French Pressure Cell Research Articles

Involvement of the cytoplasmic membrane in nitrogen fixation by Azotobacter vinelandii.

1. Azotobacter vinelandii cells were ruptured by using a French pressure cell, by lysozyme treatment, or by osmotic shock. The rates of sedimentation of nitrogenase from these extracts were compared and related to the rates of sedimentation of marker enzymes. No evidence was found for the so‐called particulate nitrogenase or an interaction between nitrogenase and cytoplasmic membrane vesicles. Nitrogenase sediments as a complex and the rate of sedimentation is comparable with that of pyruvate dehydrogenase complex.2. The oxygen stability of nitrogenase from Azotobacter is not caused by the presence of cytoplasmic membranes in extracts but by a complexation of the nitrogenase components with an Fe‐S protein. Recombination experiments showed that maximum stabilization against oxygen was only obtained at certain ratios of the Fe‐S‐protein and nitrogenase complex. It is proposed that the switch‐off state in Azotobacter cells is caused by the oxidation of flavodoxin hydroquinone rather than by a reversible inactivation of the nitrogenase.3. A membrane‐bound NAD(P)H‐flavodoxin oxidoreductase was detected. Evidence was obtained that flavodoxin might also be membrane‐bound.4. A proposal is given for electron donation to nitrogenase in Azotobacter. In this proposal the generation of reducing equivalents for nitrogenase is localized in the cytoplasmic membrane and mediated by the NADH‐flavodoxin oxidoreductase. The pH gradient which is generated by membrane energization, is used to reduce flavodoxin to its hydroquinone form in order to achieve a redox potential low enough to reduce nitrogenase.

Isolation and characterization of nuclei from Neurospora crassa

A procedure was developed for isolating nuclei from either the conidial or germinated conidial growth phase of Neurospora crassa. A frozen conidial suspension was lysed by passage through a French pressure cell, and the nuclei were freed from the broken cells by repeated homogenization in an Omni-Mixer. Pure nuclei were obtained from the crude nuclear fraction by density banding in a Ludox gradient. The final nuclear yield was 20 to 30%. The nuclei had a deoxyribonucleic acid (DNA):ribonucleic acid (RNA):protein ratio of 1:3.5:7 and were active in RNA synthesis. The nuclei, stained with the DNA stain 4,6-diamidino-2-phenylindole, appeared under fluorescence microscopy as bright blue spheres, 1 micron in diameter, essentially free from cytoplasmic attachments. Chromatin extracted from the nuclei in a 70 to 75% yield by dissociation with 2 M sodium chloride and 5 M urea had a DNA:RNA:protein ratio of 1:1.05:1.7. Chromatin reconstituted from this preparation exhibited a level of RNA polymerase template activity lower than that of pure Neurospora DNA, but the maximum level of reconstitution obtained was only 10%. Fractionation of Neurospora chromatin on hydroxylapatite separated the histones from the chromatin acidic proteins. The normal complement of histone proteins was present in both the reconstituted and dissociated chromatin preparations. The acidic protein fraction exhibited a variety of bands on sodium dodecyl sulfate gel electrophoresis ranging in molecular weight from 15,000 to 70,000. The gel pattern was much more complex for total dissociated chromatin than for reconstituted chromatin.

Ultrastructural and biochemical studies of mitoplasts and outer membranes derived from French-pressed mitochondria: Advances in mitochondrial subfractionation

Rapid separation of mitoplasts and outer membranes of rat liver mitochondria has been accomplished by extrusion of mitochondrial suspensions through a French pressure cell. Preparations were monitored using established micro-sampling techniques to determine the extent of outer membrane release, the degree of homogeneity of the mitochondrial subfractions, and the affect of French pressing on mitochondrial ultrastructure. The distribution of “marker enzymes,” monoamine oxidase (MAO), cytochrome oxidase (Cyt Ox), malate dehydrogenase (MDH), and adenylate kinase (ADK) was similar to that obtained when mitochondrial subfractionation was carried out using digitonin. For the first time a strictly physical method of removing the outer mitochondrial membrane which has yielded a mitoplast fraction retaining both acceptor control and a condensed configuration characteristic of the inner compartment of freshly isolated mitochondria is reported. Similar separation of the same components of rat heart mitochondria has also been accomplished. Thus, by this method well-defined mitochondrial subfractions, uncontaminated with detergents, have been isolated from two mammalian tissues. Digitonin treatment of mitoplasts prepared by the technique reported here resulted in the conversion of the condensed configuration to that of the digitonin-derived mitoplast. In addition, further insight into the ultrastructural nature of inner and outer membrane contact phenomena has been established.

Escherichia coli DNA-directed β-galactosidase synthesis in presence and absence of Ca2+ ion

DNA-dependent synthesis of beta-galactosidase was optimized in extracts made from cells lysed by a standard French pressure cell. Extracts made at 3200 psi synthesized up to 25-fold more beta-galactosidase than extracts made at 7500 psi. beta-Galactosidase synthesis was cyclic 3', 5' AMP dependent, as expected, and in optimal conditions transcription and translation proceeded at 8.6 nucleotides and 2.7 amino acids per s, respectively. The high pressure extracts were stimulated 3- to 5-fold by Ca2+, especially at low Mg2+ concentrations. In contrast, extracts prepared at low pressure were inhibited as much as 50-fold by Ca2+ ions. The inhibition by Ca2+ was analyzed further. Addition of kasugamycin, an antibiotic that acts on ribosomes, to reactions containing Ca2+ stimulated beta-galactosidase synthesis to nearly control levels. Extracts from a kasugamycin resistant mutant were neither inhibited by Ca2+ nor stimulated by the addition of kasugamycin to in vitro reactions containing Ca2+. The change in the mutant was ascribed to the ribosomes by testing combinations of soluble proteins, ribosome wash, and ribosomes from parental and mutant strains. These results suggest that Ca2+ ions inhibit translation by ribosomes, very likely at an initiation step; and that they enhance enzyme synthesis only in conditions where translation is inefficient (high-pressure extracts at low concentrations of Mg2+, for example). This latter effect is probably a consequence of increased RNA stability in the presence of Ca2+ (Cremer, K., and Schlessinger, D. (1974), J. Biol. Chem. 249,4730).

Studies of sperm antigenicity. 6. In vivo and in vitro cellular reactivity in guinea pigs sensitized with fractions of guinea pig spermatozoa.

Normal guinea pig spermatozoa cells were homogenized by a French pressure cell. Three soluble and three insoluble fractions were obtained by ultrascentrifugation and (emulsified in CFA) were used for guinea pig sensitization. The following were observed: 1) all fractions were immunogenic except one; 2) in vivo and in vitro delayed hypersensitivity was elicited in animals immunized with these fractions; 3) two distinctive histopathologic lesions were observed in the testes of sensitized animals: lesions of orchitis type developed in animals injected with some fractions. Other fractions induced lesions of aspermatogenic type. These results correlated well with delayed hypersensitivity results obtained by in vivo and in vitro tests. Although some other spermatozoal fractions did not cause severe changes in the testes. The lack of sperm accumulation in the epididymis was obvious.

Immunogenic activity of a ribosomal fraction obtained from Pasteurella multocida.

The cells of P. multocida strain P-1059 were destroyed with the French pressure cell; the ribosomal fraction proven to be homogeneous by analytical ultracentrifugation was obtained from the product by centrifugal fractionation, zonal electrophoresis, and Sephadex G-200 gel filtration. The ribosomal fraction exhibited intense protective antigenicity in mice and chickens, but the lipopolysaccharide (endotoxin) and the other bacterial cell fraction obtained in this experiment did not. Sodium deoxycholate treatment of the ribosomal fraction resulted in only a 13% loss in immunological activity, and ribonuclease treatment caused a 60% loss of activity.

Orientation of the protonmotive force in membrane vesicles of Escherichia coli.

AbstractMembrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D‐lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside.The latter type of vesicle, on the other hand, exhibits D‐lactate‐dependent proline transport but does not accumulate calcium with D‐lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D‐lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy‐linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).

The isolation and characterization of intact polyribosomes from a cell wall mutant of Chlamydomonas reinhardi

A technique is described for obtaining intact polyribosomes from a cell-wall mutant of Chlamydomonas reinhardi. When cells were lysed by nonionic detergent in buffers containing high salt and Mg 2+-EDTA, polyribosomes of up to 25 ribosomes per polyribosome were obtained on sucrose density gradients. Under these conditions, nascent polypeptide radioactivity was associated with the polyribosomes and not with monoribosomes, and inactive monoribosomes were dissociated to ribosomal subunits. Whole cell lysates contain a mixture of cytoplasmic and chloroplastic polyribosomes. The relative contribution of the two types of polyribosomes was evaluated by polyacrylamide gel electrophoretic analysis of ribosomal RNA extracted from polyribosomes. This analysis showed that less than 15% of the polyribosomes from detergent-lysed cells were from chloroplasts. The contribution of chloroplast polyribosomes to total polyribosomes was increased to about 30% by incubation of the cells in chloramphenicol. When cells were disrupted mechanically (in a French pressure cell), only about 10% of the resulting free polyribosomes were chloroplastic.

Thermoplasma acidophilum: intracellular pH and potassium concentration.

Thermoplasma acidophilum is a free-living thermophilic mycoplasma. Although the organism lacks a cell wall, it can grow in medium as dilute as 66 mosM. The intracellular K+ concentration can be as low as 17 mM, but varies according to the osmolality of the culture medium. The internal pH can be measured by taking advantage of the fact that T. acidophilum undergoes lysis when the pH is adjusted to neutrality. Thus, by appropriate analysis of titration curves, it is possible to conclude that the internal pH is near 5.5. This result was confirmed by a second type of experiment in which the internal pH was analyzed by rupturing the cells in a French Pressure Cell.

Babesia argentina: Immunization of cattle with a killed antigen against infection with a heterologous strain

Cattle were immunized againts infection with a heterogologous strain of Babesia argentina by the subcuntaneous inoculation of killed antigen mixed with Freund's complete adjuvant. The most effective antigen, infectted erythrocyte antigen (IEA), which confered protection comparable with that conferred by the presence of subclinical infection, was prepared by the disruption of parasitized erythrocytes in a French pressure cell. In contrast, antigen prepared from the plasma of infected animals was weakly immunogenic. The inoculation of IEA by the intravenous route without adjuvant did not confer protection but demonstrated the presence of a “kallikrein activating” substances(s) in the contents of the parasitized erythrocytes.

The subcellular distribution of carbonic anhydrase in homogenates of perfused rat brain.

Abstract— The distribution of carbonic anhydrase was examined in subcellular fractions of perfused rat brain and compared with those of markers for cytosol (lactic dehydrogenase), mitochondrial matrix (glutamic dehydrogenase), and mitochondrial membranes (succinic dehydrogenase). About half of the total carbonic anhydrase was found in particulate fractions, with the greatest part of this in the crude mitochondrial fraction. This fraction was separated into its components on a discontinuous sucrose gradient either as such or after isotonic mechanical disruption with a French pressure cell, and the resultant fractions were characterized by electron microscopy and by assay of marker enzymes.Carbonic anhydrase was solubilized by mechanical disruption, but not to the same extent as lactic dehydrogenase. The highest specific activity for carbonic anhydrase was found in the myelin fraction of the gradient. A mitochondrial locus for carbonic anhydrase is unlikely, but the presence of the enzyme in synaptosomes remains in question.Addition of soluble carbonic anhydrase did not significantly increase the activity of particulate fractions. Treatment of particulate fractions with detergent was necessary to reveal latent activity; this procedure resulted in a more than ten‐fold increase in the measurable carbonic anhydrase activity of myelin fragments.

Enzymatic activities leading to pyrimidine nucleotide biosynthesis from cell-free extracts of Rickettsia typhi.

Cell-free extracts from Rickettsia typhi were examined for the presence or absence of pyrimidine phosphotransferase enzymes and compared with the enzymes of mouse L cells and Salmonella typhimurium. The organisms were grown in mouse L cells and in the yolk sacs of chicken embryos, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The enzymes for the reutilization of uridine and thymidine, uridine kinase (EC 2.7.1.48) and thymidine kinase (EC 2.7.1.21), were not detected in R. typhi extracts with the phosphate donors effective for control enzymes. The following enzyme activities were demonstrated in R. typhi: uridine-5'-monophosphate kinase (UMPK, EC 2.7.4.4), deoxythymidine-5'-monophosphate kinase (dTMPK, EC 2.7.4.9), and nucleosidediphosphate kinase (NDPK, EC 2.7.4.6). Physicochemical and enzymatic analyses demonstrated that the pyrimidine nucleotide kinases of R. typhi were not of host origin and that the source (yolk sac and mouse L cells) did not influence the relative enzymatic activities. The specific activities of UMPK and dTMPK were higher when the rickettsiae were harvested before embryo death, whereas NDPK levels were slightly decreased. The specific activities of UMPK, dTMPK, and NDPK were comparable to those of S. typhimurium, and consequently the rickettsiae have potential for the anabolism of monophosphates, as do the host-independent bacteria. These results suggest that R. typhi cannot utilize host uridine or thymidine pools directly but must rely on themonophosphorylated molecules of the host cell or must synthesize the monophosphates de novo.

Respiratory components and oxidase activities in Alcaligenes eutrophus

1. Cells of the hydrogen bacterium Alcaligenes eutrophus are broken by gentle lysis using lysozyme treatment in hypertonic sucrose followed by osmotic shock. By this method, 93% of the in vivo activity of the H 2 oxidase is recovered and the ATPase remains particle bound. In contrast, cell disruption in a French pressure cell diminishes the in vivo activity of the H 2 oxidase by 50% and solubilizes the bulk of the ATPase. 2. The bacterium contains a periplasmic cytochrome c with bands at 418, 521 and 550 nm (difference spectrum). In addition to cytochrome aa 3 , b-560, c-553 and o, low temperature difference spectra of membranes show the presence of two further cytochromes (shoulders at 551 and 553 nm). 3. The unsupplemented membrane fraction catalyses the oxidation of hydrogen, NADH, NADPH, succinate, formate and endogenous substrate (NAD linked) at rates 2–3-fold higher than membranes obtained from cells disrupted in a French pressure cell. With the exception of the H 2 oxidase all oxidase activities in lysozyme membranes are sensitive to carbonylcyanide m-chlorophenylhydrazone (20–100% stimulation of oxygen uptake). 4. The cytoplasmic fraction contains a B-type cytochrome with absorption maxima at 436 and 560 nm, capable of combining with CO; it contains non-covalently bound protohaem. In alkaline solutions a spectral transition to the haemochrome type with bands at 423, 526 and 556 nm occurs. The addition of NADH to an aerobic suspension of this cytochrome elicits new absorption maxima at 418, 545 and 577 nm (difference spectrum), which are believed to represent an oxygenated form of the reduced cytochrome.

Ultrastructural study of Plasmodium knowlesi antigen used in vaccination of rhesus monkeys.

Material from various steps obtained in the French pressure cell technic of preparing antigen from Plasmodium knowlesi-infected red cells, was examined by elctron microscopy. A positively charged colloidal iron solution was used to differentiate between membranes of host red cells and parasites. Red cell membranes take the stain, wheras parasite membranes do not. This antigen which has been used previously to protect monkeys against P. knowlesi appears to consist almost entirely of membrane-bounded vesicles. Some of these vesicles contain a fine granular material, whereas others appear empty. The antigen failed to stain with the positively charged iron solution, which suggests that it is free of contamination by host cell membrane.

Enzymatic activities of cell-free extracts of Rickettsia typhi.

Cell-free extracts of Rickettsia typhi were tested for activities of enzymes of the tricarboxylic acid cycle, of glutamate catabolism, and of glycolysis. The organisms were grown in the yolk sacs of chicken embryos, harvested shortly before the time of embryo death, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The following enzymatic activities were demonstrated: high levels of malate dehydrogenase (MDH), moderate levels of glutamate-oxaloacetate transaminase, glutamate, succinate, and isocitrate dehydrogenases, and citrate synthase, and low levels of glutamate-pyruvate transaminase. The specific activities of some of these enzymes were higher when the rickettsiae were harvested at a time of active proliferation, 3 to 4 days prior to embryo death. Rickettsial MDH was differentiated from host MDH by its migration pattern on polyacrylamide gel electrophoresis. The activities of MDH and two other dehydrogenases, demonstrable after the cells had been disrupted, were absent from purified, intact rickettsial preparations. No activity was detected for glucose-6-phosphate, 6-phosphogluconate, glyceraldehyde-3-phosphate, lactate dehydrogenases, phosphoglucose isomerase, fructoaldolase, or pyruvate kinase. Our results suggest that extracts of R. typhi that contain demonstrable enzymes involved in the catabolism of glutamate and tricarboxylic acid cycle intermediates, unlike Coxiella burnetti, lack detectable glycolytic activity.

Membranes of Rhodospirillum rubrum: physicochemical properties of chromatophore fractions isolated from osmotically and mechanically disrupted cells.

Isolation of highly purified membrane fractions from phototrophically grown Rhodospirillum rubrum was achieved by velocity and isopyknic sedimentation under carefully controlled ionic conditions. Bacteriochlorophyll-rich and succinic dehydrogenase-rich chromatophores that were essentially devoid of contamination by non-chromatophore protein were separated from a denser fraction in extracts disrupted in a French pressure cell. Highly purified chromatophores and a nearly photopigment-free envelope fraction were also obtained from cells lysed by treatment with ethylenediaminetetraacetate-lysozyme-Brij 58. After lysis with lysozyme and ethylenediaminetetraacetate alone, about 50% of the total photosynthetic pigment was released in chromatophores similar to those isolated by the above procedures. Chromatophores prepared by each method were found to have very similar near-infrared absorption spectra, overall chemical composition, equilibrium buoyant densities in CsCl, and protein patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles of the dense, outer membrane-rich fractions were different from those of the chromatophores. The release of much of the photosynthetic apparatus as discrete chromatophores is osmotically lysed extracts necessitates a reevaluation of the concept that isolated chromatophores arise only from mechanical comminution of a larger membrane structure.

Coupling Factor Adenosine-5′-triphosphatase from Rho do spirillum rubrum: A Simple and Rapid Procedure for Its Purification

When photosynthetic membranes from Rhodospirillum rubrum, devoid of loosely bound small molecules and proteins, were passed through a French-pressure cell, the enzyme adenosine-5'-triphosphatase (EC 3.6.1.3.) (ATPase) was released into the soluble fraction. The solubilized ATPase was purified to homogeneity. In many respects it behaved like the enzyme purified by other workers, but it also hydrolyzed Mg-ATP with a small, but significant rate. Furthermore, it was much more stable. Maximal restoration of photophosphorylation in ATPase-depleted membranes was achieved by addition of about 1 mg purified ATPase per mg bacteriochlorophyll. For reconstitution of NAD+-photoreduction, about half of this amount was saturating.

Proteolytic activity of Oidiodendron kalrai

The physiochemical characteristics of the intracellular proteolytic enzymes of Oidiodendron kalari, a neuropathogenic fungus, were studied. The organism in the yeast phase was grown in a semisynthetic medium containing 1% tryptone, at 37 degrees C for 48 hr, on a gyrotory shaker. The crude extract was prepared by breaking the cells in a French pressure cell and the proteolytci activity was tested against biological substrates. The cell-free extract hydrolyzed casein, hemoglobin, lactalbumin, gelatin, elastin, collagen and purified rabbit renal basement membrane to various degrees. Optimal proteolytic activity was observed at pH 6 and at 32 degrees C. Calcium and EDTA did not affect the enzymatic activity; however, activity was partially inhibited by sulfhydryl-blocking agents and by heat-inactivated horse, calf, and human serum. The extract was totally inactivated by exposure to a temperature of 70 degrees C for 60 min. Storage at -76 degrees C or -15 degrees C for 6 months or at 4 degrees C for 4 weeks did not affect protease activity.

Constitution of the cell envelope of Haemophilus influenzae in relation to competence for genetic transformation.

Cell envelopes of Haemophilus influenzae have been prepared by breakage in a French pressure cell followed by differential centrifugation. The envelope fraction may be resolved into an inner-membrane (light) and an outer-membrane (heavy) fraction on density gradients. Envelopes from competent cells possess elevated levels of lipopolysaccharide with a composition different from that of log-phase cell envelopes. Three apparently new polypeptides have been observed in envelopes from competent cells by gel electrophoresis in sodium dodecyl sulfate; additional quantitative alterations in the profiles of membrane polypeptides also company the development of the capacity to transport deoxyribonucleic acid. Most of the polypeptide changes are confined to the outer membrane; one new polypeptide is associated with the inner cytoplasmic membrane of competent cells. Protein synthesis during competence developement is rquired for the change in lipopolysaccharides and in the envelope polypeptides to occur.

Energy-linked and energy-independent transhydrogenase activities in Escherichia coli vesicles

Active transport vesicles of Escherichia coli were shown to possess low levels of energy-independent and energy-dependent nicotinamide nucleotide transhydrogenase activities. Breakage of such vesicles in a French pressure cell resulted in a fraction which had an 8–10-fold increased respiration- and ATP-driven transhydrogenase activities. Stimulation of the ATPase activity in vesicles with Triton X-100 was also paralleled by a 2-fold increase in the energy-independent transhydrogenase. Disruption of the vesicles similarly resulted in increases in the energy-independent transhydrogenase, NADH and succinate oxidase activities but a decrease in succinate supported proline uptake. In the light of these findings, the ‘sidedness’ of the vesicle membranes is discussed.