Isolation of Relaxing Particles from Rat Skeletal Muscles in Zonal Centrifuges

Supernatants of rat skeletal muscle homogenates were fractionated by differential centrifugation and by zonal centrifugation in sucrose density gradients. Cytochrome oxidase was employed as an enzymatic marker for locating mitochondria. The subcellular fractions were also assayed for their ability to prevent the ATP-induced contraction of myofibrils. Both the mitochondrial and microsomal fractions obtained by differential fractionation were found to be rich in such relaxing activity, and the microsomal fraction was appreciably contaminated by mitochondria. In contrast to this, when fractionation was carried out by means of zonal centrifugation (4200 RPM x 205 min. to 40,000 RPM x 60 min.), relaxing activity was found to be associated only with particles having the sedimentation characteristics of microsomes (s _20,w estimated to be between 370 and 1880S). Relaxing activity was not detected in the regions of the gradient containing either the starting sample zone (soluble phase) or the mitochondrial peak. The microsomal relaxing particles showed negligible cytochrome oxidase activity.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. 3. Subfractionation of the microsomal fraction by isopycnic and differential centrifugation in density gradients

Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5''-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b ₅, and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, β-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5''-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity.     

Different Subcellular Localization of Muscarinic and Serotonin (S2) Receptors in Human, Dog, and Rat Brain

Cortex from rat, dog, and human brain was submitted to subcellular fractionation using an analytical approach consisting of a two-step procedure. First, fractions were obtained by differential centrifugation and were analyzed for their content of serotonin S2 and muscarinic receptors, serotonin uptake, and marker enzymes. Second, the cytoplasmic extracts were subfractionated by equilibration in sucrose density gradient. In human brain, serotonin and muscarinic receptors were found associated mostly with mitochondrial fractions which contain synaptosomes, whereas in rat brain they were concentrated mainly in the microsomal fractions. Density gradient centrifugation confirmed a more marked synaptosomal localization of receptors in human than in rat brain, the dog displaying an intermediate profile. In human brain, indeed, more receptor sites were found to be associated with the second peak characterized in electron microscopy by the largest number of nerve terminals. In addition, synaptosomes from human brain are denser than those from rat brain and some marker enzymes reveal different subcellular distribution in the three species. These data indicate that more receptors are of synaptosomal nature in human brain than in other species and this finding is compatible with a larger amount of synaptic contacts in human brain.     

Phosphatidate biosynthesis in mitochondrial subfractions of rat liver

1. After conventional fractionation of rat liver homogenates in 0.88m-sucrose the mitochondrial fraction was subjected to short-term water lysis followed by separation of the resulting membrane preparations. 2. Phosphatidate formation was measured in all subcellular fractions and subfractions and was compared with the distribution of succinate dehydrogenase, monoamine oxidase, rotenone-insensitive NADH cytochrome c reductase, arylsulphatase, urate oxidase, arylesterase and glucose 6-phosphatase. 3. The results obtained indicated that mitochondria were capable of synthesizing phosphatidate, though this activity was only about one-third of the total homogenate activity. 4. Mitochondrial phosphatidate formation was located predominantly in the outer mitochondrial membrane. Although this membrane preparation was found to be significantly contaminated by the microsomal fraction, this contamination was estimated to account for not more than about 20% of the total phosphatidate formation observed in preparations of outer mitochondrial membrane.     

The significance of cytochrome c redistribution during the subcellular fractionation of rat liver

1. The redistribution of mitochondrial cytochrome c during homogenization and subcellular fractionation of the liver was studied. Chromatographically homogeneous (14)C-labelled cytochrome c was added in different amounts to liver suspensions immediately before homogenization and the adsorption of radioactivity was determined in cytochrome c fractions extracted at pH4.0, first with water and then with 0.15m-sodium chloride. 2. The soluble cytochrome c remaining in the cell sap after subcellular fractionation was 7% of the calculated amount of cytochrome c passing through a soluble form during the whole process. The total amount of cytochrome c released in a soluble form and subsequently redistributed was 25-30% of the total liver cytochrome c. 3. In the standard microsomal fraction the cytochrome c extracted with water originated entirely from redistribution whereas that extracted with 0.15m-sodium chloride was 80% endogenous. In the mitochondrial fraction both cytochrome c pools were truly endogenous, so that practically none of the mitochondrial cytochrome c released to the soluble cell sap was readsorbed by the mitochondria. 4. These results support our former hypothesis that the cytochrome c extracted with 0.15m-sodium chloride at pH4.0 from the standard microsomes represents the cytochrome c newly synthesized in situ, since it does not originate from redistribution. However, the microsomal pool extracted with water cannot be an intermediate in the postulated transfer of cytochrome c from the microsomal particles to the mitochondria, since this pool arises from redistribution of mitochondrial cytochrome c.     

Subcellular distribution of iodothyronine 5′-deiodinase in cerebral cortex from hypothyroid rats

We have examined iodothyronine deiodination in subcellular fractions of cerebral cortex obtained from hypothyroid rats. Enzymatic activities were measured at 37°C in the presence of 20 mM dithiothreitol with ¹²⁵I-labeled T₄ and ¹²⁵I-labeled rT₃ as substrate for 5′-deiodination and ¹³¹I-labeled T₃ as the substrate for the 5-deiodinase. Reaction products were separated by descending paper and/or ion-exchange chromatography. Cerebral cortex subcellular fractions were also characterized by marker enzyme analysis and electron microscopy. Under optimal reaction conditions more than 80% of the 5′-deiodinase was recovered after fractionation. Both 5′-deiodinase and (Na⁺ +K⁺-ATPase showed similar subcellular distributions and were enriched approx. 3-fold in the easily sedimenting membrane fraction and nerve terminal plasma membranes. Crude microsomal membranes (6·10⁶g·min pellet) also showed 2-fold enrichment of these enzymes. Nuclei and isolated mitochondria were devoid of deiodinating activity. T₄ and T₃ 5-deiodinating activity was absent in the easily sedimenting membranes and present but not enriched in particulate fractions containing microsomal membranes. These data suggest that iodothyronine 5′-deiodinase is associated with plasma membrane fractions in the cerebral cortex.     

Differential kinetics and sensitivity to chloroquine of receptor-mediated insulin and prolactin endocytosis in liver parenchymal cells

Systemically injected [125I]prolactin or [125I]insulin was accumulated and cleared from rat liver at different rates. Quantitative subcellular fractionation indicated a predominant accumulation of [125I]insulin in liver microsomes while [125I]prolactin was found in both the light-mitochondrial and microsomal fractions. The acidotropic agent chloroquine diminished the rate and extent of loss of each ligand from liver homogenates. In chloroquine treated rats, radiolabeled insulin accumulated in both the light-mitochondrial and the microsomal fractions. Subfraction of microsomes on discontinuous sucrose gradients revealed "early' endosomes in which ligand uptake was maximal at 2-5 min. In contrast, comparable subfraction of the of light mitochondrial fraction revealed "late' endosomes in which ligand uptake was maximal at 10-20 min. Chloroquine-treated rats showed a more marked enhancement of insulin compared to prolactin uptake in the "early' endosomes. It is suggested that "early' endosomes found in the Golgi-intermediate and -heavy fractions floated from parent microsomes may selectively degrade insulin but not prolactin. This could account for the apparently different kinetics of insulin and prolactin uptake into liver parenchyma.     

The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane

We have investigated the subcellular localization of the peripheral-type benzodiazepine receptor in rat adrenal gland using the high affinity ligand 3H-labeled 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H]PK11195). The autoradiographic pattern of [3H]PK11195 binding sites in tissue sections of adrenal gland is similar to the histochemical distribution of the mitochondrial marker enzymes, cytochrome oxidase and monoamine oxidase, which are present in high concentrations only in the cortex. Subcellular fractionation studies of homogenates of adrenal gland indicate that the recovery and enrichment of [3H]PK11195 binding sites in the nuclear, mitochondrial, microsomal, and soluble fractions correlate closely with cytochrome oxidase activity, but not with markers for the nuclei, lysosomes, peroxysomes, endoplasmic reticulum, plasma membrane, or cytoplasm, indicating an association of the peripheral-type benzodiazepine receptor with the mitochondrial compartment. Titration of isolated mitochondria with digitonin results in the simultaneous release of the peripheral-type benzodiazepine receptor and of monoamine oxidase, but not cytochrome oxidase, indicating association of the peripheral-type benzodiazepine receptor with the mitochondrial outer membrane. Scatchard analysis and drug displacement studies of the binding of [3H] PK11195 to intact mitochondria and to the outer membrane-enriched digitonin extract further confirm the localization of the peripheral-type benzodiazepine receptor to the mitochondrial outer membrane.     

Subcellular distribution of delta 5-3 beta-hydroxy steroid dehydrogenase in the granulosa cells of the domestic fowl (Gallus domesticus).

1. The distribution of 3 beta-hydroxy steroid dehydrogenase was examined in the subcellular fractions of granulosa cells collected from the ovary of the domestic fowl. 2. 3 beta-hydroxy steroid dehydrogenase activity was observed in the mitochondrial (4000g for 20min) and microsomal (105 000g for 120min) fractions. 3. Approximately three times more 3 beta-hydroxy steroid dehydrogenase activity was associated with the cytochrome oxidase activity (a mitochondrial marker enzyme) in anteovulatory-follicle granulosa cells than with that of the postovulatory follicle. 4. Comparison of the latent properties of mitochondrial 3 beta-hydroxy steroid dehydrogenase with those of cytochrome oxidase and isocitrate dehydrogenase indicated that 3 beta-hydroxy steroid dehydrogenase is located extramitochondrially. 5. This apparent distribution of 3 beta-hydroxy steroid dehydrogenase is explained on the basis that the mitochondrial activity is either an artefact caused by a redistribution in the subcellular location of the enzyme, occurring during homogenization, or by the existence of a functionally heterogeneous endoplasmic reticulum that yields particles of widely differing sedimentation properties.     

A novel protocol for the subcellular fractionation of C3A hepatoma cells using sucrose density gradient centrifugation

In this paper, we describe a method to obtain a relatively pure mitochondrial and microsomal fractions by subcellular fractionation of human hepatoma cell line C3A using sucrose as the hypoosmotic medium. The cells were subjected to osmotic stress with sucrose and homogenized. Osmolarity was then restored to the cells and the organelles were separated by density gradient centrifugation. The protein profiles were examined by SDS-PAGE and the purity was analysed by marker enzymes and Western blotting. Our results indicate a good separation of mitochondrial and microsomal fractions from human hepatoma C3A cells.     

Determination of releasing hormones in subcellular fractions from porcine hypothalamic tissue

In a study of possible biosynthesis of hypothalamic hormones in mitochondria, the activities of subcellular fractions from gradient Ficoll fractionation were assayed. Mitochondrial fractions identified by qualitative assays for oxygen uptake and phosphorylation, showed both respiratory activity and release of the luteinizing hormone. Purification of solvent extracts of mitochondrial fractions by Bio-Gel P-2 and Sephadex G-25 yielded fractions which released the luteinizing and follicle stimulating hormones and somatotropin. Labeled pGlu-His-Pro-NH₂ and the release of thyrotropin served as a control.     

ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER : II. Preparation and Composition of the Microsomal Fraction

Liver homogenates have been submitted to quantitative fractionation by differential centrifugation. Three particulate fractions: N (nuclear), ML (large granules), and P (microsomes), and a final supernate (S) have been obtained. The biochemical composition of the microsomal fraction has been established from the assay and distribution pattern of 25 enzymatic and chemical constituents. These included marker enzymes for mitochondria (cytochrome oxidase), lysosomes (acid phosphatase and N-acetyl-β-glucosaminidase), and peroxisomes (catalase). The microsomal preparations were characterized by a moderate contamination with large cytoplasmic granules (only 6.2% of microsomal protein) and by a high yield in microsomal components. Enzymes such as glucose 6-phosphatase, nucleoside diphosphatase, esterase, glucuronyltransferase, NADPH cytochrome c reductase, aminopyrine demethylase, and galactosyltransferase were recovered in the microsomes to the extent of 70% or more. Another typical behavior was shown by 5'-nucleotidase, alkaline phosphatase, alkaline phosphodiesterase I, and cholesterol, which exhibited a "nucleomicrosomal" distribution. Other complex distributions were obtained for several constituents recovered in significant amount in the microsomes and in the ML or in the S fraction.     

Subcellular fractionation of isolated rat hepatocytes a comparison with liver homogenate

An improved method for the homogenization and the subsequent subcellular fractionation of hepatocytes isolated from adult rat liver is described.The homogenization procedure developed in the present study allows the preservation of the integrity of subcellular structures, as demonstrated by measurement of the activities of representative enzymes as well as by determination of their latency.The activities of representative marker enzymes, as calculated on subcellular fractions obtained by differential centrifugation of the homogenate, are identical whether the homogenate arises from isolated hepatocytes or from the whole liver.Moreover, there is a close similitude between the kinetic parameters (K_{m and} V) of two microsomal cytochrome P₄₅₀-dependent mixed-function oxidases, namely aniline hydroxylase and aminopyrine demethylase determined on microsomal preparations obtained either from isolated cells or from the whole liver.     

Differential induction of peroxisomal populations in subcellular fractions of rat liver

In rat liver, peroxisome proliferators induce profound changes in the number and protein composition of peroxisomes, which upon subcellular fractionation is reflected in heterogeneity in sedimentation properties of peroxisome populations. In this study we have investigated the time course of induction of the peroxisomal proteins catalase, acyl-CoA oxidase (ACO) and the 70 kDa peroxisomal membrane protein (PMP70) in different subcellular fractions. Rats were fed a di(2-ethylhexyl)phthalate (DEHP) containing diet for 8 days and livers were removed at different time-points, fractionated by differential centrifugation into nuclear, heavy and light mitochondrial, microsomal and soluble fractions, and organelle marker enzymes were measured. Catalase was enriched mainly in the light mitochondrial and soluble fractions, while ACO was enriched in the nuclear fraction (about 30%) and in the soluble fraction. PMP70 was found in all fractions except the soluble fraction. DEHP treatment induced ACO, catalase and PMP70 activity and immunoreactive protein, but the time course and extent of induction was markedly different in the various subcellular fractions. All three proteins were induced more rapidly in the nuclear fraction than in the light mitochondrial or microsomal fractions, with catalase and PMP70 being maximally induced in the nuclear fraction already at 2 days of treatment. Refeeding a normal diet quickly normalized most parameters. These results suggest that induction of a heavy peroxisomal compartment is an early event and that induction of 'small peroxisomes', containing PMP70 and ACO, is a late event. These data are compatible with a model where peroxisomes initially proliferate by growth of a heavy, possibly reticular-like, structure rather than formation of peroxisomes by division of pre-existing organelles into small peroxisomes that subsequently grow. The various peroxisome populations that can be separated by subcellular fractionation may represent peroxisomes at different stages of biogenesis.     

Woronin bodies fromPenicillium chrysogenum: Isolation and characterization by analytical subcellular fractionation

Differential centrifugation of whole homogenates ofPenicillium chrysogenum, disrupted by a modified Ballotini bead method, resulted in the enrichment of Woronin bodies between 800g (5 minutes) and 6000g (10 minutes). Isolated Woronin bodies are membrane-bounded, electron-opaque, approximately spherical organelles, 0.11 to 0.29 μm in diameter. Woronin bodies have a buoyant density (ϱ) of 1.21 g cm⁻³ and S_20,w values of 6300 to 37,600 in sucrose gradients. Analytical subcellular fractionation of whole homogenates in a zonal rotor showed that Woronin bodies did not cosediment with marker enzymes for lysosomes (acid phosphatase), peroxisomes (catalase), mitochondria (cytochrome c oxidase), or endoplasmic reticulum (NADPH cytochrome c reductase).     

The distribution of synaptosomal oxidative enzymes after exposure to deoxycholate

Abstract— The distributions of NADH₂ dehydrogenase, NADH, cytochrome c reductase and cytochrome oxidase have been determined utilizing synaptosomal isolation techniques. Deoxycholate was used to determine compartmentation and/or ‘latency’ of these activities. NADPH, dehydrogenase proved to be a soluble and mitochondrial enzyme and the activity of this enzyme was not appreciably changed by deoxycholate treatment. NADH_g cytochrome c reductase proved to be a mitochondrial enzyme with considerable activity in microsomal fractions. Deoxycholate treatment increased activity in the synaptosomal fraction 8.3-fold. A bimodal activation pattern was observed with synaptosomal and mitochondrial NADH, cyrochrome c reductase upon exposure to increasing concentrations of deoxycholate, with enhancement of activity at 0.25 % (w/v) and 0.50 % (w/v) deoxycholate. The enzyme was stable at concentrations of deoxycholate less than 0.25% (w/v) but was irreversibly inactivated at concentrations higher than 0.25% (w/v). The mechanism of this activation pattern appeared to be a combination of enzyme release and inactivation. Similar results were not observed in liver mitochondria. Cytochrome oxidase, a known mitochondrial marker, exhibited a 17-fold increase in synaptosomal activity with deoxycholate treatment. The synaptosomal cytochrome oxidase activity after deoxycholate treatment approached the activity in the free mitochondrial fraction. The percentage of mitochondrial protein in synaptosomal fractions was estimated to be about 30 per cent from a comparison of the respective total (deoxycholate-treated) activities. On the basis of these data we suggest that the synaptosomal fraction possesses a relatively sizable energy-producing potential which may be of significance in vivo.     

Opiate target site N-demethylase enzymes: differences from the liver N-demethylase.

Mixtures of morphine-6³H and morphine-N-¹⁴CH₃ were incubated with rat brain subcellular fractions. Isotope ratio measurements served as the marker for identification, purification and quantitation of N-nor products which were shown to consist almost solely of N-normorphine. The microsomal, synaptosomal and mitochondrial, but not the supernatant brain preparations yielded N-normorphine. The microsomal incubations were then repeated in the presence of cytochrome P-450 inhibitors which suppressed the liver reaction but did not affect the brain biotransformation. The brain N-dealkylase is therefore different from the one in the liver and is not a cytochrome P-450 linked enzyme.     

STUDIES ON ISOLATED CELL COMPONENTS : XVII. The Distribution of Cytochrome Oxidase Activity in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.     

Subcellular distribution and properties of carbonyl reductase in guinea pig lung

On subcellular fractionation, carbonyl reductase (EC 1.1.1.184) activity in guinea pig lung was found in the mitochondrial, microsomal, and cytosolic fractions; the specific activity in the mitochondrial fraction was more than five times higher than those in the microsomal and cytosolic fractions. Further separation of the mitochondrial fraction on a sucrose gradient revealed that about half of the reductase activity is localized in mitochondria and one-third in a peroxidase-rich fraction. Although carbonyl reductase in both the mitochondrial and microsomal fractions was solubilized effectively by mixing with 1% Triton X-100 and 1 M KCl, the enzyme activity in the mitochondrial fraction was more highly enhanced by the solubilization than was that in the microsomal fraction. Carbonyl reductases were purified to homogeneity from the mitochondrial, microsomal, and cytosolic fractions. The three enzymes were almost identical in catalytic, structural, and immunological properties. Carbonyl reductase, synthesized in a rabbit reticulocyte lysate cell-free system, was apparently the same in molecular size as the subunit of the mature enzyme purified from cytosol. These results indicate that the same enzyme species is localized in the three different subcellular compartments of lung.     

The subcellular localization of cerebral phosphoproteins sensitive to electrical stimulation

1. On incubating cerebral-cortex slices at 37° in an oxygenated medium marked changes resulted in the subcellular distribution of proteins and phosphoproteins in the tissue. The protein content of the nuclear fraction more than doubled, whereas the yields of microsomal and supernatant proteins were both markedly decreased. The amount of phosphoprotein/mg. of protein decreased in the microsomal and supernatant fractions, but showed little change in the nuclear and mitochondrial fractions. The loss of microsomal protein could be partly prevented by rinsing the slices briefly in cold sucrose solution before dispersion; the altered subcellular distribution was apparently related to contamination of the dispersing solution with traces of salts from the medium. 2. The subcellular location of the phosphoprotein sensitive to the effects of electrical pulses applied to cerebral slices in vitro has been reinvestigated by two different procedures. Comparison between unstimulated and stimulated slices after incubation in the presence of [³²P]orthophosphate showed that phosphoprotein radioactivity increased on stimulation to a greater extent in a membrane-rich fraction than in a mitochondria-rich fraction, these being obtained by immediate density-gradient fractionation of the tissue dispersion. With fractions isolated by differential centrifuging the percentage increase in a combined mitochondrial and nuclear fraction was 5% as compared with 24% (P<0·02) in the microsomal fraction and 30% in the original dispersion before fractionation. The sensitive phosphoprotein therefore appears to be located in structures sedimenting with the microsomal fraction, rather than with the nuclear fraction as previously claimed.