydrogenase ◽  
Stimulation Of

Summary5,-5'-diphenyl-2-thiohydantoin (DPTH) is an effective inhibitor of thyroxine (T4) stimulation of α-glycerophosphate dehydrogenase in rat liver mitochondria. Because this finding indicated a possible tool for future study of the mode of action of thyroxine, the ultrastructural and biochemical effects of DPTH and/or thyroxine on rat liver mere investigated.Rats were fed either standard or DPTH (0.06%) diet for 30 days before T4 (250 ug/kg/day) was injected. Injection of T4 occurred daily for 10 days prior to sacrifice. After removal of the liver and kidneys, part of the tissue was frozen at -50°C for later biocheailcal analyses, while the rest was prefixed in buffered 3.5X glutaraldehyde (390 mOs) and post-fixed in buffered 1Z OsO4 (376 mOs). Tissues were embedded in Araldlte 502 and the sections examined in a Zeiss EM 9S.Hepatocytes from hyperthyroid rats (Fig. 2) demonstrated enlarged and more numerous mitochondria than those of controls (Fig. 1). Glycogen was almost totally absent from the cytoplasm of the T4-treated rats.

Synthesis of Adenosine Triphosphate by a Protonmotive Force in Rat Liver Mitochondria

Nature ◽  
1966 ◽  
Vol 212 (5059) ◽  
pp. 257-258 ◽  
Author(s):  
R. A. REID ◽  
JENNIFER MOYLE ◽  
PETER MITCHELL
Keyword(s):  
Adenosine Triphosphate ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Protonmotive Force

scholarly journals Effect of micromolar concentrations of manganese ions on calcium-ion cycling in rat liver mitochondria

1983 ◽  
Vol 212 (3) ◽  
pp. 773-782 ◽  
Author(s):  
B P Hughes ◽  
J H Exton
Keyword(s):  
Rat Liver ◽  
Calcium Ion ◽  
Incubation Medium ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Manganese Ions ◽  
Dose Dependent Decrease ◽  
Incubation Temperatures ◽  
Dose Dependent

The effects of micromolar concentrations of Mn2+ on the rat liver mitochondrial Ca2+ cycle were investigated. It was found that the addition of Mn2+ to mitochondria which were cycling 45Ca2+ led to a rapid dose dependent decrease in the concentration of extramitochondrial 45Ca2+ of about 1 nmol/mg of protein. The effect was complete within 30 s, was half maximal with 10 microM Mn2+ and was observed in the presence of 3 mM Mg2+ and 1 mM ATP. It occurred over a broad range of incubation temperatures, pH and mitochondrial Ca2+ loads. It was not observed when either Mg2+ or phosphate was absent from the incubation medium, or in the presence of Ruthenium Red. These findings indicate that micromolar concentrations of Mn2+ stimulate the uptake of Ca2+ by rat liver mitochondria, and provide evidence for an interaction between Mg2+ and Mn2+ in the control of mitochondrial Ca2+ cycling.

Pathway for uncoupler-induced calcium efflux in rat liver mitochondria: inhibition by Ruthenium Red

Biochemistry ◽  
1984 ◽  
Vol 23 (8) ◽  
pp. 1645-1651 ◽  
Author(s):  
Paolo Bernardi ◽  
Venturina Paradisi ◽  
Tullio Pozzan ◽  
Giovanni Felice Azzone
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Calcium Efflux

scholarly journals The synthesis of hippurate from benzoate and glycine by rat liver mitochondria. Submitochondrial localization and kinetics

1977 ◽  
Vol 166 (1) ◽  
pp. 39-47 ◽  
Author(s):  
S J Gatley ◽  
H S A Sherratt
Keyword(s):  
Rat Liver ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
O2 Consumption ◽  
Mitochondrial Content ◽  
Presence And Absence ◽  
The Individual ◽  
Good Agreement

1. Rat liver mitochondria make hippurate at up to 4 nmol/min per mg of protein. The rate of synthesis supported by oxidation of glutamate with exogenous Pi present is identical with that supported by ATP plus oligomycin. Lower rates were obtained with other respiratory substrates, and when glutamate was used without Pi. 2. A matrix localization for hippurate synthesis is indicated by the latency of benzoyl-CoA synthetase and glycine N-acyltransferase to their extramitochondrial substrates, failure of exogenous benzoyl-CoA to inhibit incorporation of [14C]hippurate and inhibition of hippurate synthesis supported by ATP, but not glutamate, by carboxyatractyloside. 3. The relative activities of the individual enzymes and the mitochondrial content of benzoyl-CoA in the presence and absence of glycine suggest that hippurate synthesis is rate-limited by formation of benzoyl-CoA. 4. The increases in rates of ATP hydrolysis and of O2 consumption on the addition of benzoate and glycine were in good agreement with those required to support hippurate synthesis. The increase in respiration indicates that State-4 respiration [Chance & Williams (1957) Adv. Enzymol 17, 65-134] is not used, with these conditions, for ATP synthesis.

scholarly journals Stimulation of the respiration rate of rat liver mitochondria by sub-micromolar concentrations of extramitochondrial Ca2+

1987 ◽  
Vol 245 (1) ◽  
pp. 217-222 ◽  
Author(s):  
J D Johnston ◽  
M D Brand
Keyword(s):  
Respiration Rate ◽  
Rat Liver ◽  
Oxidative Metabolism ◽  
Mitochondrial Respiration ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Stimulation Of

1. The respiration rate of rat liver mitochondria was stimulated by up to 70% when the extramitochondrial Ca2+ concentration was raised from 103 to 820 nM. This occurred when pyruvate, 2-oxoglutarate, or threo-(Ds)-isocitrate was employed as substrate, but not when succinate was used. 2. Ruthenium Red prevented the stimulation of mitochondrial respiration by extramitochondrial Ca2+, showing that the effect required Ca2+ uptake into the mitochondrial matrix. 3. Starvation of rats for 48 h abolished the stimulation of mitochondrial respiration by extramitochondrial Ca2+ when pyruvate was used as substrate, but did not affect the stimulation of 2-oxoglutarate oxidation by extramitochondrial Ca2+. 4. Our findings are in accord with proposals that oxidative metabolism in liver mitochondria may be stimulated by Ca2+ activation of intramitochondrial dehydrogenases.

scholarly journals Biochemical heterogeneity of rat liver mitochondria separated by rate zonal centrifugation

1972 ◽  
Vol 129 (1) ◽  
pp. 209-218 ◽  
Author(s):  
M. A. Wilson ◽  
J. Cascarano
Keyword(s):  
Cytochrome C ◽  
Succinate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Cytochrome B ◽  
Rat Liver ◽  
Nadh Dehydrogenase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Osmotic Gradient ◽  
Glycerophosphate Dehydrogenase

1. Rat liver mitochondria were separated on the basis of their sedimentation coefficients in an iso-osmotic gradient of Ficoll–sucrose by rate zonal centrifugation. The fractions (33, each of 40ml) were collected in order of decreasing density. Fractions were analysed by spectral analysis to determine any differences in the concentrations of the cytochromes and by enzyme analyses to ascertain any differences in the activities of NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. 2. When plotted as% of the highest specific concentration, the contents of cytochrome a+a3 and cytochrome c+c1 were constant in all fractions but cytochrome b was only 65% of its maximal concentration in fraction 7 and increased with subsequent fractions. As a result, the cytochrome b/cytochrome a+a3 ratio almost doubled between fractions 7 and 25 whereas the cytochrome c+c1/cytochrome a+a3 ratio was unchanged. 3. Expression of the dehydrogenase activities as% of highest specific activity showed the following for fractions 6–26: NADH dehydrogenase activity remained fairly constant in all fractions; succinate dehydrogenase activity was 62% in fraction 6 and increased steadily to its maximum in fraction 18 and then decreased; the activity of α-glycerophosphate dehydrogenase was only 53% in fraction 6 and increased slowly to its peak in fractions 22 and 24. 4. These differences did not result from damaged or fragmented mitochondria or from microsomal contamination. 5. These results demonstrate that isolated liver mitochondria are biochemically heterogeneous. The importance of using a system for separating biochemically different mitochondria in studies of mitochondrial biogenesis is discussed.

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