scholarly journals The effects of Mg2+ and adenine nucleotides on the sensitivity of the heart mitochondrial Na+-Ca2+ carrier to extramitochondrial Ca2+. A study using arsenazo III-loaded mitochondria

1987 ◽  
Vol 244 (3) ◽  
pp. 533-538 ◽  
Author(s):  
L H Hayat ◽  
M Crompton
Keyword(s):  
Adenine Nucleotides ◽  
Mitochondrial Protein ◽  
Arsenazo Iii ◽  
Heart Mitochondria ◽  
Small Decrease ◽  
Regulatory Site ◽  
Normal Heart ◽  
Partial Inhibition ◽  
Regulatory Sites

The technique of reversible Ca2+-induced permeabilization [Al Nasser & Crompton (1986) Biochem. J. 239, 19-29, 31-40] has been applied to the preparation of heart mitochondria loaded with the Ca2+ indicator arsenazo III (2 nmol of arsenazo III/mg of mitochondrial protein). The loaded mitochondria (‘mitosomes’) were used to study the control of the Na+-Ca2+ carrier by extramitochondrial Ca2+ mediated by putative regulatory sites. The Vmax. of the Na+-Ca2+ carrier and the degree of regulatory-site-mediated inhibition were similar to normal heart mitochondria. Ca2+ occupation of the sites in mitosomes yields partial inhibition, which is half-maximal with 0.8 microM external free Ca2+. The inhibition consists of a small decrease in Vmax. and a relatively large increase in apparent Km for internal Ca2+. Mg2+ also appears to interact with the sites, but this is largely abolished by ATP and ADP (but not AMP) under conditions in which the free [Mg2+] is maintained constant. The results indicate that the regulatory sites are effective in controlling the Na+-Ca2+ carrier at physiological concentrations of adenine nucleotides, Mg2+, intra- and extra-mitochondrial free Ca2+.

scholarly journals Identification of the protein responsible for pyruvate transport into rat liver and heart mitochondria by specific labelling with [3H]N-phenylmaleimide

1981 ◽  
Vol 196 (2) ◽  
pp. 471-479 ◽  
Author(s):  
A P Thomas ◽  
A P Halestrap
Keyword(s):  
Membrane Protein ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Liver Mitochondria ◽  
Heart Mitochondria ◽  
Approximate Amount ◽  
Specific Labelling

1. N-Phenylmaleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvate. alpha-Cyanocinnamate protects the pyruvate transporter from attack by this thiol-blocking reagent. 2. In both heart and liver mitochondria alpha-cyanocinnamate diminishes labelling by [3H]N-phenylmaleimide of a membrane protein of subunit mol.wt. 15000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. Exposure of mitochondrial to unlabelled N-phenylmaleimide in the presence of alpha-cyanocinnamate, followed by removal of alpha-cyanocinnamate and exposure to [3H]N-phenylmaleimide, produced specific labelling of the same protein. 4. Both labelling and kinetic experiments with inhibitors gave values for the approximate amount of carrier present in liver and heart mitochondria of 100 and 450 pmol/mg of mitochondrial protein respectively. 5. The turnover numbers for net pyruvate transport and pyruvate exchange at 0 degrees C were 6 and 200 min-1 respectively.

scholarly journals Proton translocation coupled to quinol oxidation in ox heart mitochondria

1973 ◽  
Vol 136 (3) ◽  
pp. 711-720 ◽  
Author(s):  
Hugh G. Lawford ◽  
Peter B. Garland
Keyword(s):  
Proton Translocation ◽  
Mitochondrial Protein ◽  
Glass Electrode ◽  
Measuring System ◽  
Heart Mitochondria ◽  
Analogue Computer ◽  
Back Flow ◽  
Ubiquinol Oxidase ◽  
True Value ◽  
Proton Generation

The suitability of ubiquinol1 and duroquinol as pulse reductants for initiating respirationdriven proton translocation by aerobic ox heart mitochondria was investigated. At 25°C the Vmax. for oxidation was close to 280nmol of quinol oxidized/min per mg of protein, and the Km values were 8μm for ubiquinol1 and 28μm for duroquinol. Pulses of ubiquinol1 and duroquinol were rapidly and completely oxidized by aerobic mitochondria with a simultaneous acidification of the suspending medium as detected with a glass electrode. The →H+/2e−ratios (Mitchell, 1966) calculated from the observed extent of acidification and the amount of quinol added were 3.62 for ubiquinol1 and 2.98 for duroquinol. These values are underestimates of the true value owing to proton back-flow across the membrane. An analogue computer model was used to correct the observed extent of respirationdriven acidification for proton back-flow. The corrected →H+/2e−values were 4.01 for ubiquinol and 3.86 for duroquinol oxidation. Attempts to measure the rate of proton translocation with a pH-measuring system with a response time of 0.4s were not entirely satisfactory, owing to the relative slowness of the electrode response. Nevertheless the maximal rate of proton generation during ubiquinol1 oxidation was about 1200ng-ions of H+/min per mg of mitochondrial protein. It is concluded, contrarily to Chance & Mela (1967), that mitochondria exhibit a proton-translocating ubiquinol oxidase activity with a →H+/2e−ratio of 4.0.

scholarly journals A heart mitochondrial Ca2+-dependent pore of possible relevance to re-perfusion-induced injury. Evidence that ADP facilitates pore interconversion between the closed and open states

1990 ◽  
Vol 266 (1) ◽  
pp. 33-39 ◽  
Author(s):  
M Crompton ◽  
A Costi
Keyword(s):  
Arsenazo Iii ◽  
Heart Mitochondria ◽  
Ionophore A23187 ◽  
Relative Permeabilities ◽  
Pulsed Flow ◽  
Pore Closure ◽  
Open States ◽  
Matrix Free ◽  
Pore Number

The permeability properties of a putative Ca2(+)-activated pore in heart mitochondria, of possible relevance to re-perfusion-induced injury, have been investigated by a pulsed-flow solute-entrapment technique. The relative permeabilities of [14C]mannitol, [14C]sucrose and arsenazo III are consistent with permeation via a pore of about 2.3 nm diameter. Ca2+ removal with EGTA induced pore closure, and the mitochondria became ‘resealed’. The permeability of the unresealed mitochondria during resealing was markedly stimulated by 200 microM-ADP, and the relative permeabilities to solutes of different size were stimulated equally, indicating an increase in open-pore number, rather than an increase in pore dimensions. This is paradoxical, since ADP also stimulated the rate of resealing. The rate of EGTA-induced resealing was also stimulated by the Ca2+ ionophore A23187, which indicates that the rate of removal of matrix free Ca2+ is limiting for pore closure. An explanation for the paradox is suggested in which ADP facilitates pore interconversion between the closed and open states in permeabilized mitochondria, and pore closure in Ca2(+)-free mitochondria occurs much faster than previously thought.

scholarly journals Partial inhibition by cyclosporin A of the swelling of liver mitochondria in vivo and in vitro induced by sub-micromolar [Ca2+], but not by butyrate. Evidence for two distinct swelling mechanisms

1990 ◽  
Vol 268 (1) ◽  
pp. 147-152 ◽  
Author(s):  
A M Davidson ◽  
A P Halestrap
Keyword(s):  
Light Scattering ◽  
Cyclosporin A ◽  
Binding Sites ◽  
Mitochondrial Protein ◽  
Liver Mitochondria ◽  
Partial Inhibition ◽  
Mitochondrial Volume ◽  
Bongkrekic Acid

1. The effects of cyclosporin A on the increase in matrix PPi and consequent swelling of energized liver mitochondria incubated with 1 mM-butyrate, 30 microM-bongkrekic acid or 0.1-35 microM-Ca2+ [Halestrap (1989) Biochim. Biophys. Acta 973, 355-382] were studied. 2. Cyclosporin (1 microM) had no significant effect on the swelling induced by butyrate, bongkrekic acid or Ca2+ at concentrations of less than 0.3 microM. 3. At higher [Ca2+] (greater than 0.3 microM), swelling became progressively inhibited by cyclosporin, although the increase in matrix PPi was slightly greater in the presence than in the absence of cyclosporin. 4. Titration with cyclosporin indicated that there are 128 pmol of relevant cyclosporin-binding sites per mg of mitochondrial protein, with a Ki of about 5 nM. 5. The decrease in light-scattering by hepatocytes induced by butyrate [Davidson & Halestrap (1988) Biochem. J. 254, 379-384] was unaffected by cyclosporin, whereas that induced by vasopressin was inhibited by 20-30% without a significant change in cellular PPi content. 6. It is suggested that there are two mechanisms for the increase in mitochondrial volume induced by Ca2+: a PPi-mediated mechanism that is insensitive to cyclosporin and an additional Ca2(+)-mediated effect that is inhibited by cyclosporin. The nature of these pathways and their inter-relationship is discussed in the following paper [Halestrap & Davidson (1990) Biochem. J. 268, 153-160].

Regulation of hepatic gluconeogenesis in newborn rabbit: controlling factors in presuckling period

1985 ◽  
Vol 249 (5) ◽  
pp. E498-E505 ◽  
Author(s):  
W. A. Brennan ◽  
J. R. Aprille
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Mitochondrial Protein ◽  
Fold Increase ◽  
Insulin Injection ◽  
Pyruvate Carboxylation ◽  
Glucose Levels ◽  
Newborn Rabbit ◽  
Nucleotide Content

We have previously shown (Comp. Biochem. Physiol. 77B: 35-39, 1984) that a rapid postnatal increase in hepatic mitochondrial adenine nucleotide content activates pyruvate carboxylation and gluconeogenesis in the newborn rabbit. This study investigated factors limiting flux through the gluconeogenic pathway and examined the physiological stimuli responsible for the activation phenomenon. There is a 2.3-fold increase in total mitochondrial adenine nucleotides, along with a threefold increase in the matrix ATP/ADP ratio, by 2 h after birth, resulting overall in a sixfold increase in the amount of ATP/mg mitochondrial protein. Analysis of gluconeogenic intermediates, measured in freeze-clamped livers between birth and 4 h postnatal, suggests that pyruvate carboxylase controls gluconeogenic flux during this period. Newborn rabbits reared in an hypoxic environment (5% O2) exhibited decreased mitochondrial adenine nucleotide content, decreased rates of pyruvate carboxylation, and depressed blood glucose levels compared with littermates reared in room air or 95% O2. Manipulation of the insulin-to-glucagon ratio in vivo by injecting insulin at birth significantly delayed postnatal increases in the mitochondrial adenine nucleotide content and the rate of pyruvate carboxylation. Conversely, glucagon injection produced a supranormal increase in both mitochondrial adenine nucleotide content and pyruvate carboxylation. In addition, insulin injection prevented, whereas glucagon enhanced, the normal postnatal increase in tissue ATP/ADP. These results suggest that tissue oxygenation and a decreased insulin-to-glucagon ratio promote the rapid influx of adenine nucleotides from the liver cytosol into the mitochondrial matrix, thereby activating pyruvate carboxylation and gluconeogenesis during the presuckling period.

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.

Preparation of oligomycin-sensitive ATPase enriched submitochondrial fraction from beef heart mitochondria

1979 ◽  
Vol 44 (9) ◽  
pp. 2854-2860 ◽  
Author(s):  
Petr Svoboda ◽  
Zdeněk Drahota
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sucrose Density Gradient ◽  
Heart Mitochondria ◽  
Beef Heart ◽  
Simple Method ◽  
Beef Heart Mitochondria ◽  
Relative Molecular Weight ◽  
Protein Components

A simple method for purification of oligomycin-sensitive ATPase from beef heart mitochondria is described. The isolation procedure is based on short term solubilization of mitochondrial membrane in deoxycholate and 1M-KCl followed by sequential precipitation of hydrofobic proteins and isopycnic centrifugation of crude particulate enzyme on sucrose density gradient. The oligomycin-sesitive ATPase preparation has a specific activity 15-20μmol P/min/mg protein and contains 5% of the total mitochondrial protein which can be separated by SDS-polyacrylamide gel electrophoresis into 13 protein components of relative molecular weight from 6 000 - 65 000 daltons, respectively.

Human Adenylate Kinase 2 Deficiency Inhibits Hematopoietic Cell Differentiation towards Neutrophil and T Lymphoid Lineages.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 78-78
Author(s):  
Emmanuelle M. Six ◽  
Chantal Lagresle-Peyrou ◽  
Corinne Demerens-deChappedelaine ◽  
Frederic Rieux-Laucat ◽  
Andrea Didati ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Cell Line ◽  
Adenylate Kinase ◽  
Conflicts Of Interest ◽  
Adenine Nucleotides ◽  
Severe Combined Immunodeficiency ◽  
Mitochondrial Protein ◽  
Hematopoietic Cell ◽  
Hematopoietic Stem ◽  
Neutrophil Differentiation

Abstract Abstract 78 Reticular dysgenesis (RD), an autosomal recessive form of human Severe Combined Immunodeficiency is characterized by the absence of blood neutrophils and T lymphocytes. This pathology is due to biallelic mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of AK2 protein expression. AK2 is a mitochondrial protein which regulates the homeostasis of cellular adenine nucleotides by converting ADP into ATP and AMP. In order to understand the precise role of AK2 in hematopoiesis, we have developed a RNA interference strategy through lentiviral-mediated gene transfer of AK2 short hairpin RNAs (shAK2). The knock-down of AK2 in human or murine hematopoietic stem cells (HSC) inhibits their capacity to form granulocyte colonies in methycellulose assays and prevents them to generate mature polynucleated cells in liquid culture in the presence of G-CSF. We also determine the ability of shAK2-transduced HSC to differentiate along the T lymphoid lineage after co-culture on a OP9Delta1 stroma cell line. Our data demonstrated that the apparition of CD4+CD8+ cells was profoundly reduced in the presence of shAK2. To delineate the mechanism involved in this defect, we also studied the neutrophil differentiation of the HL60 promyelocytic cell line, following retinoic acid treatment. In this system, the absence of AK2 expression led to an arrest of neutrophil differentiation process, increased cell apoptosis and disrupt the mitochondrial membrane potential. All these data suggest a novel mechanism regulating hematopoietic cell differentiation, and involved in one of the most severe human immunodeficiency syndromes. Disclosures: No relevant conflicts of interest to declare.

Nitric oxide synthase in porcine heart mitochondria: evidence for low physiological activity

2001 ◽  
Vol 280 (6) ◽  
pp. H2863-H2867 ◽  
Author(s):  
Stephanie French ◽  
Cecilia Giulivi ◽  
R. S. Balaban
Keyword(s):  
Nitric Oxide ◽  
Physiological Activity ◽  
Mitochondrial Protein ◽  
Metabolic Effects ◽  
Heart Mitochondria ◽  
No Production ◽  
Porcine Heart ◽  
Nos Inhibitors ◽  
Oxide Synthase ◽  
Intact Heart

The capacity of isolated porcine heart mitochondria to produce nitric oxide (NO) via mitochondrial NO synthase (NOS) was evaluated. The mitochondrial NOS content and activity (0.2 nmol NO · mg mitochondrial protein−1 · min−1) were ∼10 times lower than previously reported for the rat liver. No evidence for mitochondrial NOS-generated NO was found in mitochondrial suspensions based on the lack of NO production and the lack of effect of eitherl-arginine or NOS inhibitors on the rate of respiration. The reason that even the low mitochondrial NOS activity did not result in net NO production and metabolic effects is because the mitochondrial metabolic breakdown of NO (1–4 nmol NO · mg mitochondrial protein−1 · min−1) was greater than the maximum rate of NO production measured in homogenates. These data suggest that NO production at the mitochondria via NOS is not a significant source of NO in the intact heart and does not regulate cardiac oxidative phosphorylation.

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