Isopycnic Sucrose Gradient Centrifugation Research Articles

Platelet Tetraspanin Complexes and Their Relation to Lipid Rafts.

CD63 and CD9 are members of the tetraspanin superfamily of integral membrane proteins that function as organizers of multi-molecular signaling complexes involved in cell morphology, motility and proliferation. CD63 is located in the membranes of lysosomes and dense granules in resting platelets. Following platelet activation and granule exocytosis, CD63 is expressed on the platelet plasma membrane and co-localizes with the αIIbβ3-CD9 complex. D545, a monoclonal antibody (MoAb) directed at the second extracellular loop of CD63, inhibits activated platelet spreading on immobilized fibrinogen and FAK phosphorylation in the adherent platelets. To identify CD63-associated signaling enzymes that could be involved in the signaling complex, lipid kinase assays were performed on D545 immunoprecipitates. CD63 co-immunoprecipitated with a lipid kinase with the enzymatic properties of PI4-kinase type II, confirmed by re-precipitation and immunoblotting with 4C5G (MoAb specific for the 55kDa PI4-kinase, PI4K55). The CD63-PI4K55 complex could be co-precipitated from both resting and activated platelets using anti-CD63 MoAb, and co-localized on the filopodia of thrombin-activated platelets using immuno-electron microscopy. Previous studies have demonstrated that tetraspanins associate with cholesterol-enriched membrane domains in a variety of cells including platelets. There is evidence, however, that these tetraspanin-enriched microdomains (TEMs) can be distinguished from prototypic lipid rafts on the basis of detergent solubility and protein composition. To investigate the association of the CD63-PI4K55 complex with lipid rafts in platelets, resting and thrombin-activated platelets were lysed in buffer containing either 1% Brij 35, or Triton X-100, the low- and high-density membrane fractions separated by isopycnic sucrose gradient centrifugation, and the identification of the low-density membrane fractions (LDMF) confirmed by the presence of LAT. CD63, CD9 and PI4K55 were present in the LDMF of platelets lysed in Brij 35 but not in Triton X-100; they were also present in the denser membrane fractions. CD63 and CD9 associated with cholesterol, as demonstrated by recovery of these proteins in the pellet following centrifugation of platelets lysed with 1% digitonin(a cholesterol-precipitating reagent), but not from lysates made with Brij 35/Triton X-100. Incubation of platelets with methyl-β-cyclodextrin(mβCD) to partially deplete cholesterol and disrupt the lipid rafts shifted LAT, CD63, CD9 and PI4K55 to denser fractions within the gradient. Immunoprecipitation of mβCD-treated platelets with anti-PI4K55 MoAb co-precipitated CD63 and CD9, demonstrating that the complexes were not dependent on residence within LDMFs, but remained intact in the denser fractions and pellet. Platelet tetraspanin complexes associate with cholesterol-enriched domains under conditions of mild detergent extraction. The maintenance of the complexes, however, was not dependent on their residence within lipid rafts, as the complexes remained intact following cholesterol depletion. Their presence in LDMF suggests that tetraspanin complexes may associate with platelet lipid rafts under some conditions, which could bring tetraspanin protein partners into proximity with raft residents, and facilitate the assembly and interaction of signaling complexes following platelet activation.

Constitutive expression of an endoplasmic reticulum small heat shock protein alleviates endoplasmic reticulum stress in transgenic tomato

To explore the function of endoplasmic reticulum-located small heat-shock proteins (ER-sHSPs) in ER stress, a putative ER-sHSP cDNA (the driven protein was named as LeHSP21.5 with GenBank accession No. AB026983) was isolated from tomato (Lycopersicon esculentum). Fractionation of the crude microsomes by isopycnic sucrose-gradient centrifugation revealed that LeHSP21.5 was distributed on a density corresponding to the fractions with a higher activity of ER marker enzyme, suggesting the localization of LeHSP21.5 in the ER. Overexpressing LeHSP21.5 in transgenic tomato plants (L. esculentum var. Zhongshu 4) greatly attenuated the lethal effect of tunicamycin on tomato seedlings. Moreover, under the tunicamycin treatment, transcripts of BiP, PDI and calnexin in transgenic tomato plants accumulated to a less level than those in non-transgenic tomato plants. These results suggest that LeHSP21.5 can function to alleviate the tunicamycin-induced ER stress.

Lipid composition-dependent incorporation of multiple membrane proteins into liposomes

Membrane proteins from bacteria Pasteurella multocida were used as a model for studying its incorporation into liposomes. An important step to achieve efficient high yield protein incorporation in proteoliposomes is the study of the more suitable lipid composition. To this end, we compared the amount of total protein, reconstituted by co-solubilization methods, into liposomes of phospholipids with different polar head groups and acyl chain lengths. The liposomes and proteoliposomes were characterised by isopycnic centrifugation in sucrose gradient and by dynamic light scattering. Experimental and theoretical results were compared considering the effects exerted through the hydrocarbon chain length, volume, and optimal cross-sectional area of the phospholipid (combined in the geometrical critical packing parameter, lipid–protein matching), critical spontaneous radius of curvature of the bilayer vesicle, phase transition temperature of the lipid and ratio of lipid–protein molecules present in the vesicles. The highest incorporation of multiple proteins was found with dipalmitoylphosphatidylcholine (DPPC), reaching a yield of 93% compared to the lower relative amounts incorporated in proteoliposomes of the other lipids. The incorporation of multiple proteins induces a proportional enhancement of vesicular dimension, since DPPC–proteoliposomes have an average diameter of 1850 Å, compared to the 1430 Å for pure DPPC vesicles.

In situ localization and in vitro induction of plant COPI-coated vesicles.

Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis-face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis-Golgi stack, gradually diminishing toward the trans-Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha-amylase-HDEL yielded a COPI-coated vesicle fraction that contained alpha-amylase as well as calreticulin.

Accumulation of small heat-shock protein homologs in the endoplasmic reticulum of cortical parenchyma cells in mulberry in association with seasonal cold acclimation.

Cortical parenchyma cells of mulberry (Morus bombycis Koidz.) trees acquire extremely high freezing tolerance in winter as a result of seasonal cold acclimation. The amount of total proteins in endoplasmic reticulum (ER)-enriched fractions isolated from these cells increased in parallel with the process of cold acclimation. Protein compositions in the ER-enriched fraction also changed seasonally, with a prominent accumulation of 20-kD (WAP20) and 27-kD (WAP27) proteins in winter. The N-terminal amino acid sequence of WAP20 exhibited homology to ER-localized small heat-shock proteins (smHSPs), whereas that of WAP27 did not exhibit homology to any known proteins. Like other smHSPs, WAP20 formed a complex of high molecular mass in native-polyacrylamide gel electrophoresis. Furthermore, not only WAP20 but also 21-kD proteins reacted with antibodies against WAP20. Fractionation of the crude microsomes by isopycnic sucrose-gradient centrifugation revealed that both WAP27 and WAP20 were distributed on a density corresponding to the fractions with higher activity of ER marker enzyme, suggesting localization of these proteins in the ER. When ER-enriched fractions were treated with trypsin in the absence of detergent, WAP20 and WAP27 were undigested, suggesting localization of these proteins inside the ER vesicle. The accumulation of a large quantity of smHSPs in the ER in winter as a result of seasonal cold acclimation indicates that these proteins may play a significant role in the acquisition of freezing tolerance in cortical parenchyma cells of mulberry trees.

Isolation and characterization of the outer membrane of Borrelia hermsii.

The outer membrane of Borrelia hermsii has been shown by freeze-fracture analysis to contain a low density of membrane-spanning outer membrane proteins which have not yet been isolated or identified. In this study, we report the purification of outer membrane vesicles (OMV) from B. hermsii HS-1 and the subsequent identification of their constituent outer membrane proteins. The B. hermsii outer membranes were released by vigorous vortexing of whole organisms in low-pH, hypotonic citrate buffer and isolated by isopycnic sucrose gradient centrifugation. The isolated OMV exhibited porin activities ranging from 0.2 to 7.2 nS, consistent with their outer membrane origin. Purified OMV were shown to be relatively free of inner membrane contamination by the absence of measurable beta-NADH oxidase activity and the absence of protoplasmic cylinder-associated proteins observed by Coomassie blue staining. Approximately 60 protein spots (some of which are putative isoelectric isomers) with 25 distinct molecular weights were identified as constituents of the OMV enrichment. The majority of these proteins were also shown to be antigenic with sera from B. hermsii-infected mice. Seven of these antigenic proteins were labeled with [3H]palmitate, including the surface-exposed glycerophosphodiester phosphodiesterase, the variable major proteins 7 and 33, and proteins of 15, 17, 38, 42, and 67 kDa, indicating that they are lipoprotein constituents of the outer membrane. In addition, immunoblot analysis of the OMV probed with antiserum to the Borrelia garinii surface-exposed p66/Oms66 porin protein demonstrated the presence of a p66 (Oms66) outer membrane homolog. Treatment of intact B. hermsii with proteinase K resulted in the partial proteolysis of the Oms66/p66 homolog, indicating that it is surface exposed. This identification and characterization of the OMV proteins should aid in further studies of pathogenesis and immunity of tick-borne relapsing fever.

Phagocytosis by Rat Liver: Relationships between Phagosomes and Lysosomes

To study the transfer of phagocytosed components from phagosomes to lysosomes, we have investigated phagocytosis by rat liver of killed Staphylococcus aureus labelled with 125I tyramine cellobiose. Lysosomes were identified by injecting the animals with Triton WR1339, a non ionic detergent that is endocytosed by the liver and accumulates in lysosomes, causing a marked decrease of their density; that allows these organelles to be well separated from other particles in a density gradient. Bacteria were quickly taken up by the liver; their uptake is followed by a slow degradation as ascertained by the increase of acid-soluble radioactivity in the homogenates with time. Triton WR1339 injection does not affect the uptake and the degradation of the particles. Differential centrifugation of homogenates shows that at any time after injection, most of the radioactivity is recovered in the mitochondrial fractions. Distributions of acid precipitable and acid soluble radioactivities amongst subcellular structures present in mitochondrial fractions were studied by isopycnic centrifugation in sucrose gradients, at increasing times after bacteria injection. Results show that: 1) acid-precipitable radioactivity is quasi-exclusively present in gradient fractions of high density, well separated from the fractions where there are recovered lysosomes; 2) with time, acid-soluble radioactivity is more and more associated with lysosomes, however, a significant proportion can be detected for many hours after injection, in gradient fractions where acid-precipitable radioactivity is located. The most plausible explanation of our observations is that phagocytosed particles are degraded in phagosomes and that the degradation products are delivered to lysosomes, probably by a vesicular process.

Subcellular distribution of receptor sites in human brain: Differentiation between heavy and light structures of high and low density

Studies of the subcellular localization of neuroreceptors in the rat brain have shown that most of them are associated with light and low density subcellular fractions. In two human brain areas, quite different subcellular distributions were observed. After fractionation by differential centrifugation of frontal cortex homogenates, benzodiazepine and serotonin 5-HT 2 receptors were mainly found in the heavy mitochondrial (M) fraction, whereas μ-opiate and muscarinic cholinergic receptors were mainly concentrated in the microsomal (P) fraction. In human putamen, the presynaptic markers of dopaminergic nerve terminals (neurotensin receptors, dopamine uptake sites and amine vesicular transporter-binding sites), benzodiazepine receptors and serotonin uptake sites were recovered both in the high and low density fractions, whereas the muscarinic, opiate and, to a lesser extent, dopamine D 2 receptors were mostly concentrated in the microsomal fraction. In the cerebral cortex, after isopycnic centrifugation in sucrose gradients, neuroreceptors were found in the high density fractions where the peaks of cytochrome oxidase and that of nerve endings, as identified by amine uptake and by means of electron microscopy were also found. A single peak of benzodiazepine receptors was observed in high density (1.15–1.17 g/ml) fractions suggesting that these receptors are much more concentrated in the nerve terminals or dendrites rather than in the dendritic spines or vesicles. The fact that muscarinic and opiate receptors were recovered in the P fraction with plasma membrane constituents and also in M and L fractions, which is confirmed by a bimodal distribution in sucrose gradient, suggests that they are localized in both the nerve terminals or dendrites and in the small vesicles or dendritic spines. In the putamen, much of the specific binding to uptake sites for dopamine and serotonin was recovered in the high density fractions, but the existence of another peak at a lower density indicates the presence of microsomal uptake sites. The results indicate that differential and isopycnic fractionation methods performed on human brain samples, make it possible to separate tissue fractions enriched in nerve endings, dendrites, dendritic spines, plasma membranes or vesicles.

Possible localisation of dolichol-dependent mannosyltransferase of Trypanosoma brucei to the rough endoplasmic reticulum

The glycosylphosphatidylinositol membrane anchor of variant surface glycoprotein of the African trypanosome Trypanosoma brucei contains several mannosyl residues for which dolichol phosphoryl mannose is supposed to be the precursor; this itself is probably synthesised by a dolichol-dependent mannosyltransferase. We have characterised and localised a mannosyltransferase activity of T. brucei which transfers mannose from GDP-[ 14C]mannose to exogenously added dolichyl phosphate. The enzyme was saturable for both its substrates and had a K m of 7.8 μM and 3.3 μM, respectively, for dolichyl phosphate and GDP-mannose. Mannosyltransferase was labile at 37°C in the presence of Triton X-100, but its activity remained constant for at least 60 min at temperatures between 10–15°C. The enzyme was inhibited by amphomycin and this inhibition was potentiated by the presence of 10 mM CaCl 2. After subcellular fractionation of cell homogenates by differential centrifugation, mannosyltransferase was recovered mainly in the microsomal fraction and its distribution was very similar to that of RNA, a marker for the rough endoplasmic reticulum. After isopycnic centrifugation in a linear sucrose gradient the distribution of mannosyltransferase also resembled that of RNA. Both constituents exhibited a shift towards lower densities after pre-treatment of microsomal membranes with inorganic pyrophosphate, while other membrane markers such as acid phosphatase and nucleoside diphosphatase did not. It is concluded that the formation of dolichol phosphoryl mannose from GDP-mannose and dolichyl phosphate in T. brucei occurs mainly in the rough endoplasmic reticulum.

The distribution of A1 adenosine receptor and 5'-nucleotidase in pig brain cortex subcellular fractions.

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [3H]R-PIA binding, the activity of adenosine metabolizing enzymes (5'-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A1 adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A1 adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding parameters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5'-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.

Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane

The SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COX-II). Antibodies directed against a beta-Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho0 strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.

Characterization of Glucose Transporter-Enriched Membranes from Rat Skeletal Muscle: Assessment of Endothelial Cell Contamination and Presence of Sarcoplasmic Reticulum and Transverse Tubules*

The subcellular origin of membranes from rat skeletal muscle that contain insulin-responsive glucose transporters was investigated. Rat skeletal muscle membranes were prepared by isopycnic centrifugation in sucrose gradients. In vivo insulin treatment increased the content of GLUT-4 glucose transporters in the 25% sucrose fraction (enriched in the plasma membrane marker 5'-nucleotidase) and decreased it in the 35% sucrose fraction (devoid of plasma membrane markers). The possibility of endothelial cell membrane contamination in these fractions was investigated using a mouse monoclonal antibody, MRC OX-43, raised against a cell surface protein specific to rat vascular endothelium. MRC OX-43 did not react with any of the muscle membrane fractions, but did recognize a protein of around 100 kDa in extracts of human endothelial cells and rat aorta. An antibody to the dihydropyridine receptor of skeletal muscle, IIC12, was used to determine the presence of transverse tubules in these fractions. IIC12 reacted positively with a 180-kDa protein in purified rat transverse tubules. In contrast, this antibody did not cross-react with the 25% or 35% sucrose fractions. The 25% sucrose fraction was devoid of calsequestrin and ryanodine receptor, cisternal sarcoplasmic reticulum markers. However, small amounts of these proteins were detected in the 35% sucrose fraction. The results suggest that the 25% sucrose fraction represents plasma membranes, while the 35% sucrose fraction is an insulin-sensitive intracellular fraction that contains, but is not enriched in, sarcoplasmic reticulum cisternae. The results further show that insulin-induced recruitment of GLUT-4 transporters in skeletal muscles can be demonstrated independently of GLUT-4 recruitment in endothelial cells.

Hydrogenosomes in the rumen entodiniomorphid ciliate Polyplastron multivesiculatum

The rumen entodiniomorphid ciliate protozoon Polyplastron multivesiculatum was shown, by biochemical and electron microscopic techniques, to possess hydrogenosomes. After differential centrifugation of whole cell homogenates the hydrogenosomal marker enzymes pyruvate:ferredoxin oxidoreductase and hydrogenase were recovered predominantly (61% and 70% of activity respectively) in the large granular fractions that were sedimented by centrifugation for 10(4) g-min (fraction P1) and 10(5) g-min (fraction P2). These subcellular fractions contained membrane-bound organelles that were approximately 0.4-0.6 microns in diameter and which had a mean equilibrium density of 1.22-1.24 g ml-1 after isopycnic centrifugation in sucrose gradients. Malate dehydrogenase (decarboxylating) activity, however, was predominantly non-sedimentable after centrifugation for 6 x 10(6) g-min. Numerous hydrogenosome-like organelles were present in the ectoplasm and endoplasm of the cell. Hydrogenase activity was demonstrated and localized in the protozoan cell using a novel staining procedure with distyryl nitroblue tetrazolium chloride (DSNBT).

Intracellular transport of endocytosed proteins in rat liver endothelial cells

1. Receptor-mediated endocytosis of mannose-terminated glycoproteins in rat liver endothelial cells has been followed by means of subcellular fractionation and by immunocytochemical labelling of ultrathin cryosections after intravenous injection of ovalbumin. For subcellular-fractionation studies the ligand was labelled with 125-tyramine-cellobiose adduct, which leads to labelled degradation products being trapped intracellularly in the organelle where the degradation takes place. 2. Isopycnic centrifugation in sucrose gradients of a whole liver homogenate showed that the ligand is sequentially associated with three organelles with increasing buoyant densities. The ligand was, 1 min after injection, recovered in a light, slowly sedimenting vesicle and subsequently (6 min) in larger endosomes. After 24 min the ligand was recovered in dense organelles, where also acid-soluble degradation products accumulated. 3. Immunocytochemical labelling of ultrathin cryosections showed that the ligand appeared rapidly after internalization in coated vesicles and subsequently in two larger types of endosomes. In the 'early' endosomes (1 min after injection) the labelling was seen closely associated with the membrane of the vesicle; after 6 min the ligand was evenly distributed in the lumen. At 24 min after injection the ligand was found in the lysosomes. 4. A bimodal distribution of endothelial cell lysosomes with different buoyant densities was revealed by centrifugation in iso-osmotic Nycodenz gradients, suggesting that two types of lysosomes are involved in the degradation of mannose-terminated glycoproteins in liver endothelial cells. Two populations of lysosomes were also revealed by sucrose-density-gradient centrifugation after injection of large amounts of yeast invertase. 5. In conclusion, ovalbumin is transferred rapidly through three endosomal compartments before delivering to the lysosomes. The degradation seems to take place in two populations of lysosomes.

Cellular uptake, localization and activity of fluoroquinolones in uninfected and infected macrophages

Pefloxacin, like other fluoroquinolones, accumulates in macrophages and several other types of nucleated cells (but not in erythrocytes). Upon fractionation of macrophage homogenates by isopycnic centrifugation in sucrose gradients, fluoroquinolones are not found associated with any specific cellular structure. We have compared the activities of pefloxacin and roxithromycin against intracellular Staphylococcus aureus in mouse J774 macrophages. Pefloxacin was significantly more active for equivalent intracellular drug concentrations (i.e. expressed by reference to the respective MICs of the drugs as determined in broth), suggesting differences in intracellular availability and/or capacity of the drugs to express their activity in the intracellular environment. The difference was enhanced by incubating the cells in acidic medium. We have also examined the cellular pharmacokinetics and intracellular distribution of pefloxacin in uninfected and Legionella pneumophila infected guinea pig macrophages. In contrast to uninfected cells from which pefloxacin was quickly released, macrophages infected with legionella retained approximately 20-30% of the accumulated pefloxacin after a 60-min wash-out. Cell fractionation studies indicated that the drug remaining in cells was associated with components of high buoyant density. These fractions also contained [3H] if cells had been incubated with [3H] labelled legionella (by in-vitro exposure to [3H]-thymidine, before phagocytosis). These results suggest that part of the intracellular pefloxacin becomes associated with legionella, or with legionella-containing cytoplasmic structures.

Analysis of the subcellular location of pullulanase produced by Escherichia coli carrying the pulA gene from Klebsiella pneumoniae strain UNF5023

Three different techniques, protease accessibility, cell fractionation and in situ immunocytochemistry, were used to study the location of the lipoprotein pullulanase produced by Escherichia coli K12 carrying the cloned pullulanase structural gene (pulA) from Klebsiella pneumoniae, with or without the K. pneumoniae genes required to transport pullulanase to the cell surface (secretion-competent and secretion-incompetent, respectively). Pullulanase produced by secretion-competent strains could be slowly but quantitatively released into the medium by growing the cells in medium containing pronase. The released pullulanase lacked the N-terminal fatty-acylated cysteine residue (and probably also a short N-terminal segment of the pullulanase polypeptide), confirming that the N-terminus is the sole membrane anchor in the protein. Pullulanase produced by secretion-incompetent strains was not affected by proteases, confirming that it is not exposed on the cell surface. Pullulanase cofractionated with both outer and inner membrane vesicles upon isopycnic sucrose gradient centrifugation, irrespective of the secretion competence of the strain. Examination by electronmicroscopy of vesicles labelled with antipullulanase serum and protein A-gold confirmed that pullulanase was associated with both types of vesicles. When thin-sectioned cells were examined by the same technique, pullulanase was found to be located mainly on the cell surface of the secretion-competent cells and mainly in the proximity of the inner membrane in the secretion-incompetent cells. Thus, while the results from three independent techniques (substrate accessibility, protease accessibility and in situ immunocytochemistry) show that pullulanase is transported to the cell surface of secretion-competent cells, this could not be confirmed by cell-fractionation techniques. Possible explanations for this discrepancy are discussed.

Protein Secretion by Gram-negative Bacteria: Characterization of two membrane proteins required for pullulanase secretion by escherichia coli k-12

Pullulanase secretion in Escherichia coli depends on the expression of a MalT-regulated operon called pulC. Characterization of the first two genes of this operon showed that they encode, respectively, a 31,000-Da protein (PulC) and a 70,600-Da protein (PulD) which has a putative signal peptide and that these two proteins are required for pullulanase secretion. The analysis of alkaline phosphatase hybrid proteins generated by TnphoA mutagenesis of pulC and pulD showed that both PulC and PulD contain export signals which can direct the alkaline phosphatase segment of the hybrids across the inner membrane. A representative PulC-PhoA hybrid protein fractionated mainly with the inner membrane upon isopycnic sucrose gradient centrifugation of membrane vesicles. This, together with sequencing data, suggests that PulC is an inner membrane protein. Antibodies raised against a purified PulD-PhoA hybrid protein were used to show that PulD was enriched in low density outer membrane vesicles.

Aromatic alcohol oxidase: A new membrane‐bound H2O2‐generating enzyme in alimentary tissues of the slug Arion ater

AbstractWe employed a sensitive spectrophotometric technique to detect H2O2‐generating enzymic activities capable of utilizing aldehydes or alcohols as substrates. We analysed tissue homogenates and subcellular fractions of the terrestrial slug Arion ater. Of the tissues tested, those of the alimentary system (especially crop and digestive gland) had the greatest activity and generated H2O2 when supplied with a variety of alcohol and aldehyde substrates. Aldehyde oxidase activity was soluble and had high apparent affinity for benzaldehyde. This activity is probably analogous to mammalian aldox (aldehyde oxidase; E.C.1.2.3.1), since it could use an artificial electron acceptor system and greatly preferred benzaldehyde to hypoxanthine. However, it differed from guinea pig liver aldehyde oxidase in that salicylaldehyde was a potent inhibitor rather than a substrate. Alcohol oxidase activity was particulate but not localised in mitochondria or peroxisomes. In digestive gland, it was localised differently from mannitol oxidase. In crop, the subcellular sites are either the same or inseparable by isopycnic density gradient centrifugation in sucrose gradients. Activity was at least ten times greater with aromatic alcohols than with ethanol, propanol, or butanol. No activity was detected with methanol, and the enzyme could not utilise an artificial electron acceptor system or pyridine nucleotides. Aromatic alcohol oxidase differed from mannitol oxidase with respect to temperature and pH sensitivity and in exhibiting greater apparent affinity for its substrates. The alcohol oxidase accepted a wide variety of aromatic alcohols as substrates. In several respects it resembled the corresponding enzyme of wood‐rot fungi, implying that the slug utilises dietary lignin or lignin precursors. Possible dietary, microbicidal, and detoxifying roles are considered for slug aldehyde and alcohol oxidases.

Buoyant density and lipid composition of purified myelin of aging human brain

Purified myelin of human brain from 15 young adult (below 50 years of age) and old (above 70 years of age) autopsy cases each was examined by isopycnic centrifugation in continuous sucrose gradients, and for lipid composition. The mean buoyant density of myelin was the same in both groups. Apparent features of old age were a wide range of density values, less compact myclin bands, and the dissociation of myelin into two bands in six of 15 old cases. Lipid analyses of randomly selected myelin samples of each group revealed an inverse relationship between the total lipid to protein ratio and density of myelin. In old age total lipids decreased by an average 10 mol lipid per mol. protein. This decrease was accounted for by cholestrol, phosphatidylserine and cerebrosides. Changes in fatty acid moieties mainly affected sphingolipids. C 20: 0 and C 24: 0 of sphingomyelin increased, as did even more markedly the more hydrophilic OH-fatty acids of cerebrosides. Correlations with buoyant density existed for the ratios of cholesterol to protein in young adult cases, and those of galactolipids to protein in cold cases. The results suggest that old age is associated with impaired stability and altered lipid composition of myelin.

Isolation of the outer membrane of Branhamella catarrhalis

The emergence of Branhamella catarrhalis as an important human pathogen has stimulated interest in investigations of the outer membrane (OM) of the bacterium. In this study, the OM of B. catarrhalis was isolated and partially characterized. Radiolabelled cells were lysed and fractionated by isopycnic centrifugation in a continuous sucrose gradient. Five fractions were identified. Fraction A consisted of OM fragments of varying density. Fractions B and C were OM of a discrete density containing some cytoplasmic membrane. Fraction D was cytoplasmic membrane and Fraction E contained smaller less dense fragments of cytoplasmic membrane. The protein composition of the Branhamella OM is typical for that of Gram-negative bacteria in that approximately 10 to 20 proteins were present with six to eight of these proteins predominating. Having isolated and partially characterized the OM by sucrose density centrifugation, five simpler techniques for isolating OM were employed and the preparations compared to OM isolated on the gradient. Techniques that are based on differential detergent solubility of OM and cytoplasmic membrane were ineffective in isolating OM of B. catarrhalis. By contrast, techniques that involved collection of OM vesicles were successful in isolating OM of B. catarrhalis. Collection of vesicles from broth culture supernatants and EDTA-heat-induced vesicles were identified as convenient and reliable methods for isolating OM. Isolating and partially characterizing the OM of B. catarrhalis represents an initial step in a systematic study of outer membrane antigens of the bacterium.