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.     

THE SUBCELLULAR DISTRIBUTION OF [14C]GABA AND [3H]DOPAMINE IN THE RETINA

Abstract— Rabbit retinae were homogenized in isotonic sucrose and subjected to differential and density gradient centrifugation. Preliminary electron microscopic examination of some of the fractions indicated that in addition to the subcellular particles usually observed in brain homogenates, the photoreceptor cells gave rise to several characteristic fragments. These included fragmented outer limbs, aggregations of mitochondria from the inner segments, and photoreceptor terminals. Unlike the synaptosomes formed from the conventional type of synapses in the retina, these photoreceptor terminals appeared to sediment mainly in the low speed crude nuclear pellet (P1).
Retinae were incubated with low concentrations of [¹⁴C]GABA and/or [³H]dopamine prior to subcellular fractionation and in these experiments the P2 pellet was further fractionated on sucrose density gradients. Analysis of the radioactivity in the fractions showed that labelled GABA was accumulated by osmotically sensitive particles which had the sedimentation characteristics of synaptosomes. The panicles accumulating [³H]dopamine appeared to belong to a different, slightly lighter, population than those accumulating [¹⁴C]GABA. It is tentatively suggested that the particles accumulating labelled GABA were synaptosomes because the fractions containing these particles also possessed most of the GAD activity of the gradient. In contrast, GABA-T and MAO activity was found in the dense fractions of the gradients usually associated with mitochondria.
When retinae were incubated with a high concentration of labelled GABA a'lighter'population of particles seemed to accumulate the amino acid than when a low external GABA concentration was used. These results suggest that the high and low affinity uptake processes for GABA in the retina may have different cellular sites.     

Separation of Non-filamentous Micro-organisms from Soil by Density Gradient Centrifugation in Percoll

Soil suspensions were homogenized, and desorbed non-filamentous micro-organisms were concentrated in a minimum volume of buffer by low speed centrifugation. The cells were separated from inanimate material by flotation at the interface between the buffer and a silica sol/polyvinyl pyrrolidone density gradient medium (Percoll). Cell suspensions were removed from the interface and fractionated according to density by high speed centrifugation on discriminating density gradients in Percoll.
Preliminary experiments indicated that most non-filamentous soil micro-organisms had densities in the range 1.081–1.123 g%sol;ml while Rhizobium isolated from crushed root nodules on Percoll was split into two bands of densities 1.081–1.110 and 1.041–1.073 g/ml. The lighter cells were the more pleomorphic.
The efficiency of extraction of cells from soil was governed by the extent of their desorption from inanimate particles. As rigorous desorption procedures damage cells, extraction efficiencies were low; 10–20% of cells counted microscopically in soil were recovered from density gradients. Electron microscopy of soil micro-organisms isolated by this method showed an unusual range of surface ornamentations on cell-like structures of bacterial dimensions.     

Production of Ficoll, Percoll, and albumin gradients by the freeze-thaw method.

Ficoll, sucrose, albumin, and Percoll, a modified colloidal silica centrifugation medium, all form density gradients upon freezing and thawing. The data suggest that any density gradient material will form gradients upon freezing and thawing, provided the material is stable to freezing.     

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.     

THE SUBMITOCHONDRIAL LOCALIZATION OF MONOAMINE OXIDASE : An Enzymatic Marker for the Outer Membrane of Rat Liver Mitochondria

Controlled osmotic lysis (water-washing) of rat liver mitochondria results in a mixed population of small vesicles derived mainly from the outer mitochondrial membrane and of larger bodies containing a few cristae derived from the inner membrane. These elements have been separated on Ficoll and sucrose gradients. The small vesicles were rich in monoamine oxidase, and the large bodies were rich in cytochrome oxidase. Separation of the inner and outer membranes has also been accomplished by treating mitochondria with digitonin in an isotonic medium and fractionating the treated mitochondria by differential centrifugation. Treatment with low digitonin concentrations released monoamine oxidase activity from low speed mitochondrial pellets, and this release of enzymatic activity was correlated with the loss of the outer membrane as seen in the electron microscope. The low speed mitochondrial pellet contained most of the cytochrome oxidase and malate dehydrogenase activities of the intact mitochondria, while the monoamine oxidase activity could be recovered in the form of small vesicles by high speed centrifugation of the low speed supernatant. The results indicate that monoamine oxidase is found only in the outer mitochondrial membrane and that cytochrome oxidase is found only in the inner membrane. Digitonin treatment released more monoamine oxidase than cytochrome oxidase from sonic particles, thus indicating that digitonin preferentially degrades the outer mitochondrial membrane.     

COMPARATIVE STUDIES ON MITOCHONDRIA ISOLATED FROM NEURON-ENRICHED AND GLIA-ENRICHED FRACTIONS OF RABBIT AND BEEF BRAIN

Fractions enriched in neuronal and glial cells were obtained from dispersions of whole beef brain and rabbit cerebral cortex by large-scale density gradient centrifugation procedures. The fractions were characterized by appropriate microscopic observation. Mitochondria were then isolated from these fractions by differential centrifugation of their homogenates. The two different types of mitochondria were characterized with respect to certain enzyme activities, respiratory rate, rate of protein synthesis, and their buoyant density in sucrose gradients. The mitochondria from the neuron-enriched fraction were distinguished by a higher rate of incorporation of amino acids into protein, higher cytochrome oxidase activity, and a higher buoyant density in sucrose density gradients. Mitochondria from the glia-enriched fraction showed relatively high monoamine oxidase and Na⁺- and K⁺-stimulated ATPase activities. The rates of oxidation of various substrates and the acceptor control ratios did not differ appreciably between the two types of mitochondria. The difference in the buoyant density of mitochondria isolated from the neuron-enriched and glia-enriched cell fractions was utilized in attempts to separate neuronal and glial mitochondria from the mixed mitochondria obtained from whole brain homogenates in shallow sucrose gradients. The appearance of two peaks of cytochrome oxidase, monoamine oxidase, and protein concentration in such gradients shows the potential feasibility of such an approach.     

Analysis and purification of the blood-sinusoidal domain of rat liver plasma membrane

Methods are described for the analysis and purification of the blood-sinusoidal domains of rat liver plasma membranes using a combination of sucrose and Ficoll density gradient centrifugation. Use has been made of ¹²⁵I-labelled wheat-germ agglutinin and hormone-stimulated adenylate cyclase to identify the blood sinusoidal fraction, which may be resolved from Golgi and endoplasmic reticulum markers on Ficoll gradients.     

ISOLATION AND PARTIAL CHARACTERIZATION OF FRACTIONS ENRICHED IN SYNAPTOSOMES FROM CHICK BRAIN

–From a pool of hemispheres, optic lobes and cerebellum of chick 3 fractions containing synaptosomes have been prepared. They were obtained by subcellular fractionation of a homogenate and centrifugation of a crude mitochondrial suspension on a discontinuous Ficoll density gradient in iso-osmoticsucrose. The synaptosomal fractions were isolated from bands at the interface of 5–9, 9–12 and 12–16% Ficoll. The characterization of these fractions by marker enzymes, such as lactate dehydrogenase, acetyl-cholinesterase, monoamine oxidase, acid phosphatase and rotenone-sensitive and -insensitive NADH: cytochrome c reductase is reported. Electron microscopic analyses showed that the first fraction (AB) at the 5–9% Ficoll interface contained myelin and other membrane fragments as well as synaptosomes, the second fraction (C) at the 9–12% Ficoll interface contained mainly synaptosomes, and the third fraction (D) at the 12–16% Ficoll interface contained synaptosomes and free mitochondria. A fourth fraction (E) was obtained as a pellet, and was enriched in free mitochondria. There was fair agreement between the distribution pattern of the marker enzyme activities and the particles of the fractions seen by electron microscopy. The content of glycoprotein-bound N-acetylneuraminic acid and total phospholipid of these fractions has been determined. Relative to the mitochondrial fraction (E) the synaptosome fraction contained on basis of particulate protein, respectively, 2–3 times as much protein-bound N-acetylneuraminic acid and 10–20 per cent more total phospholipid.     

A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain.

A rapid (less than 2h) method is described for the preparation of synaptosomes from rat brain by using a discontinuous Ficoll/sucrose gradient by a flotation technique. These synaptosomes are metabolically active and minimally (less than 5%) contaminated with 'free' mitochondria as judged by marker-enzyme assays and electron microscopy.     

Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla.

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules (SH (average sedimentation coefficient) = 12 400 S in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin (greater than or approximately 95% of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium. The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.     

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.     

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.     

Distribution of membranes, especially of plasma-membrane fragments, during zonal centrifugations of homogenates from glucose-repressed Saccharomyces Cerevisiae.

1. The distributions of several enzymes and other marker components were examined after zonal centrifugations of whole homogenates from glucose-repressed Saccharomyces cerevisiae on sucrose and iso-osmotic Ficoll, and the composition and morphology of the fractions were investigated. 2. After high-speed zonal centrifugation most of the protein, acid and alkaline phosphatases, alkaline pyrophosphatase, adenosine monophosphatase, beta-fructofuranosidase, alpha-mannosidase, NADPH-cytochrome c oxidoreductase and an appreciable amount of phospholipid and sterol were non-sedimentable, i.e. were at densities below 1.09 (g/cm3). Most of the RNA was at p=1.06-1.08 in Ficoll and at p=1.09-1.11 in sucrose. 3. The bulk of the Mg2+-dependent adenosine triphosphatase (Mg-ATPase) was coincident with the main peak of phospholipid and sterol, at median density 1.10, which was also rich in smooth-membrane vesicles. In Ficoll, a minor peak of phospholipid and sterol at p-1.12-1.15 contained a smaller part of the oligomycin-insensitive Mg-ATPase and heavy membrane fragments. In sucrose, several minor peaks of Mg-ATPase were in the mitochondrial density range, and a peak of oligomycin-insensitive Mg-ATPase coincident with a minor peak of phospholipid and sterol at around p-1.25 contained heavy membrane fragments of high carbohydrate content, especially mannose. 4. Further purification of the oligomycin-insensitive Mg-ATPase containing membrane preparations was performed on Urografin gradients. 5. It is argued that the oligomycin-insensitive Mg-ATPase containing membranes are fragments of the plasma membrane, but have different densities because they contain different amounts of glycoprotein particles.     

The isolation of purified neurosecretory vesicles from bovine neurohypophysis using isoosmolar density gradients

Two different density gradients are described for the isolation of highly purified fractions of neurosecretory vesicles in isoosmotic solutions (300 mosm/kg) from bovine neurohypophyses. The techniques involve differential centrifugation of neural lobe homogenates followed by density gradient centrifugation on metrizamide-sucrose or Percoll-sucrose gradients. The purified fraction contained 44 and 65 μg vasopressin/mg protein, respectively. Neurosecretory vesicles thus isolated were only slightly contaminated with other subcellular organelles, e.g., mitochondria and lysosomes. These vesicles were highly stable in isotonic sucrose solutions (pH 7.5 and 5.5) even at 37°C for at least 2 h, retaining more than 90% of their hormonal content.     

Effect of high sucrose concentrations on mitochondria: Analysis of mitochondrial populations by density-gradient centrifugation after fixation with glutaraldehyde

It has been shown previously that intact rat liver mitochondria can be separated into two populations (designated B2 and B3) with mean buoyant densities of 1·184 and 1·216 respectively, by isopycnic sucrose density gradient centrifugation. A comparison has been made of some properties of these mitochondrial fractions from density gradients with non-fractionated mitochondria. Use was made of density gradient centrifugation for analysis of preparations fixed with appropriate concentrations of glutaraldehyde. The permeability of the membranes of non-fractionated mitochondria to sucrose was increased by exposure to hypoosmotic sucrose solutions. The B3 mitochondria differed from the non-fractionated mitochondria in their response to changes in osmotic pressure of the suspending medium while the B2 mitochondria showed essentially identical behaviour with the controls. However, under conditions of energized swelling the B2 mitochondria were markedly different to the controls. This difference, which is attributed to reduced permeability of the mitochondrial membranes to metabolites brought about by exposure to the high concentrations of sucrose encountered in the density gradient, was reversed by incubation in hypo-osmotic sucrose solutions in the presence of oxidizable substrate and permeant ions.Died December, 1969.     

Purification and properties of thyrotrophin releasing hormone (TRH)-containing particles from the rat hypothalamus

The subcellular distribution of the TRH-like immunoreactivity in the rat hypothalamus and brain was studied. In differential centrifugation, the 900 g for 10 min supernatant (S1) of the hypothalamus or brain contained 61--79% of the total TRH. At 11,000 g for 20 min, 51--73% of the TRH in S1 was sedimented. When the hypothalamic S1 was fractioned under non-equilibrium conditions at 25 degrees C, two populations of TRH-containing particles were observed in several types of continuous linear density gradients. Metrizamide and sucrose gradients affected TRH-assay. TRH-particles were very light in Percol-gradients. Isotonic dextran 40,000-sucrose gradients gave the most reproducible results. In these gradients, the large TRH-particles (35%) equilibrated at 1.055--1.060 kg/l and the small ones (23%) at 1.041--1.047 kg/l. Working at 4 degrees C decreased the amount of large TRH-particles. The apparently larger particles contained cytoplasmic and mitochondrial enzymes and were sensitive to hypoosmotic shock like synaptosomes. Electron micrographs confirmed that these particles were synaptosomes. The true nature of the small particles remained unclear but morphologically a part of them were also synaptosomes. Treatment of the animals with reserpine (10 mg/kg i.p., 24 h), with 6-hydroxydopamine (100 microgram/rat i.c.v.) or with 5,7-dihydroxytryptamine (200 microgram/rat i.c.v.) did not affect significantly TRH-recovery or distribution in the hypothalamus.     

The effect of pressure on fetal and neonatal liver mitochondrial membranes

The effects of pressure on late fetal and neonatal rat liver mitochondria have been investigated. High hydrostatic pressure, as produced by isopycnic centrifugation in sucrose and glycogen gradients, altered the mitochondrial membranes of 1- and 7-day-old rats. Most of the mitochondrial enzymes, chosen for their known submitochondrial location, had a trimodal distribution in the sucrose gradients. In the glycogen gradients, a shift of the mitochondria to a lower density was noticed. Fetal liver mitochondria were resistant to the hydrostatic pressure exerted during isopycnic centrifugation experiments under different conditions such as sucrose and glycogen density gradients. The submitochondrial compartment tracer enzymes exhibited an unimodal distribution. Experimental temperatures set at 15 degrees C had a protective effect from hydrostatic pressure alterations in the neonatal liver mitochondria, whereas no effects were noticeable in the fetal mitochondria. Experiments in a hydraulic compression chamber showed that outer membranes of fetal mitochondria were more fragile and the inner membranes more resistant to compression than in the early stages after birth.     

Percoll and Ficoll self-generated density gradients by low-speed osmocentrifugation

Percoll and Ficoll self-generated density gradients can be obtained by low-speed centrifugation of their solutions within dialysis cells. Useful Percoll density gradients can be obtained after 10-30 min centrifugation at 220-2010g, within dialysis cells. Ficoll density gradients, which are more difficult to self-generate, can be obtained by the same technique. Red cell band formation in a Percoll density gradient can be done in a single step by using dialysis cells as the centrifugation solution container.     

Isolation of furanoterpene-containing particles from Ceratocystis fimbriata-infected sweet potato root tissue

Furanoterpene-containing particles were isolated by centrifugation on a discontinuous Ficoll density gradient from a homogenate of the non-infected tissue adjacent to the infected region of Ceratocystis fimbriata-infected sweet potato root tissue. The particles were recovered at a relatively high ratio in the 2% Ficoll fraction, in which there was no contamination by mitochondria and only little by endoplasmic reticulum judging from the distribution of the activities of their marker enzymes and electron micrographs. Each particle was enveloped in a single membrane, 7-10 nm thick.