Interspecific variations in proteins synthesized by mammalian mitochondria

1978 ◽  
Vol 56 (10) ◽  
pp. 939-942 ◽  
Author(s):  
Randall W. Yatscoff ◽  
Luke Aujume ◽  
Karl B. Freeman ◽  
Samuel Goldstein
Keyword(s):  
Mammalian Species ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Molecular Weights ◽  
Interspecific Variations ◽  
Established Cell Lines ◽  
Mammalian Mitochondria ◽  
Established Cell

The products of mitochondrial protein synthesis in established cell lines of various mammalian species were labelled with [35S]methionine and their number and apparent molecular weights determined by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis and fluorography. Proteins synthesized by isolated rat liver mitochondria were labelled with [3H]valine and similarly characterized. Each species had a distinctive pattern of from 10 to 13 mitochondrially synthesized proteins with apparent molecular weights between 10 000 and 50 000. No differences were detected in the number or electrophoretic mobility of the mitochondrially synthesized proteins of SV-40-transformed and nontransformed WI-38 cells.

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 The uptake of silicic acid by rat liver mitochondria

1978 ◽  
Vol 172 (3) ◽  
pp. 557-568 ◽  
Author(s):  
R N Johnson ◽  
B E Volcani
Keyword(s):  
Silicic Acid ◽  
Diffusion Mechanism ◽  
Mitochondrial Protein ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Energy Status ◽  
Matrix Space ◽  
Inner Membrane ◽  
Simple Diffusion ◽  
Mitochondrial Inner Membrane

1. To gain insight into a putative role for mitochondria in silicon metabolism, mitochondrial uptake (by which it is meant the removal from the medium) of silicic acid [Si(OH)4] was studied under conditions minimizing SI(OH)4 polymerization. 2. Measurements of mitochondrial respiration and swelling indicated indirectly a significant uptake of Si(OH)4 as a weak acid, but this was not confirmed when 31Si(OH)4 was used as a tracer. 31Si(OH)4 occupied a mitochondrial volume similar to that of 3H2O and was relatively unaffected by mitochondrial energy status and by the pH gradient across the mitochondrial inner membrane. 3. Uptake was directly proportional to Si(OH)4 concentration in the range 0-3 mM. 4. The uptake consisted of two components: under all conditions examined, the greater quantity, amounting to 1-2nmol of Si(OH)4/mg of mitochondrial protein, was bound, a major portion of it external to the inner membrane, with the lesser quantity free within the matrix space. 5. Equilibration of 31Si(OH)4 between medium and matrix was a slow process, having a half-time of approx. 10 min at 22 degrees C. 6. Mersalyl and N-ethylmaleimide inhibited the uptake by preferentially lowering the amount of Si(OH)4 bound. Their action was somewhat variable, depending on the precise nature of the suspending medium, and suggesting that the bound material may represent polymerized forms of Si(OH)4. 7. It is concluded that Si(OH)4 may penetrate the mitochondrial inner membrane by a simple diffusion mechanism.

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.

Inhibition of mitochondrial and bacterial protein synthesis by chloramphenicol

1970 ◽  
Vol 48 (4) ◽  
pp. 479-485 ◽  
Author(s):  
K. B. Freeman
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Bacterial Protein ◽  
E Coli ◽  
Inhibition Of Protein Synthesis ◽  
Acetyl Group ◽  
Bacterial Protein Synthesis ◽  
Escherichia Coli B

The structural requirements for the inhibition of protein synthesis in mitochondria and in bacterial extracts by chloramphenicol isomers and analogues are similar. D-threo-Chloramphenicol and its p-methylthio, p-methylsulfonyl, and p-sulfamoyl analogues equally inhibit protein synthesis in isolated rat-liver mitochondria and extracts of Escherichia coli B. Fifty percent inhibition is at 15 μM and 10 μM, respectively. Analogues with larger p-substituents on the phenyl ring or with an m-chloro group are less inhibitory in both systems. L-threo-Chloramphenicol and deacylated chloramphenicol do not inhibit mitochondrial protein synthesis; with a dichloroacetyl group replacing the acetyl group on chloramphenicol 50% inhibition is at 65 μM, and L-erythro-chloramphenicol is 2% as inhibitory as D-threo-chloramphenicol. The inhibition of protein synthesis in intact E. coli B is in the order: chloramphenicol > p-methylthio > p-methylsulfonyl > p-sulfamoyl, 50% inhibition being at 4 μM for chloramphenicol.

scholarly journals Partial reconstruction of in vitro gluconeogenesis arising from mitochondrial l-lactate uptake/metabolism and oxaloacetate export via novel l-lactate translocators

2004 ◽  
Vol 380 (1) ◽  
pp. 231-242 ◽  
Author(s):  
Lidia de BARI ◽  
Anna ATLANTE ◽  
Daniela VALENTI ◽  
Salvatore PASSARELLA
Keyword(s):  
Electron Flow ◽  
Mitochondrial Protein ◽  
Pyridine Nucleotide ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Inner Mitochondrial Membrane ◽  
Lactate Oxidation ◽  
Partial Reconstruction ◽  
Proton Uptake

In the light of the occurrence of l-lactate dehydrogenase inside the mitochondrial matrix, we looked at whether isolated rat liver mitochondria can take up and metabolize l-lactate, and provide oxaloacetate outside mitochondria, thus contributing to a partial reconstruction of gluconeogenesis in vitro. We found that: (1) l-lactate (10 mM), added to mitochondria in the presence of a cocktail of glycolysis/gluconeogenesis enzymes and cofactors, can lead to synthesis of glyceraldehyde-3-phosphate at a rate of about 7 nmol/min per mg mitochondrial protein. (2) Three novel translocators exist to mediate l-lactate traffic across the inner mitochondrial membrane. An l-lactate/H+ symporter was identified by measuring fluorimetrically the rate of endogenous pyridine nucleotide reduction. Consistently, l-lactate oxidation was found to occur with P/O ratio=3 (where P/O ratio is the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation) and with generation of membrane potential. Proton uptake, which occurred as a result of addition of l-lactate to RLM together with electron flow inhibitors, and mitochondrial swelling in ammonium l-lactate solutions were also monitored. l-Lactate/oxaloacetate and l-lactate/pyruvate anti-porters were identified by monitoring photometrically the appearance of l-lactate counter-anions outside mitochondria. These l-lactate translocators, which are distinct from the monocarboxylate carrier, were found to differ from each other in Vmax values and in inhibition and pH profiles, and proved to regulate mitochondrial l-lactate metabolism in vitro. The role of lactate/mitochondria interactions in gluconeogenesis is discussed.

scholarly journals CORTISONE-INDUCED ALTERATIONS IN MITOCHONDRIAL FUNCTION AND STRUCTURE

1968 ◽  
Vol 37 (1) ◽  
pp. 63-80 ◽  
Author(s):  
Daniel V. Kimberg ◽  
Alden V. Loud ◽  
Joseph Wiener
Keyword(s):  
Oxidative Phosphorylation ◽  
Mitochondrial Function ◽  
Mitochondrial Protein ◽  
Light And Electron Microscopy ◽  
Isotonic Saline ◽  
Hormone Treatment ◽  
Liver Mitochondria ◽  
Male Rats ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Volume

The effects of cortisone treatment on oxygen consumption, oxidative phosphorylation, and fine structure of rat liver mitochondria have been studied. Male rats weighing 125 g were treated for 6 days with 5 mg of cortisone acetate or isotonic saline. On the 7th day, sections of liver were excised and processed for light and electron microscopy. Mitochondrial respiration and oxidative phosphorylation were studied with mitochondria isolated from these livers. Cortisone treatment is responsible for a 14–40% decrease in the amount of oxygen consumed per mg of mitochondrial protein when succinate, α-ketoglutarate, or ß-hydroxybutyrate are used as substrates, or with ascorbate and N,N,N1,N1-tetramethyl p-phenylenediamine as electron donors. In addition, oxidative phosphorylation is uncoupled with a lowering of the P:O ratios. Randomly selected liver cells have been analyzed by quantitative morphometric techniques. The average mitochondrial volume is increased fourfold in the peripheral and midzonal regions with a commensurate decrease in the number of mitochondria per cell. These alterations are present throughout the hepatic lobule, but are most marked in midzonal cells. The total mitochondrial volume per cell and the per cent of the total cytoplasmic volume occupied by mitochondria remains relatively unaltered, as does the total amount of cristae surface per cell. While the mitochondria are enlarged, they are not "swollen." The relationships between the steroid hormone treatment and the alterations in mitochondrial function and structure are discussed.

scholarly journals Characteristics of hepatic serine-pyruvate aminotransferase in different mammalian species

1977 ◽  
Vol 161 (3) ◽  
pp. 609-614 ◽  
Author(s):  
T Noguchi ◽  
Y Takada ◽  
R Kido
Keyword(s):  
Gel Electrophoresis ◽  
Isonicotinic Acid ◽  
Mammalian Species ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Acid Hydrazide ◽  
Isonicotinic Acid Hydrazide ◽  
Enzyme Preparations ◽  
Molecular Weights ◽  
Amino Donor

1. Serine-pyruvate aminotransferase was purified from mouse, rat, dog and cat liver. Each enzyme preparation was homogeneous as judged by polyacrylamide-disc-gel electrophoresis in the presence of sodium dodecyl sulphate. However, isoelectric focusing resulted in the detection of two or more active forms from enzyme preparations from dog, cat and mouse. A single active form was obtained with the rat enzyme. All four enzyme preparations had similar pH optima and molecular weights. 2. Both mouse and rat preparations catalysed transamination between a number of L-amino acids (serine, leucine, asparagine, methionine, glutamine, ornithine, histidine, phenylalanine or tyrosine) and pyruvate. Effective amino acceptors were pyruvate, phenylpyruvate and glyoxylate with serine as amino donor. The reverse transamination activity, with hydroxypyruvate and alanine as subtrates, was lower than with serine and pyruvate for both species. Serine-pyruvate aminotransferase activities were inhibited by isonicotinic acid hydrazide. 3. In contrast, both dog and cat enzyme preparations were highly specific for serine as amino donor with pyruvate, and utilized pyruvate and glyoxylate as effective amino acceptors. A little activity was detected with phenylpyruvate. The reverse activity was higher than with serine and pyruvate for both species. Serine-pyruvate amino-transferase activities were not inhibited by isonicotinic acid hydrazide.

scholarly journals Proton translocation by cytochrome oxidase in (antimycin + myxothiazol)-treated rat liver mitochondria using ferrocyanide or hexammineruthenium as electron donor

1986 ◽  
Vol 236 (1) ◽  
pp. 15-21 ◽  
Author(s):  
I C West ◽  
R Mitchell ◽  
A J Moody ◽  
P Mitchell
Keyword(s):  
Cytochrome Oxidase ◽  
Rat Liver ◽  
Choline Chloride ◽  
Proton Translocation ◽  
Mitochondrial Protein ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Second Phase ◽  
O2 Consumption ◽  
Rapid Phase

When O2 was injected into an anaerobic suspension of valinomycin-treated rat liver mitochondria inhibited with rotenone, antimycin, and myxothiazol, a small amount of O2 (0.23-0.33 ng-atom of O/mg of protein) was reduced extremely rapidly (within the 2 s time-resolution of the oxygen electrode). The subsequent steady-state rate of flow of electrons to oxygen was very low [less than 3 nequiv. X s-1 X (g of mitochondrial protein)-1]. In the presence of valinomycin there was a rapid ejection of protons synchronous with the rapid phase of O2 consumption corresponding to 0.38-0.61 nequiv. of H+ X (mg of mitochondrial protein)-1. When valinomycin was replaced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) there was a rapid alkalification of the medium corresponding to 0.20-0.42 nequiv. of H+ X (mg of mitochondrial protein)-1. When 2 mM-Fe(CN)6(4-) was present to re-reduce endogenous cytochrome c, O2 consumption was still biphasic but the second phase of O2 consumption was very much more rapid [600 nequiv. X s-1 X (g of protein)-1], and resulted in the virtually complete consumption of the O2 in the pulse within 4 s. With 60 microM-Ru(NH3)6(2+) as reductant, O2 consumption was even faster [1200 nequiv. X s-1 X (g of protein)-1]. In a medium containing 150 mM-choline chloride with Ru(NH3)6(2+) as reductant, the proton per reducing equivalent stoichiometry (delta H+O/e-) was +0.95 in the presence of valinomycin and −0.94 in the presence of FCCP. In choline chloride medium containing Ru(NH3)6(2+) and valinomycin, there was an uptake of K+ ions corresponding to 1.86 K+/e-. It is concluded that nearly 1 proton is translocated outwards through cytochrome oxidase per oxidizing equivalent injected in this medium. In low ionic strength sucrose-based medium, with Ru(NH3)6(2+) as reductant, delta H+O/e- was 1.05 in the presence of valinomycin, and −0.71 in the presence of FCCP. It is concluded that the translocation of protons is accompanied by net acid production in this medium.

scholarly journals MITOCHONDRIAL AUTONOMY

1972 ◽  
Vol 55 (1) ◽  
pp. 147-160 ◽  
Author(s):  
H. Bruce Bosmann ◽  
Marjorie W. Myers ◽  
Delena Dehond ◽  
Richard Ball ◽  
Kenneth R. Case
Keyword(s):  
Cerebral Cortex ◽  
Sialic Acid ◽  
Outer Membrane ◽  
Electrophoretic Mobility ◽  
Rat Liver ◽  
Mitochondrial Protein ◽  
Liver Mitochondria ◽  
Rat Cerebral Cortex ◽  
Rat Liver Mitochondria ◽  
Membrane Isolation

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 ± 0.1, 0.6 mM NaHCO3, had an electrophoretic mobility of - 2.88 ± 0.01 µ/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 ± 0.02, of rat liver mitoplasts was - 1.22 ± 0.07, and of rat cerebral cortex mitoplasts - 0.91 ± 0.04 µ/sec per v per cm. Treatment of these particles with 50 µg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in µ/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 µg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pKa ∼ 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.

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