Intermediate Density Fractions Research Articles

Valorization of South African Coal Wastes through Dense Medium Separation

Sustainable management of coal waste is one of the major environmental concerns for coal mining, whether active or legacy, worldwide. Coal dump deposits demand a large physical area or footprint for disposal of solid waste, change the topography, and generate both pyritic dust and acid rock drainage (ARD) where pyritic coal waste is deposited. The beneficiation of dump deposits or, preferably, of coal waste prior to its dumping can reduce or even eliminate the liabilities related to coal waste management. In this work, dense medium separation studies of coal discards, using heavy liquids, resulted in three pooled fractions from typical South African coal waste discards from the Mpumalanga region for future use: (a) a fraction of low density with increased calorific value; (b) a fraction of intermediate density, rich in ash and acid neutralizing minerals and lower in sulfur; and (c) a fraction of high density, rich in sulfidic minerals including pyrite. The fractions were characterized using particle size analysis, sink-float studies, static tests to predict ARD potential, proximateand ultimate analysis, and gross calorific value. The results showed that approximately 70% of this discard coal is composed of a material of sufficient quality for energy generation in conventional power stations. A pyrite-rich concentrate made up 2% of the total discard mass; comprising more than 45% of the sulfidic mineral present in the feed and displaying no acid neutralizing capacity (ANC). The remaining discard fraction, with intermediate density, presented potential to be used for several ends including soil fabrication, co-disposal or as aggregate material in civil engineering; additional testing to ensure applicability for the selected re-purposing option should be chosen based on proposed use.

The role of autochthonous organic matter in radioactive cesium accumulation to riverine fine sediments

Anthropogenically released radioactive cesium (RCs) poses serious ecological and environmental concerns given its persistency in the environment. Although accumulation of RCs in aqueous and sedimentary environments is often reported to associate with organic matter (OM), the mechanisms responsible remain unclear. Here, we investigated RCs in fine sediments along the Abukuma River, the largest river near the Fukushima Daiichi Nuclear Power Plant, 1.5–4 years after the accident. Measuring the density-separated sediment fractions with a broad range of OM content (%) indicated that the RCs concentration (Bq·kg−1) is positively correlated with OM content for intermediate-density fractions in which OM is primarily characterized by autochthonous origin. This relationship, however, did not hold for light-density fractions containing a high proportion of large-size allochthonous OM. Furthermore, H2O2-assisted OM digestion and amorphous material dissolution treatments resulted in only a minor reduction in sedimentary RCs. These results along with the fact that sediments with high autochthonous OM content showed high specific surface area indicated that RCs is tightly associated with finer-grained and chemically non-labile inorganic fractions concurrently resident with autochthonous OM. Overall, our findings highlight that autochthonous OM exerts a significant control on the accumulation, transport, and fate of RCs in aqueous and sedimentary environments.

Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment

The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis.

The Use of Static and Humidity Cell Tests to Assess the Effectiveness of Coal Waste Desulfurization on Acid Rock Drainage Risk

The environmental benefits of waste desulfurization were evaluated in the Santa Catarina coal field, Brazil. Coal waste from a beneficiation plant was separated into three density fractions, using a two stage process. Characterization of these fractions indicated that the low (D 2.7 g/cm3) density fractions were potentially suitable for energy and sulfuric acid production, respectively. The waste fraction of intermediate density (2.2 < D < 2.7 g/cm3) represented 69% of the total mass studied and had a relatively low sulfide content, and it was postulated that it may be suitable for land disposal with minimum risk to the surrounding environment. This hypothesis was tested using laboratory-scale static and kinetic tests, which indicated that although the fraction remained net acid generating, the rate and net amount of metals, salts, and acidity that leached was considerably less than that of the discards before separation. It was concluded that this approach could reduce the amount of waste generated, as well as the associated pollution risk.

Nature of soil organo-mineral assemblage examined by sequential density fractionation with and without sonication: Is allophanic soil different?

Organic matter (OM) bound to soil mineral particles (higher-density particles) tends to be more stabilized, enriched in 13C and 15N, and has a lower C:N ratio. Yet how these variations in OM chemistry are linked to the nature of organo-mineral assemblage remains poorly understood, especially in allophanic soils where high amounts of OM are stabilized by interactions with reactive inorganic phases such as short-range-order (SRO) minerals. We thus assessed the extent to which the degree of aggregation and its disruption during fractionation control the distribution and chemistry of the soil organo-mineral particles across six density fractions using a volcanic soil (allophanic Andisol) based on selective dissolution, microscopy (SEM), solid-state 13C NMR spectroscopy and δ13C and δ15N analyses. Intermediate-density fractions (2.0–2.5gcm−3) accounted for 63–86% of organic C and N, 73–93% of pyrophosphate-extractable iron and aluminum (Fep, Alp), and 78–95% of oxalate-extractable metals (Feo, Alo) in the bulk soil sample. While air-drying pretreatment had little effect, sonication during fractionation led to (i) fragmentation of both plant detritus and some of the aggregates of 30–100mm sizes, (ii) release of occluded low-density fraction (<1.6gcm−3) which largely originated from the aggregates of 1.6–2.0gcm−3 density range, and (iii) redistribution of organo-mineral particles (15–16% of total OM and 7–19% of the extractable metals) within the intermediate density fractions. Positive correlation of Alp with C:N ratio and negative correlation of Alp with δ15N among the fractions suggest preferential binding of Alp phase (e.g., organo-Al complexes) to decaying plant detritus. Positive correlation of Alo and Feo with δ15N, together with theoretical density calculations of idealistic organo-mineral association modes, suggests that 15N enrichment may be coupled with OM binding to SRO minerals and with the formation of physically-stable aggregates of micron/submicron sizes in accord with our conceptual model (Asano and Wagai, 2014). The general pattern of 13C and 15N enrichment and C:N decline with increasing particle density remained largely unchanged despite the sonication effects detected, indicating that sonication-resistant organo-mineral assemblages largely control the observed patterns. The similarity in the density-dependent changes of OM chemistry between the studied Andisol and the soils with crystalline clay and metal oxide mineralogies in previous studies strongly suggests a common biogeochemical control which deserves further investigation.

Sorptive fractionation of organic matter and formation of organo-hydroxy-aluminum complexes during litter biodegradation in the presence of gibbsite

Solid and aqueous phase Al species are recognized to affect organic matter (OM) stabilization in forest soils. However, little is known about the dynamics of formation, composition and dissolution of organo-Al hydroxide complexes in microbially-active soil systems, where plant litter is subject to microbial decomposition in close proximity to mineral weathering reactions. We incubated gibbsite–quartz mineral mixtures in the presence of forest floor material inoculated with a native microbial consortium for periods of 5, 60 and 154days. At each time step, samples were density separated into light (<1.6gcm−3), intermediate (1.6–2.0gcm−3), and heavy (>2.0gcm−3) fractions. The light fraction was mainly comprised of particulate organic matter, while the intermediate and heavy density fractions contained moderate and large amounts of Al-minerals, respectively. Multi-method interrogation of the fractions indicated the intermediate and heavy fractions differed both in mineral structure and organic compound composition. X-ray diffraction analysis and SEM/EDS of the mineral component of the intermediate fractions indicated some alteration of the original gibbsite structure into less crystalline Al hydroxide and possibly proto-imogolite species, whereas alteration of the gibbsite structure was not evident in the heavy fraction. DRIFT, Py–GC/MS and STXM/NEXAFS results all showed that intermediate fractions were composed mostly of lignin-derived compounds, phenolics, and polysaccharides. Heavy fraction organics were dominated by polysaccharides, and were enriched in proteins, N-bearing compounds, and lipids. The source of organics appeared to differ between the intermediate and heavy fractions. Heavy fractions were enriched in 13C with lower C/N ratios relative to intermediate fractions, suggesting a microbial origin. The observed differential fractionation of organics among hydroxy-Al mineral types suggests that microbial activity superimposed with abiotic mineral-surface-mediated fractionation leads to strong density differentiation of organo-mineral complex composition even over the short time scales probed in these incubation experiments. The data highlight the strong interdependency of mineral transformation, microbial community activity, and organic matter stabilization during biodegradation.

Effect of different milling processes on the mineral distribution in a coal powder

Coal samples obtained from Wanbei (WB), Pingxiang (PX), Liupanshui (LP), and Datong (DT) mines were pulverized, using either a vibration mill or a ball mill, to different degrees of fineness. The effect of the different grinding methods on the mineral distribution within the pulverized coal was investigated by using proximate analysis, particle size analysis, and float-sink tests. The results show that the ash content in WB, PX, and DT coal increases with increasing particle size overall, while the ash content of the LP coal remain almost the same within each size fraction. In that case the ash in each fraction is similar to that of the raw coal. The ash versus size distributions for the same coal sample milled with the same grinding method to different degrees of fineness are similar. The ash versus size distribution of the coal powder with a 15% screen residue that was prepared with the vibration mill is different from the distribution of a similar sample prepared with a ball mill. The curves also vary between different coal samples. The grinding method has a great influence on the distribution of minerals across the various particle sizes. The float-sink tests and the laser particle size analysis results on PX and DT coal samples show that fines dominate the higher density fractions although the large +2.0g/cm3 fraction was dominated by coarse particles. The size distribution of the low density fraction and +2.0g/cm3 density fraction is bimodal. The size distribution of the intermediate density fraction is multimodal.

Pedogenic, mineralogical and land-use controls on organic carbon stabilization in two contrasting soils

Organo-mineral complexation in soils is strongly controlled by pedogenesis, but the mechanisms controlling it and its interaction with cultivation are not yet well understood. We compared the mineralogy and quality of organic carbon (C) among organo-mineral fractions from two soils with contrasting pedogenic origin. Sequential density fractionation (SDF; using 1.6, 1.8, 2.1, 2.4 and 2.6 g mL-1 sodium polytungstate solutions) followed by thermal analysis was applied to a Chernozem from Ellerslie, Alberta, and a Luvisol from Breton, Alberta, each under native and cultivated land uses. Similar clay mineralogy suggested that pedogenic controls on organic C stabilization were related to long-term vegetation cover. In addition to large differences in total organic C quantities, bulk soil and isolated fractions showed significant differences in organic C quality. Samples under native vegetation revealed greater organo-mineral complexation at Ellerslie compared with Breton, as expressed by less solubilisation, more organic C recovered in intermediate-density fractions, and exothermic differential scanning calorimetry peak signals associated with more stable forms of organic C. Long-term cultivation resulted in an overall shift to more stable organo-mineral complexes. The proportion of soil C in the 2.1-2.4 g mL-1 fraction increased under cultivation from 21 to 32% in Breton samples, and from 6 to 16% in Ellerslie samples. The quality of inherited pedogenic soil organic C stored in a soil thus appears to determine its response to long-term cultivation.Key words: Cultivation, sequential density fractionation, organic carbon, organic matter, thermal analysis

Sequential density fractionation across soils of contrasting mineralogy: evidence for both microbial- and mineral-controlled soil organic matter stabilization

Sequential density fractionation separated soil particles into “light” predominantly mineral-free organic matter vs. increasingly “heavy” organo-mineral particles in four soils of widely differing mineralogy. With increasing particle density C concentration decreased, implying that the soil organic matter (OM) accumulations were thinner. With thinner accumulations we saw evidence for both an increase in 14C-based mean residence time (MRT) of the OM and a shift from plant to microbial origin.Evidence for the latter included: (1) a decrease in C/N, (2) a decrease in lignin phenols and an increase in their oxidation state, and (3) an increase in δ13C and δ15N. Although bulk-soil OM levels varied substantially across the four soils, trends in OM composition and MRT across the density fractions were similar. In the intermediate density fractions (~1.8–2.6 g cm−3), most of the reactive sites available for interaction with organic molecules were provided by aluminosilicate clays, and OM characteristics were consistent with a layered mode of OM accumulation. With increasing density (lower OM loading) within this range, OM showed evidence of an increasingly microbial origin. We hypothesize that this microbially derived OM was young at the time of attachment to the mineral surfaces but that it persisted due to both binding with mineral surfaces and protection beneath layers of younger, less microbially processed C. As a result of these processes, the OM increased in MRT, oxidation state, and degree of microbial processing in the sequentially denser intermediate fractions. Thus mineral surface chemistry is assumed to play little role in determining OM composition in these intermediate fractions. As the separation density was increased beyond ~2.6 g cm−3, mineralogy shifted markedly: aluminosilicate clays gave way first to light primary minerals including quartz, then at even higher densities to various Fe-bearing primary minerals. Correspondingly, we observed a marked drop in δ15N, a weaker decrease in extent of microbial processing of lignin phenols, and some evidence of a rise in C/N ratio. At the same time, however, 14C-based MRT time continued its increase. The increase in MRT, despite decreases in degree of microbial alteration, suggests that mineral surface composition (especially Fe concentration) plays a strong role in determining OM composition across these two densest fractions.

CD82 endocytosis and cholesterol‐dependent reorganization of tetraspanin webs and lipid rafts

Tetraspanin CD82 suppresses cell migration, tumor invasion, and tumor metastasis. To determine the mechanism by which CD82 inhibits motility, most studies have focused on the cell surface CD82, which forms tetraspanin-enriched microdomains (TEMs) with other transmembrane proteins, such as integrins. In this study, we found that CD82 undergoes endocytosis and traffics to endosomes and lysosomes. To determine the endocytic mechanism of CD82, we demonstrated that dynamin and clathrin are not essential for CD82 internalization. Depletion or sequestration of sterol in the plasma membrane markedly inhibited the endocytosis of CD82. Despite the demand on Cdc42 activity, CD82 endocytosis is distinct from macropinocytosis and the documented dynamin-independent pinocytosis. As a TEM component, CD82 reorganizes TEMs and lipid rafts by redistributing cholesterol into these membrane microdomains. CD82-containing TEMs are characterized by the cholesterol-containing microdomains in the extreme light- and intermediate-density fractions. Moreover, the endocytosis of CD82 appears to alleviate CD82-mediated inhibition of cell migration. Taken together, our studies demonstrate that lipid-dependent endocytosis drives CD82 trafficking to late endosomes and lysosomes, and CD82 reorganizes TEMs and lipid rafts through redistribution of cholesterol.

Mitochondrial Porin VDAC 1 Seems to Be Functional in Rickettsial Cells

We have recently shown that Rickettsia prowazekii (typhus group rickettsiae) cells incorporate human mitochondrial porin VDAC1. Here, I report on the import of porin by rickettsiae of spotted fever group. It was shown that rickettsial cells of heavy band of Renografin density gradient, known as permeabilized rickettsiae, contain much more VDAC 1 compared to the cells of light band, that is, non-permeabilized rickettsiae. These data hint at a functionality of mitochondrial porin in rickettsial cells. The cells of Rickettsia canadensis were broken in French-press, and total membranes were fractionated on linear sucrose density gradient. In this way, previous data were confirmed that an outer rickettsial membrane does not contain porin, while fraction of intermediate density, which likely represents Bayer's adhesion zones, is enriched in VDAC 1. This is consistent with earlier results and their interpretation that imported porin seems to localize to contact sites between inner and outer rickettsial membranes. Based on well-known phylogenetic relationship of Rickettsiales and mitochondria, an evolutionary scenario for the origin of protein import machinery is proposed. A dependence of rickettsiae upon essential protein(s), destined to organelle, is also viewed as a nature of their obligate endosymbiotic lifestyle.

Characterization of lptA and lptB , Two Essential Genes Implicated in Lipopolysaccharide Transport to the Outer Membrane of Escherichia coli

The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic sigma factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.

Gross N transformation rates and net N mineralisation rates related to the C and N contents of soil organic matter fractions in grassland soils of different age

We investigated the relationship between soil organic matter (SOM) content and N dynamics in three grassland soils (0–10 and 10–20 cm depth) of different age (6, 14 and 50 y-old) with sandy loam textures. To study the distribution of the total C and N content the SOM was fractionated into light, intermediate and heavy density fractions of particulate macro-organic matter (150–2000 μm) and the 50–150 μm and <50 μm size fractions. The potential gross N transformation rates (mineralisation, nitrification, NH 4 + and NO 3 − immobilization) were determined by means of short-term, fully mirrored 15N isotope dilution experiments (7-d incubations). The long-term potential net N mineralisation and gross N immobilization rates were measured in 70-d incubations. The total C and N contents mainly tended to increase in the 0–10 cm layer with increasing age of the grassland soils. Significant differences in total SOM storage were detected for the long-term (50 y-old) conversion from arable land to permanent grassland. The largest relative increase in C and N contents had occurred in the heavy density fraction of the macro-organic matter, followed by the 50–150 and <50 μm fractions. Our results suggest that the heavy density fraction of the macro-organic matter could serve as a good indicator of early SOM accumulation, induced by converting arable land to permanent grassland. Gross N mineralisation, nitrification, and (long-term) gross N immobilization rates tended to increase with increasing age of the grasslands, and showed strong, positive correlations with the total C and N contents. The calculated gross N mineralisation rates (7-d incubations) and net N mineralisation rates (70-d incubations) corresponded with a gross N mineralisation of 643, 982 and 1876 kg N ha −1 y −1, and a net N mineralisation of 195, 208 and 274 kg N ha −1 y −1 in the upper 20 cm of the 6, 14 and 50 y-old grassland soils, respectively. Linear regression analysis showed that 93% of the variability of the gross N mineralisation rates could be explained by variation in the total N contents, whereas total N contents together with the C-to-N ratios of the <50 μm fraction explained 84% of the variability of the net N mineralisation rates. The relationship between long-term net N mineralisation rates and gross N mineralisation rates could be fitted by means of a logarithmic equation (net m=0.24Ln(gross m)+0.23, R 2=0.69, P<0.05), which reflects that the ratio of gross N immobilization-to-gross N mineralisation tended to increase with increasing SOM contents. Microbial demand for N tended to increase with increasing SOM content in the grassland soils, indicating that potential N retention in soils through microbial N immobilization tends to be limited by C availability.

Insights into the association of FcgammaRII and TCR with detergent-resistant membrane domains: isolation of the domains in detergent-free density gradients facilitates membrane fragment reconstitution.

Plasma membrane rafts are routinely isolated as detergent-resistant membranes (DRMs) floating in detergent-free density gradients. Here we show that both the presence and exclusion of TX-100 during the density gradient fractionation have profound effects on the location of FcgammaRII and TCR in DRM fractions. The presence of TX-100 during fractionation promoted solubilization of non-cross-linked FcgammaRII when the receptor was insufficiently dissolved upon cell lysis. In the detergent-supplemented gradients, TX-100 micelles floated, further enhancing dissociation of FcgammaRII and TCR from DRMs and promoting a shift of the receptors toward higher-density fractions. Hence, fractionation of cell lysates over the detergent-containing gradients enables isolation of DRMs devoid of weakly associated proteins, like nonactivated FcgammaRII and TCR. On the other hand, in a detergent-free gradient, non-cross-linked FcgammaRII, fully soluble in 0.2% TX-100, was recovered in DRM fractions. Moreover, employment of the TX-100-free gradient for refractionation of intermediate-density fractions, derived from detergent-supplemented gradients and containing FcgammaRII and TCR, resulted in flotation of the receptors to buoyant fractions. An analysis of the TX-100 concentration revealed that after fractionation of 0.2% TX-100 cell lysates in the absence of detergent, the level of TX-100 in DRM fractions was reduced to 0.01%, below the critical micelle concentration. Therefore, fractionation of detergent cell lysates over detergent-free gradients can mimic conditions for a membrane reconstitution, evoking association of a distinct subset of membrane proteins, including FcgammaRII and TCR, with DRMs.

Localization of p21-activated Protein Kinase γ-PAK/Pak2 in the Endoplasmic Reticulum Is Required for Induction of Cytostasis

The intracellular localization and physiological functions of the p21-activated protein kinase gamma-PAK have been examined in human embryonic kidney 293T and COS-7 cells. At 1-4 days post-transfection, cell division is inhibited by the expression of wild type (WT) gamma-PAK and the mutant S490A, whereas cells expressing S490D and the inactive mutants K278R and T402A grow exponentially, indicating a role for gamma-PAK in the induction of cytostasis. WT gamma-PAK and S490A are localized in a region surrounding the nucleus identified as the endoplasmic reticulum (ER), as determined by immunofluorescence, whereas K278R, T402A, and S490D lack localization. As shown by sucrose density gradient centrifugation, WT gamma-PAK, S490A, and endogenous gamma-PAK are distributed among the high density (ER-associated), intermediate density, and low density fractions, whereas the mutants that do not inhibit cell division are present only as soluble enzyme. The amount of endogenous gamma-PAK associated with the particulate fractions is increased 4-fold when cell division is inhibited by ionizing radiation. gamma-PAK in the ER and intermediate density fractions has high specific activity and is active, whereas the soluble form of gamma-PAK has low activity and is activable. The importance of localization of gamma-PAK is supported by data with the C-terminal mutants S490D and Delta 488; these mutants have high levels of protein kinase activity but do not induce cytostasis and are not bound to the ER. A model for the induction of cytostasis by gamma-PAK through targeting of gamma-PAK to the ER is presented in which gamma-PAK activity and Ser-490 are implicated in the regulation of cytostasis.

Carbon Turnover and Carbon‐13 Natural Abundance in Organo‐Mineral Fractions of a Tropical Dry Forest Soil under Cultivation

Soil organic matter (SOM) turnover is more rapid in tropical than in temperate soils. One possible reason is a limited ability of tropical soils to stabilize SOM. To test this, we measured C turnover resulting from 12 years' cultivation of a forest soil with sorghum [Sorghum bicolor (L.) Moench]. Carbon‐13 natural abundance (δ13C) signatures of forest‐ and sorghum‐derived C were used to quantify C losses and gains in organo‐mineral fractions separated by particle size, and further by density (for sands and silts) and magnetic susceptibility (for clays). Nearly 50% of original C was in the silt‐sized fraction, mostly in microaggregates of intermediate density; 30% was held by clays, particularly those of intermediate magnetic susceptibility; and 20% was of sand‐size, low‐density, often recognizable plant residues. The δ13C values in the forest soil showed the more humified SOM to be associated with finer, denser, and less magnetic fractions. After cultivation, total C content was 28% lower, with 59% of this reduction in the silts, 28% in the sand, and 19% in the clays. Loss of forest‐derived C amounted to 45%. The sand fraction lost 54% of its forest C, the silts 45% (mostly from intermediate density fractions), and the clays 23% (mostly from intermediate magnetic fractions). Gains in sorghum‐derived C amounted to 32% of C in the sand fraction, 12% in the silts (relatively evenly distributed among densities) and 13% in the clays (mostly in the nonmagnetic fraction). Thus, losses of forest C and gains of sorghum C occurred in different organo‐mineral fractions, indicating that there were no unique active fractions corresponding with the concept of C pools with defined turnover characteristics used in models of organic matter turnover.

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate.

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous sucrose gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.

Purification and characterization of autophagosomes from rat hepatocytes.

To investigate the properties and intracellular origin of autophagosomes, a procedure for the purification and isolation of these organelles from rat liver has been developed. Isolated hepatocytes were incubated with vinblastine to induce autophagosome accumulation; the cells were then homogenized and treated with the cathepsin C substrate glycyl-l-phenylalanine 2-naphthylamide to cause osmotic disruption of the lysosomes. Nuclei were removed by differential centrifugation, and the postnuclear supernatant was fractionated on a discontinuous Nycodenz density gradient. The autophagosomes, recognized by their content of autophagocytosed lactate dehydrogenase (LDH), could be recovered in an intermediate-density fraction, free from cytosol and mitochondria. Finally, the autophagosomes were separated from the endoplasmic reticulum and other membranous elements by centrifugation in a Percoll colloidal density gradient, followed by flotation in iodixanol to remove the Percoll particles. The final autophagosome preparation represented a 24-fold purification of autophagocytosed LDH relative to intact cells, with a 12% recovery. The purified autophagosomes contained sequestered cytoplasm with a normal ultrastructure, including mitochondria, peroxisomes and endoplasmic reticulum in the same proportions as in intact cells. However, immunoblotting indicated a relative absence of cytoskeletal elements (tubulin, actin and cytokeratin), which may evade autophagic sequestration. The autophagosomes showed no enrichment in protein markers typical of lysosomes (acid phosphatase, cathepsin B, lysosomal glycoprotein of 120 kDa), endosomes (early-endosome-associated protein 1, cation-independent mannose 6-phosphate receptor, asialoglycoprotein receptor) or endoplasmic reticulum (esterase, glucose-regulated protein of 78 kDa, protein disulphide isomerase), suggesting that the sequestering membranes are not derived directly from any of these organelles, but rather represent unique organelles (phagophores).

Enrichment of carnitine palmitoyltransferases I and II in the contact sites of rat liver mitochondria.

The submitochondrial distribution of the overt and latent carnitine palmitoyltransferases (CPT I and II respectively) of rat liver mitochondria were studied. Separation of outer and inner membranes, as well as of a fraction of intermediate density consisting of contact sites between the two membranes, was achieved, as judged by the distribution of marker enzymes. Both CPT I and CPT II were found to be enriched within the contact- site fraction of mitochondria. These data show that the two carnitine acyltransferases are distributed non-uniformly within their respective membranes, and that subpopulations of the two enzymes occur in close proximity within the mitochondrial membrane structure, while retaining their different accessibilities to cytosolic and matrix pools of metabolites. As the number of contact sites is known to vary with changes in the energy status of mitochondria, the possibility that such changes may acutely affect the proportion of CPT I within the distinctive lipid environment of the contact sites, and thus its overall kinetic characteristics, is discussed.

Distribution of inositol 1,4,5-trisphosphate receptor isoforms, SERCA isoforms and Ca2+ binding proteins in RBLm2H3 rat basophilic leukemia cells

RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) isoforms (2b and 3) as well as four Ca 2+ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP3 binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca 2+ binding proteins in fraction F3 and the sucrose density gradient fractions. Ins P3R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP3R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP3R-1 and InsP3R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP3R-1 and InsP3R-2 and the SERCs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP3R isoforms, the two SERCA isoforms and the four Ca2+ binding proteins investigated. This heterogeneity may underlie specialization of the Ca2+ stores and the subsequent initiation of intracellular Ca2+ signals.