Exchange of Cholesterol Between the Subcellular Fractions During Differential Centrifugation of Rat Adrenocortical Tissue

1979 ◽  
pp. 91-93 ◽  
Author(s):  
J. Frühling
Keyword(s):  
Subcellular Fractions ◽  
Differential Centrifugation

20 alpha-Hydroxysteroid dehydrogenase and 17 beta-estradiol dehydrogenase localize in cytosol of human term placenta

1982 ◽  
Vol 242 (3) ◽  
pp. E178-E183
Author(s):  
R. C. Strickler ◽  
B. Tobias
Keyword(s):  
Electron Microscopy ◽  
Dehydrogenase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Subcellular Fractions ◽  
Enzyme Assays ◽  
Marker Enzyme ◽  
Differential Centrifugation ◽  
Experimental Conditions ◽  
Human Term Placenta ◽  
Term Placenta

The 20 alpha-hydroxysteroid dehydrogenase activity in human term placenta has been localized by different investigators to nuclear, mitochondrial, microsomal, and cytosolic subcellular fractions. Furthermore, in the cytosol, 20 alpha-hydroxysteroid dehydrogenase activity may be a second function of the enzyme that mediates 17 beta-estradiol dehydrogenase activity. To search for a unique 20 alpha-hydroxysteroid dehydrogenase, human placental villous tissue, homogenized in three different buffer systems, was fractionated by differential centrifugation, and the 17 beta- and 20 alpha-activities were measured by radioisotope conversion assay. The enrichment and purity of the subcellular fractions were shown by marker enzyme assays and electron microscopy studies. Under all experimental conditions, 20 alpha-hydroxysteroid dehydrogenase activity was identified only in the 105,000 g placental cytosol: intact, osmotically ruptured, and acetone-extracted mitochondria, nuclei, and microsomes did not convert progesterone to 20 alpha-dihydroprogesterone. Furthermore, because 17 beta-estradiol dehydrogenase activity was in large part soluble in the cytosol, these localization studies are consistent with the hypothesis that the 20 alpha- and 17 beta-oxidoreductase activities in human placenta reside on one soluble protein.

THE SUBCELLULAR DISTRIBUTION OF 17β-HYDROXYSTEROID DEHYDROGENASE IN THE HUMAN TERM PLACENTA

1976 ◽  
Vol 82 (1) ◽  
pp. 150-163 ◽  
Author(s):  
C. M. G. Thomas ◽  
J. H. Veerkamp
Keyword(s):  
Dehydrogenase Activity ◽  
Subcellular Distribution ◽  
Medium Composition ◽  
Subcellular Fractions ◽  
Differential Centrifugation ◽  
Marker Enzymes ◽  
Cytosol Fraction ◽  
Human Term Placenta ◽  
Term Placenta ◽  
Buffer Medium

ABSTRACT Human term placenta tissue homogenates were subjected to differential centrifugation procedures. The composition of the subcellular fractions was monitored with a number of marker enzymes and the effectiveness of these enzyme systems was evaluated. The subcellular fractions were tested for their 17β-hydroxy dehydrogenase activity on testosterone, oestradiol and the synthetic substrate retrotestosterone. Buffer medium composition showed a direct influence upon enzyme distribution patterns of all fractions during the same differential centrifugation procedure. All enzyme activities tested became less sedimentable when glycerol was present in the fractionation buffer. Glycerol stabilized soluble 17β-hydroxy dehydrogenase activity during fractionation. The activity of steroid-converting enzymes was inhibited by the presence of glycerol in the medium. Subcellular distribution of marker enzymes did not sustain the presence of mitochondrial 17β-hydroxy dehydrogenase but related it to microsomal contamination. In general the proportion of 17β-hydroxy dehydrogenase activity in the particulate fractions showed a decrease in the substrate order retrotestosterone > testosterone > oestradiol which was independent from the buffer medium used. Specific activities for both particle-bound and soluble 17β-hydroxy dehydrogenase increased in the substrate order retrotestosterone < testosterone < oestradiol. The particulate enzyme activity was maximal with NAD+ for the three substrates tested, but in the cytosol fraction NADP+ was the preferential co-enzyme only when oestradiol was used as the substrate.

Differential centrifugation, calcium precipitation, and ultrasonic disruption of midgut cells of Erinnyis ello caterpillars. Purification of cell microvilli and inferences concerning secretory mechanisms

1986 ◽  
Vol 64 (2) ◽  
pp. 490-500 ◽  
Author(s):  
Custódio D. Santos ◽  
Alberto F. Ribeiro ◽  
Walter R. Terra
Keyword(s):  
Subcellular Fractions ◽  
Soluble Form ◽  
Differential Centrifugation ◽  
Posterior Midgut ◽  
Brush Borders ◽  
Columnar Cells ◽  
Differential Precipitation ◽  
Anterior Midgut ◽  
Erinnyis Ello ◽  
Calcium Precipitation

Subcellular fractions of the cells from the first and last third of midguts from Erinnyis ello caterpillars were obtained by conventional homogenization, followed by differential centrifugation or differential calcium precipitation, as well as by partial ultrasonic disruption. Aminopeptidase was enriched in the subcellular fractions, which in the electron microscope display mainly microvilli from the columnar cells (obtained by differential centrifugation and ultrasonic disruption), and also in the microvilli fraction obtained by differential precipitation. To account for the enzyme activities that sedimented with vesicles displaying brush borders, major amounts of the soluble glycosidases (cellobiase, N-acetylglucosaminidase, maltase, and trehalase) are assumed to be loosely bound to the cell glycocalyx, from where they are set free by homogenization and (or) freezing–thawing. Intracellular glycosidases seem to be bounded by membranes, which sediment together with vesicles that resemble secretory vesicles. The soluble form of amylase occurred mainly associated with the microvilli of anterior midgut cells and is supposed to be contained inside small vesicles, which are seen budding along columnar cell microvilli and fusing one with the other and with the tips of the microvilli from the anterior midgut cells. Secretory mechanisms are discussed in the light of the evidence that the posterior midgut secretes whereas the anterior midgut absorbs water.

CONVERSION OF ANDROST-4-ENE-3,17-DIONE-4-14C TO OESTROGENS BY SUBCELLULAR FRACTIONS OF THE HUMAN CORPUS LUTEUM OF THE NORMAL CYCLE

1967 ◽  
Vol 56 (2) ◽  
pp. 225-230 ◽  
Author(s):  
Rosalinda B. Arceo ◽  
Kenneth J. Ryan
Keyword(s):  
Subcellular Localization ◽  
Corpus Luteum ◽  
Paper Chromatography ◽  
Specific Activity ◽  
Subcellular Fractions ◽  
Corpora Lutea ◽  
Differential Centrifugation ◽  
Normal Cycle ◽  
Generating System ◽  
Human Corpus Luteum

ABSTRACT The subcellular localization of the aromatizing enzymes from four human corpora lutea was investigated. Subcellular fractions were prepared by differential centrifugation, and incubations of each fraction were carried out with androst-4-ene-3,17-dione-4-14C and a TPNH generating system. Oestrogen metabolites were characterized by phenolic separation, repeated paper chromatography, incubation with freshly prepared placental 17β-ol dehydrogenase, methylation and recrystallization to constant specific activity. The experimental results indicate that the aromatizing enzymes were localized mainly in the microsomal fractions of homogenates of human corpora lutea.

Diacylglycerol acyltransferase in maturing sunflower seeds

2000 ◽  
Vol 28 (6) ◽  
pp. 689-692 ◽  
Author(s):  
S. Triki ◽  
J. Ben Hamida ◽  
P. Mazliak
Keyword(s):  
Enzyme Inhibition ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Diacylglycerol Acyltransferase ◽  
Subcellular Fractions ◽  
Tween 20 ◽  
Differential Centrifugation ◽  
Sunflower Seeds ◽  
Triton X

Developing sunflower seeds exhibit a high diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) activity. The distribution of the enzyme has been studied in subcellular fractions prepared by differential centrifugation of seed homogenate. Its activity was characterized using [1-14C]oleoyl-CoA and diolein dispersed in Tween 20. Some properties of the microsomal fraction of DAGAT were investigated. Hyperbolic kinetics were observed, the apparent Km was 60 μM and the specific activity of the reaction 15 pmol/min/mg of protein. Addition of BSA (0.1%) stimulated oleate incorporation, which was not dependent on the presence of exogenous diacylglycerol. Detergents which might solubilize DAGAT, Triton X-100 and CHAPS, were tested for enzyme inhibition, and CHAPS was found to be the least denaturing.

scholarly journals Preparation of Crude Subcellular Fractions by Differential Centrifugation

2002 ◽  
Vol 2 ◽  
pp. 1638-1642 ◽  
Author(s):  
John Graham
Keyword(s):  
Density Gradient ◽  
Buoyant Density ◽  
Mitochondrial Fraction ◽  
Subcellular Fractions ◽  
Low Density ◽  
Differential Centrifugation ◽  
Density Gradient Purification

The employment of differential centrifugation to prepare crude fractions of subcellular particles from homogenates is often a necessary first step to a subsequent purification of one or more particles on a density gradient. Buoyant density gradient purification of peroxisomes or lysosomes for example is almost invariably carried out on a light mitochondrial fraction so as to eliminate smaller particles that may have similar densities. Unless they are first removed, large rapidly sedimenting particles in homogenates may also disturb shallow gradients designed to fractionate small low-density microsomes.

scholarly journals Purification of an inositol 1,4,5-trisphosphate-binding calreticulin-containing intracellular compartment of HL-60 cells

1992 ◽  
Vol 281 (3) ◽  
pp. 651-656 ◽  
Author(s):  
C Van Delden ◽  
C Favre ◽  
A Spät ◽  
E Cerny ◽  
K H Krause ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Storage Protein ◽  
Binding Sites ◽  
Subcellular Fraction ◽  
Sucrose Density Gradient ◽  
Subcellular Fractions ◽  
Differential Centrifugation ◽  
Similar Extent ◽  
Inositol 1,4,5 Trisphosphate

To investigate the identity of Ins(1,4,5)P3-sensitive intracellular Ca2+ stores in myeloid cells, we have developed a method that yields subcellular fractions highly enriched in Ins(1,4,5)P3 binding. HL-60 cells were disrupted by nitrogen cavitation, and subcellular fractions were obtained by differential centrifugation, followed by Percoll- and sucrose-density-gradient separations. A subcellular fraction enriched 26-fold in Ins(1,4,5)P3-binding sites was obtained. This fraction showed no enrichment in plasma-membrane markers and only a comparatively moderate enrichment (7-fold) in endoplasmic-reticulum markers. The ratio between specific enrichment of Ins(1,4,5)P3 binding and endoplasmic-reticulum markers in the different fractions varied over 50-fold, from less than 0.1 to greater than 5. The purified Ins(1,4,5)P3-binding fraction was enriched to a similar extent (27-fold) in the putative intravesicular Ca(2+)-storage protein calreticulin. Our results favour the concept of a distinct Ins(1,4,5)P3-binding, calreticulin-containing compartment (i.e. the calciosome) in HL-60 cells.

INCORPORATION OF RADIOACTIVE PRECURSORS INTO THE MEMBRANE AND CONTENTS OF THE NEUROSECRETORY GRANULES OF THE RAT NEUROHYPOPHYSIS AS A METHOD OF STUDYING THEIR FATE

1976 ◽  
Vol 68 (1) ◽  
pp. 95-108 ◽  
Author(s):  
R. W. SWANN ◽  
B. T. PICKERING
Keyword(s):  
Membrane Fraction ◽  
Microsomal Fraction ◽  
Subcellular Fractions ◽  
Secretory Product ◽  
Neurosecretory Granules ◽  
Differential Centrifugation ◽  
Nacl Solution ◽  
Neurohypophysial Hormones ◽  
Intracisternal Injection ◽  
Membrane Bound

SUMMARY Rat neural lobes have been separated into subcellular fractions by differential centrifugation at various times after an intracisternal injection of [35S]cysteine or [3H]choline. Both isotopes led to a rise and fall in the radioactivity of neurosecretory granules (NSG) which paralleled that found previously for the neurohypophysial hormones and the neurophysins. While the radioactivity of the NSGs resulting from [35S]cysteine injection was predominantly associated with granular contents, [3H]choline injections led to a preferential labelling of the granular membrane. There was no indication of a sequential movement of radioactivity from the NSG-membrane fraction into the microsomal fraction (containing the so-called small vesicles) which might be expected if granular membrane were recaptured as small vesicles after release of secretory product by exocytosis. When release was stimulated in injected animals by giving them 2% NaCl solution to drink, 35S disappeared from the gland as expected, but 3H was retained and, moreover, appeared in the NSG-membrane fraction; results compatible with membrane conservation occurring by recapture of large vesicles. There was an indication that some of the neurophysin in the NSG was membrane-bound and that this too was retained after release of the granular contents.

THE ROLE OF NEUROPHYSIN IN THE TRANSPORT ANDRELEASE OF NEUROHYPOPHYSIAL HORMONES

1966 ◽  
Vol 35 (3) ◽  
pp. 289-NP ◽  
Author(s):  
M. GINSBURG ◽  
M. IRELAND
Keyword(s):  
Soluble Protein ◽  
Water Soluble ◽  
Subcellular Fractions ◽  
Neurosecretory Granules ◽  
Differential Centrifugation ◽  
Particle Fraction ◽  
Neurohypophysial Hormones ◽  
Ph Values ◽  
Water Soluble Protein

SUMMARY The distribution of neurophysin in subcellular fractions obtained by differential centrifugation of bovine neurohypophyses is essentially similar to the distribution of the hormones. The neurosecretory particle fraction contained more neurophysin than other fractions and neurophysin comprised half of its water soluble protein. Estimates were derived for the concentrations of oxytocin, vasopressin and neurophysin in neurosecretory granules. In experiments in which neurosecretory granules were suspended in media at pH values between 6·2 and 8·0 it was found that release of hormones increased with increasing pH. The role of neurophysin in the transport and release of neurohypophysial hormones is discussed.

SECRETIN-LIKE ACTIVITY IN SUBCELLULAR FRACTIONS OF CHICKEN INTESTINAL MUCOSA

1974 ◽  
Vol 63 (1) ◽  
pp. 239-245 ◽  
Author(s):  
I. J. S. FIDDES ◽  
C. N. A. TROTMAN
Keyword(s):  
Hydrochloric Acid ◽  
Intestinal Mucosa ◽  
Secretory Response ◽  
Subcellular Fractions ◽  
Heat Denaturation ◽  
Differential Centrifugation ◽  
Large Proteins ◽  
The Relationship

SUMMARY Differential centrifugation of chicken intestinal mucosal homogenates showed that particles containing a material with secretin-like activity can be sedimented. The activity appeared to be contained in structures smaller than mitochondria. Approximately 70% of the stimulatory effect of the whole homogenate was sedimented in 10 min at 15000 g. The secretin-like material was released from the particles by treatment with hydrochloric acid and partially purified by heat denaturation of large proteins at pH 3. The relationship between relative dose of mucosal extract and secretory response in the anaesthetized cat was slightly different from that of porcine secretin.

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