Zonal Centrifugation In Sucrose Gradients Research Articles

Purification of Viruses and Macromolecular Assemblies for Structural Investigations Using a Novel Ion Exchange Method

We describe a novel ion exchange chromatographic technique suitable for large-scale preparation of viruses and other biomacromolecular assemblies in highly purified form. The method, which utilizes anion exchange on either of two commercially available cellulose cartridges, is applied to the Escherichia coli bacteriophage PRD1. Viral particles eluted from both QMA and DEAE cartridges retain infectivity and exhibit greater homogeneity of composition, as judged by gel electrophoresis and electron microscopy, than particles purified by rate zonal sucrose gradient centrifugation. The ion exchange protocols are rapid, requiring less than 15 min elution time, and permit retrieval of the purified viral particles at high concentration in aqueous media without centrifugal pelleting. The present method is particularly well suited to the preparation of milligram to decigram quantities of virus, sufficient for many biophysical structural analyses, including investigations by solution spectroscopic and crystal diffraction techniques. The feasibility and advantages of the ion exchange chromatographic procedure are demonstrated by application of laser Raman spectroscopy to ion exchange purified PRD1 virions and subviral assemblies.

Conservation of the Dimeric Unit of H2A and H2B Histones during the Replication Cycle

The behavior of H2A and H2B histones during the replication cycle has been investigated. H2A-H2B dimer fractions obtained from MH-134SC cells labeled with suitable precursors were fractionated by rate zonal centrifugation in sucrose gradients containing 2 M NaCl. Labeling for one round of replication cycle with an amino acid mixture enriched with dense isotopes and [ 3H]lysine as a radioactive marker yielded a distinct peak of dense dimer that sedimented faster than the normal dimer. Similar density labeling of cells did not cause measurable alteration in the sedimentation profile of the preexisting normal dimer marked with [ 14C]lysine. The data suggest conservation of the bulk of the dimeric unit during the replication cycle. Biological significance of the dimeric behavior of H2A and H2B is discussed in favor of the partially conservative model of histone octamer assembly.

Isolation, purification, and characterization of flagellar scales from the green flagellateTetraselmis striata (Prasinophyceae)

Flagellar scales from the green flagellateTetraselmis striata (Prasinophyceae) were isolated, purified by isopycnic cesium chloride-gradient and zonal sucrose gradient centrifugation and their structure and biochemical composition investigated. Three types of flagellar scales were purified to more than 90% purity, a fourth type up to 75% purity. In addition to the previously known types of flagellar scales (pentagonal scales, rod-shaped scales, hair-scales), a novel scale type (i.e., the knotted scales) was discovered. New information about the asymmetric structure of the rod-shaped scales is presented and consequently they are renamed “man scales”. Flagellar scales consist mainly of carbohydrate (50–70%), significant amounts of protein (11% of dry weight) were found only in pentagonal scales. The main sugars (90%) of the pentagonal and man scales are the unusual 2-keto-sugar acids 3-deoxy-5-O-methyl-2-octulosonic acid (5 OMeKDO), 3-deoxy-2-heptulosaric acid (DHA), and 3-deoxy-2-octulosonic acid (KDO), the knotted scales contain as major sugars galactose and arabinose in addition to KDO and 5 OMeKDO but lack DHA. 13 major polypeptides were identified in flagellar scales by one-dimensional SDS-PAGE, 11 of these are of high molecular mass (>116 kDa). While the majority of polypeptides was found associated with pentagonal scales, at least 4 polypeptides were tentatively assigned to the hair-scales and knotted scales.

Studies on the molecular organization of rat insulin secretory granules.

Secretory granule-enriched fractions prepared from isolated rat islets of Langerhans, previously labeled in culture for 18 h with [3H]leucine, have been lysed and separated into pH 5.4 soluble and insoluble fractions by zonal sucrose gradient centrifugation. A high proportion of both labeled and immunoreactive rat insulins I and II were recovered in the insoluble granule core fraction in the expected ratio of approximately 60/40, respectively. Essentially equivalent amounts of the rat C-peptides on a molar basis were recovered in the granule supernatant fractions. The proportion of labeled proinsulin in the granule core fraction was less than 2% relative to insulin, while the soluble fraction contained about 8%, which probably arose mainly from disrupted proinsulin-rich noncrystalline prosecretory vesicles. Electron microscopic examination of the granule core fraction revealed large numbers of well preserved crystalline cores exhibiting typical dimensions and regular internal spacings of normal mature rat beta-granule inclusions. These results provide direct biochemical evidence that the beta-granules are nonuniform in composition with the insulin contained mainly in a crystalline state in the electron-dense central inclusions while the C-peptide is dissolved in the fluid bathing the crystalline hormone. The significance of this structural organization of the beta-granule is discussed.

Purification and Species Distribution of Rubisco Activase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein which promotes light-dependent rubisco activation, was partially purified from spinach chloroplasts by ion-exchange and gel-filtration fast protein liquid chromatography. The protein could also be isolated using rate zonal centrifugation in sucrose gradients followed by conventional ion-exchange on DEAE-cellulose. The active enzyme was composed of 44 and 41 kilodalton subunits. Antibodies to the activase polypeptides were produced in tumor-induced mouse ascites fluid and used as probes for activase on immunoblots of soluble proteins from a number of species. One or both of the activase polypeptides were recognized in all higher plant species examined including Arabidopsis thaliana, soybean, kidney bean, pea, tobacco, maize, oat, barley, celery, tomato, pigweed, purslane, dandelion, sorghum, and crabgrass. The polypeptides were not present in a mutant of Arabidopsis which is incapable of activating rubisco in vivo. The activase polypeptides were also detected in cell extracts of the green alga Chlamydomonas reinhardii. Activase activity, which had been demonstrated previously in wild-type Arabidopsis and in spinach, was measured in protoplast extracts of Nicotiana rustica. The results suggest that control of rubisco by activase may be an ubiquitous form of regulation in eucaryotic photosynthetic organisms.

Protein structure and expression among arenaviruses.

The proteins of arenaviruses were first studies by SMADEL and his colleagues (1939, 1940, 1942), with reference to their antigenicity. These workers described the presence of a virus-specific soluble (S) antigen detectable by complement fixation (CF) in homogenates of spleen and lung from LCMV-infected guinea pigs. Soluble antigen could be separated from infectious virus by ultracentrifugation. Repeatedly washed virions reacted poorly in CF tests while the S antigen lost none of its immunoreactivity after ultracentrifugation. These studies were not extended until nearly 3 decades later when Brown and Kirk (1969), Chastel (1970), Simon (1970), and Bro-Jorgensen (1971) described antigens detectable by C F and immunodiffusion in tissues or cell cultures infected with L C M V. Bro-Jorgensen (1971) found two antigenic species by immunodiffusion using infected BHK-21 cells as the antigen source. One antigen was heat stable and resistant to proteolysis, while the second was degraded by both heating and pronase digestion. Both antigens sedimented at a rate of 3.5 S in rate zonal sucrose gradient centrifugation, and based on this S value the molecular weight of the thermolabile S antigen was estimated to be 48 000.

Isolation and characterization of syringacin W-1, a bacteriocin produced by Pseudomonas syringae pv. syringae

Syringacin W-1, a bacteriocin produced by Pseudomonas syringae pathovar syringae strain PsW-1, is a 20 × 75 nm rod-shaped particle composed of an inner core and outer sheath. Production of syringacin W-1 in broth was induced with 0.1 μLg/mL mitomycin C. The bacteriocin was purified from culture lysates using ultrafiltration, rate zonal centrifugation in sucrose gradients, and DEAE-cellulose chromatography. Purity was evaluated by subjecting syringacin W-1 preparations to electrophoresis in polyacrylamide gels under nondenaturing and denaturing conditions. The chemical composition was principally protein (67.2%), and also comigrating nonessential carbohydrate (10–35%). The physical properties of purified syringacin W-1 were a sedimentation coefficient of 104 for rod-shaped particles, pH stability of 5.2–8.2, and temperature stability from −20 to 40 °C. The bacteriocin was resistant to proteases and to 12 of 13 surfactants tested.

Improved preparative methods for isolation and purification of tobacco chloroplast ribosomes, ribosomal proteins, and rRNA

Publisher Summary This chapter describes the procedures for the isolation of chloroplast ribosomes on a preparative scale by zonal sucrose gradient centrifugation from the whole leaf extracts. Methods for extracting ribosomal proteins and ribosomal RNA (rRNA) from the purified ribosomes are also described. The protein-synthesizing machinery of chloroplasts is a class of ribosomes that are unique to the chloroplast and of a 70 S type with many structural and functional similarities to bacterial ribosomes. The prokaryotic-like ribosomes are present in green leaves in equimolar amounts, a relative to the 80 S cytoplasmic ribosomes that share common features with other eukaryotic cytosolic ribosomes. Chloroplast ribosomes differ from the 80 S cytosolic ribosomes in their susceptibility to dissociation into subunits at different levels of magnesium ion. Chloroplast ribosome monosomes can be isolated at higher magnesium concentrations. The magnesium concentration required to control the equilibrium between the monosome and its dissociated subunits depends on the concentration of the major monovalent cation present in the solution.

Structure and properties of the rapidly sedimenting replicating complex of staphylococcal phage K DNA.

Rapidly sedimenting complexes (RSCs) of replicating phage K DNA, isolated by rate zonal centrifugation in sucrose gradients, contain bacterial membrane lipids and protein. During the first half of the latent period the number of DNA molecules in a RSC increased from 1 to about 27. Digestion by Pronase caused the complexes to dissociate and release virion lengths of DNA which sedimented slowly like free mature DNA. RSCs treated with SDS disintegrated and released tangled DNA molecules, each about one virion length in size, but these structures retained their fast sedimentation characteristic. Chloramphenicol (CM) at 100 micrograms/ml did not completely inhibit complex formation or DNA replication, indicating that pre-existing host proteins were involved in these processes. CM reduced DNA replication by 50 to 80%. It is concluded that phage K DNA replicates attached to the cytoplasmic membrane of the host.

Purification and characterization of bovine rotavirus cores.

Using the chaotropic effect generated by a high concentration of CaCl2, we converted calf rotavirus particles into cores of 40 nm in diameter. These cores were purified by rate zonal centrifugation in sucrose gradients and by isopycnic gradients. They had a sedimentation coefficient of 280S +/- 20S and a density of 1.44 g/ml in CsCl. When analyzed by polyacrylamide gel electrophoresis, they contained three polypeptides (VP125, VP89, and VP78). The major internal polypeptide of the virion (VP39) was recovered in a purified and soluble form in the top fractions of the sucrose gradients. From this stepwise degradation, it appears that VP39 is the most external polypeptide of dense particles. In contrast to reovirus cores, calf rotavirus cores did not exhibit transcriptase activity. Purified VP39 also did not exhibit transcriptase activity when tested after being mixed with purified rotavirus genome RNA as a template. Transcriptase activity was partially recovered when ionic conditions were adjusted to permit the reassociation of VP39 with the cores.

Separation of the mRNAs coding for different forms of chick embryo gizzard tropomyosin.

The fractionation of gizzard tropomyosin from 17-day-old chick embryos by electrophoresis in polyacrylamide gels indicates that this protein is composed of three different polypeptides that were designated as alpha, beta, and gamma. Each one of these polypeptides is resolved into two components with different isoelectric points when tropomyosin is fractionated by two-dimensional electrophoresis; the major components were designated as tropomyosins alpha 1, beta 1, and gamma 1 and the minor ones as tropomyosins alpha 2, beta 2, and gamma 2. The results of in vitro translation of gizzard RNA indicate that only tropomyosins alpha 1, beta 1, and gamma 1 are synthesized in the cell-free system. A partial separation of the mRNAs coding for tropomyosins alpha 1 and gamma 1 was obtained by fractionation of gizzard RNA by zonal centrifugation in sucrose gradients. Altogether these results indicate that in gizzard tissue from 17-day-old chick embryos three genes coding for different forms of tropomyosin are expressed.

Clostridial apoferredoxin messenger ribonucleic acid Assay and partial purification

An assay for Clostridium pasteurianum apoferredoxin messenger ribonucleic acid (mRNA) was developed, based on the synthesis of the protein in vitro. Quantitation of apoferredoxin synthesis was accomplished by trypsinization of the cell-free incubation labeled with 3H- or 14C-labeled amino acids, separation of the products by SDS-urea polyacrylamide gel electrophoresis, and excising and counting the NCS-solubilized gel band corresponding to the unique 52-amino acid tryptic peptide derived from apoferredoxin. Its synthesis was shown to be RNA dependent, and was optimized with respect to several parameters of the in vitro protein-synthesizing system. The specificity of the assay was examined with RNA from Clostridium acidi urici, a related species the ferredoxin of which does not yield the 52-amino acid tryptic peptide, and by the use of [ 3H]leucine, which is not present in C. pasteurianum apoferredoxin. By these methods, the overestimation of apoferredoxin synthesis due to the comigration of fragments from other in vitro products with the legitimate apoferredoxin-derived peptide could be accounted for. The apoferredoxin mRNA was partially purified by the sequential zonal sucrose gradient centrifugation of total RNA followed by Sephadex G-200 chromatography of the enriched RNA, after which a fraction was obtained in which apoferredoxin mRNA was 20-fold enriched. The enriched RNA fraction can now be used for further purification of the apoferredoxin-coding sequences by cloning procedures.

The discovery of a particle unique to brain and spleen subcellular fractions from scrapie-infected mice.

SUMMARY Fresh mouse scrapie brain and frozen mouse scrapie spleen were homogenized, sized by ultrafiltration to a fraction containing particles of 30 nm to 200 nm and separated by zonal centrifugation in sucrose gradients. In both the brain and spleen, the maximum titre of scrapie infectivity banded between densities of 1.125 to 1.200 g/ml; but in spleen a second fraction of scrapie infectivity was observed between 1.200 to 1.250 g/ml. A unique 30 to 60 nm particle was found in mouse spleen gradient fractions with high scrapie infectivity titres. This particle was not observed in similar fractions isolated from normal mouse spleen or brain and was rarely found in subcellular fractions of low titre from scrapie-infected tissues. The particle was first observed by negative staining and then confirmed in thin sections after en bloc staining with ruthenium red and potassium permanganate. The previously observed ‘smearing’ of scrapie infectivity over broad ranges in sucrose density gradients may now be explained since these particles were often found as aggregates or chains of particles. The significance of these particles in scrapie infectivity remains uncertain at this time, but absence of these structures in normal tissue fractions may provide a promising new morphological approach to the purification of the infective scrapie agent.

Free cytoplasmic messenger ribonucleoprotein complexes from rabbit reticulocytes.

Free cytoplasmic globin mRNA containing mRNP-particles were isolated from rabbit reticulocytes by zonal sucrose gradient centrifugation and their properties were compared with mRNP particles isolated in the same way from EDTA-dissociated reticulocyte polyribosomes. The average poly(A)-length of 9S mRNA from free cytoplasmic mRNP was 17-20 nucleotides being about two times shorter than the average poly(A)-length of polysomal 9S mRNA. The protein composition of the free cytoplasmic mRNP particles disclosed the absence of the 76,000 dalton protein which is associated with the 3'poly(A)-segment of polysomal globin mRNA. It was concluded that free cytoplasmic mRNP-particles from rabbit reticulocytes can be classified as "old" mRNP in a post-translational phase. Free cytoplasmic mRNPs were translated in heterologous cell-free systems as well as in Xenopus laevis oocytes. Addition of hemin stimulated the synthesis of alpha-globin in all systems, while the presence of the cap analogue m7G(5')p inhibited translation of free cytoplasmic mRNA completely. The latter finding suggested that free cytoplasmic mRNA has a 5' terminal "cap". Shortening of the poly(A)-segment with concomitant loss of the 76,000 dalton protein may lead to less efficient translation of free cytoplasmic mRNP.

Purification of eukaryotic ribosomes by isopycnic centrifugation in sucrose

A simple and inexpensive procedure for the isolation and purification of ribosomes from eukaryotes is described. The method avoids pelleting of ribosomes at high centrifugal forces and involves isopyenic centrifugation of the post-mitochrondrial supernatant in sucrose, precipitation of ribosomes with 10% polyethylene glycol, and zonal sucrose gradient centrifugation. The ribosomes obtained in this way are very pure and thus especially suited for the measurements of physical properties. The isopycnic centrifugation can also be used for the purification of other macromolecules and is only limited by a maximum density of sucrose of 1.40 g/cm 3 obtained at the bottom of the centrifugation tubes.

Infectious virus-antibody complexes of sindbis virus.

Infectious virus-antibody complexes were formed when Sindbis virus was reacted with antibodies raised against purified viral envelope glycoproteins E1 and E2 as well as against preparations of intact virus. Results from rate zonal centrifugation in sucrose gradients of the complex formed with anti-E1 sera showed this complex to be about the same size as virions. A test of virus neutralization, based on direct plaque assay, by antibodies raised in rabbits and mice given virus in complete Freund adjuvant indicated the presence of antibodies able to complex but not neutralize virus. Conditions were found in which most of the virus was complexed and protected fron neutralization, suggesting that these sera may contain a mixed population of antiviral antibodies with different specificities and different avidities.

Purification and molecular characterization of adenovirus type 2 DNA-binding protein

An adenovirus type 2 (Ad2) DNA-binding protein was purified by sequential DNA-cellulose, Sephadex G-200, and DEAE-Sephadex chromatography, with a yield of 120 mug of binding protein (95 to 99% homogeneity) starting with 2 X 10(9) infected cells. By omitting the Sephadex G-200 step, 400 to 600 mug of 95% pure binding protein was obtained. To obtain high yields of highly purified binding protein, it was necessary to include deoxycholate and Nonidet P-40 at selected stages during the preparation. The highly purified binding protein appeared to have retained its native stage as indicated by: (i) binding to single-stranded but not native Ad2 DNA, (ii) almost complete precipitation by immunoglobulin G from hamsters immunized by extracts of tumors induced by Ad2-simian virus 40 hybrid viruses, and (iii) identical sedimentation coefficient with binding protein obtained from DNA-cellulose chromatography only. Zonal centrifugation in sucrose gradients and gel filtration revealed that purified binding protein has a sedimentation coefficient of 3.4S and a Stokes radius of 5.2 nm. Based on these two values, a molecular weight of 73,000 was calculated, in agreement with the estimate from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A frictional ratio of 1.88 was calculated, suggesting that the Ad2 DNA-binding protein does not have a typical globular protein structure.

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.

Isolation and chemical characterization of outer envelope of Leptospira pomona.

Cells of Leptospira interrogans serotype pomona harvested from a chemically defined medium were resuspended in 0.01 M phosphate buffer of pH 7.3. Electron microscopy showed that 90 min of exposure effectively ruptured the outer envelope, freeing it from the cells as small flakes. Both zonal centrifugation in sucrose gradients and centrifugation in isopycnic KBr and CsCl gradients could be used to separate the outer envelope from the axial filaments and protoplasmic cylinders. The latter method resulting in higher yields of purified envelope with the particular protocols used. Thin sections of isolated outer envelope showed the same trilaminar structure seen in sections of intact cells. The outer layers were 1.5 nm thick and appeared as single layers of electron-dense particles. The central electron-transparent layer was 2.0-2.5 nm thick and appeared structureless. The gross chemical composition of the purified outer envelope was 47% protein, 27% carbohydrate, and 23% lipid. Colorimetric carbohydrate determinations revealed hexose, pentose, and 6-deoxyhexose; hexosamine was identified during amino acid analysis. Muramic acid, heptose, and 2-keto-3-deoxyoctonate were not detected. Thin-layer chromatography revealed only polar lipids, about 98% phosphatidylethanolamine and 2% lysophosphatidylethanolamine. Fatty acids identified by gas-liquid chromatography were octadecanoic, octadecenoic, hexadecanoic, and hexadecenoic. Amino acid analysis revealed 17 amino acids, histidine and glutamic acid being most abundant. The outer envelope was interpreted to be comparable with the outer double-track layer found in the cell covering of gram-negative eubacteria.

DNA-dependent RNA polymerase activity associated with subviral particles of polyhedral cytoplasmic dexoyribovirus.

Polyhedral cytoplasmic deoxyribovirus virions contain a DNA-dependent RNA polymerase which catalyzes the incorporation of ribonucleotides into an acid-precipitable product. Treatment of virions with sodium deoxycholate and dithiothreitol resulted in the formation of subviral particles which could be separated from virions by rate zonal centrifugation in sucrose gradients. Subviral particles were RNA polymerase-positive and more active per unit mass of protein than virions. In vitro enzyme activity associated with subviral particles required addition of ribonucleotides, Mg(2+), and exogenous denatured DNA template. Optimal enzyme activity occurred over a broad pH (7.2 to 8.8) and Mg(2+) concentration (2 to 10 mumol) range. The specific activity of the RNA polymerase was maximal at 37 C. Addition of DNase or actinomycin D to the reaction mixture reduced the incorporation of [(3)H]UMP into an acid-precipitable product. The product of the reaction was sensitive to degradation by RNase but not to DNase or Pronase. These data suggest that the enzyme copies DNA into RNA.