The exclusion of large solutes by cartilage proteinpolysaccharide

Abstract

synopsisThe dialysis technique of Ogston and Phelps, using a Millipore filter as a membrane to measure the effect of a nondialyiable macromolecule on the distribution of dialyzable macromolecules, has been used to measure the effect of a compound of protein and polysaccharide (PP-L), isolated from cartilage, on the distribution of some dialyzable macromolecules. PP-L (1%) in buffer acts as if part of its solution volume were unavailable as follows: 55% for serum albumin, 64% for chondroitin sulfate made by alkaline degradation of PP-L, and 98% for chondroitin sulfate made by trypsin degradation of PP-L. The use of different buffers, variation of pH from 5 to 10, variation of temperature from 5 to 42"C., variation of salt concentration from 0.15 to 2N, and variation in ethanol concentration from 0 to 30% had no effect on the distribution of albumin, which was always excluded to about the same extent from the PP-L solution. This effect could account for the virtual absence of serum proteins and of free chondroitin sulfate from cartilage. At low salt concentration (0.10-0.02N) and a t 5°C. the distribution w a~ reversed and albumin behaved as if strongly bound by PP-L. At low levels of albumin this binding of PP-L increased linearly with albumin concentration. This binding was partly reversed by raising the temperature or by adding urea, and completely reversed by the addition of salt. Polystyrene sulfonate, a nondiffusible polyanion, had an effect similar to that of PP-L in excluding chondroitin sulfate from its solutions. It showed a very strong binding effect for albumin which persisted even in the presence of salt as high as 2N. A quantitative comparison has been made between the excluded volume effect of the soluble macromolecules PP-L and hyaluronate and the effect of insoluble Sephadex particles to exclude large molecules from their interiors. The terhnique of Ogston and Phelps is thus valuable not only for measuring the exclusion of diffusible macromolecules from part of the solution volume of nondiffusible macromolecules, but also for measuring the existence and extent of binding of diffusible macromolecules to nondiffusible macromolecules.

Evidence for a role of proteinpolysaccharides in regulation of mineral phase separation in calcifying cartilage

“…The virtual exclusion of serum proteins from such cartilages was postulated by Gerber and Schubert (21); thus the nature and function of this new pool of interstitial proteins is being investigated. ADDENDUM Additional support to the conclusion reached in this paper was found in analyses of well-characterized proteinpolysaccharides provided us recently by Dr. Lawrence Rosenberg.…”

Permeability of composite chondrocyte-culture-millipore membranes to solutes of varying size and shape

“…Various studies have been made into the diffusive permeability of certain connective tissues such as intervertebral disc (Paulson et al, 1951), articular cartilage (Kantor & Schubert, 1957;Brower et al, 1962;Gerber & Schubert, 1964;Maroudas, 1970;Lotke & Granda, 1972) and cornea (Hedbys, 1963). The difficulties that present themselves in studying Vol.…”

Recent Trends in the Biochemistry of the Ground Substance of the Connective Tissues

“…Application of milder methods, such as physical disintegration of the tissue by high speed homogenisation, yields the acid glycosaminoglycans linked firmly to protein moieties by covalent bonds (see for example Shatton and Schubert, 1954;Gerber, Franklin and Schubert, 1960). Erroneous implications should not be drawn from the persistent use of the generic nomenclature of polysaccharide-protein complexes for these materials when obviously the term compound would be far more appropriate.…”

“…However, it has been shown that bovine nasal septum cartilage yields two varieties of chondroitin sulphate-protein complex, distinguished by their differential sedimen-tation in the ultracentrifuge (Gerber, Franklin and Schubert, 1960). The large particle fraction (designated PP-H) has a substantially higher protein content than the lighter (socalled PP-L) fraction.…”

“…The interaction of each domain is envisaged as producing a three dimensional network, the pore size of which must obviously be governed by the degree of solvation, the intensity of charge and the chemical and physical fine structure of each complex. Experimental evidence suggests that apart from a differential ability to bind cations according to the nature of the constituent glycosaminoglycans (Dunstone, 1960(Dunstone, , 1962Mathews, 1964) such three-dimensional molecular organizations should also be able to entangle particles (Laurent, 1964) and exclude large solutes (Gerber and Schubert, 1964). Viewed in this way it is tempting to suggest that the apparently precise biological allocation of structurally different anionic polysaccharideprotein complexes is a direct function of their variable properties in controlling ion-binding, water content and even the porosity of the tissue as a whole.…”