Properties of various rotors used for zone centrifugation

The following results have been obtained from a quantitative study of zone centrifugation: (1) it is shown that the sedimentation velocity of all kinds of macromolecules is constant in 5–20% constant sucrose gradients, whatever swinging bucket or zonal rotor is being used, and at any usual temperature. (2) The proportionality constants between time of centrifugation and sedimented distance have been calculated for several rotors. They allow an estimate of relative centrifugation times. (3) An equation of the resolving power of zone centrifugation in isokinetic density gradients is used to compare the resolution of various rotors. (4) An equation of Vinograd and Bruner and Spragg and Rankin is discussed and used for the calculation of the maximum macromolecular load of the rotors. A summary of these results is presented in a table, which should help in the choice of the rotor best suited for a particular experiment.     

Sucrose density gradient sedimentation of E. coli ribosomes.

The behavior of E. coli ribosomes during sedimentation on sucrose gradients is predicted under a variety of conditions by computer simulations. Since numerous recent kinetic studies indicate equilibration in times short compared to the time of sedimentation, these simulations assume that the system attains local reaction equilibrium at every point in the gradient at all times. For any type of homogeneous equilibrating ribosome population, governed by a single formation constant at one atmosphere pressure for 70S couples, no more than two clearly defined zones will be resolved, although the presence of large dissociating effects due to pressure gradients in high speed experiments will spread the subunit zone. Normally the pattern will consist of a 30S zone and a so-called “70S” zone, which is in reality a mixture of 70S couples and 30S and 50S subunits in local equilibrium. The greater the dissociation into subunits, the more the “70S” zone will be slowed below the nominal rate of 70 Svedberg units. If ribosomes have been collected from the “70S” zone in several successive cycles of purification, the repeated deletion of resolved 30S subunits can result in a preparation with so large a molar excess of 50S subunits that the ensuing sucrose density gradient sedimentation pattern may exhibit a “70S” zone followed by zone of 50S subunits, insteadof a zone of 30S subunits. Our most important conclusion is that whenever a well-resolved 50S zone is present in a sucrose density gradient sedimentation experiment on E. coli ribosomes, in addition to a 30S and a “70S” zone, under conditions where ribosomes and subunits should be in reversible equilibrium, the preparation must be microheterogeneous, containing a mixture of “tight” and “loose” couples. Moreover in such cases the content of large subunits in the 50S zone must be derived entirely from “loose” couples whereas the 30S zone must contain small subunits derived from both “tight” and “loose” couples. Sedimentation patterns predicted for various mixtures of “tight” and “loose” couples display all the major characteristics of published experimental patterns for E. coli ribosomes, including the partial or complete resolution into three zones, depending on rotor velocity and level of Mg²⁺.     

Calculation of s20,w values using ultracentrifuge sedimentation data from linear sucrose gradients, an improved, simplified method.

A mathematical method is described for calculating the sedimentation coefficient (S20,W) with ultracentrifuge data from linear sucrose gradients. Gradient density and viscosity functions are precisely described by regression equations, which permit continuous evaluation (by integration) of the effects of gradient geometry on particle sedimentation. The results agree with previously used and more complex methods.     

Calculation of s20,w values using ultracentrifuge sedimentation data from linear sucrose gradients,an improved,simplified method

A mathematical method is described for calculating the sedimentation coefficient (s_{20, w}) with ultracentrifuge data from linear sucrose gradients. Gradient density and viscosity functions are precisely described by regression equations, which permit continuous evalution (by integration) of the effects of gradient geometry on particle sedimentation. The results agree with previously used and more complex methods.     

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.     

Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC3H1 cell line

We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC3H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, 2H, 13C, 15N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher mass (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme (s = 7.40 +/- 0.04 S, n = 36). Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme (i.e. Km for ATP and EC50 for Mn2+ or Mg2+) did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control (cells in light medium). After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value indicating that the heavy isotope-labeled enzyme replaced the pre-existing light form of the molecule. These observations show that the rapid decrease in adenylate cyclase activity and the synthesis of heavy adenylate cyclase molecules are two separate events. The relative amounts of heavy and light components of forskolin-stimulated adenylate cyclase obtained in sucrose gradient differential sedimentation were determined as a function of time beginning 24 h after the transfer into the heavy medium. The decrease of the pre-existing light form could be represented by simple first order kinetics with a half-time of 40 h. This result suggests that the metabolic renewal of forskolin-stimulated adenylate cyclase is comparable to that of most plasma membrane proteins.     

Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29.

We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with [3H]thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation. During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA. The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope. We found two major types of molecules. One consisted of unit-length duplex DNA with one single-stranded branch at a random position. The length of the single-stranded branches was similar to that of one of the double-stranded regions. The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end. Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end. These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus.     

The determination of density and molecular weight distributions of lipoproteins by sedimentation equilibrium.

A method is presented by which an experimental record of total concentration as a function of radial distance, obtained in a sedimentation equilibrium experiment conducted with a noninteracting mixture in the absence of a density gradient, may be analyzed to obtain the unimodal distributions of molecular weight and of partial molar volume when these vary concomitantly and continuously. Particular attention is given to the caracterization of classes of lipoproteins exhibiting Gaussian distributions of these quantities, although the analysis is applicable to other types of unimodal distribution. Equations are also formulated permitting the definition of the corresponding distributions of partial specific volume and of density. The analysis procedure is based on a method (employing Laplace transforms) developed previously, but differs from it in that it avoids the necessity of differentiating experimental results, which introduces error. The method offers certain advantages over other procedures used to characterize and compare lipoprotein samples (exhibiting unimodal distributions) with regard to the duration of the experiment, economy of the sample, and, particularly, the ability to define in principle all of the relevant distributions from one sedimentation equilibrium experiment and an external measurement of the weight average partial specific volume. These points and the steps in the analysis procedure are illustrated with experimental results obtained in the sedimentation equilibrium of a sample of human serum low density lipoprotein. The experimental parameters (such as solution density, column height, and angular velocity) used in the conduction of these experiments were selected on the basis of computer-simulated examples, which are also presented. These provide a guide for other workers interested in characterizing lipoproteins of this class.     

SEDIMENTATION PROPERTIES OF RAT LIVER MITOCHONDRIA : Effects of Cortisone Treatment

A prediction of the velocity of sedimentation of rat liver mitochondria in sucrose gradients is made on the basis of recent measurements of the size of isolated mitochondria suspended in sucrose medium and the model proposed by Bentzel and Solomon to describe the osmotic behavior of mitochondria. The experimentally observed velocity is extremely close to the predicted value and confirms by a different approach the estimate of mitochondrial volume made by Baudhuin and Berthet on the basis of electron microscopic measurements. Because cortisone treatment of rats is known to result in a marked increase in mitochondrial size as observed under the electron microscope, mitochondria were co-isolated from livers of control and cortisone-treated animals, and the sedimentation behavior of the mixtures was examined by sucrose density gradient centrifugation. Mitochondria from cortisone-treated animals were found to sediment 1.4 times as rapidly as those from control animals, indicating that their increased size cannot entirely be due to an increased imbibition of fluid from the surrounding sucrose medium, and that the change in size must at least in part be due to a change in content of nondiffusible mitochondrial components. Although the increase in sedimentation velocity of mitochondria from cortisone-treated animals is striking, it is less than that predicted solely on the basis of their size relative to that of control mitochondria. It is concluded that the increases in mitochondrial size and content of nondiffusible components produced by cortisone treatment are accompanied by alterations in mitochondrial composition as well.     

Growth-medium-dependent repair of DNA single-strand and double-strand breaks in X-irradiated Escherichia coli

The X-ray resistance of logarithmic phase cells of Escherichia coli K-12 is enhanced threefold by growth in rich medium versus minimal medium (N. J. Sargentini, W. P. Diver, and K. C. Smith, Radiat. Res. 93, 364-380, 1983). In this work, X-ray-induced DNA strand breaks were assayed by sedimentation in alkaline and neutral sucrose gradients to correlate the enhanced survival of rich-medium-grown cells with an enhanced capacity for DNA repair. While rich-medium-grown cells showed no enhanced capacity for repairing DNA single-strand breaks in buffer, i.e., fast, polA-dependent repair, they did show an enhanced capacity to repair both single-strand and double-strand breaks in growth medium, i.e., slow, recA-dependent repair. This enhanced capacity for DNA repair in rich-medium-grown cells was inhibited by rifampicin post-treatment, indicating the requirement for de novo RNA synthesis. Kinetic studies indicated that the repair of DNA double-strand breaks was a complex process. Relative to the sedimentation rate in neutral sucrose gradients of nonirradiated DNA, the sedimentation rate of X-irradiated DNA first changed from slow to very fast. Based on alkaline sucrose gradient sedimentation studies, all the strand breaks had been repaired during the formation of the very fast sedimenting DNA. With continued incubation, the sedimentation rate of the DNA on neutral sucrose gradients decreased to the normal rate.     

Sucrose utilization during somatic embryo development in black spruce: involvement of apoplastic invertase in the tissue and of extracellular invertase in the medium

The involvement of apoplastic invertase (Ap Inv) and sucrose synthase (SuSy) in the somatic embryo development of black spruce (Picea mariana) was investigated under different maturation conditions. Replacing 6% sucrose with 3% or 1% sucrose in the maturation medium drastically decreased Ap Inv activity and amount in embryogenic tissues. This was accompanied by a decrease in the hexose pool that resulted in a lower starch deposition and protein amount in embryogenic tissues together with a lower embryo production. Conversely, SuSy activity was stable during maturation regardless of the sucrose concentration used in the medium. The presence of an extracellular enzyme responsible for sucrose hydrolysis in the maturation medium was also verified. An immunodetection experiment with anti-acid invertase antibodies revealed the presence of an active 53 kDa polypeptide in the medium, which had a similar molecular mass to that of the Ap Inv polypeptide found in embryogenic tissues. Utilization of sucrose from the medium by the tissues was also studied using labelled 14C-sucrose. Distribution of the radioactivity between tissular sucrose, glucose, and fructose showed that sucrose was diffused into the cell wall of embryogenic tissues and partly hydrolyzed by Ap Inv. These results show that the utilization of sucrose from the medium, the Ap Inv activity in embryogenic tissues, and the release of an active invertase into the medium operate together for the utilization of the carbohydrates during somatic embryo development in black spruce.     

Isolation and purification of myotube and myoblast nuclei from cultures of embryonic chick skeletal muscle.

Methods are described for the preparation of purified myotubes from embryonic chick skeletal muscle cultures and the preparation of purified nuclei from both myotubes and myoblasts. Myotubes are released from the culture dish by digestion of their collagen substratum with collagenase, and purified by sucrose density gradient sedimentation. Nuclei are prepared from the isolated myotubes by controlled homogenization in Ca²⁺-free medium and sedimentation through 2.1 M sucrose. Nuclei are prepared from cultured myoblasts in a similar fashion, with the inclusion of the non-ionic detergent NP-40 in the homogenization medium and sedimentation through 2.4 M sucrose. Phase contrast microscopic examination showed that the nuclear preparations are free of visible cytoplasmic contamination, and are morphologically similar to nuclei observed in situ. Biochemical assays (protein/DNA and $\text{RNA}\text{DNA}$ ratios) confirm the purity of the nuclear preparations. Both nuclear preparations have been used to prepare purified chromatin which has spectral and chemical properties similar to those reported for chromatin purified directly from several chick tissues.     

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.     

Transient DNA lesions induced by benzo[a]pyrene in Chinese hamster ovary cells

Alkaline sucrose gradient sedimentation analysis was used to detect DNA lesions induced by benzo[a]pyrene B(a)P in Chinese hamster ovary cells. The number of lesions detected immediately following treatment with 10(-4) M B(a)P was related directly to the duration of treatment. When treated cells were incubated in a B(a)P-free medium, the majority of lesions disappeared rapidly and could no longer be detected 15 min following treatment. These data indicate that a population of B(a)P-induced DNA lesions may be removed by a rapid DNA-repair process. The transient nature of such lesions should be considered when assays for DNA damage or repair are designed and interpreted.     

Analysis of progress curves by simulations generated by numerical integration.

A highly flexible computer program written in FORTRAN is presented which fits computer-generated simulations to experimental progress-curve data by an iterative non-linear weighted least-squares procedure. This fitting procedure allows kinetic rate constants to be determined from the experimental progress curves. Although the numerical integration of the rate equations by a previously described method [Barshop, Wrenn & Frieden (1983) Anal. Biochem. 130, 134-145] is used here to generate predicted curves, any routine capable of the integration of a set of differential equations can be used. The fitting program described is designed to be widely applicable, easy to learn and convenient to use. The use, behaviour and power of the program is explored by using simulated test data.     

Accelerating the weighted histogram analysis method by direct inversion in the iterative subspace

The weighted histogram analysis method (WHAM) for free energy calculations is a valuable tool to produce free energy differences with the minimal errors. Given multiple simulations, WHAM obtains from the distribution overlaps the optimal statistical estimator of the density of states, from which the free energy differences can be computed. The WHAM equations are often solved by an iterative procedure. In this work, we use a well-known linear algebra algorithm which allows for more rapid convergence to the solution. We find that the computational complexity of the iterative solution to WHAM, and the closely-related multiple Bennett acceptance ratio method can be improved using the method of direct inversion in the iterative subspace. We give examples from a lattice model, a simple liquid and an aqueous protein solution.     

Changes in protein metabolism during the acquisition of tolerance to cryopreservation of carrot somatic embryos

High concentrations of sucrose are often used to cryopreserve regenerable plant cell cultures in liquid nitrogen. A 21-h pretreatment of carrot somatic embryos in medium containing 0.4 M sucrose allows 80 % of them to germinate after freezing. Substitution of sucrose by polyethylene glycol 6000 led to lower germination rates. However, a high level of freezing tolerance was restored by addition of 1 μM abscisic acid in the pretreatment medium. Using these different media, both total water soluble protein, using SDS-PAGE, and boiling-stable protein, using 2-D electrophoresis, were studied in relation to acquisition of cryopreservation tolerance. Only boiling-stable protein patterns showed some changes: five polypeptides accumulated in 0.4 M sucrose-pretreated embryos or in embryos pretreated by media containing abscisic acid. This accumulation was not detected with polyethylene glycol 6000 used as sole cryoprotectant. Although over-accumulation of polypeptides was highest with media containing ABA, the best germination rates were linked to pretreatment with 0.4 M sucrose. The addition of okadaic acid in 0.4 M sucrose medium led to embryo death after freezing, confirming the existence of a message leading to metabolic changes and acquisition of cryotolerance. Water-soluble proteins obtained from 0.4 M sucrose-pretreated embryos appeared more active than those extracted from control embryos in protecting in vitro a freeze-labile enzyme. Boiling-stable proteins, corresponding to a part of total proteins, were more active than total proteins. These results suggest that these polypeptides may be involved in a mechanism of protection needed for cell survival during freezing stress.     

High-throughput polyribosome fractionation

Polyribosome sedimentation velocity centrifugation can be used to identify differential regulation of the translation of mRNAs. However, ultracentrifugation presents practical limitations on the number of sedimentation velocity gradients that can be run simultaneously. A method for sedimentation velocity analysis of polyribosomes is presented that is based on low-speed centrifugation of sucrose gradients prepared in deep 96-well plates, the advantage of which is that hundreds of polyribosome fractionations can be performed simultaneously in a tabletop centrifuge.     

Experimental evidence for the role of long range forces in fibroblast-substrate interaction

Reflection interference microscopy was used to demonstrate that reducing the electrolyte concentration of the medium (sucrose was added to the medium to keep it isotonic) resulted in large areas of the ventral surface of the avian fibroblast moving further away from the substrate. Thus in cells 3 h after plating out the grey reflection image changed to white and in cells 24 h after plating out the predominantly white image was lost as the ventral surface moved even further away. These changes were reversible on replacing the low ionic strength solutions with 199 medium or 145 mM NaCl. This offers direct experimental evidence for the role of long range electrostatic forces in cell-substrate interactions of fibroblasts.     

Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation

A method is presented for preparing very large DNA from Bacillus subtilis protoplasts. When the DNA is characterized by sedimentation in neutral sucrose gradients, a fast-sedimenting component is found whose sedimentation coefficient varies with centrifuge speed. By use of [³H]thymine label for the DNA and a ¹⁴C-labeled amino acid, it is shown that less than 5% cellular material other than DNA is associated with this component. Irradiation of this DNA in solution with gamma rays forms a slower component, called the “main peak”, whose sedimentation coefficient also depends on centrifuge speed. More irradiation breaks down this main peak into even slower-sedimenting DNA; it is shown that for low doses, double-strand breaks are formed in both the B. subtilis DNA and in bacteriophage T2 DNA at the same rate linear in dose, 0.018 double-strand breaks per kilorad per mass equal to that of T2 DNA.The speed dependence of the DNA sedimenting at the main peak is compared with an approximate theory of the speed dependence of the sedimentation coefficient of linear DNA by B. H. Zimm (unpublished calculations). The comparison suggests that for sufficiently high centripedal acceleration, DNA molecules larger than a critical mass will sediment at much the same velocity. The theory, and data on the break-up of the DNA with gamma rays, are used to estimate that the DNA extracted is at least 13 times the mass of T2 DNA, and possibly larger.In the Appendix, data from the literature are put together with data taken during this work to make plausible the assumption that the usual theory for the sedimentation of DNA molecules, experimentally tested in salt solutions, may also be applied to sucrose solutions. If, in neutral sucrose gradients, the distance sedimented is proportional to a power α of the mass, the best value of α = 0.38.