scholarly journals Preparation of rat enterocyte mitochondria

1984 ◽  
Vol 218 (2) ◽  
pp. 441-447 ◽  
Author(s):  
B Masola ◽  
D F Evered
Keyword(s):  
Cytochrome C ◽  
Oxidative Phosphorylation ◽  
Incubation Medium ◽  
Respiratory Control ◽  
Mitochondrial Fraction ◽  
Marker Enzymes ◽  
Cell Organelles ◽  
Succinate Dehydrogenase Activity ◽  
Isolation Medium ◽  
Mitochondrial Preparation

Rat enterocyte mitochondria were prepared with respiratory control ratios of 4 or 5 and occasionally 6. When EGTA was excluded from the mitochondrial incubation medium the calculated P/O ratios were high, especially those based on the first addition of ADP. These ratios were lowered by increasing the EGTA concentration from 1 mM to 2 mM in the mitochondrial preparation medium and including 1 mM-EGTA in the incubation medium. The use of EDTA in the enterocyte isolation medium led to the mitochondria requiring added cytochrome c. Substituting EGTA for EDTA abolished this requirement. The mitochondrial fraction consisted of two components, an upper cream-coloured layer rich in DNA and a lower brown-coloured layer poor in DNA. Both components were capable of oxidative phosphorylation with succinate or the glutamate/malate couple as substrates. The mitochondrial yield was assessed by assaying succinate dehydrogenase activity, and the contamination of the mitochondrial fraction by other cell organelles was assessed by assays for appropriate marker enzymes.

Sulphide oxidation and oxidative phosphorylation in the mitochondria of the lugworm

1997 ◽  
Vol 200 (1) ◽  
pp. 83-92 ◽  
Author(s):  
S Vökel ◽  
M K Grieshaber
Keyword(s):  
Oxygen Consumption ◽  
Cytochrome C ◽  
Oxidative Phosphorylation ◽  
Cytochrome C Oxidase ◽  
Electron Flow ◽  
Respiratory Control ◽  
Sulphide Oxidation ◽  
Atp Production ◽  
Sulphide Concentration ◽  
Flow Through

Oxygen consumption, ATP production and cytochrome c oxidase activity of isolated mitochondria from body-wall tissue of Arenicola marina were measured as a function of sulphide concentration, and the effect of inhibitors of the respiratory complexes on these processes was determined. Concentrations of sulphide between 6 and 9 µmol l-1 induced oxygen consumption with a respiratory control ratio of 1.7. Production of ATP was stimulated by the addition of sulphide, reaching a maximal value of 67 nmol min-1 mg-1 protein at a sulphide concentration of 8 µmol l-1. Under these conditions, 1 mole of ATP was formed per mole of sulphide consumed. Higher concentrations of sulphide led to a decrease in ATP production until complete inhibition occurred at approximately 50 µmol l-1. The production of ATP with malate and succinate was stimulated by approximately 15 % in the presence of 4 µmol l-1 sulphide, but decreased at sulphide concentrations higher than 15­20 µmol l-1. Cytochrome c oxidase was also inhibited by sulphide, showing half-maximal inhibition at 1.5 µmol l-1 sulphide. Sulphide-induced ATP production was inhibited by antimycin, cyanide and oligomycin but not by rotenone or salicylhydroxamic acid. The present data indicate that sulphide oxidation is coupled to oxidative phosphorylation solely by electron flow through cytochrome c oxidase, whereas the alternative oxidase does not serve as a coupling site. At sulphide concentrations higher than 20 µmol l-1, oxidation of sulphide serves mainly as a detoxification process rather than as a source of energy.

Effects of 6-hydroxydopamine on oxidative phosphorylation of mitochondria from rat striatum, cortex, and liver

1988 ◽  
Vol 66 (3) ◽  
pp. 376-379 ◽  
Author(s):  
J. H. Thakar ◽  
M. N. Hassan
Keyword(s):  
Oxidative Phosphorylation ◽  
Incubation Medium ◽  
Respiratory Control ◽  
Rat Striatum ◽  
Respiratory Control Ratio ◽  
Chemical Sympathectomy ◽  
Mitochondrial Oxidative Phosphorylation ◽  
Consumption Ratio ◽  
6 Hydroxydopamine

The catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) has been used to produce cardiac chemical sympathectomy as well as a model of parkinsonism. Several mechanisms have been proposed to explain its cytotoxicity, including the productions of quinones, hydrogen peroxide, and free radicals by autooxidation and the uncoupling of mitochondrial oxidative phosphorylation. We have observed that 6-OHDA at a concentration of 0.05 mM rapidly consumes oxygen from the mitochondrial incubation medium but does not affect oxidative phosphorylation in the mitochondria from rat striatum, cortex, and liver. At the higher concentration of 0.5 mM, 6-OHDA consumes all of the available oxygen from the incubation medium. Mitochondria exposed to this concentration of 6-OHDA show decreases in the respiratory control ratio and adenosine triphosphate synthesis as measured by the consumption ratio of ADP to oxygen. Thus, only the higher (0.5 mM) concentration of 6-OHDA, which produces anoxia in vitro, also causes mitochondrial damage.

scholarly journals Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization.

1982 ◽  
Vol 93 (1) ◽  
pp. 144-154 ◽  
Author(s):  
L Marzella ◽  
J Ahlberg ◽  
H Glaumann
Keyword(s):  
Proteolytic Activity ◽  
Rat Liver ◽  
Biochemical Characterization ◽  
Secretory Granules ◽  
Density Lipoprotein ◽  
Marker Enzymes ◽  
Cell Organelles ◽  
Protein Ratio ◽  
Autophagic Vacuoles

The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial-lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42: 8-17. Marzella and Glaumann, 1980, Lab. Invest., 42:18-27). In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs). In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected. Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (approximately 90%) of intact autophagic vacuoles. These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein, particles either free or in Golgi apparatus derivatives, in particular secretory granules. The sequestered materials show ultrastructural signs of ongoing degradation. In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components. Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis. Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10- to 20-fold in the isolated AV fraction as compared to the homogenate. Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles. Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active. Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0.5) of the AV fraction suggests that phospholipids are degraded more slow than proteins. Is it concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.

scholarly journals Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts

2004 ◽  
Vol 380 (3) ◽  
pp. 919-928 ◽  
Author(s):  
Eveline HUTTER ◽  
Kathrin RENNER ◽  
Gerald PFISTER ◽  
Petra STÖCKL ◽  
Pidder JANSEN-DÜRR ◽  
...  
Keyword(s):  
Electron Transport ◽  
Oxidative Phosphorylation ◽  
Replicative Senescence ◽  
Citrate Synthase ◽  
Human Fibroblasts ◽  
Respiratory Control ◽  
Compensatory Mechanisms ◽  
Intact Cells ◽  
Atp Production ◽  
Human Diploid Fibroblasts

Limitation of lifespan in replicative senescence is related to oxidative stress, which is probably both the cause and consequence of impaired mitochondrial respiratory function. The respiration of senescent human diploid fibroblasts was analysed by highresolution respirometry. To rule out cell-cycle effects, proliferating and growth-arrested young fibroblasts were used as controls. Uncoupled respiration, as normalized to citrate synthase activity, remained unchanged, reflecting a constant capacity of the respiratory chain. Oligomycin-inhibited respiration, however, was significantly increased in mitochondria of senescent cells, indicating a lower coupling of electron transport with phosphorylation. In contrast, growth-arrested young fibroblasts exhibited a higher coupling state compared with proliferating controls. In intact cells, partial uncoupling may lead to either decreased oxidative ATP production or a compensatory increase in routine respiration. To distinguish between these alternatives, we subtracted oligomycin-inhibited respiration from routine respiration, which allowed us to determine the part of respiratory activity coupled with ATP production. Despite substantial differences in the respiratory control ratio, ranging from 4 to 11 in the different experimental groups, a fixed proportion of respiratory capacity was maintained for coupled oxidative phosphorylation in all the experimental groups. This finding indicates that the senescent cells fully compensate for increased proton leakage by enhanced electron-transport activity in the routine state. These results provide a new insight into age-associated defects in mitochondrial function and compensatory mechanisms in intact cells.

Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart

2011 ◽  
Vol 43 (12) ◽  
pp. 1729-1738 ◽  
Author(s):  
Hélène Lemieux ◽  
Severin Semsroth ◽  
Herwig Antretter ◽  
Daniel Höfer ◽  
Erich Gnaiger
Keyword(s):  
Oxidative Phosphorylation ◽  
Human Heart ◽  
Respiratory Control ◽  
Mitochondrial Respiratory

Oxidative phosphorylation in cardiac infarct. Effect of glucose-KCl-insulin solution

1965 ◽  
Vol 209 (2) ◽  
pp. 371-375 ◽  
Author(s):  
Edmundo Calva ◽  
Adela Mujica ◽  
Abdo Bisteni ◽  
Demetrio Sodi-Pallares
Keyword(s):  
Oxygen Consumption ◽  
Oxidative Phosphorylation ◽  
Respiratory Control ◽  
Low Oxygen ◽  
Normal Limits ◽  
Functional Aspects ◽  
Insulin Solution ◽  
Effect Of Glucose ◽  
Infarcted Tissue ◽  
Electrocardiographic Abnormalities

Myocardial infarction was produced in dogs by ligature of the anterior descending coronary artery. Sarcosomes were isolated from normal and infarcted tissue. Oxygen consumption was followed polarographically and adenosine triphosphate was measured as glucose 6-phosphate. One group of animals received a continuous infusion of glucose for 12 hr; another group received "polarizing solution" (glucose-KCl-insulin). Sarcosomes from the first had a low oxygen consumption, no respiratory control, and no oxidative phosphorylation. In contrast, the administration of glucose-KCl-insulin solution maintained practically within normal limits these functional aspects of the sarcosomes. The reversal of electrocardiographic abnormalities by the administration of the polarizing solution coincided with improvement of such biochemical functions. Anesthesia and surgical handling did not appear to modify the behavior of the sarcosomes.

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