Subcellular fractionation of the porcine corpus luteum: sequestration of progesterone in a unique particulate fraction

1988 ◽  
Vol 117 (3) ◽  
pp. 341-354 ◽  
Author(s):  
T. A. Bramley ◽  
G. S. Menzies
Keyword(s):  
Cytochrome C ◽  
Cell Surface ◽  
Corpus Luteum ◽  
Reductase Activity ◽  
Buoyant Density ◽  
Particulate Fraction ◽  
Luteal Cell ◽  
Cytochrome C Reductase ◽  
Intracellular Organelles ◽  
Nadh Cytochrome

ABSTRACT Homogenates of porcine corpus luteum were subjected to fractionation by differential-rate centrifugation or sucrose density gradient fractionation, with or without pretreatment with digitonin. Fractions of each gradient were assayed for a number of markers characteristic of the major intracellular organelles and cell-surface membranes, and for progesterone content. The majority of the progesterone content of homogenates of porcine corpus luteum was associated with a low-density particulate fraction which equilibrated at a buoyant density of 1·07–1·09 g/cm3. Pretreatment with digitonin increased the buoyant density of the progesterone-enriched fraction markedly (to 1·13–1·15 g/cm3) without causing release of steroid. The density distributions of progesterone content in control and digitonin-treated luteal gradient fractions were quite distinct from those of the major intracellular organelles and luteal cell-surface membranes. However, NADH–cytochrome C reductase activity (but not other endoplasmic reticulum markers) was also enriched in this fraction. The results suggest that most of the progesterone of the porcine corpus luteum is associated with a unique particulate fraction which is enriched in digitonin-reactive lipids and NADH–cytochrome C reductase activity. J. Endocr. (1988) 117, 341–354

scholarly journals Properties of a cytochrome c-enriched light particulate fraction isolated from the photosynthetic bacterium Rhodopseudomonas spheroides

1978 ◽  
Vol 174 (1) ◽  
pp. 267-275 ◽  
Author(s):  
J Barrett ◽  
C N Hunter ◽  
O T G Jones
Keyword(s):  
Cytochrome C ◽  
Sodium Dodecyl ◽  
Photosynthetic Bacterium ◽  
Particulate Fraction ◽  
Cytochrome C Reductase ◽  
Cytochrome C2 ◽  
Succinate Cytochrome C Reductase ◽  
Bulk Membrane ◽  
Nadh Cytochrome ◽  
Rhodopseudomonas Spheroides

Differential centrifugation of suspensions of French-press-disrupted Rhodopseudomonas spheroides yielded a light particulate fraction that was different in many properties from the bulk membrane fraction. It was enriched in cytochrome c and had a low cytochrome b content. When prepared from photosynthetically grown cells this fraction had a very low specific bacteriochlorophyll content. The cytochrome c of the light particles differed in absorption maxima at 77K from cytochrome c2 attached to membranes; there was pronounced splitting of the alpha-band, as is found in cytochrome c2 free in solution. Potentiometric titration at A552–A540 showed the presence of two components that fitted an n = 1 titration; one component had a midpoint redox potential of +345mV, like cytochrome c2 in solution, and the second had E0′ at pH 7.0 of +110 mV, and they were present in a ratio of approx. 2:3. Difference spectroscopy at 77K showed that the spectra of the two components were very similar. More of a CO-binding component was present in particles from photosynthetically grown cells. Light membranes purified by centrifugation on gradients of 5–60% (w/w) sucrose retained the two c cytochromes; they contained no detectable succinate-cytochrome c reductase or bacteriochlorophyll and very little ubiquinone, but they contained NADH-cytochrome c reductase and some phosphate. Electrophoresis on sodium dodecyl sulphate/polyacrylamide gels showed that the light membranes of aerobically and photosynthetically grown cells were very similar and differed greatly from other membrane fractions of R. spheroides.

Brain Regional Analysis of NADH-Cytochrome C Reductase Activity in Alzheimerʼs Disease

1990 ◽  
Vol 49 (3) ◽  
pp. 206-214 ◽  
Author(s):  
GEORGE S. ZUBENKO ◽  
JOHN MOOSSY ◽  
DIANA CLAASSEN ◽  
A. Julio Martinez ◽  
GUTTI R. RAO
Keyword(s):  
Cytochrome C ◽  
Reductase Activity ◽  
Regional Analysis ◽  
Cytochrome C Reductase ◽  
Nadh Cytochrome

Assignment of estradiol-17β dehydrogenase and of estrone reductase to cytoplasmic structures of porcine endometrium cells

1989 ◽  
Vol 121 (2) ◽  
pp. 161-167 ◽  
Author(s):  
J. Adamski ◽  
W. D. Sierralta ◽  
P. W. Jungblut
Keyword(s):  
Cytochrome C ◽  
Kinetic Data ◽  
Reductase Activity ◽  
Particulate Fraction ◽  
Marker Enzyme ◽  
Structural Element ◽  
Cytochrome C Reductase ◽  
Percoll Gradients ◽  
Cytoplasmic Structures ◽  
Estradiol 17Β

Abstract. Homogenates of porcine endometrium contain substantial activity for the dehydrogenation of estradiol-17β but little for estrone reduction. Both activities are associated with cytoplasmic structures. The dehydrogenase is characterized by a pH 7.7 optimum, Km 2.2 × 10−7 mol/l for estradiol and Km 4.4 × 10−5 mol/l for the cosubstrate NAD+. The corresponding figures for the reductase are pH 6.6, Km 1.1 × 10−6 mol/1 for estrone and Km 2.1 × 10−5 mol/l for the cosubstrate NADPH. The (mitochondrial/lysosomal) 17 000 × g particulate fraction contains a 52-fold higher dehydrogenase than reductase activity. The (microsomal) 200000 × g particulate fraction is only 16-fold richer in dehydrogenase. Isopycnic centrifugations of the two fractions in Percoll gradients reveal that estrone reductase and the coequilibrating marker enzyme cytochrome c reductase occur in constant proportions, whereas the dehydrogenase/cytochrome c reductase ratios are different. Both, the kinetic data and the structural assignments speak in favour of individual enzymes catalyzing the dehydrogenation of estradiol and the reduction of estrone. All gradient fractions exhibiting dehydrogenase activity feature small, electrondense vesicles of 0.15–0.20 μm in diameter as a common structural element which might harbour the dehydrogenase.

scholarly journals Structural and functional relationships of enzyme activities induced by nitrate in barley

1970 ◽  
Vol 119 (4) ◽  
pp. 715-725 ◽  
Author(s):  
John L. Wray ◽  
Philip Filner
Keyword(s):  
Cytochrome C ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Bottom Layer ◽  
Sucrose Density Gradient ◽  
Enzyme Complex ◽  
Cytochrome C Reductase ◽  
Functional Relationships ◽  
Nadh Cytochrome

1. Nitrate induces the development of NADH-nitrate reductase (EC 1.6.6.1), FMNH2–nitrate reductase and NADH–cytochrome c reductase activities in barley shoots. 2. Sucrose-density-gradient analysis shows one band of NADH–nitrate reductase (8S), one band of FMNH2–nitrate reductase activity (8S) and three bands of NADH–cytochrome c reductase activity (bottom layer, 8S and 3.7S). Both 8S and 3.7S NADH–cytochrome c reductase activities are inducible by nitrate, but the induction of the 8S band is much more marked. 3. The 8S NADH–cytochrome c reductase band co-sediments with both NADH–nitrate reductase activity and FMNH2–nitrate reductase activity. Nitrite reductase activity (4.6S) did not coincide with the activity of either the 8S or the 3.7S NADH–cytochrome c reductase. 4. FMNH2–nitrate reductase activity is more stable (t½ 12.5min) than either NADH–nitrate reductase activity (t½ 0.5min) or total NADH–cytochrome c reductase activity (t½ 1.5min) at 45°C. 5. NADH–cytochrome c reductase and NADH–nitrate reductase activities are more sensitive to p-chloromercuribenzoate than is FMNH2–nitrate reductase activity. 6. Tungstate prevents the formation of NADH–nitrate reductase and FMNH2–nitrate reductase activities, but it causes superinduction of NADH–cytochrome c reductase activity. Molybdate overcomes the effects of tungstate. 7. The same three bands (bottom layer, 8S and 3.7S) of NADH–cytochrome c reductase activity are observed irrespective of whether induction is carried out in the presence or absence of tungstate, but only the activities in the 8S and 3.7S bands are increased. 8. The results support the idea that NADH–nitrate reductase, FMNH2–nitrate reductase and NADH–cytochrome c reductase are activities of the same enzyme complex, and that in the presence of tungstate the 8S enzyme complex is formed but is functional only with respect to NADH–cytochrome c reductase activity.

Specific binding sites for progesterone in subcellular fractions of the porcine corpus luteum

1994 ◽  
Vol 142 (1) ◽  
pp. 101-110 ◽  
Author(s):  
G S Menzies ◽  
T A Bramley
Keyword(s):  
Corpus Luteum ◽  
Early Pregnancy ◽  
Binding Sites ◽  
Luteal Phase ◽  
Secretory Granules ◽  
Buoyant Density ◽  
Subcellular Fractions ◽  
Particulate Fraction ◽  
Luteal Cell

Abstract Subcellular fractionation of porcine corpus luteum (CL) homogenates on continuous sucrose gradients has previously demonstrated that most of the endogenous progesterone of the CL was associated with a unique particulate fraction. Exogenous radiolabelled steroids were also sequestered with some specificity by this fraction. We now report that this particulate fraction is capable of binding high levels of exogenous 3H-labelled progesterone (and pregnenolone) in vitro, but only in the presence of the saponin, digitonin. Binding was dependent on the pH, temperature and duration of incubation, and showed specificity and high affinity for progesterone (Kd, 79 nm). Androgens, oestrogens and pregnenolone competed for porcine luteal [3H] progesterone binding sites, but only at much higher concentrations, whereas cholesterol, a number of progesterone receptor agonist and antagonist analogues and inhibitors of 3β-hydroxysteroid dehydrogenase and C17-hydroxylase/C17,20-lyase did not compete. Analysis of profiles for a number of luteal cell-surface membrane and intracellular organelle markers confirmed previous studies showing the association of an NADH-cytochrome C reductase with this fraction. Moreover, the content of endogenous progesterone associated with particulate subcellular fractions isolated from porcine granulosa cell (GC) and CL homogenates at different stages of the luteal phase and early pregnancy waxed and waned with the stage of the luteal phase (and the secretory activity of the CL). Binding of [3H]progesterone in vitro equilibrated at the same buoyant density as endogenous progesterone: levels of both were highest during the mid-luteal phase and during early pregnancy, lower in early and late luteal CL, and undetectable in corpora albicantia. In contrast, relaxin secretory granules were readily resolved from progesterone binding sites. We propose that these particulate progesterone binding sites may be involved in the sequestration and/or packaging of newly-synthesized steroid for secretion by the luteal cell, or may mediate actions of progesterone within the luteal cell. Journal of Endocrinology (1994) 142, 101–110

scholarly journals Inhibition and inactivation of NADH-cytochrome c reductase activity of bovine heart submitochondrial particles by the iron(III)-adriamycin complex

1990 ◽  
Vol 265 (3) ◽  
pp. 865-870 ◽  
Author(s):  
B B Hasinoff
Keyword(s):  
Cytochrome C ◽  
Reductase Activity ◽  
Inner Mitochondrial Membrane ◽  
Submitochondrial Particles ◽  
Cytochrome C Reductase ◽  
Fast Phase ◽  
Bovine Heart ◽  
Direct Binding ◽  
Nadh Cytochrome

The NADH-cytochrome c reductase activity of bovine heart submitochondrial particles was found to be slowly (half-time of 16 min) and progressively lost upon incubation with the Fe2(+)-adriamycin complex. In addition to this slow progressive inactivation seen on incubation, a reversible fast phase of inhibition was also seen. However, if EDTA was added to the incubation mixture within 15 s, the slow progressive loss in activity was largely preventable. Separate experiments indicated that EDTA removed about one-half of the iron from the Fe2(+)-adriamycin complex in about 40 s. These results indicated the requirement for iron for the inactivation process. Since the Vmax. for the fast phase of inhibition was decreased by the inhibitor, the inhibition pattern was similar to that seen for uncompetitive or mixed-type inhibition. The direct binding of both Fe3(+)-adriamycin and adriamycin to submitochondrial particles was also demonstrated, with the Fe3(+)-adriamycin complex binding 8 times more strongly than adriamycin. Thus binding of Fe3(+)-adriamycin to the enzyme or to the inner mitochondrial membrane with subsequent generation of oxy radicals in situ is a possible mechanism for the Fe3(+)-adriamycin-induced inactivation of respiratory enzyme activity.

NADH-cytochrome c reductase activity in cultured human lymphocytes

1976 ◽  
Vol 176 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Russell A. Prough ◽  
Richard L. Imblum ◽  
Richard A. Kouri
Keyword(s):  
Cytochrome C ◽  
Reductase Activity ◽  
Human Lymphocytes ◽  
Cytochrome C Reductase ◽  
Nadh Cytochrome

Association of progesterone with a unique particulate fraction of the human corpus luteum

1988 ◽  
Vol 116 (2) ◽  
pp. 307-312 ◽  
Author(s):  
T. A. Bramley ◽  
G. S. Menzies
Keyword(s):  
Plasma Membrane ◽  
Corpus Luteum ◽  
Plasma Membranes ◽  
Buoyant Density ◽  
Particulate Fraction ◽  
Luteal Cell ◽  
Cell Plasma Membrane ◽  
Intracellular Organelle ◽  
Cell Plasma ◽  
Human Corpus Luteum

ABSTRACT Homogenates of human corpus luteum were fractionated on continuous sucrose density gradients, with and without pretreatment with digitonin to perturb plasma membranes. Fractions of each gradient were assayed for steroid content and a range of plasma membrane and intracellular organelle markers. Progesterone and oestradiol were associated with a particulate fraction (buoyant density, 1·08–1·13 g/cm3). The buoyant density distribution of these steroids was distinct from those of the luteal cell plasma membrane and intracellular organelle markers tested. Treatment with digitonin increased the buoyant density of both progesterone and oestradiol. If steroids are contained in distinct vesicles, these vesicles may be involved in the sequestration of newly synthesized steroid and its movement to the cell surface for release into the circulation. J. Endocr. (1988) 116, 307–312

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