A general and rational approach to the optimal recovery of mitochondria by differential centrifugation in homogenous media.

The sedimentation coefficients ($\text{S}$-values) of mitochondria have been determined in isotonic sucrose media for different mammalian tissues. Since the values show a relatively great variation, a general and rational approach to the optimal recovery of mitochondria from any tissue by differential centrifugation is presented. The method is primarily based on the determination of the average sedimentation coefficient at the actual isolation conditions. From the $\text{S}$-value thus determined and known centrifugation parameters, a specific procedure can be designed in each case.     

Pseudoisokinetic analytical ultracentrifugation: monitoring of bands at a fixed region in the analytical ultracentrifuge.

With the use of a Model E analytical ultracentrifuge equipped with a photoelectric scanner, a technique for obtaining isokinetic patterns of moving zones has been developed by monitoring a fixed area of the analytical cell as a function of time. Small amounts of material spread over a very wide range of sedimentation coefficients can be used in a single experiment. Since the resultant tracing is effectively isokinetic, sedimentation coefficients can be calculated by simple ratio if the S value of any component is known.     

Generation of density gradients. Approximations to equivolumetric gradients for zonal rotors

The use of a “density gradient generating function” allows the concentration profile of a density gradient to be written explicitly in terms of the required distribution of sedimentation coefficients in place of the previous implicit formulations. This function, which is easily implemented in a computer program, permits calculation of density gradients for a number of applications. This approach is applied to computation of a variety of equivolumetric gradients of sucrose for zonal rotors and yields a formula for the calibration of such gradients. An accurate approximation has been found which allows the generation of virtually all equivolumetric gradients of sucrose for a given rotor using a single program for the gradient generator employed; the adjustment for different particle densities and for different concentrations at the top of the gradient is made by varying only the initial and final concentrations of sucrose used.     

An assessment of some molecular parameters of mevalonate kinase from plant and animal sources

The molecular weights, diffusion coefficients, and sedimentation coefficients of mevalonate kinase in partially purified preparations from Hevea brasiliensis latex, Cucumis melo cotyledons, Phaseolus vulgaris cotyledons, bakers yeast, chicken liver, and rabbit liver have been determined by gel filtration in Sephadex G-100 and G-200 and by sucrose density gradient centrifugation. The enzyme had similar molecular weights (94800–103500), diffusion coefficients (5.39–5.62 × 10^?7 cm²/sec), and sedimentation coefficients (5.85–6.00 S) in the six preparations.     

The Sedimentation Pattern of D-Glucose-6-Phosphate Dehydrogenase from Bovine Fetal and Adult Erythrocytes

Erythrocytes from bovine fetuses contain about 2.4 times higher D-glucose-6-phosphate dehydrogenase activities than erythrocytes from adult cows and bulls. Studying whether this is due to the existence of a special fetal type of enzyme or an increased amount of enzyme in fetal erythrocytes, the sedimentation coefficients of the enzymes have been estimated by s-zonal ultracentrifugation, and compared to normal and deficient human erythrocyte D-glucose-6-phosphate dehydrogenase, s-zonal ultracentrifugations have been performed with a computer optimized isokinetic sucrose gradient. The mainlines in the program used for calculation of sedimentation coefficients are described. Bovine fetal and adult erythrocyte D-glucose-6-phosphate dehydrogenase was found to have the same sedimentation coefficient of 7.4 S which is different from the sedimentation coefficient of 6.4 S of both human types of the enzyme. The sedimentation coefficients of 6-phospho-D-gluconate dehydrogenase from bovine fetal, bovine adult and human erythrocytes were 6 S for all three types of this enzyme. By cellulose acetate electrophoresis bovine fetal and adult D-glucose-6-phosphate dehydrogenase show the same mobility, again differing from the normal and deficient human type. The results of these experiments show that bovine fetal and adult erythrocytic D-glucose-6-phosphate dehydrogenase with respects to molecular parameters are closely related and perhaps identical enzymes.     

Molecular weight determination of glyoxalated RNA by sedimentation centrifugation

A rapid, reliable sedimentation centrifugation technique has been developed to measure the molecular weights of rather large glyoxalated RNAs. A distinctive feature of this method is that the glyoxalated RNAs can be analyzed in sucrose gradients containing no denaturant. This feature allowed us to compute the sedimentation coefficients of glyoxalated RNAs by a comparison with those of native, untreated RNA markers. These values then were used to obtain accurate molecular weight estimates by applying the linear log-log relation between the molecular weight of an RNA and its sedimentation coefficient.     

A convenient program for the rapid calculation of sedimentation coefficients in linear salt or sucrose gradients

A program is presented for calculating s_20,w values from data obtained by zone sedimentation in linear sucrose or salt gradients in a variety of rotors at temperatures ranging from 0° to 20°C. The program can be either run with the use of high-speed computers or performed with the aid of a small calculator in a reasonably short time.     

Hydrangea mosaic virus, a new ilarvirus from Hydrangea macrophylla (Saxifragaceae)

A previously unrecognised virus isolated from Hydrangea macrophylla with chlorotic mosaic leaf symptoms in West Sussex was named hydrangea mosaic virus (HydMV). HydMV was mechanically transmitted without difficulty to four of 16 species from three of five families, and was seed transmitted in Chenopodium quinoa, but was not aphid transmitted. Although relatively unstable in vitro, HydMV was purified by clarifying leaf extracts by emulsification with chloroform and acidification with citric acid, followed by differential centrifugation and sucrose density gradient centrifugation. Purified virus incompletely separated on sucrose density gradients into three components (T. M and B) with sedimentation coefficients (s^o_20w) of 86, 97, and 105 S respectively, but all particles had buoyant densities in caesium chloride of 1.37 g/cm³. Virus contained a single polypeptide species (mol. wt 26.4 times 10³), appeared quasiisometric to ovoid or elliptical, and measured c. 28 times 30 (T), 30 times 30 (M) or 30 times 32–38 nm (B). Single-stranded RNA species or mol. wt 1–25, 1–08, 0–83, 0–36 and 0–27 (RNA-1 to 5 respectively) were obtained from virus preparations but mixtures containing RNA-1 to 3 plus either RNA-4 + 5 or the coat protein, were infective. These properties suggest that HydMV has affinities with ilarviruses, but it showed no serological relationship to any of six ilarviruses or 42 other viruses.     

Aggregation and dispersity of isolated chromaffin granules studied by intensity fluctuation spectroscopy

The aggregation and dispersity of isolated bovine adrenal secretory vesicles (chromaffin granules) were studied by intensity fluctuation spectroscopy. The degree of dispersity and the Z-average translational diffusion coefficients were calculated from the autocorrelation functions of the intensity fluctuations in lase light scattered from the granules in solution. Granules purified by sedimentation through 0.3 M sucrose/Ficoll/²H₂O showed greater dispersity than granules purified by sedimentation through 1.6 M sucrose. By monitoring the scattered light intensity and the diffusion coefficients of the granules, many of the difficulties encountered in the interpretation of absorbance measurements were avoided. Measurements over a range of granule concentrations in sucrose solutions (10 mM HEPES, pH 7.0), indicated that aggregation of the granules occurred at concentrations above 150 μg protein/ml. At low granule concentrations (15–30 μg protein/ml) Ca²⁺-induced aggregation was detected at a threshold of 2–10 mM calcium.     

A specific binding protein for 17beta-estradiol in retired breeder rat ventral prostate

A specific binding protein for 17β-estradiol has been detected in ventral prostate of normal retired breeder rats using sucrose density gradient techniques. The protein has an approximate sedimentation coefficient of 3. 5S. It is distinguishable from serum proteins which bind 17β-estradiol on the basis of binding specificity and sedimentation coefficient. It is also distinct from the cytoplasmic androgen binding protein known to be present in rat ventral prostate.     

Automatic collection and processing of data from the ultracentrifuge using a programmable desk calculator.

Previous investigators [Trautman, R., Spragg, S. P., and Halsall, H. B. (1969) Anal. Biochem.28, 396–415] have published a detailed protocol for the analysis of sedimentation velocity measurements which is adaptable to data generated by an ultraviolet scanning system. The advent of programmable desk calculators capable of sampling the output of digital measuring devices has made it possible to develop inexpensive and highly convenient systems for collecting and processing scanner data. Basing our approach on the referenced protocol, we have developed algorithms for dealing with real data, that is, data characterized by a relatively high level of noise. The techniques are applieable to both sedimentation equilibrium and sedimentation velocity measurements using the seanning system and multicell rotors. With known concentration dependence, valid estimates of weight-average sedimentation coefficients, diffusion coefficients, and heterogeneity parameters have been obtained for both simulated and actual sedimenting anddiffusing macromolecular solutes. We find, however, that concentration dependence derived internally from a single sedimentation velocity measurement is unreliable.     

Gradient design to optimize rate zonal separations

An approach to the design of gradients which maximize resolution is developed by analyzing the sedimentation of particles in linear sucrose gradients. Our analysis establishes the fundamental principles of rate separations. These principles can assist in the successful design of preparative centrifugation procedures. Rate separations are always optimal in homogeneous media or very shallow gradients of low density. In homogeneous media, resolution of particles which differ only in sedimentation coefficients is determined by the ratio of their sedimentation coefficients. Particles whose sedimentation properties oppose each other can, under certain conditions, not separate or barely separate unless conditions are carefully selected. Particle populations which differ more in density than in sedimentation coefficients clearly separate better by rate than by isopycnic banding. Rate separations in gradients are considerably improved in a type of gradient where the viscosity decreased as the density increased.     

Repair of thermal damage to the Escherichia coli nucleoid.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after heat treatment (30 min at 50 degrees C) and subsequent incubation of cells at 37 degrees C for various times. Heat treatment resulted in in vivo association of the nucleoids with cellular protein and in an increase in sedimentation coefficient. During incubation at 37 degrees C, a fraction of the nucleoids, from heated cells, because dissociated from cellular protein and regained their characteristic sedimentation coefficients. The percentage of nucleoids which returned to their control sedimentation position in the sucrose gradients corresponded to the percentage of cells able to repair thermal damage as assayed by enumeration on agar plates.     

Proteins of Vasicular Stomatitis Virus: I. Polyacrylamide Gel Analysis of Viral Antigens

Infection of L cells with vesicular stomatitis virus results in the release, into the cell-free fluid, of four antigenic components separable by rate zonal centrifugation on sucrose gradients. The largest antigens are the infectious (B) particle and a shorter noninfectious, autointerfering (T) particle. The two small antigens are characterized by sedimentation coefficients of approximately 20S and 6S. Treatment of purified B or T particles with sodium deoxycholate results in the release from the particle of a nucleoprotein core which can be purified on sucrose gradient and which has a sedimentation coefficient characteristic of the virus from which it arose. Utilizing purified antigens labeled with (14)C-amino acids during growth, we examined the protein constituents of each antigen by acrylamide-gel electrophoresis. The proteins of B and T particles are identical, each containing one minor (virus protein 1) and three major (virus proteins 2, 3, and 4) proteins, numbered in order of increasing mobility. Virus protein 3 originates from the nucleoprotein core, whereas proteins 2 and 4 come from the coat. The origin of virus protein 1 is not known. The 20S antigen contains a single protein equivalent to virus protein 3, whereas the 6S antigen shows a single protein which is similar to, but probably distinct from, virus protein 2.     

Cross-links in African swine fever virus DNA.

African swine fever virus DNA sediments in neutral sucrose density gradients as a single component with a sedimentation coefficient of 60S. In alkaline sucrose density gradients, this material shows two components with sedimentation coefficients of 85S and 95S, respectively. The sedimentation rate value of alkali-denatured virus DNA in neutral sucrose density gradients and the renaturation velocity of denatured DNA show that is reassociated much faster than expected from its genetic complexity. This behavior is compatible with the existence of interstrand cross-links in the molecule. We also present results which suggest that there are only a few such cross-links per molecule, that they are sensitive to S1 nuclease digestion, and that they are probably located next to the ends of the DNA.     

The construction and analysis of sucrose gradients for use with zonal rotors.

The rate of sedimentation of a particle in a sucrose solution depends on the viscosity and density of the medium. These two variables are related to the sucrose concentration and the temperature of the medium by new simple equations. These equations were used in a rapid iterative procedure that relates the distance moved by a zone in a continuous sucrose gradient to its sedimentation coefficient. It is shown by comparison with experiment that this iterative method allows the distance moved by a zone to be calculated rapidly. The method may therefore be used to optimize the separation of particles in a sucrose-gradient-centrifugation experiment. The method also allows the unknown sedimentation coefficients of several zones to be measured from a single sucrose-gradient-centrifugation experiment.     

Sedimentation coefficient of polyoma virus DNA

The sedimentation coefficient of the twisted circular form of polyoma virus DNA is calculated from the Kirkwood sedimentation–diffusion equation, the structure being assumed to be a rigid double superhelix. Agreement with the experimental sedimentation coefficient can be obtained, with the use of an experimental value for the number of superhelical turns, when the pitch of the superhelix is intermediate between its minimal and maximal possible values. Another model, which has been proposed for polyoma DNA at low ionic strengths, may be visualized as a superhelical structure wound about a torus. Calculations of sedimentation coefficients for this model agree qualitatively with experimental data at ionic strengths Below 10^?2M.     

Determination of sedimentation coefficients for small peptides.

Direct fitting of sedimentation velocity data with numerical solutions of the Lamm equations has been exploited to obtain sedimentation coefficients for single solutes under conditions where solvent and solution plateaus are either not available or are transient. The calculated evolution was initialized with the first experimental scan and nonlinear regression was employed to obtain best-fit values for the sedimentation and diffusion coefficients. General properties of the Lamm equations as data analysis tools were examined. This method was applied to study a set of small peptides containing amphipathic heptad repeats with the general structure Ac-YS-(AKEAAKE)nGAR-NH2, n = 2, 3, or 4. Sedimentation velocity analysis indicated single sedimenting species with sedimentation coefficients (s(20,w) values) of 0.37, 0.45, and 0.52 S, respectively, in good agreement with sedimentation coefficients predicted by hydrodynamic theory. The described approach can be applied to synthetic boundary and conventional loading experiments, and can be extended to analyze sedimentation data for both large and small macromolecules in order to define shape, heterogeneity, and state of association.     

A comparison of weight average and direct boundary fitting of sedimentation velocity data for indefinite polymerizing systems

Analysis of sedimentation velocity data for indefinite self-associating systems is often achieved by fitting of weight average sedimentation coefficients (s(20,w)) However, this method discriminates poorly between alternative models of association and is biased by the presence of inactive monomers and irreversible aggregates. Therefore, a more robust method for extracting the binding constants for indefinite self-associating systems has been developed. This approach utilizes a set of fitting routines (SedAnal) that perform global non-linear least squares fits of up to 10 sedimentation velocity experiments, corresponding to different loading concentrations, by a combination of finite element simulations and a fitting algorithm that uses a simplex convergence routine to search parameter space. Indefinite self-association is analyzed with the software program isodesfitter, which incorporates user provided functions for sedimentation coefficients as a function of the degree of polymerization for spherical, linear and helical polymer models. The computer program hydro was used to generate the sedimentation coefficient values for the linear and helical polymer assembly mechanisms. Since this curve fitting method directly fits the shape of the sedimenting boundary, it is in principle very sensitive to alternative models and the presence of species not participating in the reaction. This approach is compared with traditional fitting of weight average data and applied to the initial stages of Mg(2+)-induced tubulin self-associating into small curved polymers, and vinblastine-induced tubulin spiral formation. The appropriate use and limitations of the methods are discussed.