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.

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 Subcellular localization of oestrogen-induced uterine peroxidase

1976 ◽  
Vol 160 (2) ◽  
pp. 237-241 ◽  
Author(s):  
C R Lyttle ◽  
P H Jellinck
Keyword(s):  
Acid Phosphatase ◽  
Subcellular Localization ◽  
Succinate Dehydrogenase ◽  
Density Gradient ◽  
Peroxidase Activity ◽  
Sucrose Density Gradient ◽  
Urate Oxidase ◽  
Mitochondrial Marker ◽  
Membrane Bound

The distribution of oestrogen-induced peroxidase in the resuspended 8000g pellet of rat uterine homogenates was examined by centrifugation in a sucrose density gradient. Within 10h of treatment with oestradiol, peroxidase activity was found in a region devoid of catalase or urate oxidase (peroxisomal markers) which did not overlap the fractions containing succinate dehydrogenase (mitochondrial marker) or acid phosphatase (lysosomal marker). The induced uterine enzyme was localized in reticular membrane-bound vesicles with isopycnic density of 1.28g/ml from which it could be released by treatment with detergent.

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 Localization of α-L-Arabinofuranosidase and Acid Phosphatase in Mycelium of Sclerotinia fructigena

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 79-99 ◽  
Author(s):  
E. C. Hislop ◽  
Vivien M. Barnaby ◽  
Claire Shellis ◽  
F. Laborda
Keyword(s):  
Acid Phosphatase ◽  
Density Gradient ◽  
Azo Dye ◽  
Acid Treatment ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Soluble Form ◽  
Ammonium Tartrate ◽  
Electron Microscopes ◽  
Hyphal Wall

α-L-Arabinofuranosidase (AF) and acid p-nitrophenyl phosphatase (AP) were secreted by Sclerotinia fructigena grown in a liquid pectin/ammonium tartrate medium. ‘Gentle’ mechanical manipulation of mycelium solubilized most of the AF and much of the AP, while brief acid treatment considerably inactivated both enzymes. Both enzymes were present predominantly in a soluble form in homo-genates prepared for subcellular fractionation, but some particulate activity of both was recovered from a sucrose density gradient in a fraction which also contained mitochondria. Azo-dye techniques with appropriate 1-naphthyl derivatives as substrates and p-(acetoxymercuric) aniline diazotate as capturing agent produced similar staining patterns for both enzymes in the light and electron microscopes, but the distribution of β-glycerophosphatase activity as visualized by the Gomori technique was more variable. A proportion of the activity of the enzymes remaining after fixation was located between the plasmalemma and the hyphal wall, in vacuoles in the cytoplasm, and in spherosome-like bodies. Some evidence was obtained for structure-linked latency of both enzymes and for their secretion by a process of reverse pinocytosis.

scholarly journals Subcellular localization of NAD(P)H oxidase(s) in human neutrophilic polymorphonuclear leucocytes

1978 ◽  
Vol 176 (1) ◽  
pp. 175-178 ◽  
Author(s):  
D B Iverson ◽  
P Wang-Iverson ◽  
J K Spitznagel ◽  
L R DeChatelet
Keyword(s):  
Cell Membrane ◽  
Nadph Oxidase ◽  
Subcellular Localization ◽  
Density Gradient ◽  
Membrane Fraction ◽  
Sucrose Density Gradient ◽  
Human Neutrophils ◽  
Polymorphonuclear Leucocytes

NADH and NADPH oxidase activities in a homogenate of human neutrophils co-sediment in a linear sucrose density gradient under either velocity or isopycnic conditions of centrifugation. The position of these activities in the gradient does not correspond to any known subcellular granule or to the cell-membrane fraction. These data suggest that the oxidase activities may reside in a unique granule that has previously not been recognized.

scholarly journals THE SEDIMENTATION PROPERTIES OF THE SKIN-SENSITIZING ANTIBODIES OF RAGWEED-SENSITIVE PATIENTS

1964 ◽  
Vol 120 (1) ◽  
pp. 31-43 ◽  
Author(s):  
Burton R. Andersen ◽  
Wilton E. Vannier
Keyword(s):  
Density Gradient ◽  
Sedimentation Rate ◽  
Gradient Method ◽  
Sucrose Density Gradient ◽  
Major Portion ◽  
Density Gradient Ultracentrifugation ◽  
Cell Method ◽  
Average Value ◽  
Γ Globulins

The sedimentation coefficients of the skin-sensitizing antibodies to ragweed were evaluated by the moving partition cell method and the sucrose density gradient method. The most reliable results were obtained by sucrose density gradient ultracentrifugation which showed that the major portion of skin-sensitizing antibodies to ragweed sediment with an average value of 7.7S (7.4 to 7.9S). This is about one S unit faster than γ-globulins (6.8S). The data from the moving partition cell method are in agreement with these results. Our studies failed to demonstrate heterogeneity of the skin-sensitizing antibodies with regard to sedimentation rate.

Inhibition of Mitochondrial Electron Transfer in Rats by Ethanethiol and Methanethiol

1979 ◽  
Vol 56 (2) ◽  
pp. 147-156 ◽  
Author(s):  
T. Vahlkamp ◽  
A. J. Meijer ◽  
J. Wilms ◽  
R. A. F. M. Chamuleau
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Rat Hepatocytes ◽  
Rat Liver Mitochondria ◽  
Dimethyl Sulphide ◽  
Submitochondrial Particles ◽  
Isolated Rat Hepatocytes ◽  
The Difference ◽  
Isolated Rat

1. We have investigated the effects of ethanethiol, methanethiol and dimethyl sulphide on some metabolic processes of isolated rat hepatocytes, isolated mitochondria from liver and brain and ox-heart submitochondrial particles. 2. Ethanethiol, but not dimethyl sulphide, inhibited both gluconeogenesis and ureogenesis from various substrates in rat hepatocytes, depressed cellular ATP content and caused an increased reduction of the mitochondria. 3. Ethanethiol inhibited respiration in isolated rat-liver mitochondria with several substrates, both in the presence of ADP and phosphate or in the presence of an uncoupling agent. Ethanethiol also inhibited respiration in isolated rat-brain mitochondria. Dimethyl sulphide was much less effective in inhibiting mitochondrial respiration. 4. In ox-heart submitochondrial particles ethanethiol inhibited electron transfer between cytochrome c and oxygen. 5. Purified cytochrome c oxidase was inhibited by ethanethiol in a non-competitive manner. 6. Methanethiol inhibited cytochrome c oxidase and was an effective inhibitor of mitochondrial electron transfer, both in liver and brain. 7. The difference in inhibitory properties between ethanethiol, methanethiol and dimethyl sulphide observed in our experiments coincides with the difference in potency to elicit coma in rats. We suggest that inhibition of mitochondrial electron transfer by mercaptans may be relevant to the mechanism by which energy production in brain is depressed during hepatic coma.

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.

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