Distribution of proteins in different subcellular fractions of rat brain studied by two-dimensional gel electrophoresis

The subcellular distribution of proteins normally visible on two-dimension gels of rat brain tissue punches and crude brain homogenate was investigated using two-dimensional gel electrophoresis and computerized scanning densitometry. Seven enriched subcellular fractions (cytosol, mitochondria, microsomes, nucleus, crude synaptic vesicles, myelin and synaptic membrane) were generated from a crude extract of rat brain. Fifty microgram samples of the crude homogenate and each fraction were then taken and the proteins within these samples separated by two-dimensional gel electrophoresis. Proteins were stained with silver and the gels then analyzed by computerized scanning densitometry. Of 136 proteins visible on two-dimension gels of the crude homogenate that were quantitatively examined, a total of 73 (54%) were identified as being primarily located in a single subcellular fraction. The majority of these 73 proteins were found to be located primarily in either the cytosolic or mitochondrial fractions, while fewer proteins were identified as being primarily located in the microsomal, nuclear or crude synaptic vesicular subfractions. In contrast, the myelin and synaptic membrane fractions were found to be the primary location for only a single protein each that is clearly visible in the crude homogenate. In addition, gels of four of the subfractions (mitochondria, cytosol, nucleus and myelin) contained proteins that are not normally visible on gels generated using a crude extract. The subcellular location of a number of proteins found previously to be altered by specific experimental manipulations was also determined, providing further information on these proteins in brain. These results should prove useful in future experiments designed towards isolating and characterizing specific proteins of neurochemical interest.     

An improved procedure for the simultaneous purification in high yield of peroxisomes,lysosomes, and mitochondria from rat liver

Summary Peroxisomes, lysosomes, and mitochondria have been purified from rat liver by sucrose density gradient centrifugation without prior treatment of the animals with Triton WR-1339 or other detergents which cause hyperlipidemia. A crude organelle fraction was first prepared by differential centrifugation of a rat liver homogenate, this fraction contained approximately 70% of the mitochondrial, 40% of the peroxisomal, and 30% of the lysosomal marker enzymes measured in the homogenate. The crude organelle fraction was applied to the top of a sucrose density gradient and centrifuged. A clear separation of the organelles was obtained only when dextran was present in the gradients. Success or failure of the method was found to depend on the particular preparation of dextran used in the gradients. A method for subfractionating dextran was developed which yields dextran fractions that make the separations completely reproducible. Starting with a crude organelle fraction derived from 12 g of liver, approximately 85% of the mitochondrial, 70% of the peroxisomal, and 50% of the lysosomal activities were obtained as pure fractions. The organelle separation takes less than five hours to complete, it represents a substantial improvement over previous methods.     

盘基网柄菌细胞分化和凋亡的形态特征

本文用透射电镜和DAPI荧光染色法研究了盘基网柄菌(Dictyosteliumdiscoideum)细胞分化和柄细胞的凋亡特征,结果显示:细胞丘中绝大部分细胞的线粒体内出现一小空泡,随着发育进程,空泡逐渐增大,线粒体的嵴随之变少,直至线粒体完全空泡化,最后形成单层膜的空泡。据此我们推测前孢子细胞特有的空泡来源于线粒体,并且这种细胞器水平上的内自噬现象与前孢子细胞分化密切相关。在前柄细胞分化阶段,前柄细胞中出现数个自噬泡,最初吞噬的线粒体嵴结构完整;随着前柄细胞进一步分化,部分线粒体内出现类似于前孢子细胞中的内自噬现象,并且自噬泡只吞噬这种线粒体。在凋亡后期,细胞核内核仁消失,染色体固缩形成高电子密度斑块,自噬泡采用与细胞核膜融合的方式来完成核的清除,最后柄细胞完全空泡化且包被一层纤维素壁。作者认为前柄细胞凋亡过程实质上是一种分化过程,所以有其鲜明特点:细胞出现自噬泡,标志着凋亡开始,用自噬而不是凋亡小体来清除胞内各种细胞器,直到分化最后阶段才清除细胞核和形成纤维素壁。这些特点不仅是前柄细胞凋亡的形态学指标,也和细胞发育和分化相关。     

An NADH-tetrazolium-coupled sensitive assay for malate dehydrogenase in mitochondria and crude tissue homogenates

A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.     

Subcellular reorganization of mitochondria producing heavy DNA in aging wheat coleoptiles.

Unusual closed membrane vesicles containing one or more mitochondria were isolated from homogenates of aging wheat coleoptiles. Very similar (or the same) bodies were shown to exist in situ in vacuoles of undividing cells in the apical part of intact senescent coleoptiles. Vesicles isolated from coleoptile homogenate free of nuclei by 10 min centrifugation at 1700 x g and traditional mitochondria (sedimented at between 4300 x g and 17,400 x g) are similar in respiration rate, composition and content of cytochromes and sensitivity to respiration inhibitors. However, vesicles contain about 2-fold more Ca2+ ions than free mitochondria do. The specific feature of vesicles containing mitochondria in aging coleoptiles is an intensive synthesis of heavy (rho = 1.718 g/cm3) mitochondrial DNA (H-mtDNA). Thus, aging in plants is accompanied by an increased selective H-mtDNA production and change in subcellular organization of mitochondria.     

Isolation of a subfraction of rough endoplasmic reticulum closely associated with mitochondria : Evidence for its role in cytochrome P450 synthesis

A subfraction of rough endoplasmic reticulum (RER) structurally associated with mitochondria (mito-RER complexes) was isolated from crude nuclear fractions of rat liver homogenate. When apocytochrome P450 synthesis (which presumably occurs in RER) and mitochondrial heme synthesis was dissociated by concomitant treatment of rats with phenobarbital and cobaltous chloride, apocytochrome P450 accumulated predominantly in mito-RER complexes. These data suggest that cytochrome P450 synthesis requires structural interaction of mitochondria and RER.     

Structure of mitochondria and vacuoles of Candida utilis and Schizosaccharomyces pombe studied by electron microscopy of serial thin sections and model building

The structure of mitochondria and of vacuoles in Candida utilis and Schizosaccharomyces pombe has been studied by electron microscopy of serial thin sections and subsequent model building. The models of the two cells of C. utilis which were studied confirmed our earlier findings, made by high voltage electron microscopy of thick sections, that there is a single, branched and continuous mitochondrial network in the cell (Davison & Garland, 1975). A model of a S. pombe cell showed that the mitochondrial structure was far more continuous than expected from inspection of thin sections, there being but two large and two small mitochondria. The models demonstrated that the few large vacuoles in C. utilis were interconnected into a single cluster, whereas in S. pombe there were two separate complexes of interconnected vacuoles towards each pole of the cell.     

An electron microscopic study of the adrenal cortical tissue of the domestic fowl

Summary The fine structure of the adrenal cortex of the domestic fowl has been studied from the 14th day of embryonic life to 980 days after hatching, using the electron microscope.Many mitochondria and vacuoles, round or oval, are observed in all cortical cells. The cristae mitochondriales are not laminar but villous. In the embryo, the mitochondria, whose cristae are not as well developed as those in chick and hen, are very low in electron density. Cytoplasmic vacuoles, either rough or smooth-surfaced, and with a homogeneous content of low electron density, are enclosed by a similar limiting membrane. Some of them are formed by the outer nuclear membrane and are characterized by many small particles on their outer surfaces. These vacuolated structures are perhaps parts of the endoplasmic reticulum. In the 980 day old hen, a number of osmiophilic droplets, round or irregularly shaped, are seen. Some of these are formed by mitochondria, while others accumulate within or around cytoplasmic vacuoles. In the embryo and in the chick, such droplets are never seen. It is assumed that the droplets are substances related to ageing, and that the lipid implicated in cortical hormone is not osmiophilic in the young chick and the embryo. It is perhaps produced in the cytoplasmic vacuoles with the aid of mitochondria. A perisinusoidal space and interparenchymatous cell spaces similar to those described for mammals by other workers, are observed in the adrenal gland of the domestic fowl. I assume that the secretory substance of the cortical and the medullary cell is first secreted into these spaces and then infiltrates into the capillary lumen.     

Selective flocculation of nucleic acids, lipids, and colloidal particles from a yeast cell homogenate by polyethyleneimine, and its scale-up.

Nucleic acids, lipid, and colloidal particulate material can be selectively flocculated from a yeast cell homogenate by the cationic polymer polyethyleneimine (PEI). Flocculation can occur from a crude homogenate, a homogenate clarified centrifugally, or by the prior use of sodium tetraborate (borax). Flocculation from a homogenate previously clarified by the use of borax is best suited for large-scale operation. The supernatant obtained following centrifugation is effectively free of nucleic acid, lipid, and particulate material with essentially 100% soluble enzyme recovery. Enzyme specific activity increases by approximately 45% compared to a zero PEI control.     

Rapid Isolation of Metabolically Active Mitochondria from Rat Brain and Subregions Using Percoll Density Gradient Centrifugation

Two procedures are described for isolating free (nonsynaptosomal) mitochondria from rat brain. Both procedures employ a discontinuous Percoll gradient and yield well coupled mitochondria which exhibit high rates of respiratory activity and contain little residual contamination by synaptosomes or myelin. The procedures are considerably more rapid than methods described previously for the isolation of brain mitochondria and do not require an ultracentrifuge or swing-out rotor. The first method separates mitochondria by gradient centrifugation from a P2 (crude mitochondrial) fraction and is likely to be widely applicable for studies in which at least 500 mg of tissue are available as starting material. In the second method, the unfractionated homogenate is subjected directly to gradient centrifugation. This method requires the preparation of more gradients (per gram of tissue) than the first method and yields a subcellular fraction with slightly more synaptosomal contamination. However, this second procedure is more rapid, requires less manipulation of the tissue, and is suitable for obtaining mitochondria with well preserved metabolic characteristics from subregions of single rat brains.     

Invaginated apical vacuoles in the cells of the proximal convoluted tubule in the rat kidney

Summary Following perfusion fixation of the rat kidney with glutaraldehyde the proximal tubule cells display small apical vacuoles, large apical vacuoles, and apical vacuoles in which a part of the limiting membrane is invaginated into the vacuole. These invaginated apical vacuoles occur more frequently in proximal convoluted tubules than in proximal straight tubules. One tubular cell may contain apical vacuoles of different sizes and stages of invagination, ranging from larger vacuoles with a wide lumen and a small area of invaginated membrane to smaller elements with no apparent lumen and a large area of invaginated membrane. Invaginated apical vacuoles lie either singly in the cytoplasm or close to the membranes of other apical vacuoles, but never in contact with the cell membrane or the membranes of lysosomes, endoplasmic reticulum, Golgi apparatus, mitochondria and peroxisomes.These findings suggest that the invaginated apical vacuoles are not fixation artifacts, but rather develop in living state in cells of the proximal tubule from spherical endocytotic elements.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Neuronal pigmented autophagic vacuoles: lipofuscin, neuromelanin, and ceroid as macroautophagic responses during aging and disease

The most striking morphologic change in neurons during normal aging is the accumulation of autophagic vacuoles filled with lipofuscin or neuromelanin pigments. These organelles are similar to those containing the ceroid pigments associated with neurologic disorders, particularly in diseases caused by lysosomal dysfunction. The pigments arise from incompletely degraded proteins and lipids principally derived from the breakdown of mitochondria or products of oxidized catecholamines. Pigmented autophagic vacuoles may eventually occupy a major portion of the neuronal cell body volume because of resistance of the pigments to lysosomal degradation and/or inadequate fusion of the vacuoles with lysosomes. Although the formation of autophagic vacuoles via macroautophagy protects the neuron from cellular stress, accumulation of pigmented autophagic vacuoles may eventually interfere with normal degradative pathways and endocytic/secretory tasks such as appropriate response to growth factors.     

Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis

Endosomes are prelysosomal organelles that serve as an intracellular site for the sorting, distribution, and processing of receptors, ligands, fluid phase components, and membrane proteins internalized by endocytosis. Whereas the overall functions of endosomes are increasingly understood, little is known about endosome structure, composition, or biogenesis. In this paper, we describe a rapid procedure that permits analytical and preparative isolation of endosomes from a variety of tissue culture cells. The procedure relies on a combination of density gradient centrifugation and free flow electrophoresis. It yields a fraction of highly purified, functionally intact organelles. As markers for endosomes in Chinese hamster ovary cells, we used endocytosed horseradish peroxidase, FITC-conjugated dextran, and [35S]methionine-labeled Semliki Forest virus. Total postnuclear supernatants, crude microsomal pellets, or partially purified Golgi fractions were subjected to free flow electrophoresis. Endosomes and lysosomes migrated together as a single anodally deflected peak separated from most other organelles (plasma membrane, mitochondria, endoplasmic reticulum, and Golgi). The endosomes and lysosomes were then resolved by centrifugation in Percoll density gradients. Endosomes prepared in this way were enriched up to 70-fold relative to the initial homogenate and were still capable of ATP-dependent acidification. By electron microscopy, the isolated organelles were found to consist of electron lucent vacuoles and tubules, many of which could be shown to contain an endocytic tracer (e.g., horseradish peroxidase). SDS PAGE analysis of integral and peripheral membrane proteins (separated from each other by condensation in Triton X-114) revealed a unique and restricted subset of proteins when compared with lysosomes, the unshifted free flow electrophoresis peak, and total cell protein. Altogether, the purification procedure takes 5-6 h and yields amounts of endosomes (150-200 micrograms protein) sufficient for biochemical, immunological, and functional analysis.     

5'-Nucleotidase activity and adenosine production in rat liver mitochondria.

The controversial subject of mitochondrial 5'-nucleotidase in the liver was studied employing density gradient fractionation combined with a method for analyzing the distribution profiles of marker enzymes based on multiple regression analysis. Triton WR-1339 was used to improve the separation of mitochondria from lysosomes by the gradient centrifugation technique. Adenosine production was examined further using acetate to increase intramitochondrial AMP, and thus adenosine production, in incubations with gradient centrifugation-purified mitochondria. Distribution analysis of the crude homogenate showed that 5'-nucleotidase activity exists in the mitochondrial fraction. To increase the resolution of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied. In this case the relative mitochondrial activity decreased but 5'-nucleotidase activity was still clearly detectable. The mitochondrial 5'-nucleotidase exhibited a Km of 94 microM and a Vmax of 31 nmol/min per mg protein for AMP. The kinetic data for the Mg2+, ATP, ADP and AOPCP sensitivity of the enzyme showed that it differs from the plasma membrane, lysosome and cytosol 5'-nucleotidases. AOPCP was only a moderate inhibitor, and ATP was a more potent inhibitor than ADP at a 1 mM concentration. The enzyme also showed a requirement of Mg2+. Acetate caused the conversion of intramitochondrial adenylates to AMP and the formation of adenosine. Adenosine concentration increased in the extramitochondrial space in a time-dependent manner, but only trace amounts of nucleotides were detected. The data show that 5'-nucleotidase activity producing adenosine exists in rat liver mitochondria and a concentration-dependent adenosine output from mitochondria by diffusion or facilitated diffusion is also suggested.     

A method for isolating intact mitochondria and nuclei from the same homogenate,and the influence of mitochondrial destruction on the properties of cell nuclei

1. An improved type of ground glass homogenizer for soft tissues has been described which brings about a high degree of cell disruption and liberation of nuclei without causing appreciable damage to mitochondria. The gentleness and effectiveness of the new homogenizer in respect to isolation of mitochondria have been ascertained by comparing the ATP-ase activities of mitochondria isolated in 0.25 M sucrose solution without pH adjustment using a previous type of homogenizer with those of mitochondria isolated under the same conditions with the aid of the new homogenizer. In these experiments sucrose of 0.25 molarity without pH adjustment has been used in order to maintain the mitochondria in a rather sensitive state so as to make slightly deleterious effects of homogenization readily apparent. 2. A new method is described for the isolation of morphologically intact mitochondria and cell nuclei from the same homogenate. In this procedure the pH of the homogenate in 0.44 M sucrose is maintained at 6.0-6.2 with citric acid during the homogenization. An alternative method employing 0.44 M sucrose plus 0.005 M CaCl(2) is given for the isolation of nuclei from tumor cells. However, the latter method does not produce unaltered mitochondria. 3. The alpha-ketoglutarate, malate, succinate, and hexanoate oxidases of the "intact" mitochondria isolated in 0.44 M sucrose adjusted to pH 6.0-6.2 with very dilute citric acid as described in this paper have been investigated, and it has been shown that the mitochondria compare favorably to those isolated in 0.25 M sucrose by a previously described method. 4. Mitochondria have been found to contain an enzyme which causes nuclei to lose their ability to form gels in dilute alkali. This enzyme is released from the mitochondria when the latter are disrupted. 5. Some properties of nuclei isolated by the new method have been briefly discussed.     

Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H(2) O(2) ) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress.     

Why are plastids maternally inherited in epilobium? Ultrastructural observations during microgametogenesis

We have investigated the sequential stages of microgametogenesis by electron microscopy, to determine the basis of maternal inheritance of plastids in Epilobium. The development of both the vegetative and generative cells has been followed using a semi-artificial growth system for pollen tubes. The generative cells inside the pollen grain contains numerous mitochondria, 5–8 proplastids, and, in contrast to the vegetative cytoplasm, only a few vacuoles. When the generative cell has divided into the two sperm cells inside the pollen tube, small vesicles deriving from dicytosome cisternae become abundant. These vesicles appear to form vacuoles by fusion which then contain remnants of fibrillar, globular or membranaceous material. It is suggested that this material derives from proplastids as the proplastids disappear either before or shortly after the generative cell has divided, concurrently with the appearance of the ‘remnants’ in the vacuoles. The mitochondria of the sperm cells remain intact.     

Identification and mitotic partitioning strategies of vacuoles in the unicellular red alga <Emphasis Type="Italic">Cyanidioschyzon merolae</Emphasis>

Cyanidioschyzon merolae is considered as a suitable model system for studies of organelle differentiation, proliferation and partitioning. Here, we have identified and characterized vacuoles in this organism and examined the partitioning of vacuoles using fluorescence and electron microscopy. Vacuoles were stained with the fluorescent aminopeptidase substrate 7-amino-4-chloromethylcoumarin l-arginine amide, acidotrophic dyes quinacrine and LysoTracker, and 4′,6-diamidino-2-phenyl indole, which, at a high concentration, stains polyphosphate. Vacuoles have been shown to be approximately 500 nm in diameter with a mean of around five per interphase cell. The vacuolar H⁺-ATPase inhibitor concanamycin A blocked the accumulation of quinacrine in the vacuoles, suggesting the presence of the enzyme on these membranes. Electron microscopy revealed that the vacuoles were single membrane-bound organelles with an electron-dense substance, often containing a thick layer surrounding the membrane. Immunoelectron microscopy using an anti-vacuolar-H⁺-pyrophosphatase antibody revealed the presence of the enzyme on these membranes. In interphase cells, vacuoles were distributed in the cytoplasm, while in mitotic cells they were localized adjacent to the mitochondria. Filamentous structures were observed between vacuoles and mitochondria. Vacuoles were distributed almost evenly to daughter cells and redistributed in the cytoplasm after cytokinesis. The change in localization of vacuoles also happened in microtubule-disrupted cells. Since no actin protein or filaments have been detected in C. merolae, this result suggests an intrinsic mechanism for the movement of vacuoles that differs from commonly known mechanisms mediated by microtubules and actin filaments.     

Some properties of vacuoles isolated from Neurospora crassa slime variant

A method for the isolation of vacuoles based on polybase induced lysis of protoplasts of the cell wall deficient Neurospora crassa slime variant is described. Isolated vacuoles are characterized by 12 to 50 times increased specific activities of several hydrolases as compared with the total homogenate of protoplasts. Total -amino nitrogen, arginine, and polyphosphate are also greatly enriched in these vacuoles. Vacuoles are equipped with a permease for the transport of basic amino acids across the tonoplast.Non-Standard Abbreviation DEAE-dextran diethylaminoethyl-dextran     

THE MECHANISM OF MITOCHONDRIAL EXTRUSION FROM PHENYLHYDRAZINE-INDUCED RETICULOCYTES IN THE CIRCULATING BLOOD

The mechanism of mitochondrial extrusion from reticulocytes was studied in whole blood from dogs made anemic by treatment with phenylhydrazine hydrochloride. The initial stage of preparation for mitochondrial extrusion was attraction of vesicles to mitochondria. There was subsequent encirclement of the organelle and other bodies, such as ferritin, by coalesced vesicles forming double membrane-limited vacuoles. Large vacuoles were formed from the union of single vacuoles, and they were usually situated near the periphery of the cell. Fusion of the outer membrane of vacuoles with the plasmalemma of the reticulocyte provided a route for exposure and release of mitochondria and other material to an extracellular location. An extracellular mitochondrion, therefore, was confined by its original double membrane, and a third membrane was derived from the internal boundary of vacuoles.