Macro and micro rate zonal analytical centrifugation of polydisperse and slowly diffusing sedimenting systems in isovolumetric density gradients. Application to cartilage proteoglycans

A method to study the polydispersity of zonally sedimenting and slowly diffusing macromolecules or particles in isokinetic or isovolumetric density gradients is presented. First, a brief theory is given for predicting the zonal profile after a "triangular" (or "inverse") zone is centrifuged. This type of zone is essential to preserve hydrodynamic stability of the very slowly diffusing polydisperse solutes. It is proven, both by semitheoretical considerations and by computer calculations, that the resulting concentration profile of macrosolute is almost identical with that obtainable with a rectangular zone coextensive with the triangular one and carrying the same total mass. Next, practical procedures are described for the convectionless layering of very small triangular zones (50 microL or less). The linearity and stability of the zones are experimentally tested and verified. Finally, the method is applied to cartilage proteoglycan preparations that included either the monomeric molecules only or both the monomeric and the aggregated ones. The zonal results are compared with those obtained by using conventional boundary sedimentation. The two sets of results are seen to coincide fairly well, thus proving that the present technique can add to preparative zonal centrifugation the analytical precision of boundary sedimentation. A multimodal polydisperse system is suggested to describe the aggregated proteoglycan macromolecules.     

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.     

Band centrifugation of macromolecules in self-generating density gradients. III. Conditions for convection-free band sedimentation

The conditions for convection-free hand sedimentation are analyzed in terms of the negative density gradients associated with the leading edge of a band and the positive density gradients generated during the experiment. The amount of material necessary to perform a band-centrifugation experiment depends on the diffusion coefficient of the macromolecules, which determines the rate at which the concentration at band maximum decreases, and on the extinction coefficient at the wavelength of observation. The maximum negative gradients in bands of macromolecules with diffusion and extinction coefficients typical of proteins, nucleic acids, and viruses are calculated. Positive density gradients generated by diffusion of small molecules between the thin lamella and the bulk solution are calculated for bulk solutions such us 1M NaCl or 95% D₂O. These “diffusion gradients” are generally adequate to stabilize bands of the above macromolecules. Positive density gradients generated by sedimentation of salts within the bulk solution may be significant in providing stability near the bottom of the cell. The effects of inadequate stabilizing gradients are discussed, and are found to cause forward spreading of the band.     

Respective roles of high and low molecular weight serum growth factors in the metabolism of proteoglycans and other cartilage macromolecules

35S radiolabeling allowed an evaluation to be made of neosynthesized macromolecules in chick embryo cartilage cultures. Activities for growth factors of high (serum retentate) or low (ultrafiltrate below 1,000) molecular weight (MW) were assessed in pelvic cartilage explants and in corresponding incubation media. In the absence of growth factor, 35S was mostly incorporated in glycosaminoglycans (GAGs) as regards the medium and for cartilage, in guanidinium chloride unextractable material. In retentate-enriched medium, 35S incorporation was enhanced in all cartilage GAGs while in the medium, stimulation essentially occurred in macromolecules other than GAGs. Low MW growth factors exclusively enhanced cartilage levels of macromolecules which were insoluble in guanidinium chloride. In the medium, these factors did not display any significant effect. These results indicate that human serum growth factors with high and low MW possess different metabolic targets at the cellular level.     

Mathematics of band centrifugation: Concentration-independent sedimentation and diffusion in shallow density gradients

Integral expressions for concentration as a function of time and distance are derived from the continuity equation for centrifugation in a sector-shaped cell for a macro-molecular solute initially contained in a finite upper layer and a solute of low molecular weight in the supporting liquid. Computer patterns based on the sedimentation and diffusion coefficients of sucrose and of spherical and randomly coiled model solutes illustrate: (1) the time course of redistribution of both banded and supporting solutes from initial uniform concentrations; (2) the influence of the initial concentration, width, and solute concentration of the upper band; and (3) the effect of restricted diffusion at the meniscus on subsequent band shape. A Gaussian, approximation to band shape is derived and graphically tested. Rapid methods, not requiring computers, are out lined for the estimation of sedimentation and diffusion coefficients, where their concentration dependence is negligible, by band centrifugtion. The theoretical resolution of mixtures attainable by this technique is compared with moving-boundary centrifugation, with the use of both integral (interferotmetric or absorption) and derivative (schlieren) optics.     

Preparative density gradient centrifugation of RNA and DNA in cesium sulfateurea mixture

A new procedure is described for a large scale separation and purification of unfixed DNA and RNA from a mixture of partially extracted nucleic acids and lysates of subcellular fractions by centrifugation to equilibrium in cesium sulfate-urea mixture. Optimum conditions are described for the separation and quantative recovery of both RNA and DNA in a pure form. The procedure allows determination of peak buoyant densities of 4–5s RNA, 7–11s mRNA and total cytoplasmic RNA. The procedure also allows fractionation of small molecular weight classes of cytoplasmic RNAs from the 18s and 28s rRNAs.     

Biosynthesis of proteoglycans in cartilage slices. Fractionation by gel chromatography and equilibrium density-gradient centrifugation

The kinetics of incorporation of [(35)S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ;pulse-chase' radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ;pulse-chase' experiments over 5h did not demonstrate any precursor-product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ;pulse-chase' experiments there was again no evidence for precursor-product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.     

RNA isolation from cartilage using density gradient centrifugation in cesium trifluoroacetate: an RNA preparation technique effective in the presence of high proteoglycan content.

An efficient method for the isolation of RNA from cartilage is described. The difficulties in obtaining RNA from cartilage, a tissue of low cell density and high proteoglycan content, were overcome by making several modifications to the guanidine thiocyanate/cesium chloride method of RNA extraction. Cartilage tissue is frozen, crushed, and homogenized in a 4 M guanidine thiocyanate lysis buffer. The RNA is then pelleted by ultracentrifugation through a cesium trifluoroacetate density gradient. The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA isolated from cartilage. The ultracentrifugation has been adapted to the Beckman TL100 tabletop centrifuge and is complete in 3 h. This fast, simple method produces high quality RNA, suitable for use in RNase protection assays, polymerase chain reaction analysis, and Northern analysis. This purification procedure may be applicable to other sources, from which RNA isolation is complicated by the presence of abundant cell wall or matrix components.     

Distribution in cesium chloride gradients of proteoglycans of chick embryo brain and characterization of a large aggregating proteoglycan

Proteoglycans were extracted from 14-day chick embryo brains, which had been labelled in vitro with [35S]sulfate or 3H-labelled amino acids. 4.0 M guanidinium chloride (containing proteinase inhibitors) extracted 94% of the 35S-labelled glycoconjugates. Following cesium chloride equilibrium centrifugation, the proteoglycans in each fraction were characterized by chromatography on Sepharose CL-2B. The most dense fraction (D1), which contained no detectable non-proteoglycan proteins, contained a large, aggregating chondroitin sulfate proteoglycan in addition to small chondroitin sulfate and heparan sulfate proteoglycans. The less dense fractions (D2-D6) contained both small chondroitin sulfate and heparan sulfate proteoglycans. Removal of hyaluronate from the D1 sample by digestion with Streptomyces hyaluronidase in the presence of proteinase inhibitors showed that aggregation of the large chondroitin sulfate proteoglycan is hyaluronate-dependent. Aggregation was restored by re-addition of hyaluronate. Reduction and alkylation, which blocked aggregation of a cartilage A1 proteoglycan, did not interfere with aggregation of the large brain proteoglycan.     

Isolation and characterization of cartilage proteoglycans immunoreactive with an antibody to skin proteodermatan sulfate core protein

Low density proteoglycans showing cross-reaction with an antibody to skin proteodermatan sulfate (PDS) core protein were isolated from the bovine articular cartilage, by CsC1 density gradient centrifugation followed by repeated DEAE-cellulose chromatography. The size and amino acid composition of the core proteins of the immunoreactive proteoglycans, eluted at 0.25M and 0.5M NaC1 on DEAE-cellulose column, were quite similar to that of PDS. The glycosaminoglycan components of both proteoglycans were shown to be composed of a hybrid structure of chondroitin sulfate and dermatan sulfate, based on chondroitinase treatments followed by two-dimensional electrophoresis.     

From barriers to bridges: chondroitin sulfate proteoglycans in neuropathology

Emerging studies have revealed new roles for the neural extracellular matrix in neuropathologies. The structure of this matrix is unusual and uniquely enriched in chondroitin sulfate proteoglycans, particularly those of the lectican family. Historically, lecticans have attracted considerable interest in the normal and injured brain for their prominent roles as inhibitors of cellular motility, neurite extension and synaptic plasticity. However, these molecules are structurally heterogeneous, have distinct expression patterns and mediate unique interactions, suggesting that they might have other functions in addition to their traditional role as chemorepulsants. Here, we review recent work demonstrating unique modifications and structural microheterogeneity of the lecticans in the diseased CNS, which might relate to novel roles of these molecules in neuropathologies.     

Changes in the cartilage proteoglycans in relation to age and osteoarthrosis

Changes in the extracellular components of various connective tissues with time and continuous use, as well as within experimentally-induced and human pathological conditions, were studied by morphological, chemical and biochemical methods. Osteoarthrotic lesions were produced surgically by implantation of polyethylene sheets, by continuous compression of the knee joint, and by intraarticular papain injection. Results of the model experiments showed that, irrespective of the method used for the production of experimental cartilage lesions, the alterations are strikingly similar. Within connective tissues a gradual deterioration takes place, as measured by marked differences in the histological and histochemical patterns. The degradative changes occur primarily in the matrix, resulting in a significant decrease in proteoglycan content. Under conditions of stress, in young animals, chondrocytes seem to revert to the chondroblastic state, showing mitotic figures, and are capable of producing matrix much more rapidly than is normally seen. This increased activity might be regarded as a nonspecific "feed-back" response of cells, leading to a reparative normalization of the various destructive tissue alterations.     

Microvilli of the human term placenta. Isolation and subfractionation by centrifugation in sucrose density gradients.

Human placental microvilli were isolated and separated into two fractions by centrifugation in sucrose density gradients. Electron-microscopic morphology and morphometry, the distribution of enzymic activities and the results of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of proteins were used to assess the purity of the final preparations and to define their properties. The combined evidence strongly suggested that the preparations contained negligible material that was not plasma membrane. The two fractions of microvilli differed in buoyant density, protein composition, enzyme specific activities and microscopic appearance. Some of these differences were explained by the absence of internal structure in the microvilli of the lighter fraction.     

Binding of renin and angiotensinogen to macromolecules in plasma studied by sedimentation equilibrium centrifugation

Plasma from normal and aggressive mice was subjected to sedimentation equilibrium centrifugation in an air-driven centrifuge. The aim was to study the apparent molecular weight of renin and angiotensinogen in undiluted plasma. Both renin and angiotensinogen appeared heterogeneous with respect to molecular size, suggesting binding to plasma macromolecules. Subsequent high pressure liquid chromatography, using a size exclusion column, demonstrated molecular homogeneity and molecular weights as found in noncentrifuged plasma, indicating that the binding is easily reversible. It is concluded that renin and angiotensinogen in undiluted and unfractionated plasma are weakly bound to plasma macromolecules. This may reduce their activity and to some extent explain the previously observed apparent inhibition of the enzymatic activity.     

The dermatan sulfate proteoglycans of bovine sclera and their relationship to those of articular cartilage. An immunological and biochemical study

Dermatan sulfate proteoglycans were isolated from adult bovine sclera and adult bovine articular cartilage. Their immunological relationships were studied by enzyme-linked immunosorbent assays using polyclonal antibodies raised against the large and small dermatan sulfate proteoglycans from sclera and a polyclonal and monoclonal antibody directed against the small dermatan sulfate proteoglycans from cartilage. The small dermatan sulfate proteoglycans from sclera and cartilage displayed immunological cross-reactivity while there was no convincing evidence of shared epitope(s) with the larger dermatan sulfate proteoglycans, nor did these larger proteoglycans share any common epitopes with each other. A hyaluronic acid binding region was detected immunologically on the larger scleral dermatan sulfate proteoglycan but was absent from the larger dermatan sulfate proteoglycan of cartilage and both the small dermatan sulfate proteoglycans. These antibodies were used in immunofluorescence microscopy to localize the scleral proteoglycans and molecules containing these epitopes in the eye. The large scleral dermatan sulfate proteoglycan was restricted to sclera while molecules related to the small scleral and cartilage proteoglycans were found in the sclera, anterior uveal tract, iris, and cornea. Amino acid sequencing of the amino-terminal regions of the core proteins of the small dermatan sulfate proteoglycans from sclera and articular cartilage showed that all the first 14 amino acids analyzed were identical and the same as reported earlier for the small bovine skin and tendon dermatan sulfate proteoglycans. These studies demonstrate that the larger dermatan sulfate proteoglycans of sclera and cartilage are chemically unrelated to each other and to the smaller dermatan sulfate proteoglycans isolated from these tissues. The latter have closely related core proteins and probably represent a molecule with a widespread distribution in which the degree of epimerization of glucuronic acid and iduronic acid varies between tissues.