Physiochemical properties of rat liver mitochondrial ribosomes

In the present study, the physiochemical properties of rat liver mitochondrial ribosomes were examined and compared with Escherichia coli ribosomes. The sedimentation and translational diffusion coefficients as well as the molecular weight and buoyant density of rat mitochondrial ribosomes were determined. Sedimentation coefficients were established using the time-derivative algorithm (Philo, J. S. (2000) Anal. Biochem. 279, 151-163). The sedimentation coefficients of the intact monosome, large subunit, and small subunit were 55, 39, and 28 S, respectively. Mitochondrial ribosomes had a particle composition of 75% protein and 25% RNA. The partial specific volume was 0.688 ml/g, as determined from the protein and RNA composition. The buoyant density of formaldehyde-fixed ribosomes in cesium chloride was 1.41 g/cm(3). The molecular masses of mitochondrial and E. coli ribosomes determined by static light-scattering experiments were 3.57 +/- 0.14 MDa and 2.49 +/- 0.06 MDa, respectively. The diffusion coefficient obtained from dynamic light-scattering measurements was 1.10 +/- 0.01 x 10(-7) cm(2) s(-1) for mitochondrial ribosomes and 1.72 +/- 0.03 x 10(-7) cm(2) s(-1) for the 70 S E. coli monosome. The hydration factor determined from these hydrodynamic parameters were 4.6 g of water/g of ribosome and 1.3 g/g for mitochondrial and E. coli ribosomes, respectively. A calculated hydration factor of 3.3 g/g for mitochondrial ribosomes was also obtained utilizing a calculated molecular mass and the Svedberg equation. These measurements of solvation suggest that ribosomes are highly hydrated structures. They are also in agreement with current models depicting ribosomes as porous structures containing numerous gaps and tunnels.     

Altered hepatic mitochondrial ribosome structure following chronic ethanol consumption.

Chronic ethanol consumption decreases the synthesis of all 13 polypeptides encoded by the hepatic mitochondrial genome. This alteration in mitochondrial protein synthesis is due to modifications in mitochondrial ribosomes. In the current study, the nature of these alterations was investigated by determining some of the hydrodynamic properties, namely sedimentation coefficient, shape, and mass of mitochondrial ribosomes. The effect of ethanol consumption on the capacity for mitochondrial ribosomes to translate proteins was also determined using an in vitro Poly (U) assay system. Rats were fed the Lieber-DeCarli diet for 31 days with ethanol as 36% of total calories. The sedimentation coefficient, measured by sedimentation velocity analyses, was slightly, but significantly lower in ethanol mitochondrial ribosomes (53.2 +/- 0.5S) when compared with pair-fed controls (54.1 +/- 0.5S) (P = 0.04). Mitochondrial ribosomes from ethanol-fed animals also had a greater tendency to dissociate into subunits. The diffusion coefficient, determined by dynamic light scattering, was lower in mitochondrial ribosomes from ethanol-fed rats than pair-fed controls and this indicated a significantly greater diameter for ethanol ribosomes (42.1 +/- 0.2 nm) than for preparations from pair-fed controls (39.1 +/- 0.5 nm; P = 0.008). These alterations to ethanol mitochondrial ribosomes occurred despite no change in molecular mass, which suggested a significant ethanol-related shape change in the ribosomes. The translation capacity of mitochondrial ribosome preparations from ethanol-fed animals was markedly reduced due to dissociation of the monosome into light and heavy subunits. In summary, these observations demonstrate that chronic ethanol consumption causes significant structural and functional alterations to mitochondrial ribosomes. The loss in ribosome function leads to impaired mitochondrial polypeptide synthesis and is an example of a pathology giving rise to an alteration in the mitochondrial ribosome structure.     

messenger RNA-containing ribonucleoprotein from mitochondrial polyribosomes of rat liver

A ribonucleoprotein was released from carefully purified rat liver mitochondrial polyribosomes after dissociation with 1 M potassium chloridepuromycin. This ribonucleoprotein was characterized by a sedimentation coefficient ranging from 10-14 S and buoyant density of 1.48 g cm(-3) in cesium chloride equilibrium centrifugation differing in these parameters from the subunits of mitochondrial ribosomes. Poly(A)-containing RNA constituted more than 30% of the total RNA content in this non-ribosomal ribonucleoprotein.     

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

1. At 0-4 degrees C mitochondrial ribosomes (55S) dissociate into 39S and 29S subunits after exposure to 300mm-K(+) in the presence of 3.0mm-Mg(2+). 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 25.5nmx20.0nmx21.0nm, 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 degrees C have a buoyant density of 1.45g/cm(3); from the rRNA content calculated from buoyant density and from the rRNA molecular weights it is confirmed that the two subparticles have weights of 2.0x10(6) daltons and 1.20x10(6) daltons; the weights of the two subunits of cytosol ribosomes are 2.67x10(6) and 1.30x10(6) 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 degrees 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-(471.2x1/rho(CsCl)), where 1/rho(CsCl)=[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.     

Physical properties of Artemia salina ribosomes.

Eukaryotic ribosomes were isolated from the cryptobiotic embryos and from the further-developed free-swimming nauplii of the brine shrimp Artemia salina. Analytical boundary sedimentation and photon correlation spectroscopy yielded, respectively, the standard sedimentation and diffusion coefficients at infinite dilution, s degrees 20,w = 81 +/- 1 S and D degrees 20,w = (1.41 +/- 0.02) x 10(-7) cm2/s, for the unfixed and formaldehyde-fixed ribosomes from different developmental stages and for ribosomes attached to a messenger RNA fragment. Also, the density increment was determined, from which the partial specific volume was derived (0.63 +/- 0.01 cm3/g). Combination of the different measured parameters gives accurate values for the molecular weight (3.8 +/- 0.1) x 106 and for size and solvation parameters. These results are compared with their counterparts for the smaller ribosomes from the prokaryote Escherichia coli.     

MITOCHONDRIAL AND CYTOPLASMIC RIBOSOMES FROM TETRAHYMENA PYRIFORMIS : Correlative Analysis by Gel Electrophoresis and Electron Microscopy

Mitochondrial and cytoplasmic ribosomes from Tetrahymena pyriformis have been isolated and studied by the techniques of polyacrylamide gel electrophoresis and electron microscopy used in conjunction. Although the two ribosome types show the same coefficient of sedimentation (80S) in sucrose gradients, they can be distinguished by gel electrophoresis: mitoribosomes migrate in a single band, considerably slower than the cytoribosome band. Electron microscope observations of negatively stained cytoribosomes show typical rounded or triangular profiles, about 275 x 230 Å; mitoribosome profiles are much larger and clearly elongate, about 370 x 240 Å. An electron-opaque spot delimits two nearly equal size subunits. In mixtures of mito- and cytoribosomes, each type can be recognized by its characteristic electrophoretic mobility and by its distinctive fine structure. Cytoribosomal 60S and 40S subunits each produce a distinct electrophoretic band. On the contrary, neither electrophoretic analysis, using a variety of conditions, nor electron microscopy is able to discern two different subunit types in the single 55S mitoribosomal subunit peak. Electrophoretic analysis of RNA shows that both ribosomal RNA species are present in the mitoribosomal subunit fraction. These results establish that mitoribosomes from T. pyriformis dissociate into two subunits endowed with the same sedimentation coefficient, the same electrophoretic mobility, and a similar morphology.     

The translation system of mammalian mitochondria

Oligoribonucleotides and mRNA were used to define properties of the bovine mitoribosomal mRNA binding site. The RNA binding domain on the 28 S subunit spans approx. 80 nucleotides of the template, based on ribosome protection experiments, but the major interaction with the ribosome occurs over a 30 nucleotide stretch. The binding site for E. coli IF3 is conserved in bovine mitoribosomes, but mitochondrial factors appear essential for proper interaction of mRNA with mitoribosomes. The small subunit of bovine mitoribosomes contains a high-affinity binding site for guanyl nucleotides, further indication of specialized mechanisms for initiation complex formation and function of mammalian mitochondrial ribosomes.     

Quaternary structure of erythrocruorin from the nematode Ascaris suum. Evidence for unsaturated haem-binding sites.

The quaternary structure of erythrocruorin from the nematode Ascaris suum was studied. The native protein had a sedimentation coefficient, at a protein concentration of 1 mg/ml, of 11.6 +/- 0.3 S and an Mr, as determined by sedimentation equilibrium, of 332,000 +/- 17,000. SDS/polyacrylamide-gel electrophoresis gave one band with a mobility corresponding to an Mr of 43,000 +/- 2000. The Mr of the polypeptide chain was determined to be 41,600 +/- 1,500 by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol. Cross-linking with glutaraldehyde followed by SDS/polyacrylamide-gel electrophoresis yielded a maximal number of eight bands. The haem content of Ascaris erythrocruorin was observed to vary from one preparation to another. This finding was shown to be due to non-realization of the full binding capacity for haem. By titration with haemin, the haem content was found to attain a maximal value of 2.86 +/- 0.14%, corresponding to a minimal Mr per haem group of 21,000 +/- 1,000. Our findings indicate that Ascaris suum erythrocruorin is composed of eight identical polypeptide chains, carrying two haem sites each.     

Mitochondrial ribosomes of the phytoflagellata Astasia longa

The physico-chemical properties of ribosomes and rRNA isolated from the mitochondria of the phytoflagellata Astasia longa were studied. It was shown that the mitochondrial ribosomes of A. longa have the sedimentation coefficient of 81S (those of the cytoplasm-82S); upon a decrease of Mg2+ concentration in the medium they dissociate into subparticles with sedimentation coefficients of 60 and 45S. The relative protein content in the mitochondrial ribosomes of A. longa is equal to 42% (rho = 1,60 g/cm3), that of cytoplasmic ribosomes-49%. The molecular weights of mitochondrial rRNA are equal to 1,05 . 10(6) and 0,71 . 10(6) and differ from those for cytoplasmic rRNA (1,32 . 10(6) and 0,94 . 10(6)). It was shown that the GC-content in mitochondrial rRNA is equal to 32,0 mol. %, that in cytoplasmic rRNA-55,9 mol. %. Thus, the mitochondrial ribosomes of A. longa differ in some of their properties from both procaryotic and eucaryotic ribosomes and are probably related to a special type of mitochondrial ribosomes.     

Mitoribosomes from Candida utilis. Morphological, physical, and chemical characterization of the monomer form and of its subunits

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     

Bacteriophage phiNS11: a lipid-containing phage of acidophilic thermophilic bacteria. IV. Sedimentation coefficient, diffusion coefficient, partial specific volume, and particle weight of the phage.

The particle weight (molecular weight) of phiNS11 was determined from the sedimentation coefficient, diffusion coefficient, and partial specific volume of the phage. The sedimentation coefficient of the phage (S(0)20, W) is 416 +/- 2.7S. The diffusion coefficient D(0)20, W), which was determined by quasielastic light scattering measurement, is (0.57 +/- 0.03) x 10(-7) cm2/s. The partial specific volume was determined by the mechanical oscillation technique to be 0.747 +/- 0.007 cm3/g. Based on these values, the particle weight of the phage was calculated to be (70.3 +/- 4.3) x 10(6) daltons, which agrees well with the particle weight (69--72 x 10(6) daltons) estimated from the molecular weight of phage DNA and the content of DNA. The Stokes radius of the phage particle was calculated to be 37.7 +/- 2 nm and hydration of the phage was estimated to be 1.18 cm3/g of dry phage. From the particle weight and the chemical composition of the phage, we estimated that one phage particle contains one double-stranded DNA molecule, 16,000 residues of fatty acid, 72 protein I molecules, 920 protein II, 42 protein III, 48 protein IV, 290 protein V molecules, and 3,700 molecules of polyamines.     

Purification of the JS-3 isolate of Herpesvirus ovis (Bovid herpesvirus 4) and some properties of its DNA.

A procedure incorporating the use of heparin was developed to purify Herpesvirus ovis. The viral DNA has a buoyant density of 1.706 +/- 0.001 g/cm3, and the sedimentation constant was estimated to be 47.5 +/- 1.5S; from the latter, the molecular weight was calculated as 67.3 +/- 5.4 X 10(6). Estimates of the guanine-plus-cytosine content made from the buoyant density and melting point (72 degrees C) gave levels of 47 and 46%, respectively.     

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

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.     

Characterization of Inducible Bacteriophages in Bacillus licheniformis

Two morphologically distinct and physically separable defective phages have been found in Bacillus licheniformis NRS 243 after induction by mitomycin C. One of them (PBLB) is similar to the defective phage PBSX of B. subtilis, which has a density of 1.373 g/cm(3) in CsCl and a sedimentation coefficient of 160S. PBLB incorporates into its head mainly bacterial deoxyribonucleic acid (DNA) which has a sedimentation coefficient of 22S and a buoyant density in CsCl of 1.706 g/cm(3). The other phage (PBLA) has a morphology similar to the temperate phage phi105 of B. subtilis; the head diameter is about 66 nm, and it possesses a long and noncontractile tail. PBLA has a density of 1.484 g/cm(3) in CsCl and the phage-specific DNA, which is exclusively synthesized after induction by mitomycin C, has a density of 1.701 g/cm(3). PBLA DNA is double-stranded and has a sedimentation coefficient of 36S, corresponding to a molecular weight of 34 x 10(6) to 35 x 10(6) daltons. The phage DNA has one interruption per single strand, giving single-stranded segments with molecular weights of 13 x 10(6) and 4 x 10(6) daltons. Common sequences between the two phage DNA species and with their host DNA have been demonstrated by DNA-DNA hybridization studies. Both phage particles kill sensitive bacteria. However, all attempts thus far to find an indicator strain to support plaque formation have been unsuccessful.     

Sedimentation behavior of chloroplast ribosomes from Chlamydomonas reinhardtii.

The identity of peaks generated by chloroplast ribosomes of Chlamydomonas reinhardtii were determined by zone velocity sedimentation on sucrose density gradients, and analysis of distribution of ribosomal RNAs in the gradients. The sedimentagion coefficient of the principal peak was 66-70 S (usually 69 S), in good agreement with previously reported values for chloroplast ribosomes of C. reinhardtii, and other organisms. The fast sedimenting side of the 69 S peak contained an excess of chloroplast large subunit. When ribosome dissociation was prevented by sedimentation at low velocity, by aldehyde fixation, or by the presence of nascent polypeptide chains, the principal peak had a sedimentation coefficient of about 75 S. Thus the 69 S peak was an artifact caused by dissociation during centrifugation. Peaks that contained chloroplast ribosomal RNAs were also observed at '60 S' and '45 S' when chloroplast ribosomes were centrifuged unfixed at high velocity. The amounts of '60 S' and '45 S' components were decreased by centrifugation at low speed, or fixation, but sedimentation coefficients remained unchanged. The '60 S', and '45 S' components were identified as large, and small subunits of chloroplast ribosomes, respectively. The artifacts produced by centrifugation of chloroplast ribosomes, are similar to the artifacts produced by centrifuging ribosomes of Escherichia coli. Similar explanations appear to apply to both. We concluded that the 69 S chloroplast ribosome peak occurs because of dissociation of 'tight' couples, and incomplete separation of subunits. Subunit peaks (60 S and 45 S) arise from free subunits, and/or from dissociation of 'loose' couples.     

Mitochondrial and cytoplasmic ribosomes and their activity in blood and culture form Trypanosoma brucei

Ribosomes of Trypanosoma brucei, a parasitic, flagellated protozoan (order Kinetoplastida), were identified on sucrose density gradients by their radioactively labeled nascent peptides. Ultraviolet absorption revealed only cytoplasmic ribosomes which served as internal sedimentation markers. Synthesis on cytoplasmic ribosomes was completely inhibited by cycloheximide. In the presence of this antibiotic, nascent peptides were associated with ribosomes of lower sedimentation coefficient than the cytoplasmic ribosomes. Chloramphenicol blocked synthesis on these ribosomes which are probably the mitochondrial ribosomes. These ribosomes differed from the cytoplasmic ribosomes in several ways. Their sedimentation coefficient was about 72S rather than 84S. The stability of the 72S ribosomes was less sensitive to pancreatic ribonuclease and low Mg-++ concentrations, dissociating below 0.1 mM Mg++. The 72S ribosomes were more sensitive to elevated KCl concentrations, dissociation above 0.25 M. Protein synthetic activity associated with the 72S class of ribosomes was found in trypanosomes grown in rats. Under these conditions no cytochromes or fully active Krebs cycle is present in these cells and respiration is insensitive to cyanide.     

Protein synthesis by brain-cortex mitochondria. Characterization of a 55S mitochondrial ribosome as the functional unit in protein synthesis by cortex mitochondria and its distinction from a contaminant cytoplasmic protein-synthesizing system

Homogenates of rat brain cortex were fractionated by conventional methods of velocity sedimentation and separated into a microsomal and a washed mitochondrial fraction. By electron microscopy the mitochondrial fraction was shown to be rich in synaptosomes. The mitochondria-synaptosome fraction synthesized protein in vitro by a route that was partially inhibited by cycloheximide and partly by chloramphenicol. The relative effectiveness of the two inhibitors varied greatly with the medium used. In the mitochondria-synaptosome fraction active 80S cytoplasmic ribosomes and active 55S mitochondrial ribosomes were detected; these were also seen in the electron microscope. Mild osmotic shock of the mitochondria-synaptosome fraction followed by velocity sedimentation in sucrose-EDTA allowed isolation of a mitochondrial fraction free of synaptosomes. Protein synthesis in this fraction was entirely inhibited by chloramphenicol, but was completely resistant to cycloheximide both in a medium promoting oxidative phosphorylation and in ATP-generating medium. Ouabain had no inhibitory effect on protein synthesis in a purified mitochondrial preparation. It is concluded that brain-cortex mitochondria synthesize protein entirely on 55S mitochondrial ribosomes.     

Physical properties of human polynucleotide kinase: hydrodynamic and spectroscopic studies.

Human polynucleotide kinase (hPNK) is a putative DNA repair enzyme in the base excision repair pathway required for processing and rejoining strand-break termini. This study represents the first systematic examination of the physical properties of this enzyme. The protein was produced in Escherichia coli as a His-tagged protein, and the purified recombinant protein exhibited both the kinase and the phosphatase activities. The predicted relative molecular mass (M(r)) of the 521 amino acid polypeptide encoded by the sequenced cDNA for PNK and the additional 21 amino acids of the His tag is 59,538. The M(r) determined by low-speed sedimentation equilibrium under nondenaturing conditions was 59,600 +/- 1000, indicating that the protein exists as a monomer, in contrast to T4 phage PNK, which exists as a homotetramer. The size and shape of hPNK in solution were determined by analytical ultracentrifugation studies. The protein was found to have an intrinsic sedimentation coefficient, s(0)(20,w), of 3.54 S and a Stokes radius, R(s), of 37.5 A. These hydrodynamic data, together with the M(r) of 59 600, suggest that hPNK is a moderately asymmetric protein with an axial ratio of 5.51. Analysis of the secondary structure of hPNK on the basis of circular dichroism spectra, which revealed the presence of two negative dichroic bands located at 218 and 209 nm, with ellipticity values of -7200 +/- 300 and -7800 +/- 300 deg x cm(2) x d(mol(-1), respectively, indicated the presence of approximately 50% beta-structure and 25% alpha-helix. Binding of ATP to the protein induced an increase in beta-structure and perturbed tryptophan, tyrosine, and phenylalanine signals observed by aromatic CD and UV difference spectroscopy.