Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01.

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35,000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [¹⁴C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35 000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Permeability of Rat Liver Microsomes to Sucrose and Carboxypoly-glucose in vitro

A study was made of the permeability of the microsomes to C¹⁴-sucrose and to C¹⁴-carboxypolyglucose, a branch-chained glucose polymer with a molecular weight of approximately 50,000. It was concluded that the microsomal membranes are permeable to sucrose on the basis of the following evidence: the volume of distribution of C¹⁴-sucrose was 84 per cent of the total microsomal pellet water; the sucrose unavailable volume, the per cent dry weights of the microsomal pellets, and the optical density of microsomal suspensions were independent of the concentration of sucrose in the suspending medium. It is suggested that the microsomal water which is unavailable to sucrose may be bound to protein and/or ribonucleic acid of the microsomes. The volume of distribution of C¹⁴-carboxypolyglucose was 44 per cent of the total pellet water, and it is considered that the microsomal membranes may be impermeable to this compound. Pretreatment with ribonuclease resulted in small increases in the volumes of distribution of both C¹⁴-sucrose and C¹⁴-carboxypolyglucose.     

PERMEABILITY OF MICROSOMAL MEMBRANES ISOLATED FROM RAT LIVER

Water compartments, permeability, and the possible active translocation of various substances in rat liver microsomes were studied by using radioactive compounds and ultracentrifugation. The total water of the microsomal pellet, 3.4 µl/mg dry weight, is the sum of water in the extramicrosomal and intramicrosomal spaces, or 56 and 44%, respectively. Sucrose space accounts for 77% of the intramicrosomal water and the hydration water ~ 14%, leaving almost no sucrose-impermeable space when using the ultracentrifugation approach. With increasing sucrose concentration, microsomes do not show an osmotic response. The intramicrosomal water decreases greatly in the presence of Cs⁺ and Mg⁺⁺ in rough but not in smooth microsomes. Uncharged substances of molecular weight of up to at least 600 freely penetrate microsomal membranes, which already become impermeable to charged substances at a molecular weight of 90. These substances also induce an osmotic response. The vesicles can be made permeable to charged substances after water treatment and cooling, which, however, does not increase glucose-6-phosphatase and inosine diphosphatase (IDPase) activities, and these enzymes can still be activated by deoxycholate. IDPase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation reactions, performed in vitro, also disproved the hypothesis of an accumulation of charged substances inside of vesicles of being a major pathway. The products of the enzymic reactions as well as the glucuronidated form of a hydroxylated product can be recovered on the cytoplasmic side of membranes, and little accumulation occurs in the intravesicular compartment.     

The mechanism of acceptor reactions of leuconostoc mesenteroides B-512F dextransucrase

Reactions of dextransucrase and sucrose in the presence of sugars (acceptors) of low molecular weight have been observed to give a dextran of low molecular weight and a series of oligosaccharides. The acceptor reaction of dextransucrase was examined in the absence and presence of sucrose by using d-[¹⁴C]glucose, d-[¹⁴C]fructose, and ¹⁴C-reducing-end labeled maltose as acceptors. A purified dextransucrase was pre-incubated with sucrose, and the resulting d-fructose and unreacted sucrose were removed from the enzyme by chromatography on columns of Bio-Gel P-6. The enzyme, which migrated at the void volume, was collected and referred to as “charged enzyme”. The charged enzyme was incubated with ¹⁴C-acceptor in the absence of sucrose. Each of the three acceptors gave two fractions of labeled products, a high molecular weight product, identified as dextran, and a product of low molecular weight that was an oligosaccharide. It was found that all three of the acceptors were incorporated into the products at the reducing end. Similar results were obtained when the reactions were performed in the presence of sucrose, but higher yields of labeled products were obtained and a series of homologous oligosaccharides was produced when d-glucose or maltose was the acceptor. We propose that the acceptor reaction proceeds by nucleophilic displacement of glucosyl and dextranosyl groups from a covalent enzyme-complex by a specific, acceptor hydroxyl group, and that this reaction effects a glycosidic linkage between the d-glucosyl and dextranosyl groups and the acceptor. We conclude that the acceptor reactions serve to terminate polymerization of dextran by displacing the growing dextran chain from the active site of the enzyme; the acceptors, thus, do not initiate dextran polymerization by acting as primers.     

The reversible Ca2+-induced permeabilization of rat liver mitochondria.

Rat liver mitochondria became permeabilized to sucrose according to an apparent first-order process after accumulating 35 nmol of Ca2+/mg of protein in the presence of 2.5 mM-Pi, but not in its absence. A fraction (24-32%) of the internal space remains sucrose-inaccessible. The rate constant for permeabilization to sucrose decreases slightly when the pH is decreased from 7.5 to 6.5, whereas the rate of inner-membrane potential (delta psi) dissipation is markedly increased, which indicates that H+ permeation precedes sucrose permeation. Permeabilization does not release mitochondrial proteins. [14C]Sucrose appears to enter permeabilized mitochondria instantaneously. Chelation of Ca2+ with EGTA restores delta psi and entraps sucrose in the matrix space. With 20 mM-sucrose at the instant of resealing, about 21 nmol of sucrose/mg of protein becomes entrapped. The amount of sucrose entrapped is proportional to the degree of permeabilization. Entrapped sucrose is not removed by dilution of the mitochondrial suspension. Resealed mitochondria washed three times retain about 74% of the entrapped sucrose. In the presence of Ruthenium Red and Ca2+ buffers permeabilized mitochondria reseal only partially with free [Ca2+] greater than 3 microM. [14C]Sucrose enters partially resealed mitochondria continuously with time, despite maintenance of delta psi, in accordance with continued interconversion of permeable and impermeable forms. Kinetic analyses of [14C]sucrose entry indicate two Ca2+-sensitive reactions in permeabilization. This conclusion is supported by the biphasic time courses of resealing and repolarization of permeabilized mitochondria and the acute dependence of the rapid repolarization on the free [Ca2+]. A hypothetical model of permeabilization and resealing is suggested and the potential of the procedure for matrix entrapment of substances is discussed.     

The Incorporation of d-Glucosamine into Glycolipids and Glycoproteins of Membrane Preparations from Phaseolus aureus Hypocotyls

Radioactivity from d-glucosamine-(14)C is incorporated into particulate fractions of hypocotyls of Phaseolus aureus (mung bean) seedlings. Polyacrylamide gel electrophoresis of the sodium dodecyl sulfate-solubilized materials revealed that several polypeptide components varying considerably in molecular weight had become radioactive during the incubation. A considerable amount of (14)C was also recovered in lipid. Equilibrium centrifugation of the particulate material, isolated by initial centrifugation at 100,000 times gravity on sucrose density gradients revealed that radioactivity was recoverable in all of the membrane fractions along the gradient. It is suggested that glycoproteins and glycolipids containing amino sugar are normal constituents of such membranes. The ability of the particulate preparations to catalyze the transfer of N-acetyl-d-glucosamine from uridine diphospho-N-acetyl-d-glucosamine to endogenous acceptor material was also tested. Transfer was optimal at around pH 9 and in the presence of 10 mm Mg(2+), and it occurred largely into an unidentified lipid fraction. After equilibrium centrifugation of crude membrane material on sucrose gradients, a number of distinct fractions could be detected which would catalyze the transfer reaction. Uridine diphospho-d-glucose transferase activity showed a similar but not identical distribution along the gradient.     

Evidence for active sodium transport across alveolar epithelium of isolated rat lung

We have previously presented evidence that cultured alveolar epithelial cell monolayers actively transport sodium from medium to substratum, a process that can be inhibited by sodium transport blockers and stimulated by beta-agonists. In this study, the isolated perfused rat lung was utilized in order to investigate the presence of active sodium transport by intact adult mammalian alveolar epithelium. Radioactive tracers (22Na and [14C]sucrose) were instilled into the airways of isolated Ringer-perfused rat lungs whose weight was continuously monitored. The appearance of isotopes in the recirculated perfusate was measured, and fluxes and apparent permeability-surface area products were determined. A pharmacological agent (amiloride, ouabain, or terbutaline) was added to the perfusate during each experiment after a suitable control period. Amiloride and ouabain resulted in decreased 22Na fluxes and a faster rate of lung weight gain. Terbutaline resulted in increased 22Na flux and a more rapid rate of lung weight loss. [14C]sucrose fluxes were unchanged by the presence of these pharmacological agents. These data are most consistent with the presence of a regulable active component of sodium transport across intact mammalian alveolar epithelium that leads to removal of sodium from the alveolar space, with anions and water following passively. Regulation of the rate of sodium and fluid removal from the alveolar space may play an important role in the prevention and/or resolution of alveolar pulmonary edema.     

Permeability of the peroxisomal membrane to cofactors of beta-oxidation. Evidence for the presence of a pore-forming protein

Peroxisomes were purified from livers of clofibrate-treated rats. Permeability measurements on the isolated organelles revealed that peroxisomes are permeable to small solutes, including sucrose and the cofactors for fatty acid oxidation NAD+, CoA, ATP, and carnitine. The intraperoxisomal distribution volume was equal for all solutes. Peroxisomal solute uptake was rapid, not saturable and not visibly influenced by temperature. NAD+ and carnitine uptake in the solute accessible volume was not diminished by a variety of analogs and inhibitors. Subfractionation of peroxisomes and reconstitution of the subfractions into liposomes preloaded with solutes made the liposomes reconstituted with the integral membrane protein fraction, but not those reconstituted with the other subperoxisomal protein fractions, permeable to the same solutes that entered intact peroxisomes. Solute leakage from the preloaded liposomes was rapid and not visibly influenced by temperature. Leakage activity was destroyed by heat treatment of the integral membrane protein fraction and was not present in lipid extracts of the membrane. Separation of the integral membrane proteins on sucrose density gradients and reconstitution of the gradient fractions into liposomes indicated that the leakage activity was caused by a polypeptide of rather low molecular weight. The gradient distribution of leakage activity corresponded most closely to the presence of a 22- and a 28-kDa polypeptide. Our experiments indicate that the nonspecific permeability of the peroxisomal membrane to small solutes is based on the presence in the membrane of a nonselective pore-forming protein.     

SOME OBSERVATIONS ON THE PERMEABILITY OF MITOCHONDRIA TO SUCROSE

In experiments carried out with sucrose-isolated rat liver mitochondria, C¹⁴-sucrose seems to penetrate most of the mitochondrial space rapidly, whereas it seems to penetrate the remainder relatively slowly. However, taking into account the wide size distribution found in isolated preparations, these rates have been found consistent with the kinetics expected from the assumption of a single compartment per mitochondrion. These data cannot be readily interpreted on the basis of two mitochondrial spaces, one highly permeable, and another relatively impermeable to sucrose.     

Evidence for copurification of micronuclei in sucrose density gradient-enriched plasma membranes from cell lines

Sucrose density gradient-enriched membrane preparations and membrane fraction enrichment through affinity purification techniques are commonly used in proteomic analysis. However, published proteomic profiles characterized by the above methods show the presence of nuclear proteins in addition to membrane proteins. While shuttling of nuclear proteins across cellular compartments and their transient residency at membrane interfaces could explain some of these observations, the presence of nuclear proteins in proteomic profiles generated with crude and enriched membranes could be the result of nonspecific contamination of nuclear debris during cell fractionation procedures. We hypothesized that micronuclei arising from the genomic instability inherent to cancer cells may copurify with plasma membrane fractions on sucrose gradients. Using sucrose gradient-enriched plasma membranes from breast cancer cell lines derived from the MCF-7 cell line, we provide experimental evidence to indicate that micronuclei are present in fresh preparations of plasma membranes. The origin of these micronuclei was traced to budding of nuclei in intact cells. Furthermore, mass spectrometric analysis confirmed the presence of nuclear proteins as well as membrane and associated signaling proteins in sucrose gradient-enriched preparations.     

Permeability Changes in Senescing Tissue

Measurements of K⁺ leakage and apparent free space (A.F.S.)to ¹⁴C glycerol and ¹⁴C sucrose indicated permeability changesin senescing Phaseolus multiflorus cotyledons, and Arum maculatumspadix. A.F.S. to glycerol was found to increase in two steps,the first of which took place early in development. A comparativestudy of respiration showed that permeability changes occurbefore any changes in the rate of respiration. They are an earlysign of senescence. The similarity of the increasing changesin leakage of K⁺, A.F.S. to glycerol and sucrose, and permeabilityacross the membrane surrounding the osmotic volume in the twodifferent materials suggests that they are of general occurrence.     

Adenylate cyclase and protein kinase C mediate opposite actions on endothelial junctions.

To determine whether the endothelial paracellular pathway is regulated, the effect of intracellular messengers on the transendothelial flux of inert radiolabeled molecules of diverse molecular size was examined in bovine aortic endothelial cells grown on collagen-coated filters. The endothelial monolayers showed a modest electrical resistance (21 +/- 10 delta.cm2; m +/- SD) and restricted the passage to 14C-sucrose, 3H-inulin, 14C-dextran (70 kDa), and 125I-polyvinyl pyrrolidone (125I-PVP, 360 kDa) according to their molecular mass. 8-Bromoadenosine 3'-5' cyclic monophosphate (8-Br-cAMP) reduced by more than 30% the permeability coefficients of 14C-sucrose and 3H-inulin but had no effect on the permeability of 125I-PVP. The permeabilities of 14C-sucrose and of 14C-inulin were strikingly increased by activating protein kinase C (PKC) by phorbol 12-myristate-13-acetate or sn-1,2-dioctanoly-glycerol whereas the latter compound had no effect on the permeability of 125I-PVP. In addition, the permeability of 14C-sucrose was unchanged by a phorbol ester that does not activate PKC. Increasing intracellular calcium with ionomycin had no effect on the permeability of 14C-sucrose. None of these maneuvers significantly affected the protein content of the endothelial monolayers. The results indicate that 8-Br-cAMP and PKC activators modulate a pathway across the endothelial monolayer that excludes 125I-PVP (360 kDa) but readily accepts 14C-sucrose and 3H-inulin, suggesting that this pathway is the paracellular pathway. Hence, low molecular weight molecules such as sucrose and inulin can be used to probe the behavior of the paracellular pathway of endothelial monolayers grown in vitro. The results also indicate that the paracellular pathway in endothelium is regulated and suggest that endothelial junctions can be closed by simulating adenylate cyclase and opened by stimulating protein kinase C.     

Isolation of latent phenoloxidase from prepupae of the housefly,Musca domestica

Latent phenoloxidase was purified from prepupae of the housefly, Musca domestica vicina Maquart. The purification procedures included DEAE-cellulose column chromatography, sucrose density gradient centrifugation adn second sucrose density gradient centrifugation. The final preparations appear to be homogeneous based on results obtained from polyacrylamide gel electrophoresis in the presence of EDTA. Electrophoresis in the absence of EDTA caused spontaneous activation of latent phenoloxidase. While latent phenoloxidase was fairly stable over the range of temperatures between 0 and 40°C, it was quite sensitive to changes in pH, being stable only around pH 6.0. The molecular weight of latent phenoloxidase was estimated to be 178,000, as determined by gel filtration and sucrose density gradient centrifugation. Furthermore, phenoloxidase formed by the activation of latent phenoloxidase indicated a higher molecular weight (340,000) than that of latent phenoloxidase. Thus, it appears that the mechanism of the activation of latent phenoloxidase involves the association and disassociation system.     

THE ISOLATION OF CELL NUCLEI FROM RAT BRAIN

A method for preparing highly pure cell nuclei from adult rat brain, using both differential and isopycnic centrifugation in sucrose media, is described. The morphology of these preparations was examined by both phase contrast and electron microscopy. The isolated nuclei retained many aspects of their in situ morphology; in particular, the nuclear envelope was double layered and interrupted by pore-like discontinuities, and the nucleoli consisted of irregular masses of densely packed granules. Analyses of these nuclear preparations for cytochrome oxidase and cholinesterase activity, as well as RNA/DNA ratio, indicated minimal contamination with mitochondria and microsomes. Problems involving the homogenization technique, choice of ionic conditions in the homogenization medium, and choice of optimal density of the sucrose solution used for the final purification of nuclei are discussed. Results of application of the technique to isolation of adult rat liver nuclei are also reported.     

Thyroid lysosomes: the stability of the lysosomal membrane

To determine the integrity of lysosomes during their isolation from rat thyroid glands and their subsequent incubation at 37 degrees C, the sedimentability of lysosomal acid phosphatase and thyroglobulin (amount of undisrupted lysosomes) and the latency of sedimentable acid phosphatase (permeability of undisrupted lysosomes) were measured concomitantly. The following results were obtained: (a) During isolation of lysosomes in 0.25 M sucrose medium, mild homogenization of thyroid tissue or cholesterol addition did not modify the amount of undisrupted lysosomes but reduced their permeability. Homogenization in 0.5 M sucrose decreased both the amount and the permeability of undisrupted lysosomes. It also reduced their content of recently iodinated thyroglobulin (Tg). Cholesterol addition had no effect in 0.5 M sucrose medium. (b) During incubations at 37 degrees C of lysosomes, the amount of undisrupted lysosomes decreased progressively while their permeability increased. According to the incubation pH, the permeability of lysosomes prepared in 0.25 M sucrose was either more (pH 8) or less (pH 6) extensively increased than that of lysosomes prepared in 0.5 M sucrose. From these results, we concluded: (a) that isolation and incubation of the thyroid lysosomal fraction induce increased permeability of lysosomes prior to their complete disruption: (b) that recently formed lysosomes (high content of recently iodinated Tg) and aged lysosomes (low content of recently iodinated Tg) differ significantly. Recently formed lysosomes are more permeable, are stabilized by cholesterol and are more extensively disrupted in 0.5 M sucrose medium. During incubations, the permeabilities of these two classes of lysosomes are also differently affected by external pH.     

Properties of the leak permeability induced by a cytotoxic protein from Pseudomonas aeruginosa (PACT) in rat erythrocytes and black lipid membranes

A cytotoxic protein, isolated from Pseudomonas aeruginosa (PACT), was tested on red blood cells of rats and on black lipid membranes for changes of membrane permeability. In rat erythrocytes PACT induces lysis indicative of the formation of a leak permeable to monovalent ions. The dose response curve for the PACT-induced hemolysis demonstrates that the rate of lysis as well as the fraction of lytic cells increases with increasing toxin concentration. Furthermore, the leak pathway discriminates hydrophilic non-electrolytes according to their molecular weight. The findings indicate formation by PACT of a pore with an apparent radius of about 1.2 nm. In pure lipid membranes PACT forms hydrophilic pathways with moderate selectivity for small cations over small anions. The presence of cholesterol is a prerequisite for the occurrence of these PACT-induced permeability changes.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing sucrose: relationship with posthypertonic hemolysis and solute movements

Human red blood cells were frozen at temperatures down to ?9 °C in solutions containing sucrose, and the hemolysis on thawing was measured. This was compared with the hemolysis caused by exposing the cells to high concentrations of sucrose and then resuspending them in more dilute solutions at 4 °C. The effects of the hypertonic solutions of sucrose on potassium, sodium, and sucrose movements were also investigated. It was found that sucrose does not prevent damage to the cells by very hypertonic solutions (whether during freezing and thawing or at 4 °C) but it does reduce hemolysis of cells previously exposed to these solutions if present in the resuspension (or thawing) solution. Evidence is presented that the damaging effects of the hypertonic solutions of sucrose occurring during freezing are associated with changes in cell membrane permeability but that posthypertonic hemolysis is not primarily associated with a “loading” of the cells with extracellular solutes in the hypertonic phase. It is concluded that sucrose may reduce hemolysis of red blood cells by slow freezing and thawing by reducing colloid osmotic swelling of cells with abnormally permeable membranes.     

Biochemical characterization of subcellular particles in fetal and neonatal rats.

By a variety of methods, such as ultracentrifugation in different media, hypoosmotic activation and hydrostatic compression, subcellular particles were characterized at different perinatal ages and compared to the adult rats. Fetal mitochondria elicited a higher density, an increased osmotic space and a greater resistance to compression. The size of these particles was larger than in the adults. Mitochondria in the 1-day-old animals were freely permeable to sucrose and their external membrane was more resistant to hypoosmotic activation. Lysosomes were shown to decrease their sucrose permeability and their resistance to hypoosmotic activation with development. Moreover, the size of the lysosomes increased with development.     

Isolation and characterization ofEuglena nuclei

Summary A novel method for isolatingEuglena gracilis Z. nuclei, based on pretreatment of cells in concentrated glycerol buffer before homogenization, is described. Such a treatment weakens the tough cell pellicle facilitating cell disruption, and avoids nuclear damage induced by detergents and by freezing and thawing the cells in aqueous media. Nuclei, purified by centrifugation in dense sucrose, are obtained with a 30% yield, and only small amounts of cell wall fragments contaminate the nuclear pellets. The purified nuclei retain their ultrastructural characteristics. High molecular weight DNA, as well as undegraded RNA species and histones, can be extracted from these nuclei. Nuclease digestions and spread preparations show an unaltered nucleosomal structure of chromatin. This method has been applied to cell samples at any stage of the cell cycle, including mitosis, since inEuglena the nuclear envelope persists during cell division.