Characterization of partially purified microbodies from hydrocarbon-grown cells of Cladosporium resinae

1989 ◽  
Vol 35 (5) ◽  
pp. 565-572 ◽  
Author(s):  
David B. Carson ◽  
Joseph J. Cooney
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Density Gradient ◽  
Gradient Centrifugation ◽  
Urate Oxidase ◽  
Marker Enzyme ◽  
Thin Sections ◽  
Cell Extracts ◽  
Mitochondrial Fractions ◽  
A Minor

Cells of the filamentous fungus Cladosporium resinae synthesize many more microbodies when they are grown on an n-alkane than when they are grown on glucose. Cladosporium resinae was grown on n-dodecane and spheroplasts were prepared, disrupted, and fractionated by differential and density gradient centrifugation. A fraction was isolated which was enriched in catalase, a marker enzyme for microbodies. Another fraction was isolated which was enriched in cytochrome c oxidase, a marker for mitochondria. Urate oxidase, a second marker for microbodies, was not detected in cell extracts. The microbody and mitochondrial fractions were relatively free of contamination from the endoplasmic reticulum and cytosol as indicated by the amounts of glucose-6-phosphatase and glucose-6-phosphate dehydrogenase present, respectively. Transmission electron microscopy revealed that the catalase-enriched fraction contained intact microbodies, with mitochondria as a minor contaminant. Catalase was localized in microbodies by staining with 3,3′-diaminobenzidine. Mitochrondria were present in the cytochrome c oxidase enriched fraction and took up the vital stain Janus green B. In similar preparations from cells grown on glucose, catalase was largely nonparticulate. Microbodies were not observed in thin sections prepared from density gradient fractions, but mitochondria were present in a cytochrome c oxidase enriched fraction.Key words: Cladosporium resinae, microbodies, mitochondria, catalase, cytochrome c oxidase.

scholarly journals Observations on iron uptake, iron metabolism, cytochrome c content, cytochrome a content and cytochrome c-oxidase activity in regenerating rat liver

1965 ◽  
Vol 97 (2) ◽  
pp. 532-539 ◽  
Author(s):  
ARL Gear
Keyword(s):  
Cytochrome C ◽  
Liver Regeneration ◽  
Cytochrome C Oxidase ◽  
Density Gradient ◽  
Rat Liver ◽  
Oxidase Activity ◽  
Specific Content ◽  
Cytochrome C Reductase ◽  
Regenerating Rat Liver ◽  
Mitochondrial Fractions

1. Differential and density-gradient centrifugation were used to fractionate mitochondria and fluffy layer from normal and regenerating rat liver. The iron, cytochrome a and cytochrome c contents and cytochrome c-oxidase activity were studied as well as the uptake of (59)Fe into protein and cytochrome c. 2. A certain degree of heterogeneity was evident between the heavy-mitochondrial and light-mitochondrial fractions, and in their behaviour during liver regeneration. 3. The specific content of light-mitochondrial iron and cytochrome a was 1.3-1.4 times that of heavy mitochondria. Changes in cytochrome c-oxidase activity closely followed those of cytochrome a content during liver regeneration, but not for light mitochondria after 10 days. 4. Radioactive iron ((59)Fe) was most actively taken up by well-washed light mitochondria during early liver regeneration. After 22 days fluffy layer became preferentially labelled. This substantiates the view that fluffy layer partially represents broken-down mitochondria. 5. During early regeneration, light-mitochondrial fractions separated along a density gradient were about 3 times as radioactive, and showed distinct heterogeneity of (59)Fe-labelling, in contrast with near homogeneity for heavy mitochondria. 6. Immediately after partial hepatectomy fractions corresponding to density 1.155 were 5-10 times as radioactive as particles of greater density. The radioactivity decreased sharply after 6 days. 7. These particles of low density possessed higher NADH-cytochrome c-reductase (1.5-5-fold) and succinate-dehydrogenase (1.1-2-fold) activities than typical mitochondrial fractions. Their succinate-cytochrome c-reductase and cytochrome c-oxidase activities were slightly lower. 8. The results are discussed in relation to mitochondrial morphogenesis, and a possible route from submitochondrial particles is suggested.

scholarly journals Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria.

1977 ◽  
Vol 72 (3) ◽  
pp. 714-725 ◽  
Author(s):  
G C Shore ◽  
J R Tata
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Structural Damage ◽  
Gradient Centrifugation ◽  
Mitochondrial Protein ◽  
Enzyme Analysis ◽  
Marker Enzyme

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KCl and 20 mM EDTA, and subjected to shearing. Excluding KCl, EDTA, or shearing from the procedure resulted in incomplete separation. Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way fromanimals injected with [35S]methionine +/- cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.

scholarly journals Oxidative phosphorylation during glycollate metabolism in mitochondria from phototrophic Euglena gracilis

1975 ◽  
Vol 150 (3) ◽  
pp. 373-377 ◽  
Author(s):  
N Collins ◽  
R H Brown ◽  
M J Merrett
Keyword(s):  
Cytochrome C ◽  
Oxygen Uptake ◽  
Oxidative Phosphorylation ◽  
Cytochrome C Oxidase ◽  
Electron Acceptor ◽  
Euglena Gracilis ◽  
Gradient Centrifugation ◽  
Antimycin A ◽  
Isolated Mitochondria ◽  
Glycollate Dehydrogenase

Mitochondria were isolated by gradient centrifugation on linear sucrose gradients from broken cell suspensions of phototrophically grown Euglena gracilis. An antimycin A-sensitive but rotenone-insensitive glycollate-dependent oxygen uptake was demonstrated in isolated mitochondria. The partial reactions of glycollate-cytochrome c oxidoreductase and cytochrome c oxidase were demonstrated by using Euglena cytochrome c as exogenous electron acceptor/donor. Isolated mitochondria contain glycollate dehydrogenase and glyoxylate-glutamate aminotransferase and oxidize exogenous glycine. A P:O ratio of 1.7 was obtained for glycollate oxidation, consistent with glycollate electrons entering the Euglena respiratory chain at the flavoprotein level. The significance of these results is discussed in relation to photorespiration in algae.

scholarly journals Subcellular localization of ferritin and iron taken up by rat hepatocytes

1989 ◽  
Vol 262 (2) ◽  
pp. 685-688 ◽  
Author(s):  
J C Sibille ◽  
M Ciriolo ◽  
H Kondo ◽  
R R Crichton ◽  
P Aisen
Keyword(s):  
Acid Phosphatase ◽  
Cytochrome C ◽  
Subcellular Localization ◽  
Cytochrome C Oxidase ◽  
Density Gradient ◽  
Rat Hepatocytes ◽  
Sucrose Density Gradient ◽  
Gel Chromatography ◽  
Density Gradient Ultracentrifugation

The subcellular localization of ferritin and its iron taken up by rat hepatocytes was investigated by sucrose-density-gradient ultracentrifugation of cell homogenates. After incubation of hepatocytes with 125I-labelled [59Fe]ferritin, cells incorporate most of the labels into structures equilibrating at densities where acid phosphatase and cytochrome c oxidase are found, suggesting association of ferritin and its iron with lysosomes or mitochondria. Specific solubilization of lysosomes by digitonin treatment indicates that, after 8 h incubation, most of the 125I is recovered in lysosomes, whereas 59Fe is found in mitochondria as well as in lysosomes. As evidenced by gel chromatography of supernatant fractions, 59Fe accumulates with time in cytosolic ferritin. To account for these results a model is proposed in which ferritin, after being endocytosed by hepatocytes, is degraded in lysosomes, and its iron is released and re-incorporated into cytosolic ferritin and, to a lesser extent, into mitochondria.

scholarly journals Changes in enzyme activities and distributions during glucose de-repression and respiratory adaptation of anaerobically grown Saccharomyces carlsbergensis

1973 ◽  
Vol 132 (3) ◽  
pp. 609-621 ◽  
Author(s):  
T. G. Cartledge ◽  
D. Lloyd
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Respiration Rate ◽  
Enzyme Activities ◽  
Specific Activity ◽  
Antimycin A ◽  
Saccharomyces Carlsbergensis ◽  
Whole Cells ◽  
Nadh Cytochrome ◽  
A Minor

1. During anaerobic glucose de-repression the respiration rate of whole cells of Saccharomyces carlsbergensis remained constant and was insensitive to antimycin A but was inhibited by 30% by KCN. Aeration of cells for 1 h led to increased respiration rate which was inhibited by 80% by antimycin A or KCN. 2. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucose de-repressed cells and the distribution of marker enzymes was investigated after zonal centrifugation on sucrose gradients containing MgCl2. These homogenates contained no detectable cytochrome c oxidase or catalase activity. The complex density distributions of NADH– and NADPH–cytochrome c oxidoreductases and adenosine triphosphatase(s) [ATPase(s)] were very different from those of anaerobically grown, glucose-repressed cells. 3. The specific activity of total ATPase was lowered and sensitivity to oligomycin decreased from 58 to 7% during de-repression. 4. Cytochrome c oxidase and catalase activities were detectable in homogenates of cells after 10min aeration. Zonal centrifugation indicated complex, broad sedimentable distributions of all enzyme activities assayed; the peaks of activity were at 1.27g/ml. 5. Centrifugation of homogenates of cells adapted for 30min and 3 h indicated a shift of density of the major sedimentable peak from 1.25g/ml (30min) to 1.235g/ml (3 h). After 30min adaptation a minor zone of oligomycin-sensitive ATPase and 15% of the total cytochrome c oxidase activities were detected at ρ=1.12g/l; these particles together with those of higher density containing cytochrome c oxidase, ATPase and NADH–cytochrome c oxidoreductase activities were all sedimented at 105g-min. 6. Electron microscopy indicated that the mitochondria-like structures of anaerobically grown, glucose-de-repressed cells were similar to those of repressed cells. After 10min of respiratory adaptation highly organized mitochondria were evident which resembled the condensed forms of mitochondria of aerobically grown, glucose-de-repressed cells. High-density zonal fractions of homogenates of cells after adaptation also contained numerous electron-dense vesicles 0.05–0.2μm in diameter. 7. The possibility that the `promitochondria' of anaerobically grown cells may not be the direct structural precursors of fully functional mitochondria is discussed.

scholarly journals Lysosomes, but not mitochondria, accumulate iron and porphyrins in porphyria induced by hexachlorobenzene

1986 ◽  
Vol 235 (3) ◽  
pp. 671-675 ◽  
Author(s):  
A Tangerås
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Iron Status ◽  
Gradient Centrifugation ◽  
Female Rats ◽  
Percoll Density Gradient Centrifugation ◽  
Mitochondrial Fractions ◽  
Percoll Density Gradient ◽  
Haem Iron

In female rats with porphyria induced by hexachlorobenzene, the amounts of non-haem iron and porphyrins in liver mitochondrial fractions were increased almost 3-fold and greater than 500-fold respectively compared with that of untreated animals. A considerable fraction of both iron and porphyrins in this fraction was shown to be located in lysosomes. Thus mitochondrial preparations, which were further depleted of lysosomes by Percoll-density-gradient centrifugation, contained 2.78 +/- 0.75 and 2.99 +/- 0.49 nmol of non-haem iron/mg of protein when isolated from the liver of control rats and hexachlorobenzene-treated rats respectively. Mitochondria isolated from the liver of hexachlorobenzene-treated animals contained a pool of iron (about 1 nmol/mg of protein) that was available for haem synthesis in vitro. This pool is similar to that previously reported for mitochondria isolated from the liver of rats with normal haem synthesis. Hexachlorobenzene treatment, therefore, does not affect the iron status of the mitochondria.

scholarly journals THE ISOLATION OF RETINAL OUTER SEGMENT FRAGMENTS

1965 ◽  
Vol 27 (3) ◽  
pp. 459-473 ◽  
Author(s):  
David G. McConnell
Keyword(s):  
Cytochrome C ◽  
Density Gradient ◽  
Respiratory Chain ◽  
Lipid Content ◽  
Outer Segment ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Mitochondrial Respiratory Chain ◽  
Centrifugal Field ◽  
High Lipid

Bovine retinal outer segment fragments were isolated by density gradient centrifugation in a high centrifugal field. Assays of the final preparation for enzymes of the mitochondrial respiratory chain indicated mitochondrial contamination not in excess of 1 per cent. Glucose-6-phosphatase and TPNH-cytochrome c reductase activities, presumably diagnostic for microsomes, were also absent. Electron micrographs did not disclose the presence of significant numbers of particles other than fragments of the outer segment discs. The red fragments were characterized by an ascorbate-oxidizing system and a high lipid content.

Isolation and characterization of subcellular membranes from canine stomach smooth muscle

1981 ◽  
Vol 59 (12) ◽  
pp. 1260-1267 ◽  
Author(s):  
Y. Sakai ◽  
J. McLean ◽  
A. K. Grover ◽  
R. E. Garfield ◽  
J. E. T. Fox ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Density Gradient ◽  
Circular Muscle ◽  
Sucrose Density Gradient ◽  
Mitochondrial Fraction ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Isolation And Characterization ◽  
Nadph Cytochrome C Reductase

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5′-nucleotidase and NADPH–cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18–32% (w/w) sucrose was maximally enriched (13-fold) in 5′-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH–cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32–40% (w/w) sucrose, was maximally enriched in NADPH–cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive, ATP-dependent Ca2+ uptake correlated very well with that of 5′-nucleotidase but less well with NADPH–cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role in regulating intracellular Ca2+ concentration in canine corpus circular muscle.

scholarly journals A procedure for the rapid preparation of mitochondria from rat liver

1982 ◽  
Vol 204 (3) ◽  
pp. 731-735 ◽  
Author(s):  
P H Reinhart ◽  
W M Taylor ◽  
F L Bygrave
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Calcium Content ◽  
Mitochondrial Fraction ◽  
Protonmotive Force ◽  
Marker Enzyme ◽  
Rapid Preparation ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient

A technique for the rapid preparation of mitochondria from rat liver is described. Tissue fractionation is performed by a single centrifugation step with a discontinuous Percoll density gradient. Total preparation times of 5-6 min are achieved by using this method. The mitochondrial fraction obtained is relatively free of contaminating organelles, as judged by marker-enzyme activity determinations. Mitochondria isolated by Percoll-density-gradient centrifugation differ from mitochondria obtained by differential centrifugation [Taylor, Prpić, Exton & Bygrave (1980) Biochem. J. 188, 443-450] in that the former exhibit a higher acceptor control ratio and a higher calcium content. Values obtained for the protonmotive force are not significantly different between the two preparations. The technique described may be widely applicable for studies requiring the rapid preparation of functionally intact and relatively uncontaminated mitochondria.

scholarly journals A new procedure for the purification of monodisperse highly active cytochrome c oxidase from bovine heart

1987 ◽  
Vol 242 (2) ◽  
pp. 417-423 ◽  
Author(s):  
Y Li ◽  
A Naqui ◽  
T G Frey ◽  
B Chance
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Gel Filtration ◽  
Homogeneous State ◽  
Bovine Heart ◽  
Highly Active ◽  
Solubilized Enzyme

A simple and rapid method for the isolation of a large quantity of cytochrome c oxidase from bovine heart mitochondria was developed, based on selective solubilization of mitochondrial protein with first Triton and then lauryl maltoside. Gel filtration shows that the lauryl maltoside-solubilized oxidase preparation is in a hydrodynamically homogeneous state with a Stokes radius of 7.5 +/- 0.2 nm. It contains 8.0 mumol of haem (with an a/a3 ratio of 1)/g of protein. The catalytic constant (maximum turnover number) with respect to cytochrome c approaches 600 S-1. After further purification of the solubilized enzyme on a sucrose-gradient centrifugation, the purified enzyme has a haem content of 10.3 mumol/g of protein and eight major polypeptide bands shown on SDS/polyacrylamide-gel electrophoresis.

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