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INTRODUCTION |
The mitochondrial genome encodes 13 polypeptides of the oxidative
phosphorylation system, 22 tRNAs, and the 12 and 16 S rRNAs (1).
Mitochondria DNA deletions or mutations of mitochondrial tRNAs can lead
to impairment of polypeptide synthesis and defects in energy production
(2, 3). Likewise, in animal models of alcoholic liver disease, chronic
ethanol consumption decreases the mitochondrial synthesis of ATP due to
lowered concentrations of all 13 polypeptides encoded by the
mitochondrial genome (4). However, in this latter case, the depressed
functioning of the oxidative phosphorylation system is due to
alterations in mitochondrial ribosome function and structure (5, 6).
These observations provide another type of mitochondrial disorder that
may play a role in pathologies associated with alterations in the
mitochondrial protein synthesizing mechanism. Our previous studies
demonstrated the need to characterize rat liver mitochondrial ribosomes
(mitoribosomes)1 more
rigorously to determine whether alterations in function are accompanied
by changes in the physical properties, i.e. molecular weight
and overall shape. In the present study, a combination of approaches
have been employed to determine the physiochemical properties of rat
liver mitoribosomes. These included sedimentation velocity measurements
with an analytical ultracentrifuge, light-scattering analyses, and
electron microscopy.
In previous studies of rat liver mitoribosomes (7-9), the
sedimentation coefficient was determined on a sucrose density gradient utilizing Escherichia coli ribosomes as a standard. In the
present investigation, we have applied a rigorous approach in
determining the sedimentation coefficient, namely the time-derivative
algorithm as described by Stafford (10) and Philo (11). This procedure has been used to determine the distribution of sedimenting species from
sedimentation velocity data obtained in an analytical ultracentrifuge. The time derivative has several advantages when compared with conventional analyses, including a higher signal-to-noise ratio, and
the ability to resolve the components of a mixture (10). This latter
characteristic was useful in the present study in evaluating whether
the intact ribosome dissociated during sedimentation velocity measurements.
Static and dynamic light scattering were employed to measure the
molecular weight and translational diffusion coefficient for the rat
liver mitoribosomes. The latter parameter can be utilized to determine
the Stokes radius of a particle and was employed with electron
microscopic analyses to estimate the shape and water content of rat
liver mitoribosomes. The light-scattering analyses were particularly
useful in this study because measurements can be carried out quickly
under conditions that precluded ribosome dissociation. To validate the
techniques employed to characterize the rat liver mitoribosomes, we
applied the same procedures to characterize the physiochemical
properties of the well described E. coli ribosome (12,
13).
The sedimentation velocity analyses and the light-scattering
measurements revealed a molecular weight for rat liver mitoribosomes that is higher than that reported previously for either rat liver (8)
or beef liver (14). This difference in mass between rat and beef liver
mitoribosomes is consistent with the earlier finding that only 15% of
mitoribosomal proteins had conserved electrophoretic properties (15)
and also with the suggestion that mitoribosomal proteins have a high
evolutionary rate (15). Furthermore, the present investigation yielded
a diffusion coefficient for mitoribosomes, which had not been reported
previously. From this parameter an estimation of the solvation was
determined, which indicated that these ribosomes are highly hydrated particles.
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EXPERIMENTAL PROCEDURES |
Materials
Male Sprague-Dawley rats were obtained from Zivic Miller
Laboratories, Inc. (Zelienople, PA). Rats were allowed 1 week to acclimatize prior to experimental studies. Ultrapure sucrose was obtained from ICN Pharmaceuticals (Aurora, OH). All other chemicals were of analytical grade.
Methods
Preparation of Mitochondrial Ribosomes--
The isolation of
ribosomes was as described by Cahill et al. (9), except that
a mixture of protease inhibitors (phenylmethylsulfonyl fluoride (40 µg/ml), leupeptin (5 µg/ml), pepstatin A (7 µg/ml), and aprotonin
(5 µg/ml)) was added to the buffers used in the isolation. After
fractionation by sucrose density gradient centrifugation (9), the
fraction exhibiting the highest absorbance at 260 nm was collected for
subsequent analyses by analytical ultracentrifugation and
light-scattering measurements. This fraction typically exhibited a 260 nm/280 nm absorbance ratio of 1.4-1.5. The concentration of ribosomes
was determined utilizing an
E
= 7.5 (see
"Results" for extinction coefficient determination). The activity
of purified mitochondrial 55 S ribosomes was measured using a poly(U)
directed [3H]phenylalanine polymerization assay as
described previously (9), with the exception that activity was measured
using ribosomes obtained from the peak maxima of sucrose density gradients.
The purified ribosomes were analyzed for possible contamination with
the
-ketoglutarate (OGDC) and pyruvate dehydrogenase complexes (PDC)
by monitoring for dehydrogenase activities (16) and dehydrogenase
complex subunits by immunoblotting technology (17) employing the Pierce
SuperSignal West Pico chemiluminescence kit. Antibody against the E1
subunit of PDC and purified PDC E1 subunit were kindly provided by Dr.
M. S. Patel (State University of New York, Buffalo, NY). Dr. G. Lindsay (University of Glasgow, Glasgow, Scotland) generously provided
purified OGDC and the antiserum against OGDC.
Preparation of E. coli Ribosomes--
E. coli
ribosomes were prepared according to Rheinberger et al. (18)
utilizing E. coli strain MRE600 (an RNase I
strain) to minimize ribonuclease levels. Ribosomes were shock-frozen and stored at
80 °C until utilized. For sedimentation velocity analysis in buffer A (30 mM NH4Cl, 10 mM MgCl2, 10 mM Tris-HCl, pH 7.5, 6 mM
-mercaptoethanol), ribosomes were thawed on ice, diluted with buffer A to give absorbances of 0.7-1.2 units/ml, and
subsequently used for analytical ultracentrifugation. For sedimentation
velocity analysis in sucrose, ribosomes were immediately loaded onto a
10-30% sucrose gradient prepared in buffer A and centrifuged at
45,000 × g for 14 h in a Beckman SW27 rotor. The gradients
were fractionated and absorbances monitored at 260 and 280 nm. Peaks
corresponding to purified E. coli ribosomes were subsequently used for analytical ultracentrifugation and
light-scattering analyses. The concentration of ribosomes was
determined utilizing an
E
= 14.5 (19,
20). The published value of 0.639 ml/g was used for the partial
specific volume (
) (19, 21).
Buoyant Density Analysis in Cesium Chloride--
The purified 55 S peak fraction from the sucrose density gradient was dialyzed
overnight in buffer B (100 mM KCl, 20 mM
MgCl2, 20 mM triethanolamine, pH 7.5, 5 mM
-mercaptoethanol), containing 1% formaldehyde. The
refractive index of the sample was measured to ensure no sucrose was
present. Buoyant density measurements of formaldehyde-fixed ribosomes
in cesium chloride were carried out in buffer B according to Spirin
et al. (22). This procedure reliably stabilizes ribosomes by
fixing the particles with formaldehyde. Inadequate fixation results in
loss of ribosomal proteins and artificially high buoyant density values
(22). Centrifugation at 187,000 × g in a Beckman SW41
Ti rotor was carried out at 4 °C for 48 h. Fractions were
collected, and the density was determined according to Ifft et
al. (23). Absorbance readings of the fractions were measured at
260 nm.
Sedimentation Velocity--
Sedimentation velocity experiments
were carried out at 4 °C using a Beckman Optima XL-A analytical
ultracentrifuge. Bovine serum albumin samples with an absorbance at 280 nm of 0.5, 1.0, and 1.5 were utilized to determine the linearity of the
XL-A's optical system. Ribosome boundary movements at a rotor speed of 21,000 rpm, and at fixed time intervals (3 min) as a function of the
radius (0.003 radial step size), were obtained over a period of 5 h. Ribosome sample concentrations between 0.05 and 0.16 mg/ml were
sufficiently dilute such that they behaved as ideal, noninteracting species.
Data were analyzed by several software applications. First, DCDT+
version 1.02 (Dr. John S. Philo, Thousand Oaks, CA) was applied using
the time-derivative algorithm (11). This software was used to analyze
sequential files selected to conform to the criteria defined by
Stafford (10) to obtain apparent g(s*)
distribution plots of sedimenting species (i.e. uncorrected
for diffusion). This analysis can also resolve more than one species by
fitting the data to a Gaussian function. Second, the Fujita-MacCosham function based on the Lamm equation (24) using the software SVEDBERG,
version 6.23 (Dr. John S. Philo, Thousand Oaks, CA) was also employed.
This analysis can resolve sedimentation coefficients of more than one
species and can fit data obtained over a long time range. Third, the
van Holde-Weischet procedure (25) using the software UltraScan 4.1 (Dr.
Borries Demeler, University of Texas Health Science Center, Houston,
TX) was used to determine the sedimentation coefficient distribution of
the sample corrected for diffusion and to provide information on the
homogeneity of the sample.
Experimentally determined sedimentation coefficients were corrected for
the effects of temperature, solvent density (buffer A, 0.9997 g/cm3; buffer B, 1.008 g/cm3) and viscosity
(buffer A, 1.57 centipoise; buffer B, 1.57 centipoise). Sedimentation
coefficients of samples in sucrose were corrected for solvent density
and viscosity as a function of concentration and temperature using
standard tables (26). Sedimentation coefficients obtained with the
software described above were converted from 4 °C to water at
20 °C to obtain s20,w values (27). The molecular weight of the intact ribosome was calculated from the
Svedberg equation (28) utilizing the
s20,w and the translational diffusion
coefficient (Dt) corrected to water at 20 °C
(i.e. D20,w). The
D20,w was obtained from dynamic
light-scattering analysis (see below).
Static Light-scattering Analysis--
The molecular weight of
purified 55 S ribosomes was determined by static light scattering,
using a Brookhaven Instrument BI-2030AT correlator, operated together
with a BI-200SM light-scattering goniometer/photon counting detector
(Brookhaven Instruments, Holtsville, NY) and a Spectra Physics 127 helium-neon laser (35 milliwatts, equipped with a vertical polarization
rotator; Spectra Physics, Mountain View, CT) (29, 30). The fraction
with maximal absorbance at 260 nm obtained from the sucrose density
gradient was used for molecular weight determinations. Light-scattering
measurements were made at an angle of 90o in specially
formulated microcuvettes (Hellma Cells, Inc., White Plains, NY)
maintained at 15 °C in a refractive index matching bath (containing
50% glycerol). Samples were passed through a 0.2-µm filter, with the
filtrate collected into acid-washed, dust-free microcuvettes. Intensity
measurements from duplicate or triplicate runs were averaged and used
for molecular weight calculations. An aliquot of the sample was also
utilized for sedimentation velocity studies to provide information on
the intactness of the particle. As a consequence, only preparations
showing no dissociation were utilized in all analyses. The viscosity
and concentration of sucrose in the ribosome sample was determined from
the refractive index of sucrose using an Abbe refractometer.
Corrections for solvent (i.e. sucrose) scattering were
obtained by subtracting the intensity measurement determined of blank
samples (which had the same sucrose concentration as the ribosome
sample) obtained from a control 10-30% sucrose gradient. Sample
scattering intensities were expressed relative to a benzene standard
(29, 30).
Ribosome concentrations were determined from 260 nm absorbance
measurements using an extinction coefficient of mitoribosomes determined from the protein and RNA composition (Ref. 31; see "Results"). The published refractive index increment
(dn/dc) for E. coli ribosome particles
(30, 50, and 70 S) of 0.20 cm3/g (32) was used in molecular
weight calculations. Weight-average molecular weights were determined
from the solvent corrected relative scattering intensity (at
90o) using Rayleigh-Gans-Debye theory (33).
Dynamic Light-scattering Analysis--
Dynamic light scattering
was carried out simultaneously with the same sample used for static
light scattering. Measurements were carried out with the detector
aperture set at 0.4 or 0.8 mm to optimize the signal to noise ratio
(which ranged from 0.13 to 0.25). The intensity-normalized photocount
autocorrelation function was used to determine the translational
diffusion coefficient Dt by cumulants analysis
(33-35). The Dt was corrected for temperature and solvent viscosity to water at 20 °C (i.e.
D20,w) (36). The
D20,w was subsequently employed to
determine the particle size (Stokes radius) utilizing the
Einstein-Sutherland equation (37). Data analysis of the autocorrelation
function also provided an index of the homogeneity of each sample (see "Results"). Additional information about sample heterogeneity was
obtained by CONTIN analysis (38) to obtain plots of the distribution of
scattering components.
Electron Microscopy--
Ribosomes obtained from the peak maxima
of sucrose gradients were utilized for electron microscopy. Aliquots of
particles were placed on carbon-coated grids and negatively stained
with uranyl acetate (39). Micrographs were taken at a magnification of
70,800 with a Phillips 400 transmission electron microscope operated at
80 kev. Particle diameter measurements were randomly recorded from
digitized images of 70-100 ribosomes. The length and width of
particles were also assessed when ribosomes were orientated in the
frontal view.
Unless otherwise stated, results from all experiments are expressed as
the mean ± S.E. of six observations.
 |
RESULTS |
Sedimentation Velocity: Mitochondrial Ribosomes--
The rat liver
mitochondrial ribosomes utilized in this study displayed a sucrose
density gradient sedimentation profile identical to that reported by
Cahill et al. (9). The peak absorbance fraction gave a
260:280 ratio of between 1.4-1.5, which is in good agreement with
previous observations (9). Since OGDC and PDC complexes have
sedimentation coefficients of 36 and 70 S, respectively, the
preparation was checked for possible contamination. No pyruvate
dehydrogenase activity was detected. Measurement of OGDC activity
indicated that 1.4% of the protein in the ribosomal preparation could
be this enzyme complex. Analyses of immunoblots were also carried out
using purified OGDC and purified E1 subunits of PDC as standards. These
analyses suggested that no more than 0.05% and 1.2% of the total
protein in the ribosome preparation could be PDC and OGDC,
respectively. This indicates that the ribosome preparation is not
significantly contaminated with these enzyme complexes, an observation
also substantiated by the residual analyses of sedimentation velocity
and light-scattering measurements (see below). The translation activity
of the purified ribosome, measured by polymerization of phenylalanine,
was 0.02 pmol of phenylalanine polymerized/min/pmol of ribosomes. These
preparations demonstrated no activity in the absence of soluble
translation factors.
The sedimentation properties of ribosome samples taken from the peak
maxima at 260 nm of the sucrose gradient were analyzed by various
algorithms. Using the time-derivative method, sedimentation data were
transformed into an overall distribution of sedimenting species to
obtain the g(s*) plot shown in Fig.
1A. Fig. 1B shows data from E. coli ribosomes that will be discussed in a
subsequent section. Fig. 1 A demonstrates the application of
the DCDT+ analysis, which shows the monosome fitted to a one-species
Gaussian distribution centered at 54.0 S with no detectable amounts of
its 39 and 28 S subunits. The peak sedimentation coefficient determined
by this procedure was 55.1 S (Table I).
The ribosome sample in sucrose showed very little dissociation, which
was verified with the SVEDBERG analysis that resulted in a single
species fit (55 S) from six out of eight preparations. In addition,
from the SVEDBERG analysis, the residuals of this typical fit resulted
in a root-mean-square deviation of 0.02 absorbance units. A summary of
the sedimentation coefficients of ribosomes utilizing the DCDT+ and
SVEDBERG software procedures is shown in Table I. In all preparations,
the sedimentation coefficient was independent of concentration over the
range of 0.05-0.16 mg/ml.

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Fig. 1.
A g(s*)
plot of mitochondrial and E. coli ribosomes fitted to
one-species Gaussian function. The ribosome sample was taken from
the absorbance peak maxima of a 10-30% sucrose density gradient. The
sedimentation coefficient distribution (i.e.
g(s*)) was calculated from the time-derivative
algorithm of the concentration profile using the software DCDT+ fitted
to a one-species Gaussian function. A, mitochondrial
ribosomes. The inset in A pertains to the
residuals (difference between the experimental data and the fitted data
for each point). The residuals from this typical fit resulted in a
root-mean-square deviation of 0.003 absorbance units/Svedberg.
B, E. coli ribosomes.
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Table I
Summary of sedimentation coefficients of mitochondrial and E. coli
ribosomes determined by various software analyses
The numbers in parentheses are the numbers of preparations analyzed.
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To further validate the DCDT+ analysis procedure, sedimentation
velocity data were also analyzed using the van Holde-Weischet method.
Fig. 2A illustrates absorbance
scans taken at fixed time intervals to monitor the movement of sample
through the cell. From the extrapolation plot observed in Fig.
2B, the convergence of apparent sedimentation coefficients
on the y intercept indicates that the mitoribosome sample is
nearly homogeneous. This finding was reflected in the integral
distribution of s-values plot (Fig. 2C), where
94% of the sample sedimented at ~54-55 S. A small amount of sample
(6%) corresponded to dissociated ribosome particles, as verified from
the SVEDBERG analysis. There was no evidence of any other particle
sedimenting higher than the 55 S monosome. This procedure therefore
confirms the findings obtained from the DCDT+ and SVEDBERG
analyses.

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Fig. 2.
van Holde-Weischet analysis of
mitoribosomes. A, the particle boundary movement
through the cell. The sample was centrifuged in sucrose in an AnTi 60 rotor at 4 °C. Absorbance measurements were taken at fixed time
intervals to monitor the sedimentation rate. A period of 5 h at
21,000 rpm was utilized to obtain an adequate number of scans for
determination of the sedimentation rate. In this figure representative
scans at 3-min intervals are shown. Since a spike was observed in one
absorbance scan, this was removed from the analyses. B, an
extrapolation plot to obtain the diffusion-corrected
S-values (y axis intercept). In this
procedure the apparent sedimentation coefficients are extrapolated to
infinite time. The 49 absorbance scans shown in Fig. 4A were
analyzed with 50 equally spaced boundary fractions using the software
UltraScan 4.1. C, plots of boundary fraction
versus the corrected S-values (from Fig.
4B) yielded the integral distribution of sedimentation
coefficients.
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Two of eight preparations showed the presence of a small fraction
sedimenting at 39 S. This was attributed to a minor amount of
dissociation in these two preparations. The effect of dissociating the
mitochondrial monosome by increasing the potassium and decreasing the
magnesium concentrations resulted in two overlapping peaks, with peak
sedimentation coefficients of 39 S (large subunit) and 28 S (small
subunit) (data not shown). This dissociation pattern is similar to that
obtained with bovine liver mitochondrial ribosomes (7).
Sedimentation Velocity: E. coli Ribosomes--
The well
characterized E. coli ribosomes (12, 13, 19, 20) were
analyzed in this study to confirm the applicability of the recently
developed analyses to mitochondrial ribosomes. E. coli
ribosomes were applied to sucrose density gradients, and the peak
material obtained was subsequently used for sedimentation velocity and
light-scattering experiments.
Fig. 1B shows a representative g(s*)
plot of E. coli ribosomes in sucrose obtained from the peak
maxima of density gradients applying the DCDT+ analysis to a
one-species Gaussian fit. In three out of four preparations, a
one-species model was obtained from the DCDT+ analysis. The average
peak sedimentation coefficient was 67 S, obtained in sucrose (Table I).
When analyzed in buffer A, the sedimentation coefficient for E. coli ribosomes was 70 S (data not shown). All preparations fitted
a single species model in the SVEDBERG analysis. Furthermore, the
sedimentation coefficient was independent of concentration over the
range utilized (0.05-0.08 mg/ml). A summary of sedimentation
coefficients is shown in Table I. Using the van Holde-Weischet
procedure, 96% of E. coli ribosomes had a sedimentation
coefficient of ~67-68 S with a small amount (4%) corresponding to
dissociated particles (data not shown).
Mitoribosome Composition, Partial Specific Volume, and Buoyant
Density Measurements--
The ribosome composition was determined
using a mass of rat liver mitochondrial ribosomal proteins of 2.6 MDa
(9) and the sum of rat liver mitoribosomal 12 S (0.324 MDa) and 16 S
(0.527 MDa) rRNA species (40). Utilizing these values, the protein content was 75% and RNA, 25%. This assumes one molecule of each polypeptide per ribosome and is therefore the minimum amount of protein
contained in each ribosome. The low yield of mitoribosomes (1 absorbance unit/ml at 260 nm/25 g of rat liver) precluded direct measurement of
and the extinction
coefficient. The protein (75%) and RNA (25%) composition was used to
determine the ribosome extinction coefficient from the relationship
proposed by Hamilton and Ruth (31).
|
(Eq. 1)
|
An extinction coefficient
(E
) of 7.5 was
determined from this equation. This value was utilized in calculating
ribosome concentrations in the light-scattering measurements. The
partial specific volume can be determined from a particle's chemical
composition by assuming additivity for the values for RNA (25%) and
protein (75%) from the following relationship (31, 41).
|
(Eq. 2)
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In Equation 2,
= partial specific
volume of ribosome,
RNA = 0.53 ml/g (41, 42), and
Protein = 0.74 ml/g (41, 43). A partial specific volume of 0.688 ml/g was
calculated from this relationship. This value was compared with the
partial specific volume estimated using the reciprocal of the buoyant density determined experimentally for formaldehyde fixed ribosomes. The
buoyant density of ribosomes in cesium chloride was 1.41 ± 0.01 g/cm3 (n = 3) (Fig.
3). In previous studies of ribosomes (41,
43, 44), the inverse of the buoyant density has been utilized to estimate the partial specific volume. With the mitoribosomes the reciprocal gave a value of 0.709 ml/g, which is within 3% of the value
calculated above. The calculated partial specific volume was utilized
in determining s20,w and particle
hydration values.

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Fig. 3.
Buoyant density measurements of rat
mitochondrial ribosomes. Purified ribosomes were fixed in
formaldehyde and centrifuged in a cesium chloride gradient to determine
the buoyant density. The gradient formed was from 1.26 to 1.55 g/cm3. See "Experimental Procedures" for further
details.
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Molecular Weight Determinations for Mitochondrial and E. coli
Ribosomes--
Molecular weight determinations of mitochondrial and
E. coli ribosomes obtained from static light-scattering
analyses are shown in Table II. The
scattering intensity exhibited a linear dependence on concentration
over the range utilized (0.03-0.09 mg/ml). Thus, a molecular mass of
3.57 ± 0.14 MDa was obtained for the 55 S mitochondrial monosome.
Employing the Svedberg equation and sedimentation coefficients as shown
in Fig. 1A and diffusion coefficients from the same
ribosomal preparation (Fig.
4B), the molecular mass was
calculated to be 3.79 MDa. Using the calculated weight of ribosomal
proteins (9) and the weight of the ribosomal 12 and 16 S RNAs (39), a
theoretical mass of 3.45 MDa was determined. This is the minimal value
since it assumes one molecule of each of the 86 polypeptides per
ribosome (9). The molecular mass of E. coli ribosomes
determined from light-scattering experiments was 2.49 ± 0.06 MDa
(Table II); here also, the scattering intensity was linear with
concentration over the range of 0.02-0.26 mg/ml. From the Svedberg
equation, a molecular mass of 2.61 MDa was obtained utilizing
sedimentation coefficients as shown in Fig. 1B and diffusion coefficients from the same ribosome preparation (Fig.
4B).
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Table II
Summary of physiochemical properties of mitochondrial and E. coli
ribosomes
The numbers in parentheses are the numbers of preparations analyzed.
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Fig. 4.
Diffusion coefficient measurements of
mitochondrial and E. coli ribosomes. Dynamic
light-scattering analysis was carried out on ribosomes obtained from
the 260-nm absorbance peak of sucrose density gradients. A,
reduced first-order autocorrelation function for mitoribosomes. The
first 64 channels of the BI-2030 correlator were divided into four
blocks, each of 16 channels, using sample times of 1, 2, 4, and 8 µs;
the last 8 channels were used to define the base line and were extended
to 85 ms. Data were collected for 3 min with the detector aperture set
at 0.4 or 0.8 mm. Signal/noise ratios of 0.13-0.25 were obtained. The
solid line corresponds to the autocorrelation
function calculated using Dt determined from a
second-order cumulant analysis (see "Experimental Procedures"). The
inset corresponds to intensity distributions of scattering
components obtained from CONTIN analysis (38). B,
translational diffusion coefficients for mitochondrial
(triangles) and E. coli (circles)
ribosomes. Each point represents the mean of at least two
determinations. The straight line between each
set of data points indicates the mean diffusion coefficient.
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Determination of Diffusion Coefficients by Dynamic Light
Scattering--
Fig. 4A shows the reduced first-order
autocorrelation function (29, 33-35), which was used to obtain the
translational diffusion coefficient. We routinely analyzed these data
with a second order cumulants analysis, as this approach consistently
yielded excellent agreement with the experimental data, as shown by the
solid line in Fig. 4A. Size
distribution analysis (CONTIN) revealed a narrow distribution of
scattering components, as shown by the inset in Fig.
4A. The translational diffusion coefficients obtained from dynamic light-scattering experiments for mitochondrial and E. coli ribosomes were 1.10 × 10
7
cm2 s
1 and 1.72 × 10
7 cm2
s
1, respectively (Table II). The
D20,w was independent of
concentration over the range 0.04-0.08 mg/ml for mitochondrial ribosomes (Fig. 4B) and 0.02-0.26 mg/ml for E. coli ribosomes (Fig. 4B) and therefore behaved as an
ideal noninteracting system within the above concentration range.
The diameter obtained from calculating the Stokes radius was 39.1 ± 0.3 nm for mitoribosomes and 25.1 ± 0.4 for E. coli
ribosomes. In both cases the CONTIN analyses indicated that less than
1% of the total sample was present at a diameter greater than 100 nm
(see inset in Fig. 4A). Therefore, it is unlikely
that the diameter determined from the
D20,w contains a significant contribution from any larger species.
Electron Microscopy of Mitoribosomes and E. coli
Ribosomes--
Fig. 5A shows
electron micrographs of negatively stained mitoribosomes. The
dimensions were 26.2 nm × 23.6 nm, resulting in an axial ratio of
1.11. For the corresponding E. coli ribosomes (Fig.
5B), the dimensions were 21.0 nm × 19.9 nm, resulting
in an axial ratio of 1.06. In both cases, ribosomes demonstrated a
cleft, which delineates the large and small subunits.

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Fig. 5.
Electron micrographs of mitochondrial and
E. coli ribosomes. Electron microscopy was
carried out on ribosomes obtained directly from the peak maxima of
sucrose density gradients. Ribosomes were negatively stained with
uranyl acetate and visualized at a magnification of 70,800. A, mitochondrial ribosomes. B, E. coli
ribosomes.
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 |
DISCUSSION |
The aim of the present study was to determine the physiochemical
properties of rat liver mitochondrial ribosomes. The molecular weight
was measured by light scattering and also by the Svedberg equation with
data obtained from sedimentation velocity and diffusion coefficient
measurements. The sedimentation coefficients for the complete monosome,
large subunit, and small subunit were 55, 39, and 28 S, respectively,
as determined by the time-derivative sedimentation velocity (DCDT+)
protocol (11). The other protocols for determining sedimentation
coefficients, listed in Table I and Fig. 2, provided s20,w values for the intact monosome
very close to that obtained by the DCDT+ analysis. For E. coli ribosomes, the sedimentation coefficient of the monosome was
~70 S in buffer and 67 S in sucrose, as indicated by the DCDT+
analyses. These values are the first reports of sedimentation
properties of ribosomes using the DCDT+, SVEDBERG, and van
Holde-Weischet analyses developed for analytical ultracentrifugation.
Additionally, the homogeneity of sample preparations was examined using
above mentioned software analyses. In some preparations, a small amount
of the 39 S subunit was observed, which never exceeded 6% of the total
sample. The van Holde-Weischet analysis also indicated no species
larger than the 55 S particle.
The chemical composition of the mitochondrial ribosomes was calculated
using the reported mass of rat mitochondrial ribosomal proteins (9) and
the particle weights for the rRNA species (40). Based on these data,
ribosome particles contained 75% protein and 25% RNA. This is in
agreement with the composition determined by chemical analyses of rat
liver mitochondrial ribosomes (8, 41). It is also identical to the
ratio of rRNA and protein of mitoribosomes from Xenopus
laevis (45). Because our estimate of the ratio of RNA and protein
agreed with the value obtained experimentally (8, 41), we calculated a
for rat liver mitoribosomes. This
calculated
agreed closely with that
obtained from buoyant density analysis of mitoribosomes (3%
difference), allowing its use in molecular weight determinations.
The calculated molecular mass of the mitochondrial ribosomes from
light-scattering experiments was 3.57 MDa, and a similar value of 3.79 MDa was obtained from the sedimentation and diffusion coefficients
(Table II). These values are similar to 3.5 MDa obtained for X. laevis (45) but are higher than the 3.2 MDa earlier estimated for
rat liver (8). In the study by Sacchi et al. (8), the molecular weight was estimated from measurements of the RNA content of
the ribosome and estimates of the masses of the two rRNAs, which in
turn were estimated from molecular weights of cytosolic rRNAs. When the
sequences of the rRNAs were established (40), their estimates of the
molecular weights of the rRNAs were shown to be too low. This would
explain, at least in part, the small difference (<10%) between our
measurements of molecular weight and their earlier estimate. The
molecular mass determined by sedimentation equilibrium experiments for
bovine mitoribosomes has been reported as 2.8 MDa (14), which is
significantly lower than the molecular weight obtained in this study
for the rat liver mitoribosomes. The difference in molecular weights
between bovine and rat liver ribosomes is unlikely to be due to the
primary structure of the individual proteins, as analyses to date of
mitochondrial ribosomal proteins from different mammalian species
indicate significant sequence similarities (46-48).
Since the molecular mass for mitoribosomes obtained in this study by
static light scattering was higher than that reported earlier for
mitoribosomes from both rat liver (8) and beef liver (14), preparations
from E. coli were utilized to evaluate the applicability of
the light-scattering measurements for analysis of ribosomes. A
molecular mass of 2.5 MDa was determined (Table II), which agrees very
well with published values of 2.3 MDa (49), 2.5 MDa (50), and 2.6 MDa
(19, 20). E. coli ribosomes were also analyzed by analytical
ultracentrifugation. The molecular mass derived from the Svedberg
equation was 2.61 MDa, which is only 4% higher from the value obtained
by light scattering. These results suggest that the approaches used
provide accurate measures of the molecular mass of rat liver mitoribosomes.
From dynamic light-scattering experiments, we obtained the
translational diffusion coefficient. This hydrodynamic parameter, combined with other independently determined physical parameters, was
used to determine the frictional ratio using Stokes-Einstein equation.
The 55 S ribosome particle has a frictional ratio
(f/fmin) of 1.97 based on the
hydrodynamic diameter (39.1 nm) compared with that of an anhydrous
spherical particle diameter (19.8 nm), which indicates either a highly
asymmetric and/or highly hydrated structure. If a large particle
contaminated the ribosome preparations, then the
D20,w would be lower and this would
lead to a higher diameter. However, the plots of the distribution of
scattering components indicated that less than 1% of the particle was
larger than the monosome (see inset in Fig. 4A).
Furthermore, the data from sedimentation velocity experiments suggest
that the sample is nearly homogeneous, with only a small amount of
dissociation indicated by the presence of the 39 S subunit. Homogeneity
was verified by the van Holde-Weischet analysis which gave a precise picture of the S-value distribution (25). Aggregation of the ribosome particle could also explain the high frictional ratio. This
again appears unlikely, as from our experience rat liver mitoribosomes
have a greater tendency to dissociate which would lead to faster moving
species and a lower diameter.
In comparison to mitoribosomes, the
D20,w for E. coli
ribosomes was considerably higher (1.72 × 10
7 cm2
s
1) and agreed closely with published values
of 1.71 × 10
7 cm2
s
1 (50, 51), also obtained by dynamic
light-scattering measurements. The diameter derived from
Stokes-Einstein equation was 25.1 nm, which is also similar to
published values for bacterial ribosomes (12, 52). The frictional ratio
(f/fmin) was 1.46, which corresponded to either an asymmetric structure and/or a hydrated particle. This
observed frictional ratio agreed closely with reported
(f/fmin) values of 1.42 (21), 1.47 (51), and 1.50 (50). Since the observed values for E. coli
ribosomes corresponded closely with those published previously, this
confirms that the dynamic light-scattering measurements used in this
study for determining the diameter and frictional ratio for
mitoribosomes were reliable.
Electron microscopy images of mitoribosomes revealed a particle with
the dimensions of 26.2 × 23.6 nm, which results in an axial ratio
of 1.11. This is similar to the axial ratio (1.06) obtained from
analysis of electron microscopy images of E. coli ribosomes.
The axial ratio obtained for E. coli ribosomes by electron microscopy is consistent with the structure established by high resolution cryo-electron microscopy (13, 53), which demonstrates an
axial ratio close to unity.
This agreement on overall shape, as demonstrated by electron microscopy
of negatively stained particles and cryo-electron microscopy of
E. coli ribosomes (13, 53), indicates that the images we
obtained on negatively stained mitoribosomes reflect their structure in
solution. Thus, the electron microscopy indicates that the high
frictional ratio obtained by dynamic light scattering cannot be
attributed to the overall shape of the particle. The frictional ratio
of a particle is also affected by the amount of hydration
(
). If it is assumed that the rat
mitoribosome is spherical and that the excess friction can be
attributed to hydration, then the

is 4.5 g of water/g of
ribosome, as calculated from the equation 
= (
2/
1[(f/fmin)3
(1.11)] (36). Application of the same equation with E. coli ribosomes yields a 
of
1.3 g of water/g of ribosome, which agrees with earlier reports of
water content determined by independent measurements (19, 54). This
agreement validates the approach utilized in this study to estimate
hydration of ribosome particles.
Using the measured value for molecular weight determined by
static light scattering and the sedimentation coefficient
(s20,w), a diffusion coefficient was
also calculated using the Svedberg equation (28). This calculated
D20,w (1.20 × 10
7 cm2
s
1) is slightly larger (9%) that that
obtained from dynamic light scattering (1.10 × 10
7 cm2
s
1). Utilizing the calculated diffusion
coefficient, a value of 3.3 g of water/g of ribosome was
estimated. Either of these two estimates of water content for
mitoribosomes indicate a highly hydrated particle. It is notable that
these estimates of hydration for the mitochondrial ribosome are in the
same range as that reported for rat liver cytoplasmic ribosomes (3.7 g/g of ribosome) (55) and its large subunit (3.3 g/g) (56). The degree
of hydration of mitoribosomes may explain the finding that, despite
possessing a higher molecular weight than E. coli ribosomes
(Table II), the sedimentation coefficient is much lower (Table I).
In the past decade, there have been major advances in determining the
fine structure of the ribosome applying advanced techniques such as
electron cryomicroscopy, neutron scattering, small angle x-ray
scattering, and x-ray crystallography (12, 13, 52). The structures
obtained have provided considerable detail on the relationship between
ribosomal structure and the translation mechanism (57, 58). However,
there are two diverging views on the compactness of the ribosomal
structure. Frank and co-workers propose a rather compact structure
(59), whereas van Heel favors a more porous structure, characterized as
looking like "a swiss cheese, full of hollows, voids, gaps and
tunnels" (58). This porous structure is supported by the recognition
that the E. coli ribosome has a high water content (18, 54).
In this study, we have verified this high level of solvation of
E. coli ribosomes (1.3 g of water/g of ribosome) using
light-scattering techniques. This independent observation of a highly
solvated particle provides additional support for the ribosome existing
as a porous structure.
Structural analysis of eukaryotic ribosomes suggests the possibility
for greater water immobilization than with prokaryotic particles (57,
60), as has been observed in this and earlier studies (55, 56). The
outer surface of eukaryotic ribosomes "show more complex extended
structures" (57). It is possible that this more complex surface could
have the effect of immobilizing water in addition to that which fills a
porous structure apparently similar in architecture to that of the
prokaryotes (57).