Boundary centrifugation in isovolumetric and isokinetic cesium sulfate density gradients: application to cartilage proteoglycans and other macromolecules

A boundary sedimentation methodology is described that avoids plateau dilution and simplifies the calculation of centrifugal parameters. The technique is designed for the preparative ultracentrifuge and uses a newly developed sectorial cell. It is based on previous developments of the transport method and depends on isokinetic or isovolumetric Cs2SO4 density and viscosity gradients. These gradients are prepared with a single-chamber mixing device, and the only two parameters required for their calculations are presented in a tabulated form for general use with most available rotors and cell sizes. Conditions are specified (1) to assure that the density and shape of the sedimenting molecules remain invariant through the selected electrolytic gradient, (2) to monitor the gradient profiles, and (3) to verify attainment of isokinetic or isovolumetric sedimentations. A set of equations is presented to calculate the average and transport sedimentation coefficients and the differential sedimentation coefficient distribution for both the isokinetic and isovolumetric centrifugal regimes. The method was applied to slowly diffusing polydisperse proteoglycan monomers, to a paucidisperse DNA from bacteriophage PM2, and to a diffusible monodisperse system (purified bovine serum albumin). In all cases, the expected results were obtained.     

Variable nature of cartilage proteoglycans.

Cartilage proteoglycan aggregates are separated from collagen and other non-proteoglycan protein by preparative rate zonal sedimentation under associative conditions. Dissociative rate zonal sedimentation produces sedimented proteoglycan of lower protein content with a corresponding increase in the amount of less sedimentable protein-rich proteoglycan. An extensive number of sequential rate zonal sedimentations discloses that the proceess of disaggregation involves the separation of proteoglycans varying continuously in composition with no apparent discontinuities in distribution to indicate the presence of distinctively different macromolecules. The variations encompass proteoglycans of low protein content containing less than 2% keratan sulfate and proteoglycans with keratan sulfate as the predominant polysaccharide (present in concentrations greater than 2-fold that of the chondroitin sulfate) and more than a 10-fold increase in protein content.     

Selective aggregation of proteoglycans with hyaluronic acid.

Conditions were established to separate proteoglycan aggregate (AH1) from a bovine nasal septum extract by associative rate zonal sedimentation on a NaCl gradient. The AH1 has a higher protein content than the mixed aggregate-monomer (A1) isolated by conventional associative CsCl density gradient centrifugation from a portion of the same extract. The same associative rate zonal conditions separated the A1 fraction into aggregated AH1 containing hyaluronic acid and nonaggregated proteoglycan monomer (N1) essentially free of hyaluronic acid. The AH1 fraction is richer in protein and keratin sulfate than is N1. Dissociative rate zonal sedimentation of A1 under conditions which totally sedimented most of the disaggregated monomer (AH1-D1) and the nonaggregated monomer N1 separated a less sedimentable protein and keratan sulfate-rich proteoglycan monomer (AH1-D2). Chromatography on Sepharose 2B under dissociative conditions demonstrated that the nonaggregated N1 monomer is intermediate in size between the disaggregated monomers AH1-D1 and AH1-D2. N1 has a buoyant density higher than AH1 and is practically equivalent to AH1-D1. All are dense fractions so that separation by CsCl density gradient equilibration is not feasible.     

Resolution in zonal rotors

We have studied “static” factors affecting resolution in zonal rotors, that is, factors that are independent of diffusion or sedimentation of the sample.For most zonal rotors under the recommended conditions, there is proportionately very little zone broadening of sample volumes larger than 10 cc. There appears to be a correlation between the height of the rotor and resolution.We show that step gradients surrounding the sample can result in zone broadening in the case of slowly sedimenting particles. We did not detect any deleterious effects from increasing the initial sample radius.     

Quantitation of autoradiographic grains in different zones of articular cartilage with image analyzer

A novel method is introduced for the estimation of grain numbers in autoradiographic sections of articular cartilage with an image analyzer. It is based on separation of grains from the underlying structures by gray level thresholding and determination of the percentage of total area occupied by grains in a relatively large measuring field. The mean grain size is used as a reference to calculate grain numbers per cell profile and per unit area of tissue in various zones of bovine articular cartilage labelled with 35S-sulphate in tissue culture. The results demonstrate considerable zonal differences as well as site related topographic variation in the rate of 35S-sulphate incorporation. The largest site-related variation in the grain counts was observed in the superficial zone, suggesting a delicate control of proteoglycan synthesis in this zone.     

Determination os sedimentation coefficient distributions for cartilage proteoglycans

Sedimentation coefficient distributions of widely polydisperse proteoglycan preparations were made using a previously developed transport sedimentation methodology. Boundary stability was improved by centrifuging samples in a preformed CsCl density gradient (0.016 g/cm⁴). The results were compared with the distributions obtained with an interferometric analytical centrifugation method. When these two techniques were applied to analyze A1 and A1–D1 proteoglycan preparations, results were in substantial agreement with respect to the mean sedimentation coefficients of the peaks, average S value, sedimentation coefficient distribution, skewness, proportion of monomer and aggregates, and linearity of the plot ln(s) versus C extrapolations to zero concentration. The lower solute concentration compatible with the transport (velocity gradient) method makes this technique particularly suitable for studying the details of proteoglycan distribution of molecular sizes, especially for aggregates.     

Quantitation of autoradiographic grains in different zones of articular cartilage with image analyzer

Summary A novel method is introduced for the estimation of grain numbers in autoradiographic sections of articular cartilage with an image analyzer. It is based on separation of grains from the underlying structures by gray level thresholding and determination of the percentage of total area occupied by grains in a relatively large measuring field. The mean grain size is used as a reference to calculate grain numbers per cell profile and per unit area of tissue in various zones of bovine articular cartilage labelled with ³⁵S-sulphate in tissue culture. The results demonstrate considerable zonal differences as well as site related topographic variation in the rate of ³⁵S-sulphate incorporation. The largest site-related variation in the grain counts was observed in the superficial zone, suggesting a delicate control of proteoglycan synthesis in this zone.The IBAS program used in this work is available from Dr. J.J. Parkkinen or through Bitnet or EARN mail: MLAMMI at FINKUO     

A sectorial centrifuge cell for swinging bucket rotors--application to a velocity gradient centrifugation methodology

A sectorial cell of 1.55 ml capacity, designed to be used in swinging bucket rotors, is introduced and applied to boundary sedimentation studies. The cell, made of polycarbonate by injection molding, represents an improvement over previously existing models in terms of resistance and attainable speeds. Its use has been extended to polydispersity determination by evaluation of the s value distribution function g(s) = dC/ds. The latter can be corrected to standard conditions (water at 20 degrees C), by using newly derived equations in connection with a cesium sulfate linear density and viscosity gradient which is introduced for boundary stabilization. The cell performance is illustrated with the centrifugal characterization of a polydisperse hyaluronate solution and a paucidisperse proteoglycan A1 preparation. In the latter case a distinctive distribution of aggregates into two polydisperse families of molecules, hitherto not reported in the literature but previously observed with cylindrical cells in this laboratory, has been clearly confirmed. Analysis of plateau dilution during centrifugation indicated absence of artifacts.     

Size-separation of yeast mitochondria in the zonal centrifuge

Mitochondria, released from yeast spheroplasts and subjected to rate separation through sorbitol gradients in the zonal centrifuge, migrated in a wide symmetrical zone. Electron micrographs showed that the mitochondria had been resolved within the zone according to size. The mean mitochondrial diameter at the leading edge was approximately twice that at the trailing edge of the particle zone. Activities of the enzymes cytochrome oxidase, malate dehydrogenase, and reduced nicotinamide adenine dinucleotide- and d-lactate cytochrome c reductases were essentially uniform throughout the mitochondrial zone. Mitochondria from a vegetative-petite mutant had almost the same size distribution as the isogenic wild type, but with somewhat larger mean diameter and either absent or markedly reduced enzyme activities. Mixtures of wild-type and petite mitochondria produced sedimentation profiles showing overlap of particle populations with respect to mean sedimentation rates and mitochondrial diameters, as well as intermediate levels of enzyme activities. Both cristate and noncristate organelles were present throughout the mitochondrial zone from these mixtures. Mitochondria centrifuged in sorbitol density gradients were well-preserved and yielded consistent sedimentation profiles, whereas particles in sucrose density gradients migrated more slowly, produced varied sedimentation profiles, and often showed spurious peaks, presumably due to particle aggregations.     

Band centrifugation of macromolecules in self-generating density gradients. II. Sedimentation and diffusion of macromolecules in bands

Three approaches to the simultaneous sedimentation and diffusion of hands or zones of noninteracting homogeneous macromolecules are examined: (1) The authors' method of moments: (2) the transport me of Sehumaker and Rosenbloom; and (3) the stochastic solution of the Lamm equation due to Gehatia and Katehalski. All three methods indicate that the motion of the maximum of the hand may be used to evaluate the sedimentation coefficient. The moment, method provides relations which appear to be useful for measuring diffusion coefficients. Relations are given for the analysis of resolved components. The problem of measuring sedimentation coefficients of macromolecules with concentration-dependent sedimentation coefficients is examined. Methods are described for evaluating the sedimentation coefficient in these systems and for obtaining the sedimentation coefficient at infinite dilution. Methods are described for determining the weight-average sedimentation coefficient in Multi-component systems, and the differential and integral distribution of sedimentation coefficients of macromolecules with low-diffusion coefficients.     

Molecular weight distribution profile of cartilage proteoglycan in aqueous guanidinium hydrochloride before and after carboxyl group modification.

The molecular weight distribution profile of a proteoglycan preparation (s^o_20.w = 23.1 S), isolated from bovine nasal cartilage in the presence of protease inhibitors, was studied by equilibrium sedimentation in 4 m guanidinium hydrochloride. Apparent reduced molecular weights ranged from 0.8 to 2.2 × 10⁶ and their concentration dependence appeared to be compatible with the presence of a heterogeneous population of self-associating macromolecules. Carbodiimide-induced modification of about 20% of the total carboxyl groups of the complex resulted in a shift of the molecular weight distribution profile, the new values ranging from 6 to 9 × 10⁵, with a marked predominance of the larger species. Exposure of the proteoglycan to carbodiimide or methylamine alone produced only a small shift of the apparent molecular weights. Moreover, chondroitin 4-sulfate molecules subjected to the same carbodiimide-promoted modification showed no significant change in their average molecular weights. It is therefore considered that the changes observed after carboxyl group modification cannot be attributed to cleavage of either the protein or the carbohydrate moiety of the proteoglycan complex. Rather, the evidence suggests that these functions are essential to the stabilization of the oligomeric species, which constitute a large proportion of the preparation even in 4 m guanidinium hydrochloride.     

Preconstruction of density gradients in cells of the analytical ultracentrifuge

The principles and techniques of zonal centrifugation are now well established (1), but despite the advantage of requiring smaller amounts of material than conventional experiments, this procedure has not been widely applied to the analytical ultracentrifuge. In general, the method has been limited to experiments where separations are based on differences in density. Usually, this involves the generation in situ of gradients, and has been widely used in the analysis of nucleic acids (3,4); recently, the scope of this technique was enlarged by a method for fractionating the zones produced by this type of separation (5). However, Rosenbloom and Schumaker (2) showed that a preformed gradient can be constructed in the analytical cell prior to running, and this stabilised the sedimentation of a boundary or a zone of nucleic acid. Although the use of a preformed density gradient (containing initially a uniform distribution of the macromolecule(s)) could reduce the time to reach isopycnic equilibrium it is not a prerequisite for the experiment, however, a preformed gradient is essential when measuring the velocity of a zone, as in Cohen, Giraud, and Messiah (8). This communication describes a simple technique for generating density gradients in the analytical cell prior to running and with the minimum of disturbance.     

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.     

Isolation and characterization of high-buoyant-density proteoglycans from bovine femoral-head cartilage.

Proteoglycans were extracted from bovine (15-18 months old) femoral-head cartilage. The heterogeneity of the A1D1 proteoglycan fraction was examined by gel chromatography, sedimentation velocity, sucrose rate-zonal centrifugation and CS2SO4 isopycnic centrifugation. In all cases polydisperse but unimodal distributions were obtained. Chemical analysis of the preparation yielded a galactosamine/glucosamine molar ratio of 7:1, and 13C n.m.r. spectroscopy showed that the chondroitin sulphate comprised equal proportions of the 4- and 6-sulphate isomers. Gel chromatography of a papain and Pronase digest of the proteoglycan indicated that the chondroitin sulphate chains had a Mn of approx. 10500. The mean buoyant density of the proteoglycan in pure CS2SO4 was 1.46 g/ml. Physical characterization of the proteoglycan preparation in 4M-guanidine hydrochloride, pH 7.4, by using conventional light-scattering gave a radius of gyration of 42 nm and a Mw of 0.96 X 10(6). Quasi-elastic light-scattering in the same solvent yielded a translational diffusion coefficient, D020, of 5.41 X 10(-8) cm2 X S-1, and ultracentrifugation gave a sedimentation coefficient, S020, of 12.0S. Thus from sedimentation-diffusion studies a Mw of 1.36 X 10(6) was calculated. The possible origins for the differences in the two molecular-weight estimates are discussed. It is concluded that the high-buoyant-density proteoglycans from bovine articular cartilage are significantly smaller than those from bovine nasal septum, and that this is largely due to the smaller size of their chondroitin sulphate chains.     

Effect of oxygen-derived reactive species on cartilage proteoglycan-hyaluronate aggregates

Proteoglycan-hyaluronate aggregates were incubated with oxygen-derived reactive species generated enzymatically by the action of xanthine oxidase upon hypoxanthine. Analysis of the products of the incubation by caesium sulphate zonal sedimentation revealed that degradation of aggregate had occurred. This effect was reversed by inclusion of superoxide dismutase, catalase or diethylenetriaminepentaacetic acid in the incubations suggesting that hydroxyl radicals were the active species. Separate analysis by gel filtration chromatography on Sepharose CL-2B of proteoglycan monomer subjected to a similar treatment indicated that the molecule is minimally degraded. These results are discussed with reference to the well established degradation of hyaluronate by oxygen-derived reactive species.     

Zones of influence for soil organic matter dynamics: A conceptual framework for data and models

Soil organic matter (SOM) is an indicator of sustainable land management as stated in the global indicator framework of the United Nations Sustainable Development Goals (SDG Indicator 15.3.1). Improved forecasting of future changes in SOM is needed to support the development of more sustainable land management under a changing climate. Current models fail to reproduce historical trends in SOM both within and during transition between ecosystems. More realistic spatio‐temporal SOM dynamics require inclusion of the recent paradigm shift from SOM recalcitrance as an ‘intrinsic property’ to SOM persistence as an ‘ecosystem interaction’. We present a soil profile, or pedon‐explicit, ecosystem‐scale framework for data and models of SOM distribution and dynamics which can better represent land use transitions. Ecosystem‐scale drivers are integrated with pedon‐scale processes in two zones of influence. In the upper vegetation zone, SOM is affected primarily by plant inputs (above‐ and belowground), climate, microbial activity and physical aggregation and is prone to destabilization. In the lower mineral matrix zone, SOM inputs from the vegetation zone are controlled primarily by mineral phase and chemical interactions, resulting in more favourable conditions for SOM persistence. Vegetation zone boundary conditions vary spatially at landscape scales (vegetation cover) and temporally at decadal scales (climate). Mineral matrix zone boundary conditions vary spatially at landscape scales (geology, topography) but change only slowly. The thicknesses of the two zones and their transport connectivity are dynamic and affected by plant cover, land use practices, climate and feedbacks from current SOM stock in each layer. Using this framework, we identify several areas where greater knowledge is needed to advance the emerging paradigm of SOM dynamics—improved representation of plant‐derived carbon inputs, contributions of soil biota to SOM storage and effect of dynamic soil structure on SOM storage—and how this can be combined with robust and efficient soil monitoring.     

A new approximate whole boundary solution of the Lamm differential equation for the analysis of sedimentation velocity experiments

Sedimentation velocity is one of the best-suited physical methods for determining the size and shape of macromolecular substances or their complexes in the range from 1 to several thousand kDa. The moving boundary in sedimentation velocity runs can be described by the Lamm differential equation. Fitting of suitable model functions or solutions of the Lamm equation to the moving boundary is used to obtain directly sedimentation and diffusion coefficients, thus allowing quick determination of size, shape and other parameters of macromolecules. Here we present a new approximate whole boundary solution of the Lamm equation that simultaneously allows the specification of sedimentation and diffusion coefficients with deviations smaller than 1% from the expected values.     

Hydrodynamics of concentrated proteoglycan solutions

The dynamics of water transport in proteoglycan compartments has been studied in relation to osmotic flow (proteoglycan diffusion) and hydraulic permeability (proteoglycan sedimentation) in concentrated solutions of proteoglycan subunit and native proteoglycan aggregate isolated from Swarm rat chondrosarcoma. A central parameter that describes the kinetics of both types of water movement is the hydrodynamic frictional coefficient of water with proteoglycan. The frictional coefficient is markedly concentration dependent, increasing with increasing concentration, and highlights important structural features and types of organization of the proteoglycans in concentrated solutions. These include the requirements that proteoglycans in the extracellular matrix not to be immobilized but to have translational diffusive mobility and concentration gradients to be osmotically active, that chondroitin sulfate segmental mobility describing translational motion largely determines osmotic flow and hydraulic permeability of the proteoglycans, and that the proteoglycans exhibit an enhanced ability to resist flow as compared to other macromolecules. Additional dynamic studies suggest the formation of transient super-aggregate structures may occur at high concentrations which endows the proteoglycan subunit hydrodynamic properties similar to proteoglycan aggregate.     

Ultrastructure of elastic cartilage in the rat external ear

Summary The structure of elastic cartilage in the external ear of the rat was investigated by transmission and scanning electron microscopy.The narrow subperichondrial, boundary zone contains predominantly ovoid cells rich in cell organelles: mitochondria, Golgi complex, granular endoplasmic reticulum and small (40–100 nm) vesicles. Scarce glycogen granules and bundles of 6–7 nm cytoplasmic filaments are also present. Deeper in the boundary zone, one or more cytoplasmic lipid droplets appear and cytofilaments become more abundant.Fully differentiated chondrocytes in the central zone of the cartilage plate resemble white adipose cells. They are globular and contain a single, large cytoplasmic lipid droplet. The cytoplasm is reduced to a thin peripheral rim; it contains a flattened nucleus, few cytoplasmic organelles and abundant, densely packed, cytoplasmic filaments.The intercellular matrix is very sparse. The pericellular ring consists of collagen fibrils about 20 nm in diameter and a proteoglycan cartilage matrix in the form of a stellate reticulum. The complex of these two structures appears in the scanning electron micrographs as a network of randomly oriented, ca 100 nm thick fibrils. Spaces between pericellular rings of matrix also contain thick elastic fibers or plates, apparently devoid of microfibrils. In scanning electron micrographs elastic fibers could be detected only in a few areas, in which they were not obscured by other constituents of the matrix. Immature forms of elastic fibers, oxytalan (pre-elastic) and elaunin fibers, were found in the perichondrial and boundary zones.     

Secretion pattern, ultrastructural localization and function of extracellular matrix molecules involved in eggshell formation

The chicken eggshell is a composite bioceramic containing organic and inorganic phases. The organic phase contains, among other constituents, type X collagen and proteoglycans (mammillan, a keratan sulfate proteoglycan, and ovoglycan, a dermatan sulfate proteoglycan), whose localization depends on a topographically defined and temporally regulated deposition. Although the distribution of these macromolecules in the eggshell has been well established, little is known about their precise localization within eggshell substructures and oviduct cells or their pattern of production and function during eggshell formation. By using immunofluorescent and immuno-ultrastructural analyses, we examined the distribution of these macromolecules in oviduct cells at different post-oviposition times. To understand the role of proteoglycan sulfation on eggshell formation, we studied the effects of inhibition of proteoglycan sulfation by treatment with sodium chlorate. We showed that these macromolecules are produced by particular oviduct cell populations and at precise post-oviposition times. Based on the precise ultrastructural localization of these macromolecules in eggshell substructures, the timing of the secretion of these macromolecules by oviduct cells and the effects on eggshell formation caused by the inhibition of proteoglycan sulfation, the putative role of mammillan is in the nucleation of the first calcite crystals, while that of ovoglycan is to regulate the growth and orientation of the later forming crystals of the chicken eggshell.