Distribution of estrogen receptors in subfractions of hen oviduct chromatin.

Hen oviduct chromatin was digested with DNase II and separated into two fractions. The MgCl2 insoluble chromatin fraction (43% of the total DNA) was enriched in nucleosome-like particles, which sedimented at 11 S and contained 185 base pairs of DNA. The MgCl2 soluble chromatin fraction (5% of the total DNA) was characterized by 5 S and 14 S peaks in sucrose gradients; Estrogen receptors in the chromatin fractions were labelled with [3H] estradiol using the steroid exchange assay. The concentration of receptors in the MgCl2 soluble chromatin was 4;5 times higher than that in the MgCl2 insoluble chromatinmin sucrose gradient analysis the 11 S particles displayed a negligible specific radioactivity suggesting that estrogen receptors mainly bind to extranucleosomal chromatin.     

The localization of glycollate-pathway enzymes in Euglena.

Isolation of organelles from broken-cell suspensions of phototrophically grown Euglena gracilis Klebs was achieved by isopycnic centrifugation on sucrose gradients. 2. Equilibrium densities of 1.23g/cm3 for peroxisome-like particles, 1.22g/cm3 for mitochondria and 1.17g/cm3 for chloroplasts were recorded. 3. The enzymes glycollate dehydrogenase, glutamate-glyoxylate aminotransferase, serineglyoxylate aminotransferase, aspartate-alpha-oxoglutarate aminotransferase, hydroxy pyruvate reductase and malate dehydrogenase were present in peroxisome-like particles. 4. Unlike higher plants glycollate dehydrogenase and glutamate-glyoxylate aminotransferase were present in the mitochondria of Euglena. 5. Rates of glycollate and D-lactate oxidation were additive in the mitochondria, and, although glycollate dehydrogenase was inhibited by cyanide, D-lactate dehydrogenase activity was unaffected. 6. Glycollate oxidation was linked to O2 uptake in mitochondria but not in peroxisome-like particles. This glycollate-dependent O2 uptake was inhibited by antimycin A or cyanide. 7. The physiological significance of glycollate metabolism in Euglena mitochondria is discussed, with special reference to its role in photorespiration in algae.     

Studies on the sub-cellular location of particulate nitrate and nitrite reductase,glutamic dehydrogenase and other enzymes in barley roots

Summary The distribution of nitrate and nitrite reductase, glutamic dehydrogenase, cytochrome oxidase, fumarase, peroxidase and catalase in particular fractions of barley roots, separated by differential and density gradient centrifugation, has been determined. Evidence obtained suggests that there are three separate groups of particles, one, the mitochondria, containing cytochrome oxidase, fumarase and glutamic dehydrogenase, one containing catalase, and one containing nitrate and nitrite reductase. The results show that, under certain conditions, the high osmotic pressures obtained in sucrose density gradients may cause artefacts due to the release of enzymes, especially nitrite reductase, from the particles.     

Estradiol-17β receptors in the immature rat ovary

Estradiol binding components in the cytosol and nuclear fractions of the ovary from immature rats (22–28 days old) were characterized by $\text{in vitro}$ methods. Several of the biochemical characteristics of the estradiol binding components in the ovarian tissue were compared with the estradiol receptor from the uterus. The results suggest that the ovarian estradiol binding components are similar to the specific high affinity estradiol receptors in the uterus. In the cytosol of intact rat ovary a significant fraction of the total binding sites was found to be occupied, presumably by the endogenous estrogen. Following hypophysectomy there was a significant increase in the available cytosol binding sites. Evidence for translocation of cytosol receptor-estrogen (RE) complex to the nucleus was obtained for the ovary. The sedimentation properties of the RE complex of the ovary and the uterus are similar. The ovarian cytosol RE complex sediments at 7-8S in glycerol gradients at low ionic strength and at 4S in sucrose gradients at high ionic strength. Following extraction with 0.4 M KCl the ovarain nuclear RE complex sediments at 5S in sucrose gradients which is identical to that of the uterine nuclear receptor.     

Identification of a catalase-negative sub-population of peroxisomes induced in mouse liver by clofibrate.

The peroxisomal compartment in mouse liver was investigated using rate sedimentation of liver subfractions on sucrose density gradients. Treatment of mice with clofibrate, a hypolipidemic agent and peroxisome proliferator, resulted in the formation of small particles which were devoid of catalase and urate oxidase, but which were identified as peroxisomal on the basis of content of the clofibrate-induced peroxisomal beta-oxidation enzymes (fatty acyl-CoA oxidase, hydratase/dehydrogenase bifunctional protein, and thiolase) and the 68 kDa peroxisomal integral membrane protein. Immunoelectron microscopy confirmed the membrane-bound organellar nature and enzyme composition of these particles. These particles were absent in normal mice, and were increased to a maximal level within 2 days of clofibrate treatment. These data have been taken as indicative of a role of these particles in the mechanism of drug-induced peroxisome proliferation.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.     

SEPARATION OF CATECHOLAMINE STORING SYNAPTOSOMES IN COLLOIDAL SILICA DENSITY GRADIENTS

Abstract— Catecholamine storing particles mainly from rat brain hypothalamus and corpus striatum have been isolated by isopycnic centrifugation in density gradients made of colloidal silica. As markers, tritium-labelled noradrenaline, endogenous noradrenaline and dopamine were measured. Cytochrome oxidase was determined as an indicator of mitochondria.
Two distinct populations of amine containing particles were recognized with densities of 1 , 03–1.04 g/ml and 1 , 045–1.065 g/ml in continuous isotonic gradients made of silica sol and a polymer. The light fraction was assumed to contain myelin fragments, light synaptosomes and possibly also catecholamine storage vesicles, while the other one was probably a heavy population of synaptosomes containing more mitochondria. Free mitochondria were found in a band at a density of 1 , 09–1.11.
The distribution pattern in isotonic gradients was compared with that in density gradients made of silica sol and sucrose or sucrose alone. The heavy population of the catecholamine particles was found to have a higher density in hypertonic gradients. Furthermore these synaptosomes seemed to lose more mitochondria and catecholamines than those in isotonic gradients probably due to the hypertonicity.
The present results confirm similar findings by other workers separating brain sub- cellular particles in isotonic gradients of Ficoll and sucrose.
Colloidal silica solutions might be of value for analytical centrifugation of brain sub-cellular particles, since it has a lower tonicity than sucrose, lower viscosity than Ficoll and furthermore it is very easy to handle. The silica sol is inexpensive and allows large scale work.     

Subcellular fractionation of striatum: Sedimentation properties of dopaminergic synaptosomes

Linear sucrose density gradient centrifugation of a crude synaptosomal-mitochondrial preparation of rat striatum was performed at 82, 500g for 7.5, 15 and 30 min and 1, 4 and 20 h. After centrifugation various marker enzyme activities were measured throughout the gradients, viz. tyrosine hydroxylase (TH) and DOPA decarboxylase (DD) as markers of dopaminergic synaptosomes, lactate dehydrogenase (LDH) as a general synaptosomal marker and monoamine oxidase (MAO) as a mitochondrial marker. At all centrifugation times the distribution patterns of TH and DD activity coincided almost perfectly. Notable differences were found between the sedimentation properties of these TH/DD-containing particles and LDH-containing particles: TH and DD were symmetrically distributed in the gradient much sooner than LDH, at all centrifugation times the top of the TH and DD curves was lying deeper in the gradient than the highest LDH activity, and Th and DD became enriched in the gradients to a much greater extent than LDH. It is concluded that rat striatal dopaminergic synaptosomes form a relatively homogenous population of particles sedimenting faster into the gradients than the bulk of striatal synaptosomes does. This distinct sedimentation behaviour of the dopaminergic synaptosomes can be usefully applied for analytical purposes.     

Identification of an androgen receptor in the adult chicken oviduct

The chicken oviduct androgen receptor was characterized by sucrose density gradient centrifugation, Scatchard analysis, competition studies, and affinity labeled with dihydrotestosterone 17 beta-bromoacetate. A specific 8.5 S peak was seen on 0.01 M KCl sucrose density gradients when the receptor was labeled with [3H]5 alpha-dihydrotestosterone. Specific 4.6 S peaks were seen when receptor labeled with [3H]5 alpha-dihydrotestosterone or [3H]dihydrotestosterone 17 beta-bromoacetate was analyzed on 0.3 M KCl sucrose density gradients. Scatchard analysis of [3H]5 alpha-dihydrotestosterone binding by oviduct cytosol was consistent with two binding sites. A Kd of 0.13 nM was found for the high affinity androgen receptor. Competition studies showed the following order of ligand affinity: 5 alpha-dihydrotestosterone greater than dihydrotestosterone 17 beta-bromoacetate greater than progesterone greater than estradiol. A 61.2 kDa protein was specifically covalently labeled with [3H]dihydrotestosterone 17 beta-bromoacetate. The chicken oviduct androgen receptor possesses characteristics similar to other androgen receptors, and provides a good source of androgen receptor for physicochemical studies of the native receptor protein.     

Properties of Intracytoplasmic A Particles Isolated from Oncornavirus-Producing Human Cells

A particles with the diameter of 70 to 80 nm were isolated from the cytoplasm of HEp-2, HeLa, and AO cells producing oncornavirus of Mason-Pfizer-like type. Most of the A particles banded at 1.23 to 1.24 g/ml, whereas 3 to 10% banded at 1.29 g/ml in equilibrium sucrose gradients. They banded at 1.30 g/ml in CsCl gradients suggesting that they contained 8% RNA. Individual A particles sedimented at 200 to 250S in velocity sucrose gradients, but their significant part was found aggregated and sedimented at more than 300S. They were resistant to RNase digestion. A particles possessed polymerase activity which was preferentially activated by Mn(2+) rather than by Mg(2+), the RNA template being 60S RNA. Cross-hybridization with two DNA products and immunoassay showed that A particles and Mason-Pfizer-like oncornavirus produced by the same cells contained neither homological RNA sequences nor common antigens, suggesting that A particles are not intracellular precursors of Mason-Pfizer-like oncornavirus but represent an independent oncornavirus. Hybridization of A particle RNA with excess of cellular DNA revealed about 20 proviral copies per HEp-2 cell genome and no proviral copies in human embryo and placenta cell genomes.     

Zonal electrophoresis and isoelectric focusing of proteins and virus particles in density gradients of small volume

Proteins and virus particles were separated by zonal electrophoresis or isoelectric focusing in glass tubes of small volume. The tubes were covered at the bottom with dialysis membranes and sucrose gradients containing either buffer or ampholytes were generated directly into them. When ampholytes were employed, reproducible pH gradients were generated during electrophoresis. After the separations were finished, dense sucrose was pumped into the bottom of each tube and the gradient was fractionated from the top; the recovery of virus was nearly complete.     

INTRACELLULAR LOCALIZATION OF PHENOL SULPHOTRANSFERASE IN RAT BRAIN

—The intracellular localization of phenol sulphotransferase in rat brain was studied The distribution pattern found after differential centrifugation closely resembles that of lactate dehydrogenase and does not change during postnatal development. The distribution of the enzyme in discontinuous and continuous sucrose gradients, however, shows a deviation from the lactate dehydrogenase pattern and a shift towards a higher sucrose concentration during development. In the adult the phenol sulphotransferase coincides with monoamine oxidase, succinate dehydrogenase and β-glucuronidase. Disruption experiments, purification of mitochondria and electron microscopy exclude localization of phenol sulphotransferase in mitochondria. These studies support the idea of phenol sulphotransferase as a cytoplasmic enzyme with a preferential binding to or localization in oligodendroglial cells or, more probably, a specific type of synaptosomes.     

Studies of the housefly virus structure and disruption during purification procedures

When purifying preparations of the newly discovered virus from the housefly Musca domestica, the virus particles were rendered undetectable by electron microscopic examination, following attempted separation on density gradients of sucrose prepared in distilled water. When this procedure was repeated using gradients consisting of sucrose in phosphate buffer (pH 7.2), virus fragments were found during E.M. examination of the virus fractions. Ficoll 400 density gradients in phosphate buffer were also used in attempts to purify the virus. These resulted in breakdown of the virus and the production of small virus-like particles, which were interpreted as being the virus nucleoprotein cores. The structures revealed in partially degraded virions during the extraction procedures, with special reference to the second treatment with carbon tetrachloride and subsequent purification procedures, have suggested a possible structure for the virion. From these studies it is emphasized that a proteinaceous middle layer exists between the inner shell and the virus nucleoprotein core.     

Presence of three different estradiol binding proteins in rat prostate cytosol

Depending upon the experimental conditions, three distinct [³H]-17β-estradiol binding entities can be detected in rat prostate cytosol. A protein with characteristics similar to other estrogen receptors is found in prostate cytosol from intact rats. This protein has a sedimentation coefficient of 8S on sucrose gradients. It has a high affinity only for natural and synthetic estrogens. It decreases rapidly after castration (less than 20% of binding activity after 24 hr) and reappears progressively after 8–10 days.A second binding entity with a sedimentation coefficient of 4–5S on sucrose gradients is also observed. It is found both in intact and castrated rats. At low temperatures, the binding of estradiol increases progressively and reaches a maximum after 24–48 hr of incubation as determined by charcoal assay. This binding species is highly specific for natural estrogens but not for diethylstilbestrol and is probably identical with the 4–5S binder.Finally, a third binding entity is found in 24 hr castrated rats with short incubations at 0°C This binding is labile even at low temperatures. Natural and synthetic androgens compete more strongly than estradiol for this binding. This behaviour suggests that the third estradiol binding entity is identical with the androgen receptor.     

Subcellular localisation of purine-metabolising enzymes in Leishmania mexicana mexicana

Leishmania mexicana mexicana cultured promastigotes were fractionated by isopycnic centrifugation on linear sucrose gradients. Guanine, hypoxanthine and xanthine phosphoribosyltransferase activities were found to be associated with glycosomes, whereas adenine phosphoribosyltransferase was cytosolic. 3'- and 5'-nucleotidases and IMP dehydrogenase were shown to be particulate, the former two possibly being associated with the plasma membrane, IMP dehydrogenase with the endoplasmic reticulum. Nucleosidases and deaminases were found to be cytosolic. The results demonstrate that intracellular separation of enzymes could play a part in the regulation of the parasite's purine metabolism.     

Regional and subcellular distribution of choline acetyltransferase in the brain of rats

—Homogenates of corpus striatum, cerebral cortex and hypothalamus excised from rat brain were fractionated on discontinuous Ficoll and sucrose density gradients, and the distribution of choline acetyltransferase (ChAc) in the mitochondrial and synaptosomal fractions was determined. In the hypothalamic and cortical regions the fractions enriched in synaptosomes showed much higher activity of ChAc than those containing mainly mitochondria. On the other hand, the corpus striatum showed an equal distribution of ChAc activity in those two fractions. The localization of ChAc was also studied in the postnuclear supernatants obtained from three brain regions, using continuous sucrose density gradients. The distribution of ChAc was compared to that of monoamine oxidase (MAO), potassium and protein. When the pellets obtained from the fractions collected from the gradient were suspended in sucrose, the peak of ChAc activity was close to that of MAO in all three brain regions. When 0.1 mm EDTA +1% butanol was used in order to liberate the occluded form of ChAc, the maximum liberation occurred in lighter fractions, resulting in a shift of the activity peak toward the top of the gradient. This was found with fractions from hypothalamic and cortical regions. In the striatum, the liberated ChAc remained in the same fractions as the occluded enzyme. The results indicate that ChAc is liberated only in those fractions where it is present in synaptosomes. In agreement with the results on the discontinuous gradients this occurs in particles of lower density than mitochondria in cortex and hypo-thalamus, but in particles of similar density to mitochondria in the corpus striatum, indicating regional differences in the distribution of ChAc in the brain. K+ containing particles centrifuged in less dense fractions than those containing ChAc, indicating that synaptosomes are heterogeneous with respect to these two marker substances.     

Use of dextran-40 gradients for separation of pea cotyledon mitochondria into different fractions

A crude pea (Pisum sativum L. var. Homesteader) mitochrondrial preparation was divided into two equal parts. One part was layered on a Dextran-40 step gradient, and the other on a sucrose step gradient, and they were centrifuged to obtain different bands of particles. The densities at which the particles banded and the mitochondrial respiratory activities of the particles were determined. Dextran-40 density gradient centrifugation resulted in a better separation of mitochondrial populations than did sucrose density gradient centrifugation. Separation by sucrose density gradient centrifugation may not be according to the true densities of the particles. On the other hand, the use of gradients of Dextran-40, a solute of low osmotic potential, facilitated separation of particles acording to their true densities. Such mitochondria showed better respiratory control ratio and ADP:0 values, than those isolated by sucrose density gradient centrifugation.     

Adenovirus type 2 assembly analyzed by reversible cross-linking of labile intermediates.

Dimethyl-4,4'-dithiobisbutyrimidate dihydrochloride was used as a cleavable cross-linking reagent to maintain the structure of labile intermediates in adenovirus type 2 assembly. Analysis on sucrose gradients of nuclear adenovirus particles revealed two size classes, with sedimentation rates of 750 and 600S. After reversible fixation with diimido ester, the different classes were further separated on CsCl gradients and characterized with regard to their buoyant density, DNA content, and polypeptide composition. The 750S particles banded at 1.345 g/cm3 in CsCl, contained a DNA with a sedimentation coefficient of 34S in alkaline sucrose gradients, and had a polypeptide composition similar to that of young virions. The 600S population consisted of two types of particles with buoyant densities of 1.315 and 1.37 g/cm3. The 1.315-g/cm3 particles contained a DNA fragment of 7--11S and lacked the core proteins V and VII. In their place were found precursors P VI and P VIII and two nonvirion proteins with molecular weights of 50,000 (50K) and 39,000 (39K). 34S DNA was present in the 1.37-g/cm3 particles, which also lacked core proteins V and VII, as well as the 50K and 39K. Pulse-chase labeling kinetics suggested that the 1.315-g/cm3 particles were anterior to the 1.37-g/cm3 particles, themselves preceding the 1.345-g/cm3 young virions, and that the release of both 50K and 39K, possible scaffolding proteins, was required for entry of viral DNA.     

A Survey of Plants for Leaf Peroxisomes

Leaves of 10 plant species, 7 with photorespiration (spinach, sunflower, tobacco, pea, wheat, bean, and Swiss chard) and 3 without photorespiration (corn, sugarcane, and pigweed), were surveyed for peroxisomes. The distribution pattern for glycolate oxidase, glyoxylate reductase, catalase, and part of the malate dehydrogenase indicated that these enzymes exist together in this organelle. The peroxisomes were isolated at the interface between layers of 1.8 to 2.3 m sucrose by isopycnic nonlinear sucrose density gradient centrifugation or in 1.95 m sucrose on a linear gradient. Chloroplasts, located by chlorophyll, and mitochondria by cytochrome c oxidase, were in 1.3 to 1.8 m sucrose.In leaf homogenates from the first 7 species with photorespiration, glycolate oxidase activity ranged from 0.5 to 1.5 mumoles x min(-1) x g(-1) wet weight or a specific activity of 0.02 to 0.05 mumole x min(-1) x mg(-1) protein. Glyoxylate reductase activity was comparable with glycolate oxidase. Catalase activity in the homogenates ranged from 4000 to 12,000 mumoles x min(-1) x g(-1) wet weight or 90 to 300 mumoles x min(-1) x mg(-1) protein. Specific activities of malate dehydrogenase and cytochrome oxidase are also reported. In contrast, homogenates of corn and sugarcane leaves, without photorespiration, had 2 to 5% as much glycolate oxidase, glyoxylate reductase, and catalase activity. These amounts of activity, though lower than in plants with photorespiration, are, nevertheless, substantial.Peroxisomes were detected in leaf homogenates of all plants tested; however, significant yields were obtained only from the first 5 species mentioned above. From spinach and sunflower leaves, a maximum of about 50% of the marker enzyme activities was found to be in these microbodies after homogenization. The specific activity for peroxisomal glycolate oxidase and glyoxylate reductase was about 1 mumole x min(-1) x mg(-1) protein; for catalase. 8000 mumoles x min(-1) x mg(-1) protein, and for malate dehydrogenase, 40 mumoles x min(-1) x mg(-1) protein. Only small to trace amounts of marker enzymes for leaf peroxisomes were recovered on the sucrose gradients from the last 5 species of plants. Bean leaves, with photorespiration, had large amounts of these enzymes (0.57 mumole of glycolate oxidase x min(-1) x g(-1) tissue) in the soluble fraction, but only traces of activity in the peroxisomal fraction. Low peroxisome recovery from certain plants was attributed to particle fragility or loss of protein as well as to small numbers of particles in such plants as corn and sugarcane.Homogenates of pigweed leaves (no photorespiration) contained from one-third to one-half the activity of the glycolate pathway enzymes as found in comparable preparations from spinach leaves which exhibit photorespiration. However, only traces of peroxisomal enzymes were separated by sucrose gradient centrifugation of particles from pigweed. Data from pigweed on the absence of photorespiration yet abundance of enzymes associated with glycolate metabolism is inconsistent with current hypotheses about the mechanism of photorespiration.Most of the catalase and part of the malate dehydrogenase activity was located in the peroxisomes. Contrary to previous reports, the chloroplast fractions from plants with photo-respiration did not contain a concentration of these 2 enzymes, after removal of peroxisomes by isopycnic sucrose gradient centrifugation.     

Size-separation of yeast mitochondria in the zonal centrifuge

Mitochondria, released from yeast spheroplasts and subjected to rate separation through sorbitol gradients in the zonal centrifuge, migrated in a wide symmetrical zone. Electron micrographs showed that the mitochondria had been resolved within the zone according to size. The mean mitochondrial diameter at the leading edge was approximately twice that at the trailing edge of the particle zone. Activities of the enzymes cytochrome oxidase, malate dehydrogenase, and reduced nicotinamide adenine dinucleotide- and d-lactate cytochrome c reductases were essentially uniform throughout the mitochondrial zone. Mitochondria from a vegetative-petite mutant had almost the same size distribution as the isogenic wild type, but with somewhat larger mean diameter and either absent or markedly reduced enzyme activities. Mixtures of wild-type and petite mitochondria produced sedimentation profiles showing overlap of particle populations with respect to mean sedimentation rates and mitochondrial diameters, as well as intermediate levels of enzyme activities. Both cristate and noncristate organelles were present throughout the mitochondrial zone from these mixtures. Mitochondria centrifuged in sorbitol density gradients were well-preserved and yielded consistent sedimentation profiles, whereas particles in sucrose density gradients migrated more slowly, produced varied sedimentation profiles, and often showed spurious peaks, presumably due to particle aggregations.