The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitrificans.

1. A method is described for preparing spheroplasts from Paracoccus denitrificans that are substantially depleted of dissimilatory nitrate reductase (cytochrome cd) activity. Treatment of cells with lysozyme + EDTA together with a mild osmotic shock, followed by centrifugation, yielded a pellet of spheroplasts and a supernatant that contained d-type cytochrome. The spheroplasts were judged to have retained an intact plasma membrane on the basis that less than 1% of the activity of a cytoplasmic marker protein, malate dehydrogenase, was released from the spheroplasts. In addition to a low activity towards added nitrite, the suspension of spheroplasts accumulated the nitrite that was produced by respiratory chain-linked reduction of nitrate. It is concluded that nitrate reduction occurs at the periplasmic side of the plasma membrane irrespective of whether nitrite is generated by nitrate reduction or is added exogenously. 2. Further evidence for the integrity of the spheroplasts was that nitrate reduction was inhibited by O2, and that chlorate was reduced at a markedly lower rate than nitrate. These data are taken as evidence for an intact plasma membrane because it was shown that cells acquire the capability to reduce nitrate under aerobic conditions after addition of low amounts of Triton X-100 which, with the same titre, also overcame the permeability barrier to chlorate reduction by intact cells. The close relationship between the appearance of chlorate reduction and the loss of the inhibitory effect of O2 on nitrate reduction also suggests that the later feature of nitrate respiration is due to a control on the accessibility of nitrate to its reductase rather than on the flow of electrons to nitrate reductase.     

Organization of the glycolytic enzymes in Escherichia coli

Differential centrifugation of osmotically lysed lysozyme-EDTA spheroplasts from Escherichia coli sedimented 50–70% of the glycolytic activities examined in a low speed pellet; the remaining activity, occurring in a high speed supernatant, contained the soluble enzymes of the cell. The distribution pattern of the enzymes could be altered by extrusion of the spheroplasts through the French Press or by lysis at different pH values. Electron micrographs of the pellet fraction revealed lysed spheroplasts mostly devoid of cellular constituents but consisting of cytoplasmic membranes surrounded by partially degraded cell wall fragments. Washing of the pellet showed that the enzymes were not all bound to the same degree to the membrane fraction. Throughput activity of the glycolytic pathway was demonstrated for the membrane fraction, but none was observed for the soluble fraction of the cell (i.e. for enzymes present in the supernatants) unless these were first concentrated by ultrafiltration. The supernatant from the lysed spheroplasts, together with a further supernatant obtained by washing the membrane pellet, was concentrated by ultrafiltration and chromatographed on a Bio-Gel column. The eluate contained glycolytic activities both in fractions corresponding to relatively high and relatively low molecular weight material The high molecular weight species, containing a proportion of all the enzymes studied, had a molecular weight of at least 1.2 × 10⁶. A multienzyme aggregate containing one each of the glycolytic enzymes would have a molecular weight of ～ 1.3 × 10⁶. The specific rate of pyruvate formation from glucose by the high molecular weight species was similar to that obtained from a preparation in which the fractions containing all the low molecular weight material enzyme activities were pooled and concentrated by ultrafiltration. Using the high molecular weight material, studies were made of the ability of added unlabelled glycolytic intermediates to compete for catalytic sites with intermediates produced endogenously from [¹⁴C₆] glucose. The relatively weak competition observed indicated a high degree of protection afforded the labelled intermediates derived from [¹⁴C₆] glucose.     

In vitro reconstitution of intercompartmental protein transport to the yeast vacuole

Toward a detailed understanding of protein sorting in the late secretory pathway, we have reconstituted intercompartmental transfer and proteolytic maturation of a yeast vacuolar protease, carboxypeptidase Y (CPY). This in vitro reconstitution uses permeabilized yeast spheroplasts that are first radiolabeled in vivo under conditions that kinetically trap ER and Golgi apparatus-modified precursor forms of CPY (p1 and p2, respectively). After incubation at 25 degrees C, up to 45% of the p2CPY that is retained in the perforated cells can be proteolytically converted to mature CPY (mCPY). This maturation is specific for p2CPY, requires exogenously added ATP, an ATP regeneration system, and is stimulated by cytosolic protein extracts. The p2CPY processing shows a 5-min lag period and is then linear for 15-60 min, with a sharp temperature optimum of 25-30 degrees C. After hypotonic extraction, the compartments that contain p2 and mCPY show different osmotic stability characteristics as p2 and mCPY can be separated with centrifugation into a pellet and supernatant, respectively. Like CPY maturation in vivo, the observed in vitro reaction is dependent on the PEP4 gene product, proteinase A, which is the principle processing enzyme. After incubation with ATP and cytosol, mCPY was recovered in a vacuole-enriched fraction from perforated spheroplasts using Ficoll step-gradient centrifugation. The p2CPY precursor was not recovered in this fraction indicating that intercompartmental transport to the vacuole takes place. In addition, intracompartmental processing of p2CPY with autoactivated, prevacuolar zymogen pools of proteinase A cannot account for this reconstitution. Stimulation of in vitro processing with energy and cytosol took place efficiently when the expression of PEP4, under control of the GAL1 promoter, was induced then completely repressed before radiolabeling spheroplasts. Finally, reconstitution of p2CPY maturation was not possible with vps mutant perforated cells suggesting that VPS gene product function is necessary for intercompartmental transport to the vacuole in vitro.     

Interaction of the isolated DNA of lambda phage with spheroplasts of E. coli treated with sturine]

The method of centrifugation in sucrose density gradient (30-55%) of the spheroplast membrane preparations treated and untreated with sturine and infected with phage lambda DNA demonstrated that sturine, treatment increased the phage lambda DNA absorption three-fold. About 50% of the lambda DNA molecules adsorbed by spheroplasts are bound with the cytoplasmic membrane of spheroplasts treated with sturine; 50% of the lambda DNA molecules are bound with the cell wall membrane on the sturine-untreated spheroplasts. The data obtained allow to conclude that the stimulating effect of sturine in E. coli spheroplasts transfection by lambda DNA is connected with redistribution of phage DNA absorbed on spheroplasts from the cell wall to the cytoplasmic membrane facilitating the penetration of DNA and its fastening on the membrane.     

A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.

We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY). Yeast spheroplasts are lysed by extrusion through polycarbonate filters. After differential centrifugation, a 125,000-g pellet is enriched for radiolabeled proCPY and is used as "donor" membranes. A 15,000-g pellet, harvested from nonradiolabeled cells and enriched for vacuoles, is used as "acceptor" membranes. When these membranes are incubated together with ATP and cytosolic extracts, approximately 50% of the radiolabeled proCPY is processed to mature CPY. Maturation was inhibited by dilution of donor and acceptor membranes during incubation, showed a 15-min lag period, and was temperature sensitive. Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from a PEP4 yeast strain, indicating intercompartmental transfer. Cytosol made from a yeast strain deleted for the VPS33 gene was less efficient at driving transport. Moreover, antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays. This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of a VPS gene product.     

Characteristics of chromosomal DNA isolated from Escherichia coli spheroplasts

The chromosomal DNA of Escherichia coli spheroplasts induced by penicillin G was studied biochemically and electron microscopically. Although the spheroplasts were unable to divide, they continued to synthesize chromosomal DNA for several hours even in the presence of penicillin G. Some differences were observed between the chromosomal DNA of the parent cells and that of the spheroplasts in sucrose gradient centrifugation and electron microscopy; two types of chromosomal DNA, a slower sedimenting form and a faster sedimenting form, were released from the gently lysed parent cells. The former was membrane-free folded chromosome and the latter was membrane-associated chromosome. In contrast, the chromosome from the spheroplast showed a single intermediate value of sedimentation coefficient between those of the chromosomal DNA from the parent cell. Cytochrome spreading for electron microscopy showed that the spheroplast chromosomal DNA formed an aggregated mass consisting of several chromosome-molecules of the parent cell.     

Isolation problems and structural organization of membrane units in Mycobacterium sp. smegmatis

Summary Membrane units from lysed spheroplasts induced by lysozyme or glycine from Mycobacterium spec. smegmatis were isolated in a biological active state by differential centrifugation and by density gradient technique. They were compared morphologically with membraneous fractions obtained from mycobacterial cells disintegrated under a high hydrostatic pressure.Higher homogeneity of membraneous structures isolated from spheroplasts was confirmed. Three types of membraneous structures could be distinguished. They include empty ghosts of spheroplasts, tubular structures containing cytoplasmic material and fragments of typical membraneous structures relatively free of contaminants. By studying protoplasts in the process of lysis it was determined that these structures correspond with cytoplasmic membranes and mesosomes.Differences between lysozome and glycine induced spheroplasts as a starting material for isolation of membraneous structures include the proportion of contamination by other cellular components, reasons of which are discussed.     

Fractionation by differential and zonal centrifugation of spheroplasts prepared from a glucose-repressed fission yeast Schizosaccharomyces pombe 972h-.

A method is described for the preparation of spheroplasts in high yield from Schizosaccharomyces pombe, by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome c oxidase activity and spectroscopically-detectable cytochromes a + a3) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with Saccharomyces carlsbergensis, cytochrome c oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mM-MgCl2 or 0-4 mM-EDTA. Mitochondria were apparently smaller and of lower buoyant density in gradients containing EDTA. The bulk of the total units of malate dehydrogenase and NADH; cytochrome c oxidoreductase sedimented with mitochondria, whereas NADPH: cytochrome c oxidoreductase was located in fractions containing no mitochondria. The distributions of mitochondrial enzymes were heterogeneous in populations of mitochondria separated on the basis of size or density. The possible origins of mitochondrial heterogeneity in extracts of S. pombe are discussed with special reference to changes in the enzyme activities of cells during the cell cycle.     

Stability of Candida utilis Cells and Spheroplasts Toward Gravitational Forces

Candida utilis cells and spheroplasts containing uric acid crystals in their vacuoles were not damaged by the mechanical stress of centrifugation at 20,000 x g for 10 min, as judged by plating, microscopy, and spectrophotometry.     

Studies of cell pellets: I. Electrical properties and porosity.

Cell pellets formed by centrifugation provided a good system to study the osmotic behavior, electroporation, and interaction between cells. Rabbit erythrocyte pellets were used in this study because they were simpler than nucleated cells to model analytically. Structurally, cell pellets possessed properties of porous solid bodies and gels. Electrically, cell pellets were shown to behave as a parallel set of resistance, Rp, and capacitance, Cp. Information on pellet structures was obtained from electric measurements. The pellet resistance reflected the intercellular conductivity (porosity and gap conductivity), whereas the pellet capacitance depended mostly on membrane capacitance. The pellet resistance was more sensitive to experimental conditions. The intercellular gap distance can be derived from pellet porosity measurements, providing the cell volume and surface area were known. Rp increased and relaxed exponentially with time when centrifugation started and stopped; the cycles were reversible. When supernatants were exchanged with solutions containing hypotonic electrolytes or macromolecules (such as PEG) after the pellets were formed, complicated responses to different colloidal osmotic effects were observed. A transient decrease followed by a large increase of Rp was observed after the application of a porating electric pulse, as expected from a momentary membrane breakdown, followed by a limited colloidal-osmotic swelling of pelleted cells. The equilibrium values of Rp, Cp, pellet porosity, and intercellular distances were measured and calculated as functions of cell number, centrifugation force, and ionic strength of the exchanged supernatant. Thus, the structure and properties of cell pellets can be completely characterized by electrical measurements.     

Stable L-forms of Clostridium perfringens and their growth on glass surfaces.

L-forms of Clostridium perfringens were induced in brain heart infusion broth containing 10% sucrose and 2 units of penicillin. After a few hours of growth, spheroplasts, granules, and elongated bacilli were apparent. At 24-h intervals, serial subcultures were made in the above medium which resulted in a culture composed entirely of spheroplasts (or protoplasts) and granules. Upon the withdrawal of penicillin these L-form cultures grew well and, after 100 passages, there was no reversion to the bacillary form. Sucrose could also be withdrawn from the medium. The effects of centrifugation, osmotic stabilizer, ultraviolet light, temperature, pH, and lyophilization upon stable L-forms were examined. L-forms were found to attach to the walls of culture tubes during trowth and sheets of L-form growth were obtained on cover slips in Leighton tubes and on the sides of medicine bottles.     

Activation of guinea pig polymorphonuclear leukocytes with soluble stimulators leads to nonrandom distribution of NADPH oxidase in the plasma membrane

Guinea pig polymorphonuclear leukocytes (PMN) were briefly activated with soluble stimulators such as sodium myristate (SM) or phorbol myristate acetate (PMA) and then disrupted by the nitrogen cavitation method to study the subcellular distribution of NADPH oxidase, which is responsible for O2 - generation. Fc-receptor and 5'-nucleotidase activities were measured as plasma membrane markers. 1) The homogenate was first fractionated by differential centrifugation. The O2- -generating activity of PMN activated either by SM or PMA was recovered in a 2 X 10(4) g pellet which contained a large amount of granules and about 50% of the plasma membrane markers, but not in a 1 X 10(5) g pellet which consisted of plasma membranes and few granules. 2) Further separation of the 2 X 10(4) g pellet from PMA-activated PMN was attempted by an iso-osmotic Percoll density gradient centrifugation. The O2- -generating activity was recovered in light fractions in which plasma membrane markers were found, but neither in specific nor in azurophil granules. The 1 X 10(5) g pellet showed a similar distribution of the plasma membrane markers to that of the 2 X 10(4) g pellet, except that the peak of the O2- -generating activity was much smaller on an identical density gradient. The results showed that NADPH oxidase is located in the plasma membranes precipitated by centrifugation at 2 X 10(4) X g but not in the ones precipitated at 1 X 10(5) X g. The results suggest that the plasma membrane of activated PMN has a mosaic distribution of NADPH oxidase.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : III. Electron Microscopy and Analysis of the Cytochromes

The membranous nature of pellets obtained from broken Escherichia coli spheroplasts by successive centrifugation at 3500 g (P₁), 20,000 g (P₂), and 105,000 g (P₃), has been established by electron microscopy. Spectrophotometric analysis has shown that about 90% of the cytochromes are concentrated in the particulate fractions. The crude ribosomal pellet (P₃) contained as much of the total cytochromes as did the pellet obtained at 20,000 g (P₂). The high cytochrome content of P₃ is consistent with its high oxidative activity (1) and the presence of membrane vesicles in this fraction. Analysis at 77°K intensified the optical extinction of all the cytochrome absorption bands, but the degree of intensification was not uniform for each fraction nor for each band within a given fraction. Carbon monoxide had little or no inhibiting effect on NADH oxidation. Reduced plus carbon monoxide difference spectra yielded artifactual absorption bands in the wave length regions where reduced vs. oxidized absorption bands normally occur. Succinate and NADH, either together or separately, reduced nearly all of the cytochromes, indicating that the cytochrome portion of the electron-transport chain is shared by both substrates. A tentative formulation of the electron-transport chain is presented.     

Location of three key enzymes of gluconeogenesis in baker's yeast

The subcellular location of hexose diphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase in baker's yeast (Saccharomyces cerevisiae) was investigated by density gradient centrifugation of spheroplast lysates obtained by osmotic shock treatment of spheroplasts and centrifugation for 10000 g x min. On the evidence obtained from zonal separations these three enzymes of gluconeogenesis are most probably located in the soluble cytosol.List of Abbreviations HDPase Hexose diphosphatase - PEPCK Phosphoenolpyruvate carboxykinase - PC Pyruvate carboxylase     

Trimethylamine oxide respiration in Proteus sp. strain NTHC153: electron transfer-dependent phosphorylation and L-serine transport

Cells of Proteus sp. strains NTHC153 grown anaerobically with glucose and trimethylamine oxide (TMAO) were converted to spheroplasts by the penicillin method. The spheroplasts were lysed by osmotic shock, and the membrane vesicles were purified by sucrose gradient centrifugation. Vesicles energized electron transfer from formate to TMAO displayed active anaerobic transport of serine. An anaerobic cell-free extract of Proteus sp. disrupted in a French pressure cell reduced TMAO with formate and NADH with the concomitant formation of organic phosphate. The net P/2e- ratios determined were 0.1 and 0.3, respectively. The NADH- and TMAO-dependent phosphorylation was sensitive to uncouplers of oxidative phosphorylation (protonophores), and the formate- and TMAO-dependent serine transport was sensitive to ionophores and protonophores. We conclude that TMAO reduction in Proteus sp. fulfills the essential features of anaerobic respiration.     

A method to maintain mammalian cells for days alive at 4 °C

This paper describes a method for the temporary storage of cultured cells. Cells from recently completed cell monolayers were trypsinized and then centrifuged. After centrifugation, the supernatant and pellet were kept at 4 °C for one week. After storage, the supernatant was discarded, the cells were resuspended and used for seeding new flasks and for titration of virus. The cells not only remained viable, but also rapidly formed new monolayers and allowed immediate infection and growth of viruses. We conclude that this method can be a helpful asset to cell culture experiments.     

An ultra scale‐down analysis of the recovery by dead‐end centrifugation of human cells for therapy

An ultra scale‐down method is described to determine the response of cells to recovery by dead‐end (batch) centrifugation under commercially defined manufacturing conditions. The key variables studied are the cell suspension hold time prior to centrifugation, the relative centrifugal force (RCF), time of centrifugation, cell pellet resuspension velocities, and number of resuspension passes. The cell critical quality attributes studied are the cell membrane integrity and the presence of selected surface markers. Greater hold times and higher RCF values for longer spin times all led to the increased loss of cell membrane integrity. However, this loss was found to occur during intense cell resuspension rather than the preceding centrifugation stage. Controlled resuspension at low stress conditions below a possible critical stress point led to essentially complete cell recovery even at conditions of extreme centrifugation (e.g., RCF of 10000 g for 30 mins) and long (~2 h) holding times before centrifugation. The susceptibility to cell loss during resuspension under conditions of high stress depended on cell type and the age of cells before centrifugation and the level of matrix crosslinking within the cell pellet as determined by the presence of detachment enzymes or possibly the nature of the resuspension medium. Changes in cell surface markers were significant in some cases but to a lower extent than loss of cell membrane integrity. Biotechnol. Bioeng. 2015;112: 997–1011. © 2014 Wiley Periodicals, Inc.     

Isolation and properties of two classes of low-density vesicles from Saccharomyces cerevisiae.

A mixture of small (0.43-mum diameter) and large (0.62-mum diameter) low-density vesicles from spheroplasts of Saccharomyces cerevisiae was fractionated by rate centrifugation in a gradient of 0 to 8% (wt/vol) Ficoll to yield fractions rich (90 to 95%) in small or large vesicles. The large, but not small, vesicles swelled when diluted into mannitol solutions containing less than 0.4 M mannitol. The pH-electrophoretic mobility curve of the large vesicles showed that they are probably enclosed in a phospholipid-protein membrane. The dyes neutral red and toluidine blue, accumulated into large vesicles by intact cells and spheroplasts, were largely lost from large vesicles when these were separated from stained spheroplasts. Sudan black III stained small and large vesicles, both classes of vesicle retaining the stain on separation. Fractions rich in large vesicles contained proportionately more phospholipid and less free sterols, diacylglycerols, and free fatty acids compared with those enriched in small vesicles. The two classes of vesicles contained about the same proportions of esterified sterols and triacylglycerols. The free fatty acids in both small and large vesicles were free from unsaturated fatty-acyl residues; diacylglycerols and triacylglycerols contained appreciable proportions of unsaturated fatty-acyl residues. Small vesicles were richer in lipase activity, whereas the larger vesicles contained greater beta-glucanase and alpha-mannosidase activities. Phospholipase activity could not be detected in any of the fractions.     

Subcellular structure of bovine thyroid gland. VII. A study on the distribution of bovine thyroid plasma membranes by density gradient centrifugation in zonal rotors.

In order to obtain plasma membrane-rich fractions two methods were tried. Approach A was based on differential pelleting followed by discontinous gradient centrifugation in a B-XIV zonal rotor. In approach B homogeneization was performed in buffered water (NaHCO3, pH 7.4). The 73 300 X g pellet from this homogenate was subjected to buoyant density equilibrium in a HS zonal rotor (continuous sucrose gradient). Using approach A, the highest relative specific activity for plasma membrane markers was found at the 30-37% sucrose interphase. However, an increase for glucose 6-phosphatase (endoplasmic reticulum marker) was also found at that interphase. Using approach B marker profiles different from approach A were found. Approach B results in a subdivision of membrane material in four distinct regions. These regions do not contain completely pure membrane species, although region I seems to be essentially derived from plasma membranes. It is also concluded from approach A that plasma membranes from bovine thyroid tissue are heterogeneous.     

Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae

Intact vacuoles are released from spheroplasts of Saccharomyces cerevisiae by means of a gentle mechanical disintegration method. They are purified by centrifugation in isotonic density gradients (flotation and subsequent sedimentation), and analyzed for their soluble amino acid content. The results indicate that about 60% of the total amino acid pool of spheroplasts is contained in the vacuoles. This may be an underestimate, as it presupposes no loss of amino acids from the vacuoles during the purification procedure. The amino acid concentration in the vecuoles is calculated to be approximately 5 times that in the cytoplasm if the total volumes of the two compartments are used for the calculation. The vacuolar amino acid pool is rich in basic amino acids, and in citrulline and glutamine, but contains a remarkably small amount of glutamate. Radioactive labeling experiments with spheroplasts indicate that the vacuolar amino acids are separated from the metabolically active pools located in the cytoplasm. This is particularly evident for the basic amino acids and glutamine; in contrast, the neutral amino acids and glutamate appear to exchange more rapidly between the cytoplasmic and the vacuolar compartments of the cells.