scholarly journals Mitochondrial and cytoplasmic ribosomes from mammalian tissues. Further characterization of ribosomal subunits and validity of buoyant-density methods for determination of the chemical composition and partial specific volume of ribonucleoprotein particles

1977 ◽  
Vol 168 (2) ◽  
pp. 245-259 ◽  
Author(s):  
Ada Sacchi ◽  
Ugo Ferrini ◽  
Paola Londei ◽  
Piero Cammarano ◽  
Nadir Maraldi
Keyword(s):  
Chemical Composition ◽  
Specific Volume ◽  
Buoyant Density ◽  
Volume Ratio ◽  
Partial Specific Volume ◽  
Ribosomal Subunits ◽  
Molecular Weights ◽  
Mitochondrial Ribosomes ◽  
Density Methods ◽  
Cytoplasmic Ribosomes

1. At 0–4°C mitochondrial ribosomes (55S) dissociate into 39S and 29S subunits after exposure to 300mm-K+ in the presence of 3.0mm-Mg2+. When these subunits are placed in a medium containing a lower concentration of K+ ions (25mm), approx. 75% of the subparticles recombine giving 55S monomers. 2. After negative staining the large subunits (20.3nm width) usually show a roundish profile, whereas the small subunits (12nm width) show an elongated, often bipartite, profile. The dimensions of the 55S ribosomes are 25.5nm×20.0nm×21.0nm, indicating a volume ratio of mitochondrial to cytosol ribosomes of 1:1.5. 3. The 39S and 29S subunits obtained in high-salt media at 0–4°C have a buoyant density of 1.45g/cm3; from the rRNA content calculated from buoyant density and from the rRNA molecular weights it is confirmed that the two subparticles have weights of 2.0×106 daltons and 1.20×106 daltons; the weights of the two subunits of cytosol ribosomes are 2.67×106 and 1.30×106 daltons. 4. The validity of the isodensity-equilibrium-centrifugation methods used to calculate the chemical composition of ribosomes was reinvestigated; it is confirmed that (a) reaction of ribosomal subunits with 6.0% (v/v) formaldehyde at 0°C is sufficient to fix the particles, so that they remain essentially stable after exposure to dodecyl sulphate or centrifugation in CsCl, and (b) the partial specific volume of ribosomal subunits is a simple additive function of the partial specific volumes of RNA and protein. The RNA content is linearly related to buoyant density by the equation RNA (% by wt.)=349.5−(471.2×1/ρCsCl), where 1/ρCsCl=[unk]RNP (partial specific volume of ribonucleoprotein). 5. The nucleotide compositions of the two subunit rRNA species of mitochondrial ribosomes from rodents (42% and 43% G+C) are distinctly different from those of cytoplasmic ribosomes.

scholarly journals The proteins of Xenopus ovary ribosomes

1971 ◽  
Vol 125 (4) ◽  
pp. 1091-1107 ◽  
Author(s):  
P J Ford
Keyword(s):  
Potassium Chloride ◽  
Ribosomal Proteins ◽  
Large Subunit ◽  
Buoyant Density ◽  
Small Subunit ◽  
Chemical Methods ◽  
Ribosomal Subunits ◽  
Molecular Weights ◽  
Caesium Chloride ◽  
Chloride Treatment

1. The preparation of ribosomes and ribosomal subunits from Xenopus ovary is described. 2. The yield of once-washed ribosomes (buoyant density in caesium chloride 1.601g·cm-3; 44% RNA, 56% protein by chemical methods) was 10.1mg/g wet wt. of tissue. 3. Buoyant density in caesium chloride and RNA/protein ratios by chemical methods have been determined for ribosome subunits produced by 1.0mm-EDTA or 0.5m-potassium chloride treatment and also for EDTA subunits extracted with 0.5m-, 1.0m- or 1.5m-potassium chloride, 4. Analysis of ribosomal protein on acrylamide gels at pH4.5 in 6m-urea reveals 24 and 26 bands from small and large EDTA subunits respectively. The actual numbers of proteins are greater than this, as many bands are obviously doublets. 5. Analysis of the proteins in the potassium chloride extract and particle fractions showed that some bands are completely and some partially extracted. Taking partial extraction as an indication of possible doublet bands it was found that there were 12 and 20 such bands in the small and large subunits respectively, making totals of 36 and 46 proteins. 6. From the measured protein contents and assuming weight-average molecular weights for the proteins of large and small subunits close to those observed for eukaryote ribosomal proteins it is possible to compute the total numbers of protein molecules per particle. It appears that too few protein bands have been identified on acrylamide gels to account for all the protein in the large subunit, but probably enough for the small subunit.

scholarly journals ELECTRON MICROSCOPE STUDY OF MITOCHONDRIAL 60S AND CYTOPLASMIC 80S RIBOSOMES FROM LOCUSTA MIGRATORIA

1974 ◽  
Vol 62 (3) ◽  
pp. 860-875 ◽  
Author(s):  
W. Kleinow ◽  
W. Neupert ◽  
F. Miller
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope Study ◽  
Flight Muscle ◽  
Locusta Migratoria ◽  
Volume Ratio ◽  
Negative Staining ◽  
Mitochondrial Ribosomes ◽  
Tissue Sections ◽  
Shallow Groove ◽  
Cytoplasmic Ribosomes

Purified mitochondrial ribosomes (60S) have been isolated from locust flight muscle. Purification could be achieved after lysis of mitochondria in 0.055 M MgCl2. Mitochondrial 60S and cytoplasmic 80S ribosomes were investigated by electron microscopy in tissue sections, in sections of pellets of isolated ribosomes, and by negative staining of ribosomal suspensions. In negatively stained preparations, mitochondrial ribosomes show dimensions of ∼270 x 210 x 215 Å; cytoplasmic ribosomes measure ∼295 x 245 x 255 Å. From these values a volume ratio of mitochondrial to cytoplasmic ribosomes of 1: 1.5 was estimated. Despite their different sedimentation constants, mitochondrial ribosomes after negative staining show a morphology similar to that of cytoplasmic ribosomes. Both types of particles show bipartite profiles which are interpreted as "frontal views" and "lateral views." In contrast to measurements on negatively stained particles, the diameter of mitochondrial ribosomes in tissue sections is ∼130 Å, while the diameter of cytoplasmic ribosomes is ∼ 180–200 Å. These data suggest a volume ratio of mitochondrial to cytoplasmic ribosomes of 1:3. Subunits of mitochondrial ribosomes (40S and 25S) were obtained by incubation under dissociating conditions before fixation in glutaraldehyde. After negative staining, mitochondrial large (40S) subunits show rounded profiles with a shallow groove on a flattened side of the profile. Mitochondrial small subunits (25S) display elongated, triangular profiles.

scholarly journals Particle weights and protein composition of the ribosomal subunits of the extremely thermoacidophilic archaebacterium Caldariella acidophila

1983 ◽  
Vol 209 (2) ◽  
pp. 461-470 ◽  
Author(s):  
P Londei ◽  
A Teichner ◽  
P Cammarano ◽  
M De Rosa ◽  
A Gambacorta
Keyword(s):  
Molecular Weight ◽  
Average Molecular Weight ◽  
Buoyant Density ◽  
Protein Composition ◽  
Equilibrium Density ◽  
Number Average Molecular Weight ◽  
Ribosomal Subunits ◽  
Molecular Weights ◽  
Protein Components ◽  
Protein Number

1. The ribosomal subunits of one thermoacidophilic archaebacterium (Caldariella acidophila) and of two reference eubacterial species (Bacillus acidocaldarius, Escherichia coli) were compared with respect to ribosome mass and protein composition by (i) equilibrium-density sedimentation of the particles in CsCl and (ii) gel-electrophoretic estimations of the molecular weights of the protein and the rRNA. 2. By either procedure, it is estimated that synthetically active archaebacterial 30S subunits (52% protein by wt.) are appreciably richer in protein than the corresponding eubacterial particles (31% protein by wt.) 3. The greater protein content of the archaebacterial 30S subunits is accounted for by both a larger number and a greater average molecular weight of the subunit proteins; specifically, C. acidophila 30S subunits yield 28 proteins whose combined mass is 0.6×10(6) Da, compared with 20 proteins totalling 0.35×10(6) Da mass for eubacterial 30S subunits. 4. No differences in protein number are detected among the large subunits, but C. acidophila 50S subunits exhibit a greater number-average molecular weight of their protein components than do eubacterial 50S particles. 5. Particle weights estimated by either buoyant-density data, or molecular weights of rRNA plus protein, agree to within less than 2%. By either procedure C. acidophila 30S subunits 1.15×10(6) Da mass) are estimated to be about 300 000 Da heavier than their eubacterial counterparts (0.87×10(6) Da mass); a smaller difference. 0.15×10(6) Da, exists between the archaebacterial and the eubacterial 50S subunits (respectively 1.8×10(6) and 1.65×10(6) Da). It is concluded that the heavier-than-eubacterial mass of the C. acidophila ribosomes resides principally in their smaller subunits.

scholarly journals MITORIBOSOMES FROM CANDIDA UTILIS

1972 ◽  
Vol 54 (3) ◽  
pp. 468-492 ◽  
Author(s):  
Pierre V. Vignais ◽  
Barbara J. Stevens ◽  
Janine Huet ◽  
Jean André
Keyword(s):  
Candida Utilis ◽  
Buoyant Density ◽  
Electron Microscopic Observation ◽  
Electron Microscopic ◽  
Mitochondrial Ribosomes ◽  
Characteristic Views ◽  
Polypeptide Chains ◽  
Complete Dissociation ◽  
Cytoplasmic Ribosomes ◽  
Made In

Highly purified mitochondrial ribosomes (mitoribosomes) have been obtained from the yeast Candida utilis. Sedimentation analysis in sucrose gradients made in 5 mM MgCl2, 1 mM Tris, pH 7.4 and 50 mM KCl clearly distinguishes mitoribosomes (72S) from cytoplasmic ribosomes (cytoribosomes) (78S). Mitoribosomes are completely dissociated into 50S and 36S subunits at 10-4 M MgCl2 whereas complete dissociation of cytoribosomes into 61S and 37S subunits occurs only at 10-6 M MgCl2 Electron microscopy of negatively stained mitoribosomes (72S peak) shows bipartite profiles, about 265 x 210 x 200 A Characteristic views are interpreted as frontal, dorsal, and lateral projections of the particles, the latter is observed in two enantiomorphic forms Mitoribosome 50S subunits display rounded profiles bearing a conspicuous knoblike projection, reminiscent of the large bacterial subunit. The 36S subunits show a variety of angular profiles. Mitoribosomal subunits are subject to artifactual dimerization at high Mg2+ concentration Under these conditions, a supplementary 80S peak arises. Electron microscopic observation of the 80S peak reveals closely paired particles of the 50S type Buoyant density determinations after glutaraldehyde fixation show a single peak at ρ = 1.48 for mitoribosomes and 1.53 for cytoribosomes In the presence of ethylenediaminetetraacetate (EDTA), two species of RNA, 21S and 16S, are obtained from mitoribosomes, while 25S and 17S RNA are obtained from cytoribosomes It is established that the small and large RNA species are derived from the 36S and 50S subunits, respectively, by extraction of the RNA from each subunit The G + C content of the RNA is lower for mitoribosomes (33%) than for cytoribosomes (50%). Incubation of C utilis mitochondria with leucine-14C results in the labeling of 72S mitoribosomes. The leucine-14C incorporation is inhibited by chloramphenicol and resistant to cycloheximide Puromycin strips the incorporated radioactivity from the 72S mitoribosomes, which is consistent with the view that leucine-14C is incorporated into nascent polypeptide chains at the level of mitoribosomes

scholarly journals Macromolecular distribution near the limits of density-gradient columns. Some applications to the separation and fractionation of glycoproteins

1977 ◽  
Vol 161 (3) ◽  
pp. 449-463 ◽  
Author(s):  
M J Creeth ◽  
J R Horton
Keyword(s):  
Molecular Weight ◽  
Density Distribution ◽  
Density Gradient ◽  
Specific Volume ◽  
Buoyant Density ◽  
Apparent Molecular Weight ◽  
Partial Specific Volume ◽  
Macromolecular Component

1. Expressions are derived for the distribution at density-gradient equilibrium of macromolecules whose densities are (a) close to the values characterizing the solution limits or (b) outside the span of the gradient. 2. Density-distribution predicted by the expressions agree with those obtained by rigorous methods. 3. The distribution equations are applied to hypothetical mixtures of proteins and glycoproteins in commonly used density-gradient media to simulate separation and fractionation conditions. 4. It is shown that CsBr, although less efficient than CsCl for fractionation, is nevertheless adequate for most purposes; in analytical experiments it may often have advantages over CsCl. Limitations on the use of LiBr are explored. 5. An expression is derived which allows the variance of the partial specific volume of the macromolecular component to be determined from the variance of the buoyant density. It is shown that the relative resolving powers of different salts is expressed by their values of the quantity (formula: see text). 6. The equations are applied to a well-characterized glycoprotein preparation at equilibrium in CsCl and in Cs2SO4:it is shown that the much wider distribution in CsCl than in Cs2SO4 is explicable in terms of the variance in buoyant density and the solvation properties of the salts. 7. Limitations of the expressions arise when dispersity in density is represented by a low apparent molecular weight; realistic simulations can then only be obtained when the component is fully banded.

Cycloheximide and heat shock induce new polypeptide synthesis in Neurospora crassa

1982 ◽  
Vol 2 (10) ◽  
pp. 1167-1173
Author(s):  
J Perlman ◽  
J F Feldman
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Neurospora Crassa ◽  
Temperature Shift ◽  
Molecular Weights ◽  
Mitochondrial Ribosomes ◽  
New Synthesis ◽  
Mechanisms Of Adaptation ◽  
Cytoplasmic Ribosomes ◽  
Polypeptide Band

Treatment of Neurospora crassa with 0.1 microgram of cycloheximide per ml, a concentration which inhibited protein synthesis by about 70%, resulted in the greatly enhanced synthesis of at least three polypeptide bands with estimated molecular weights of 88,000, 30,000, and 28,000. A temperature shift from 25 to 37 degrees C resulted in the appearance of a single new polypeptide band of 70,000 daltons, the same size as the major heat shock-induced proteins observed in species of Drosophila and Dictyostelium. Synthesis of the cycloheximide-stimulated polypeptide bands was on cytoplasmic ribosomes rather than on mitochondrial ribosomes, as incorporation of isotope into the polypeptide bands was inhibited by 1.0 microgram of cycloheximide per ml but not by 1 mg of chloramphenicol per ml. In a mutant with cycloheximide-resistant ribosomes, 0.1 microgram of cycloheximide per ml failed to alter the pattern of protein synthesis from that of the controls. It is suggested that the new synthesis of the polypeptide bands reflects specific mechanisms of adaptation to different kinds of environmental stress, including inhibition of protein synthesis and temperature increases.

scholarly journals Cycloheximide and heat shock induce new polypeptide synthesis in Neurospora crassa.

1982 ◽  
Vol 2 (10) ◽  
pp. 1167-1173 ◽  
Author(s):  
J Perlman ◽  
J F Feldman
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Neurospora Crassa ◽  
Temperature Shift ◽  
Molecular Weights ◽  
Mitochondrial Ribosomes ◽  
New Synthesis ◽  
Mechanisms Of Adaptation ◽  
Cytoplasmic Ribosomes ◽  
Polypeptide Band

Treatment of Neurospora crassa with 0.1 microgram of cycloheximide per ml, a concentration which inhibited protein synthesis by about 70%, resulted in the greatly enhanced synthesis of at least three polypeptide bands with estimated molecular weights of 88,000, 30,000, and 28,000. A temperature shift from 25 to 37 degrees C resulted in the appearance of a single new polypeptide band of 70,000 daltons, the same size as the major heat shock-induced proteins observed in species of Drosophila and Dictyostelium. Synthesis of the cycloheximide-stimulated polypeptide bands was on cytoplasmic ribosomes rather than on mitochondrial ribosomes, as incorporation of isotope into the polypeptide bands was inhibited by 1.0 microgram of cycloheximide per ml but not by 1 mg of chloramphenicol per ml. In a mutant with cycloheximide-resistant ribosomes, 0.1 microgram of cycloheximide per ml failed to alter the pattern of protein synthesis from that of the controls. It is suggested that the new synthesis of the polypeptide bands reflects specific mechanisms of adaptation to different kinds of environmental stress, including inhibition of protein synthesis and temperature increases.

Preparation of graft copolymers by the reaction of polymeric acyl chlorides with monohydroxy-terminated polymers: An introductory study

1988 ◽  
Vol 53 (8) ◽  
pp. 1735-1744 ◽  
Author(s):  
Jitka Horská ◽  
Jaroslav Stejskal ◽  
Pavel Kratochvíl ◽  
Aubrey D. Jenkins ◽  
Eugenia Tsartolia ◽  
...  
Keyword(s):  
Light Scattering ◽  
Chemical Composition ◽  
Statistical Model ◽  
Graft Copolymers ◽  
Coupling Reaction ◽  
Molecular Characteristics ◽  
Gel Formation ◽  
Acyl Chlorides ◽  
Molecular Weights

An attempt was made to prepare well-defined graft copolymers by the coupling reaction between acyl chloride groups located along the backbone chain and monohydroxy-terminated grafts prepared separately. The molecular weights and the parameters of heterogeneity in chemical composition of the products were determined by light scattering and osmometry. The determination of molecular characteristics revealed that the degree of grafting was low. The results therefore could not be confronted with a statistical model at this stage. The problems encountered in the synthesis, e.g., gel formation, and the data relating to the soluble products are discussed.

Sign in / Sign up

Export Citation Format

Share Document