The isolation of endoplasmic reticulum from barley aleurone layers

Techniques for the isolation and purification of endoplasmic reticulum (ER) from aleurone layers of barley (Hordeum vulgare L.) were assessed. Neither differential centrifugation nor density gradient centrifugation of a homogenate separate the ER or other organelles of this tissue from the lipidcontaining spherosomes. Isopycnic sucrose gradient centrifugation of organelles first purified by molecular sieve chromatography on Sepharose 4B, however, results in separation of the organelles based on their differing buoyant densities. Manipulation of the magnesium concentration of the isolation media and density-gradient solutions affords isolation of ER at a density of 1.13–1.14 g cc^-1 and 1.17–1.18 g cc^-1. Electron microscopy shows that the membranes sedimenting at 1.13–1.14 g cc^-1 are devoid of ribosomes and are characteristic of smooth ER, while those sedimenting at 1.17–1.18 g cc^-1 are studded with ribosomes and have the features of rough ER. Endoplasmic reticulum isolated by isopycnic density gradient centrifugation can be further purified by rate-zonal centrifugation.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid - Trizma tris(hydroxymethyl)aminomethane     

Electron microscopic demonstration and isolation of ribosomes in mesosomes from Staphylococcus aureus

Mesosomes of Staphylococcus aureus 209P were observed to be extruded as tubules upon protoplast formation by electron microscopy and isolated under hypertonic conditions to maintain their structural integrity by differential centrifugation followed by sucrose density gradient centrifugation. Isolated mesosomes were composed of long, branched tubules of irregular sizes and they were shortened during purification. Thin sections of isolated mesosomes showed that the mesosomal tubule was surrounded by a triple-layered membrane and contained ribosome-like particles in diameter of about 15 to 20 nm. These particles were isolated from purified mesosomal preparation by disrupting the mesosomal tubule with deoxycholate and Triton X-100 under hypotonic conditions followed by a linear sucrose density gradient centrifugation. Negatively stained preparations of the isolated particles revealed the same appearance as those of the ribosomes isolated from the cytoplasm. The mesosomal particles sedimented at 70S in sucrose gradients in the presence of 10 mM Mg2+, but they were dissociated into two subparticles, 50S and 30S subunits, upon lowering the Mg2+ concentration to 1 mM. These findings indicate that the mesosomal tubule is packed with ribosomes.     

Membrane-bound ribosomes in kidney: methods of estimation and effect of compensatory renal growth

Membrane-bound ribosomes are thought to secrete protein for export and free ribosomes to secrete protein for intracellular use. The proportion of the total ribosomes that is bound to membranes in normal mouse kidneys has been estimated by three different methods, and the results have been compared with those obtained by a fourth method used by us previously. The most valid estimates appear to be those obtained (a) by comparison of radioactivity in peaks representing free and membrane-bound ribosomes on linear sucrose gradients after labeling for 24 hr with ¹⁴C-orotic acid, and (b) by measurements of optical density in free and bound ribosomes that had been separated by centrifugation on discontinuous gradients of 0.5 M/2.0 M sucrose. Analyses by these methods show that about 20–25% of the ribosomes in a postnuclear supernatant prepared from mouse kidneys, but only 10–15% of the ribosomes in a post-mitochondrial supernatant, are membrane-bound. About 75% of the bound ribosomes sediment as polysomes of many different sizes. The proportion of membrane-bound ribosomes and their aggregation into polysomes were unchanged in kidneys undergoing compensatory hypertrophy after removal of the opposite kidney. These experiments show that, unlike liver, kidney has a predominance of free ribosomes compared to bound ribosomes; those ribosomes that are membrane-bound do not become free during compensatory renal growth.     

A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli

With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3′-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)₆-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)₆-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.     

ISOLATION OF CYTOPLASMIC AND CHLOROPLAST RIBOSOMES AND THEIR DISSOCIATION INTO ACTIVE SUBUNITS FROM CHLAMYDOMONAS REINHARDTII

A mixture of cytoplasmic (80S) and chloroplast (70S) ribosomes from Chlamydomonas reinhardtii was freed of contaminating membranes by sedimentation of the postmitochondrial supernatant through a layer of 1.87 M sucrose. The purified ribosomes were separated into 80S and 70S fractions by centrifugation at a relatively low speed on a 10–40% sucrose gradient containing 25 mM KCl and 5 mM MgCl₂. Both the 80S and 70S ribosomes were dissociated into compact subunits by centrifugations in 5–20% high-salt sucrose gradients. The dissociations of both ribosomal species under these conditions were not affected by the addition of puromycin, indicating that the ribosomes as isolated were devoid of nascent chains. Subunits derived from the 80S ribosomes had apparent sedimentation coefficients of 57S and 37S whereas those from the 70S ribosomes had apparent sedimentation coefficients of 50S and 33S. In the presence of polyuridylic acid and cofactors, the 80S and 70S ribosomes incorporated [¹⁴C]phenylalanine into material insoluble in hot TCA. The requirements for incorporation were found to be similar to those described for eukaryotic and prokaryotic ribosomes. Experiments with antibiotics showed that the activity of the 80S ribosomes was sensitive to cycloheximide, whereas that of the 70S ribosomes was inhibited by streptomycin. The isolated subunits, when mixed together in an incorporation medium, were also active in the polymerization of phenylalanine in vitro.     

The product of ribosome-associated RNA-dependent RNA polymerase in immature chicken erythrocytes

The radioactively labelled product obtained after incubation of chicken erythrocyte ribosomes with [³H]-UTP was shown by sucrose density gradient centrifugation to have a sedimentation coefficient of 4–5S; the ratio of UMP to uridine incorporated was 6.09. The product synthesized and isolated after incubation of intact cells in the presence of [³H]-uridine and actinomycin D was shown to be very similar with respect to sedimentation coefficient and the ratio of UMP/uridine incorporated, providing for the first time evidence of a ribosome-associated terminal ribonucleotidyl-transferase activity in intact cells.     

A method for visualizing and quantifying the total complement of free and membrane-bound ribosomes in rat liver.

A method is described for both visualization and quantification of the total complement of rat liver free and membrane-bound ribosomes, undegraded by nucleolysis and unaggregated by pelleting. The method involves: (a) differential centrifugation of liver homogenate which separates free and membrane-bound ribosomes; (b) treatment of the fractions with detergents to solubilize membranes and remove nuclei; (c) centrifugation of a portion of each fraction to remove all the ribosomes; (d) sedimentation of the samples and blanks on sucrose gradients; and (e) difference photometric scanning of the gradients, sample minus ribosome-free blank, to detect the ribosomes free of interference from nonribosomal materials. The use of the SW 56 rotor in the initial centrifugation and of a high Mg²⁺ concentration (20 mm) in the medium used to suspend the bound fraction prior to detergent treatment were found to be essential in obtaining bound polysomes of large size (～19-somes). The difference scanning technique is shown to be a sensitive, accurate, and reproducible means of eliminating interference from nonribosomal materials, principally detergents and protein, and of quantifying ribosomes in both fractions. The method is rapid (3.5 h), simple to perform, and well suited for the analysis of multiple liver samples. It can be used to assess the concentration, distribution, organization, and average size of the total complement of rat liver free and membrane-bound ribosomes in a single experiment.     

RIBOSOMAL SUBUNITS FROM NEONATAL MOUSE BRAIN HIGHLY ACTIVE IN POLYPHENYLALANINE SYNTHESIS

Abstract— A highly active subcellular protein synthesising system is described, in which uncomplexed ribosomes isolated from 5 to 7 day old mouse brain can be reprogrammed with polyuridylic acid. Either purified free polyribosomes or microsomes were used as the starting material for the preparation of uncomplexed ribosomes by treatment with 0.5 m -KCl and puromycin. After reduction of the salt concentration 80S ribosomes were isolated by washing through sucrose. When, subsequently, zonal centrifugation in equivolumetric sucrose gradients containing 0.5 m -KCI was performed, purified ribosomal subunits were obtained. Cross-contamination of subunits was less than 5%. Re-associated ribosomes and recombined isolated ribosomal subunits both showed high activities in vitro. Incorporation levels of 50–60 phenylalanine residues per ribosome could be reached, at a rate of 0.5–2.0 residues/min/ribosome, depending on the activity of the high speed supernatant enzymes added. It was shown by paper chromatography of the cell-free product that only oligophenylalanine formation takes place. It was estimated that 6&70% of the ribosomes present in vitro were actively participating in the protein synthesis process.     

Isolation of Ribonucleic Acids Having Template Activities from Particulate Components of Soybean Seeds

Ribonucleic acids having template activities were obtained from particulate components prepared from the postribosomal supernatant of soybean seeds. These RNA were 9 S and 18 S in size, and these corresponded to the components (9 S, 18 S) of high molecular weight RNA (H–RNA) prepared from the supernatant of 100,000×g centrifugation. The sizes of the particulate components were 37 S and 59 S, respectively. Larger particles contained 18 S and 9S RNA, and smaller particles contained 9S RNA, but not 18 S RNA. Those particulate components differed in absorption pattern and in the behaviour on sucrose gradient centrifugation depending on the concentration of Mg²⁷⁺ from the subunits of ribosomes.     

An efficient preparative isopycnic flotation method for the isolation of chitosomes

Summary Chitin synthetase, a key enzyme in fungal cell wall biosynthesis, is located in chitosomes (microvesicles). To produce large quantities of chitosomes for immunochemical and biochemical characterization, we developed a two-step purification procedure in which isopycnic sucrose density gradients were centrifuged at ultra-high gravitational forces (fixed-angle rotor at 361,000×g R_av). Chitosomes from yeast cells ofMucor rouxii were separated from the soluble proteins and from the larger membranes by isopycnic centrifugation of the cell-free extract. The resulting crude chitosome sample was adjusted to a higher sucrose concentration, and a sucrose gradient was layered over the sample. Upon recentrifugation, the chitosomes moved up into the gradient and equilibrated at their buoyant density (1.15–1.16). This accelerated flotation separated contaminating particles of higher buoyant density (larger vesicles, ribosomes, and other miniorganelles) and yielded a large population of microvesicles with a mean diameter of 48.9±13.8 nm. This preparation contained vesicles essentially free of other particulate contaminants; more than 99% of the vesicles were smaller than 100 nm. When required, an additional velocity centrifugation step was added to remove the larger vesicles from the chitosome samples. This streamlined method for chitosome isolation was much simpler and faster than earlier isolation procedures, gave a high yield of functional chitosomes, and made the large scale isolation of these organelles possible.     

A STUDY OF THE MOLECULAR SIZES AND STABILITY OF CHOLINE ACETY LTRANSFER ASE

Aggregation of the enzyme acetyl-CoA: choline-O-acetyltransferase (ChAc, EC 2.3.1.6) which appears to be homogeneous has been observed. The molecular weight of the most abundant form of ChAc was estimated by gel filtration and sucrose gradient centrifugation to be in the range 58,000-62,000. The most frequently encountered aggregates were much larger and eluted in the void volume from Sephadcx® G-100 and G-150 indicating molecular weights in excess of 400,000. In fact, they were subsequently found to be 1.2 × 10⁶ and 1.9 × 10⁶ by sucrose gradient centrifugation. The percentage of activity associated with high molecular weight ChAc increased with purification, but these aggregates disappeared after storage for 2-3 weeks at ?20°C. The loss occurred independently of any fall in enzymic activity in the preparations examined.     

ISOLATION OF TWO DISTINCT CLASSES OF POLYSOMES FROM A NUCLEAR FRACTION OF RAT LIVER

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-¹⁴C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-¹⁴C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.     

ISOLATION OF SMOOTH VESICLES AND FREE RIBOSOMES FROM RAT LIVER MICROSOMES

Microsomes, isolated from rat liver homogenate in 0.88 M sucrose, have been fractionated by differential centrifugation. The 2nd microsomal fraction, sedimented between 60 minutes at 105,000 g and 3 hours at 145,000 g, consists mainly of smooth vesicles, free ribosomes, and ferritin. By utilizing the differences in density existing between the membranes and the granular elements it has been possible to separate the smooth membranes from the free ribosomes and ferritin. The procedure is to resuspend the 2nd microsomal fraction in a sucrose solution of 1.21 or 1.25 density and centrifuge it at 145,000 g for 20 or 40 hours. A centripetal migration of membranes and a centrifugal sedimentation of granular elements are obtained. Phospholipids, as well as the enzymatic activities DPNH-cytochrome c reductase, glucose-6-phosphatase and esterase are localized in the membranes. The free ribosomes have been purified by washing. A concentration of 200 µg RNA per mg nitrogen has been reached. RNA is also present in the membranes. These results are discussed in relation to current views on microsomal structure and chemistry.     

The cytoplasmic ribonucleoprotein particles of rat sarcoma cells

Summary The metabolism of the cytoplasmic ribosomal RNP-particles from rat sarcoma cells have been studied after intraperitoneal injection in animals of¹⁴C-leucine alone or together with H₃P³²O₄. By investigating the change of the specific activity of the ribosomes with respect to the time after injection of¹⁴C-leucine we have demonstrated that the half-life of ribosomes is 35 hours.To study the metabolic activity of ribosomes not taking part in protein synthesis, the sucrose gradient centrifugation method was used for their separation from polyribosomes. Four, seven, twelve hours after injection of the isotopes free ribosomal subunits and monoribosomes were isolated and their radioactivity was determined. As expected the label was found in the ribosomal subunits at the beginning and later on in the monoribosomal fraction. At the same time it was observed that the incorporation of H₃P³²O₄ into ribosomal subunits occurred at a much greater rate as compared with the incorporation of¹⁴C-leucine. These results indicate the existence of a pool of ribosomal proteins in sarcoma cells whose role deserves attention.an invited article     

Isolation and some characteristics of cell nuclei from brain cortex of adult cat

A rapid detergent method for the isolation of nuclei from cat brain cortex is described. It involves the homogenization of the tissue in buffered 0.34 M sucrose with the addition of the non-ionic detergent Cemulsol NPT 12 and the subsequent low speed centrifugal sieving of the nuclei through two layers of sucrose (0.68 M and 1.0 M). The final purification is achieved by high speed centrifugation (40,000 g) of the nuclear suspension layered over 1.8 M sucrose. Observations by light microscopy indicate that highly purified and well preserved nuclei are obtained from neurons and glial cells. Electron microscopy reveals some microsomal contaminants adhering to the nuclear membrane. According to an analysis of the nuclear size distribution, a considerable loss of smaller nuclei (10 to 20µ²), mainly from glial cells, occurs during the purification procedure. The action of different detergents is compared, the best results being obtained with Cemulsol NPT 12 or Triton X-100. Chemical analyses of the purified nuclear fraction give the following content expressed in picograms per nucleus: DNA, 6.54; RNA, 2.94; cholesterol, 1.50; and protein, 97.5. The sucrose density gradient centrifugation of nuclei isolated from cat brain cortex shows that their density is equal to or higher than that of 2.2 M sucrose and is thus similar to the density of nuclei from other tissues. The observation of a varying influence of different suspending media on the density of brain cell nuclei resolves the conflicting data in the literature on the density of these nuclei.     

Isolation of 80S ribosomes from spores of Aspergillus fumigatus and their antigenicity in rabbits

Ribosomes were obtained from spores of Aspergillus fumigatus by mechanical disruption and differential centrifugation. The initial preparation (crude ribosomes) contained spore components which appeared to be broken fragments of plasmalemma with or without organelles. Purified ribosomes free of membranous material were prepared by gel filtration chromatography on Sepharose CL-4B. Monomeric 80S ribosomes consisting of 40% protein and 60% RNA, and morphologically characteristic of fungal ribosomes were isolated.Serological reactions in sera from rabbits injected with crude or purified ribosomes were similar indicating that the purification process did not change or eliminate antigens that stimulated antibody detectable either by indirect hemagglutination (IHA) or in gel precipitin tests.     

Isolation,Purification, and Properties of Peroxisomal Malate Dehydrogenase from Maize Mesophyll

Peroxisomal malate dehydrogenase (EC 1.1.1.37) with a specific activity of 533 U/mg (144-fold purification) and a yield of 5% was obtained in a homogeneous state by a purification scheme including sucrose gradient centrifugation from maize mesophyll. The Michaelis constants for the forward and reverse reactions were determined to be 11.6 mM and 256 μM, and the pH optimum was 9.5 and 9.0, respectively. Analysis of the molecular weight of the native enzyme and its subunits showed that the peroxisomal malate dehydrogenase was a homodimer. It was established that the isolated and purified isoform of the enzyme had a higher affinity for malate and NAD⁺ in comparison with the mitochondrial and cytoplasmic isoforms.     

Presence of an endopeptidase activity in rat liver ribosomes

Preparation of ribosomes using different procedures (treatment of postmitochondrial-postlysosomal supernatant or microsomes with 1% triton in 0.15 or 0.5 M KCl and subsequent sucrose gradient centrifugation; treatment of microsomes with 1.5% deoxycholate/2% triton) results in purified ribosomes which contain an endopeptidase activity detectable by breakdown of ribosomal proteins to trichloroacetic acid soluble split products. The proteolytic activity can be recovered also in the extracted proteins of whole ribosomes. With ribosomes the pH optimum of proteolytic breakdown is at about 7. The inhibition of the activity by leupeptin, DIFP and soya bean trypsin inhibitor suggests a serine type of the proteolytic activity.     

Cellular content of ribonucleic acid and protein in Saccharomyces cerevisiae as a function of exponential growth rate: calculation of the apparent peptide chain elongation rate.

The average cellular content of ribonucleic acid and protein was determined in cultures of Saccharomyces cerevisiae growing exponentially at different rates in a variety of media. Estimations of the proportion of total cellular ribonucleic acid that is made up of ribosomal ribonucleic acid were used to calculate the average number of ribosomes per cell at the different growth rates. The fraction of ribosomes actively engaged in translation was estimated by sucrose gradient centrifugation of ribosomes and polysomes. These data were used in a calculation of the apparent time taken for the addition of an amino acid to the growing polypeptide chain; this value was found to vary linearly with growth rate over a fivefold range of doubling times.     

Partial purification of mitochondrial ribosomes from broad bean and identification of proteins encoded by the mitochondrial genome

An approach towards the identification at the protein level of the ribosomal proteins encoded by the mitochondrial genome of broad bean (Vicia faba) has been developed. After Triton X-100 treatment of isolated mitochondria, a fraction enriched in mitochondrial ribosomes was obtained by successive centrifugation, first onto a sucrose cushion, and then in a sucrose gradient. Mitochondrial translation products were labelled in isolated mitochondria with [³⁵S]methionine and added to the enriched mitochondrial ribosomal proteins before separation by two-dimensional gel electrophoresis. Six spots, identified both by Coomassie blue staining and autoradiography, were analysed by protein micro-sequencing. Two of these were shown to correspond to ribosomal proteins S10 and S12. We conclude that these two proteins are encoded by the mitochondrial genome of broad bean and that the method described here can be used to identify other proteins encoded by the mitochondrial genome. Received: 4 September 1996 / Accepted: 30 November 1996