Minimal size of Nitrobacter membrane frangments retaining nitrite oxidizing activity

Summary The minimal size of active membrane fragments from sonic homogenates of a Nitrobacter cell suspension is about 22 m. Nitrite oxidizing activity is absent from particles smaller than that although the distribution of cytochrome a, a link in the respiratory chain responsible for nitrite oxidation, does not show any abnormality below the critical size indicated.Two independent estimates of the parameters of the cumulative distribution function for particle size in sonic homogenates have been made using either statistics from electron micrographs or sedimentation pattern observed after density gradient centrifugation. The distribution function was found to be of the log-normal type which was considered to be a consequence of the observed kinetics of sonic comminution. Another estimate of the parameters of the distribution function was made by fixed angle centrifugation, a method much less accurate with respect to size determination but which allowed a rapid estimate of nitrite oxidizing capacity remaining in particles smaller than the given size.Results from the first two determinations of the distribution function for particle size and of its parameters were then used for correcting the error in the estimate of the critical size from the activity found in supernatants from centrifugations in fixed angle rotors.     

A diffusion driven instability in systems that separate particles by velocity sedimentation.

Velocity sedimentation has been used extensively to separate particles according to the magnitude of their sedimentation velocity in suitable media. This technique has been used over a wide range of particle size from protein molecules, viruses, subcellular particles to whole cells. Successful separation demands that collective particle motion should not occur. In practice it is observed that such systems may, under certain circumstances, suffer from a particular type of instability which destroys the normal dependence of sedimentation velocity on particle size and density. The aim of this paper is to identify the critical parameters that determine the development of this instability. Stability criteria are deduced and predictions of the theory compared with published observations. Satisfactory agreement between theory and observation is obtained. It is concluded that the simple stability criterion, namely that stable sedimentation will occur if the total density gradient is in the direction of the sedimenting force, grossly overestimates the particle load that can be separated in practice. Some specific recommendations for optimum particle loading are included. Earlier theoretical and experimental works are briefly reviewed.     

Settling of fixed erythrocyte suspension droplets

The fractionation of micron-size particles according to physical properties of size, density and surface characteristics by centrifugation and electrophoresis is hindered when the particles behave collectively rather than individually. The formation and sedimentation of droplets containing particles is an extreme example of collective behavior and a major problem for these separation methods when large quantities of particles need to be fractionated. In this paper, experiments that measured droplet sizes and settling rates for a variety of particles and droplets are described. Expressions are developed relating the particle concentration in a drop to measurable quantities of the fluids and particles. The number of particles in each droplet was then estimated along with the effective droplet density and certain trends are noted. Since a major application of this work is the purification of biological cells in the range of 10 microns, for which monodisperse inert particles are not available, red blood cells from different animals fixed in glutaraldehyde provided model particle groups with the necessary size range, visibility and stability for these fluid dynamical studies.     

Simultaneous measurements of sedimentation and diffusion coefficients using photon correlation spectroscopy

A simple experimental demonstration is described to show that photon correlation spectroscopy can be used to measure simultaneously the diffusion coefficient and, by the Doppler effect, the sedimentation coefficient of a small suspended particle. Calculation shows that the method should be readily extendable to particles with sedimentation coefficients of 5 S.     

Fractionation of rat fibroblasts in a zonal rotor by means of a viscosity barrier.

Cell fractionation procedures involving differential sedimentation followed by resuspension of pellets and isopycnic centrifugation are very difficult to apply to the small amounts of material available from tissue culture cells. We have explored the possibility of successive differential and isopycnic sedimentation in a zonal rotor using a short viscosity barrier for the differential sedimentation. The marker enzymes used were cytochrome oxidase, acid phosphatase, catalase, and 5′-nucleotidase. The results of these procedures are compared to the results of one-step isopycnic separations in gradients of sucrose and Stractan. The Stractan gradient was much more effective than the sucrose gradient in separating the marker enzymes from the proteins of a postnuclear supernatant, but neither type of gradient could significantly purify the marker enzymes one from another. A two-step procedure using a viscosity barrier was effective in separating particles carrying catalase from the other marker enzymes assayed and from most of the protein. A three-step procedure resulted in similar purification of mitochondria. Modification of barrier composition and centrifugation times would probably result in further improvement of separations according to individual requirements for yield, purification, and freedom from specific contamination by other subcellular particles.     

Purification of filamentous plant viruses by thin layer centrifugation.

The construction of a modified thin layer ultracentrifuge rotor is described. This rotor was used in the purification of five filamentous plant viruses, viz. TMV, SCMV, PVX, SGV and YMC. The purification and concentration of these viruses in their monomeric forms is hazardous when conventional "tube" rotors are used since they invariably result in dissociation and aggregation of the virus particles. Using the thin layer rotor these infective agents may be concentrated in volumes of fluid equal to approximately 1% of the starting suspension and not as pellets obtained after ultracentrifugation is conventional "tube" rotors. Electron microscopy revealed that the virus particles concentrated by thin layer centrifugation were not aggregated and that only few fragments of the virus filaments were present in the final preparations.     

19. Purification of Filamentous Plant Viruses by Thin Layer Centrifugation (Applied to TMV,SCMV, PVX,SCV, and YMC Viruses)

ABSTRACT

The construction of a modified thin layer ultracentrifuge rotor is described. This rotor was used in the purification of five filamentous plant viruses, viz. TMV, SCMV, PVX, SCV and YMC. The purification and concentration of these viruses in their monomeric forms is hazardous when conventional "tube" rotors are used since they invariably result in dissociation and aggregation of the virus particles. Using the thin layer rotor these infective agents may be concentrated in volumes of fluid equal to approximately 1% of the starting suspension and not as pellets obtained after ultracentrifugation in conventional "tube" rotors. Electron microscopy revealed that the virus particles concentrated by thin layer centrifugation were not aggregated and that only few fragments of the virus filaments were present in the final preparations.     

Inhibition of bacterial growth by metal salts. The accumulation of ribonucleic acid during inhibition of Escherichia coli by cobalt chloride

During inhibition of the growth of Escherichia coli by cobalt chloride protein synthesis was decreased more than the synthesis of RNA. Three species of particle accumulated during the incubation. These had sedimentation coefficients of about 44s, 33s and 23s in tris buffer containing 10 mm-magnesium acetate and 100 mm-potassium chloride, but their sedimentation properties were susceptible to changes in buffer composition. The particles contained RNA but were more readily degraded by ribonuclease than were the ribosomes. RNA isolated from the particles differed slightly in sedimentation properties from the major species of ribosomal RNA. The particles are likely to be closely related to ribosome precursors that have been detected in other circumstances. Changes in the polyribosome fraction during inhibition by cobalt chloride, nickel chloride and chloramphenicol provided further evidence that inhibition by Co(2+) involves specific effects on the protein-synthesizing machinery.     

Analog modeling of glucagon-induced autophagy in rat liver. II. Evaluation of iron labeling as a means for identifying telolysosome, autophagosome and autolysosome populations.

To evaluate the potential usefulness of iron labeling as a means for identifying the telolysome, autophagosome and autolysosome populations of rat liver, animals treated with Jectofer (iron-citric acid-sorbitol complex), or with Jectofer followed by glucagon, have been studied with a variety of biochemical and morphological methods. Differential centrifugation studies of liver homogenates revealed that the sedimentation velocity and mechanical fragility of acid phosphatase bearing particles increase with the duration of Jectofer treatment and that iron accumulates in the mitochondrial and nuclear fractions. Rate sedimentation studies confirmed the change in sedimentation velocity, which was shown to be due in part to a marked increase in particle density. Quantitative morphological analysis of liver M + L and N + M + L fractions revealed a nearly complete absence of pericanalicular dense bodies after 6–7 days of Jectofer treatment. In these fractions a new type of particle containing fine electron dense granules was seen. The mean volume of these particles was decreased and their number increased when compared to dense bodies but the general morphology and overall size distribution of the two particle classes were similar. In animals given both Jectofer and glucagon, autophagic vacuole formation was similar to that found in animals receiving only glucagon. However, the increase in osmotic fragility of acid phosphatase bearing particles usually seen after glucagon administration occurred at a significantly slower rate. Examination of paniculate fractions revealed the presence of autophagic vacuoles with (autolysosomes) and without (autophagosomes) fine dense granules. The number of autolysosomes and their relative proportion in the autophagic vacuole population were correlated with an increase in the osmotic fragility of the acid phosphatase bearing particles in the same fraction. Organelle degeneration was observed more frequently in autolysosome profiles. These results support the contention that iron labeling can be used to separate the principal particle populations participating in the autophagic response induced by glucagon.     

Resolution in zonal rotors

We have studied “static” factors affecting resolution in zonal rotors, that is, factors that are independent of diffusion or sedimentation of the sample.For most zonal rotors under the recommended conditions, there is proportionately very little zone broadening of sample volumes larger than 10 cc. There appears to be a correlation between the height of the rotor and resolution.We show that step gradients surrounding the sample can result in zone broadening in the case of slowly sedimenting particles. We did not detect any deleterious effects from increasing the initial sample radius.     

Purification of Large Quantities of Influenza Virus by Density Gradient Centrifugation

New zonal centrifuges can conveniently process as much as five orders of magnitude (10(5)) greater sample volumes than conventional swinging-bucket rotors. The continuous-sample-flow-with-banding versions may be used in series with ancillary purification procedures. Here we have studied the combined process: absorption and elution of influenza virus with barium sulfate followed by concentration and isopycnic banding of the virus in a buffered sucrose gradient. Kilogram quantities of impurity have been rapidly separated from grams of purified virus, which have been conveniently concentrated several hundred-fold by the purification process. Experimental vaccines made by these procedures are being evaluated.     

Acid hydrolases in the suckling rat small intestine I. Fractionation of mucosal homogenates

Synopsis Small intestine mucosal homogenates of suckling rats have been fractionated by centrifugation and analyzed for acid hydrolases and for biochemical markers of subcellular organelles. The results indicate that the acid hydrolases are associated with particles having sedimentation properties similar to those of mitochrondria. The acid hydrolases exhibited latent activity. Subfractionation on a continuous density gradient of sucrose in deuterium oxide demonstrated that these enzymes are associated with particles distinct from other subcellular organelles. Electron micrographs of the acid hydrolase-rich region of the gradient show the presence of numerous small electron dense bodies bounded by a unit membrane.     

Size-separation of yeast mitochondria in the zonal centrifuge

Mitochondria, released from yeast spheroplasts and subjected to rate separation through sorbitol gradients in the zonal centrifuge, migrated in a wide symmetrical zone. Electron micrographs showed that the mitochondria had been resolved within the zone according to size. The mean mitochondrial diameter at the leading edge was approximately twice that at the trailing edge of the particle zone. Activities of the enzymes cytochrome oxidase, malate dehydrogenase, and reduced nicotinamide adenine dinucleotide- and d-lactate cytochrome c reductases were essentially uniform throughout the mitochondrial zone. Mitochondria from a vegetative-petite mutant had almost the same size distribution as the isogenic wild type, but with somewhat larger mean diameter and either absent or markedly reduced enzyme activities. Mixtures of wild-type and petite mitochondria produced sedimentation profiles showing overlap of particle populations with respect to mean sedimentation rates and mitochondrial diameters, as well as intermediate levels of enzyme activities. Both cristate and noncristate organelles were present throughout the mitochondrial zone from these mixtures. Mitochondria centrifuged in sorbitol density gradients were well-preserved and yielded consistent sedimentation profiles, whereas particles in sucrose density gradients migrated more slowly, produced varied sedimentation profiles, and often showed spurious peaks, presumably due to particle aggregations.     

Properties of various rotors used for zone centrifugation

The following results have been obtained from a quantitative study of zone centrifugation: (1) it is shown that the sedimentation velocity of all kinds of macromolecules is constant in 5–20% constant sucrose gradients, whatever swinging bucket or zonal rotor is being used, and at any usual temperature. (2) The proportionality constants between time of centrifugation and sedimented distance have been calculated for several rotors. They allow an estimate of relative centrifugation times. (3) An equation of the resolving power of zone centrifugation in isokinetic density gradients is used to compare the resolution of various rotors. (4) An equation of Vinograd and Bruner and Spragg and Rankin is discussed and used for the calculation of the maximum macromolecular load of the rotors. A summary of these results is presented in a table, which should help in the choice of the rotor best suited for a particular experiment.     

Studies on the polyhedrosis virus forming polyhedra in the midgut-cell nucleus of the silkworm,Bombyx mori: I. Purification procedure and form of the virion

A procedure for the isolation and purification of a new polyhedrosis virus that forms polyhedra in the midgut-cell nucleus is described. The method is a modification of that used by Hayashi and Bird (1970) for the isolation of free cytoplasmic-polyhedrosis virus (CPV). The purified virions were spherical in shape with several projections that measured 62 nm in diameter. The sedimentation coefficient of this virion was 430 S. These features are very similar to those of CPV. No significant difference was observed between this virus and CPV in the sedimentation profiles of preparations. Electron microscope observations indicated that the purified virus preparations contained many more empty particles than those of CPV.From these results, it is concluded that this virus is quite similar to CPV.     

Production and purification of hybrid Ty-VLPs

This article describes how pure Ty-VLPs (virus-like particles) can be prepared from hybrid Ty-VLPs. Many different hybrid Ty-VLPs have been produced and may be easily purified. Since the sedimentation properties of different hybrid Ty-VLPs are similar, a simple purification process can be used for any VLP. This fast, versatile, and easy process allows for the production of a variety of recombinant proteins.     

The production of hybrid Ty:IFN virus-like particles in yeast.

The yeast retrotransposon Ty encodes proteins that assemble into virus-like particles (Ty-VLPs) which can be readily purified. We have recently shown that expression of the pl protein encoded by the TYA gene of Ty is sufficient for particle formation. In this paper we show that when a heterologous coding sequence, human interferon-alpha 2 (IFN), is fused in frame to the TYA gene, the resulting p1-IFN fusion protein is still assembled into VLPs. These Ty:IFN-VLPs can be easily purified to near homogeneity and furthermore, they induce an antibody response to interferon when they are injected into rabbits. Therefore, these data show that hybrid Ty-VLPs can be used as a convenient system for the efficient purification of fusion proteins in yeast.     

The calculation of sedimentation coefficients in vertical rotors

A program for the calculation of sedimentation coefficients of molecules centrifuged in sucrose in vertical rotors has been developed. The program has been tested with both protein and RNA of known sedimentation coefficients. The preparation can accept any shape of gradient in the 0–70% sucrose and any temperature in the range of 0–60°C. The program can be used with any vertical rotor for which the dimensions are known.     

The sedimentation potential and the boycott effect

The sedimentation potential or the Dorn effect occurs when heavy particles fall in a liquid. An electrode near the bottom of the vessel acquires a potential difference with respect to another identical electrode placed near the upper surface of the solution. The Boycott phenomenon enhances the sedimentation in inclined vessels. In this investigation, fixed erythrocytes at 2-3% concentration were studied. The shapes include discs, oblate spheroids, spheres and spindles. From the sedimentation potentials, the zeta potentials were calculated and compared with those determined by laser Doppler velocity. By using a technique of reduced variables, it is shown that all of the data could be placed on a single curve, combining particle flexibility, shape, concentration and angle of inclination.