Direct measurement of osmotic pressure of glycosaminoglycan solutions by membrane osmometry at room temperature

Articular cartilage is a hydrated soft tissue composed of negatively charged proteoglycans fixed within a collagen matrix. This charge gradient causes the tissue to imbibe water and swell, creating a net osmotic pressure that enhances the tissue's ability to bear load. In this study we designed and utilized an apparatus for directly measuring the osmotic pressure of chondroitin sulfate, the primary glycosaminoglycan found in articular cartilage, in solution with varying bathing ionic strength (0.015 M, 0.15 M, 0.5 M, 1 M, and 2 M NaCl) at room temperature. The osmotic pressure (pi) was found to increase nonlinearly with increasing chondroitin sulfate concentration and decreasing NaCl ionic bath environment. Above 1 M NaCl, pi changes negligibly with further increases in salt concentration, suggesting that Donnan osmotic pressure is negligible above this threshold, and the resulting pressure is attributed to configurational entropy. Results of the current study were also used to estimate the contribution of osmotic pressure to the stiffness of cartilage based on theoretical and experimental considerations. Our findings indicate that the osmotic pressure resulting from configurational entropy is much smaller in cartilage (based on an earlier study on bovine articular cartilage) than in free solution. The rate of change of osmotic pressure with compressive strain is found to contribute approximately one-third of the compressive modulus (H(A)(eff)) of cartilage (Pi approximately H(A)(eff)/3), with the balance contributed by the intrinsic structural modulus of the solid matrix (i.e., H(A) approximately 2H(A)(eff)/3). A strong dependence of this intrinsic modulus on salt concentration was found; therefore, it appears that proteoglycans contribute structurally to the magnitude of H(A), in a manner independent of osmotic pressure.     

The distributions and diffusivities of small ions in chondroitin sulphate, hyaluronate and some proteoglycan solutions

The distributions and diffusivities of Na+, Ca2+ and Cl- in chondroitin sulphate (CS), hyaluronate (HA) and proteoglycan solutions were measured using equilibrium dialysis and a capillary tube method. Measurements were made for a range of glycosaminoglycan (GAG) concentrations up to those normally found in dense connective tissue (10% CS, 2.5% HA), ionic strengths up to normal physiological concentrations (0.15 M) and for different combinations of monovalent and divalent cations. The partition coefficients, Ki, of the positive ions increased with increasing matrix concentration and with decreasing ionic strength but with one exception the selectivity coefficient KCaNa = square root of KCa/KNa was close to unity, indicating nearly ideal Donnan distributions. The ionic diffusivities decreased very much like those of small neutral solutes with increasing matrix concentration and with one exception were relatively independent of ionic strength, The exception in both cases was low matrix concentrations and low ionic strengths for which the diffusivity of Ca2+ was an order of magnitude lower and selectivity coefficients were approximately 2. We conclude that at physiological ionic strengths and GAG concentrations the distributions of small ions are determined by simple electrostatic interactions, without binding or condensation, and the diffusivities are not affected by the electrostatic field.     

The sulphation of chondroitin sulphate in embryonic chicken cartilage

1. Whole tissue preparations and subcellular fractions from embryonic chicken cartilage were used to measure the rate of incorporation of inorganic sulphate into chondroitin sulphate in vitro. 2. In cartilage from 14-day-old embryos, [(35)S]sulphate is incorporated to an equal extent into chondroitin 4-sulphate and chondroitin 6-sulphate at a rate of 1.5nmoles of sulphate/hr./mg. dry wt. of cartilage. 3. Microsomal and soluble enzyme preparations from embryonic cartilage catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate into both chondroitin 4-sulphate and chondroitin 6-sulphate. 4. The effects of pH, ionic strength, adenosine 3'-phosphate 5'-sulphatophosphate concentration and acceptor chondroitin sulphate concentration on the soluble sulphotransferase activity were examined. These factors all influence the activity of the sulphotransferase, and pH and incubation time also influence the percentage of chondroitin 4-sulphate formed.     

Osmotic pressure of DNA solutions and effective diameter of the double helix

A simple osmometer with nuclear filters (polymer films with pores of a preset diameter) were used to measure the osmotic pressure of Col E1 plasmid DNA solutions in the concentration range of 1-4 mg/ml DNA. Linear and open circular DNA forms proved to have the same osmotic pressure within the experimental accuracy. The results of the measurements were used for calculating the second virial coefficient A2 of the solution of DNA segments and the effective chain diameter d eff in the ionic strength range of 10(-2)-0.1 M. As the ionic strength is lowered from 0.1 to 10(-2) M the effective diameter of DNA increases from 80 to 220 A. The results are in rather good agreement with theory and with other experimental data.     

Autoactivation of prolegumain is accelerated by glycosaminoglycans

The cysteine protease legumain participates in several biological and pathological processes including tumour invasion and metastasis. Legumain is synthesized as a zymogen and undergoes pH-dependent autoactivation of the proform in order to reach an enzymatically active form. Here we demonstrate that the naturally occurring polyanionic glycosaminoglycans (GAGs) chondroitin 4-sulphate (C4S), chondroitin 6-sulphate (C6S), chondroitin 4,6-sulphate (C4,6S), heparin, heparan sulphate (HS) as well as chondroitin sulphate (CS)-derived decasaccharides accelerated the autocatalytic activation of prolegumain through ionic interactions in a concentration-, size- and time-dependent manner at pH 4.0. In contrast, at pH 5.0 only C4S and C4,6S were able to promote prolegumain activation, while CS-derived decasaccharides, C6S, heparin and HS lost their effect at this pH.     

Osmotic pressure measurements of ovalbumin and lysozyme mixtures

Ovalbumin and lysozyme have been reported to undergo a mixed association in solutions of low ionic strength. Osmotic pressure experiments were performed on ovalbumin and on lysozyme solutions in 0.06 M cacodylate buffer (I = 0.02, pH = 5.8) at 30 and at 37 degrees C. The individual proteins did not undergo any self-associations at either temperature; these measurements indicated that each of the solutions was nonideal. Osmotic pressure experiments on three blends of lysozyme and ovalbumin at 30 and 37 degrees C could be interpreted in two ways. One interpretation was that a nonideal, nonassociating mixture of A and B was present; for the three solutions the mixed nonideal term BAB was negative. A negative nonideal term is usually interpreted as indicating an association. The other interpretation of the data was as a quasi-ideal mixed association of the type A + B in equilibrium AB.     

Glycosaminoglycans in porcine lung: an ultrastructural study using Cupromeronic Blue

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix contributing to the mechanical properties of connective tissues as well as to cell recognition and growth regulation. The ultrastructural localization of GAGs in porcine lung was studied by means of the dye Cupromeronic Blue in the presence of 0.3 M MgCl₂ according to Scott's critical electrolyte concentration technique. GAGs were observed in locations described as follows. Pleura: Dermatan sulphate (DS) and chondroitin sulphate (CS) attached in the region of the d-band of collagen fibrils, interconnecting the fibrils; heparan sulphate (HS) at the surface of elastic fibers and in the basement membrane of the mesothelium and blood vessels. Bronchial cartilage: Abundant amounts of GAGs were observed in three zones: pericellular, in the intercellular matrix and at the perichondrial collagen. By enzyme digestion a superficial cartilage layer with predominantly CS could be distinguished from a deep zone with CS and keratan sulphate. The structure of the large aggregating cartilage proteoglycan was confirmed in situ. Airway epithelium: HS at the whole surface of cilia and microvilli and in the basement membrane of the epithelial cells. Alveolar wall: CS/DS at collagen fibrils, HS at the surface of elastic fibers and in the basement membranes of epithelium and endothelium.     

Osmotic shock and the strength of viral capsids

Osmotic shock is a familiar means for rupturing viral capsids and exposing their genomes intact. The necessary conditions for providing this shock involve incubation in high-concentration salt solutions, and lower permeability of the capsids to salt ions than to water molecules. We discuss here how values of the capsid strength can be inferred from calculations of the osmotic pressure differences associated with measured values of the critical concentration of incubation solution.     

Binding of substrates and other anions to yeast phosphoglycerate kinase.

1. The binding of all four substrates to yeast phosphoglycerate kinase has been studied using a gel filtration technique. The binding of phosphate and sulphate anions has also been investigated. 2. Two sites for each adenine nucleotide were found, one site being weaker than the other by between 30 and 50-fold. Only one binding site for the phosphoglycerate substrates was found. 3. 1,3-Bisphosphoglycerate (1,3-P2-glycerate) bound to the enzyme approximately 1000 times tighter than the other three substrates, its dissociation constant being 0.06 micrometer at ionic strength 0.15 M. 4. Sulphate and phosphate were mutually competitive and sulphate competed with the binding of all substrates except MgADP. MgADP bound to the enzyme more weakly in the presence of sulphate. The dissociation constant for sulphate binding was 1.6 mM at ionic strength of 0.15 M, and 0.05 mM at ionic strength 0.015 M. 5. These results are consistent with sulphate acting as a competitive inhibitor, as found by kinetic studies at high sulphate concentrations. The activatory effect of sulphate at lower concentrations and the substrate activation phenomea displayed by this enzyme, are interpreted in terms of a two-step dissociation of 1, 3-P2-glycerate. The presence of moderate concentrations of MgATP, 3-phosphoglycerate or sulphate causes acceleration of the rate of dissociation of the product, 1, 3-P2-glycerate, this being the rate-limiting step in the overall enzyme reaction.     

Chondroitin sulfate reduces the friction coefficient of articular cartilage

The objective of this study was to investigate the effect of chondroitin sulfate (CS)-C on the frictional response of bovine articular cartilage. The main hypothesis is that CS decreases the friction coefficient of articular cartilage. Corollary hypotheses are that viscosity and osmotic pressure are not the mechanisms that mediate the reduction in the friction coefficient by CS. In Experiment 1, bovine articular cartilage samples (n=29) were tested in either phosphate buffered saline (PBS) or in PBS containing 100mg/ml of CS following 48h incubation in PBS or in PBS+100mg/ml CS (control specimens were not subjected to any incubation). In Experiment 2, samples (n=23) were tested in four different solutions: PBS, PBS+100mg/ml CS, and PBS+polyethylene glycol (PEG) (133 or 170mg/ml). In Experiment 3, samples (n=18) were tested in three solutions of CS (0, 10 and 100mg/ml). Frictional tests (cartilage-on-glass) were performed under constant stress (0.5MPa) for 3600s and the time-dependent friction coefficient was measured. Samples incubated or tested in a 100mg/ml CS solution exhibited a significantly lower equilibrium friction coefficient than the respective PBS control. PEG solutions delayed the rise in the friction coefficient relative to the PBS control, but did not reduce the equilibrium value. Testing in PBS+10mg/ml of CS did not cause any significant decrease in the friction coefficient. In conclusion, CS at a concentration of 100mg/ml significantly reduces the friction coefficient of bovine articular cartilage and this mechanism is neither mediated by viscosity nor osmolarity. These results suggest that direct injection of CS into the joint may provide beneficial tribological effects.     

The sulphatase of ox liver. XIX. On the nature of the polymeric forms of sulphatase A present in dilute solutions.

Weight-average elution volumes of sulphatase A (an arylsulphate sulphohydrolase, EC 3.1.6.1) from Sephadex G-200 have been determined as functions of protein concentration, pH, ionic strength and temperature. The results are used to calculate the apparent association equilibrium constants for tetramer formation and the associated standard-state thermodynamic parameters. While the apparent association constant decreased from 10(28) to 10(21) M-3 on increasing the pH from 4.5 to 5.6 at ionic strength 0.1, at any particular pH value studied it was relatively insensitive to temperature variation so that deltaH is close to zero and tetramer formation in solution is associated with a positive entropy change. At pH 5.0, increasing the ionic strength from 0.1 to 2 decreased the association constant by a factor of 100. Methylumbelliferone sulphate has no effect on the association of sulphatase A. The equilibrium results are used to define the degree of association of sulphatase A likely to encountered in experiments designed to elucidate its kinetic properties. In the liver lysosome, the tetramer is probably the dominant species. The monomer and tetramer of sulphatase A have similar, or identical, specific activities with nitrocatechol sulphate and 4-methylumbelliferone sulphate as substrates. With nitrocatechol sulphate, sulphatase A shows Michaelis kinetics under conditions where the monomer is the dominant species and non-Michaelis kinetics where the tetramer is dominant. There is apparently a negative cooperativity between the monomer units in the tetramer. In 2 mM sodium taurodeoxycholate and 0.035 M MnCl2, but not in 0.1 M NaCl, the tetramer shows Michaelis kinetics. This is not due to dissociation of the tetramer. The critical micellar concentration of sodium taurodeoxycholate is about 0.8 mM in both 0.1 M NaCl and 0.035 M McCl2 but the aggregation number is greater in the latter.     

Protein interactions in the calf eye lens: interactions between β-crystallins are repulsive whereas in γ-crystallins they are attractive

Non-specific interactions in beta- and gamma-crystallins have been studied by solution X-ray scattering and osmotic pressure experiments. Measurements were carried out as a function of protein concentration at two ionic strengths. The effect of temperature was tested between 7 degrees C and 31 degrees C. Two types of interactions were observed. With beta-crystallin solutions, a repulsive coulombic interaction could be inferred from the decrease of the normalized X-ray scattering intensity near the origin with increasing protein concentration and from the fact that the osmotic pressure increases much more rapidly than in the ideal case. As was previously observed with alpha-crystallins, such behaviour is dependent upon ionic strength but is hardly affected by temperature. In contrast, with gamma-crystallin solutions, the normalized X-ray scattering intensity near the origin increases with increasing protein concentration and the osmotic pressure increases less rapidly than in the ideal case. Such behaviour indicates that attractive forces are predominant, although we do not yet know their molecular origin. Under our experimental conditions, the effect of temperature was striking whereas no obvious contribution of the ionic strength could be seen, perhaps owing to masking by the large temperature effect. The relevance of the different types of non-specific interactions for lens function is discussed.     

OSMOTIC ADJUSTMENT OF PLANTS TO SALINE MEDIA. I. STEADY STATE

Bernstein , Leon . (U. S. Salinity Lab., Riverside, Calif.) Osmotic adjustment of plants to saline media. I. Steady state. Amer. Jour. Bot. 48(10): 909–918. Illus. 1961.—The osmotic pressure (OP) of roots, as well as the OP of aboveground parts of cotton and pepper plants, increase pari passu with increases in the OP of the medium over as wide a range of salinity as would permit any growth. New plasmolytic and cryoscopic techniques yield comparable OP values for roots when dilution of cell sap by the free-space solution and by endosmosis during rinsing of roots are taken into account. Since OP differentials between plant parts and root media are maintained, turgor does not decrease and growth inhibition by salinity cannot be attributed to water stress in the sense of lowered plant turgor. Some alternative mechanisms for osmotic inhibition of growth are considered and the osmotic adjustment process itself is indicated as a likely limiting factor for growth under saline conditions. Osmotic adjustment of cells to salinity depends in part on increased accumulation of ions and also on substitution of monovalent for polyvalent ions. The relative importance of increased salt accumulation, ionic substitution and increased non-electrolyte concentration varies with organ (leaf or root), leaf age and salinity level.     

The strain-dependent osmotic pressure and stiffness of the bovine nucleus pulposus apportioned into ionic and non-ionic contributors

Elucidation of the load-bearing mechanism of the nucleus pulposus (NP) facilitates understanding of the mechanical and metabolic functioning of the intervertebral disc and provides key data for mathematical models. Negatively charged proteoglycans in the NP generate an ionic osmotic pressure, pi(i), which contributes to the tissue's resistance to load and, moreover, is the main mechanism by which the unloaded disc rehydrates. Functionally important, pi(i) has seldom been investigated in situ and, crucially, its variation with strain has not been reported. In a confined compression apparatus, we aimed to apportion the strain-dependent load-bearing mechanism of the NP at equilibrium to the tissue matrix and ionic osmotic pressure; and to determine whether any proteoglycan loss occurs during confined compression testing. Forty-eight confined compression experiments were conducted in isotonic (0.15M NaCl) and hypertonic (3.0 and 6.1M NaCl) external solutions in single and multiple step-strain protocols. The 6.1M NaCl external solution was needed to eliminate as much of the ionic effects as possible. The ionic osmotic pressure was well described by pi(i)=19.1lambda(-1.58) (R(2)=0.992), and was approximately 70% of the applied load at equilibrium, independent of lambda. The effective aggregate modulus, H(A)(eff), also increased with strain: H(A)(eff)=59.0lambda(-2.18). Concentrations of sulphated glycosaminoglycans were obtained for the samples tested in isotonic NaCl with no proteoglycan loss detected from the confined compression tests. These results highlight the non-linearity of the stress-strain response of NP tissue and the necessity to include a non-linear function for osmotic pressure in mathematical models of this tissue.     

Proteoglycan inhibition of calcium phosphate precipitation in liposomal suspensions.

The major proteoglycan in cartilage (aggrecan) is a complex macromolecule with numerous chondroitin sulphate, keratan sulphate, and oligosaccharide substituents. It has been proposed that this macromolecule has an important role in regulating mineralization in this tissue, a process which is initiated by the deposition of apatite in matrix vesicles. We have used a liposome-centred endogenous precipitation method as a model for matrix vesicle mineralization to study the effect of the rat chondrosarcoma aggrecan and its chondroitin sulphate and core protein components on apatite formation from solution. Precipitation was initiated by encapsulating buffered (pH 7.4) 50 mmol/l KH2PO4 solutions in the aqueous centres of 7:2:1 phosphatidylcholine:dicetylphosphate:cholesterol liposomes, adding 2.25-2.65 mmol/l Ca2+ and 1.5 mmol/l total inorganic phosphate (PO4) to the suspending medium (pH 7.4, 22 degrees C), then making the intervening lipid membranes permeable to the Ca2+ ions with the calcium ionophore X-537A. Aggrecan (0.5%) in the suspending medium had no effect on intraliposomal precipitation, but severely reduced (approximately 70% reduction at 24 h) its subsequent spread into the medium. The chondroitin sulphate and core protein were similarly inhibitory. The degree to which aggrecan and its constituent parts inhibited precipitation correlated with their capacity to bind Ca2+ ions. These findings suggest that functional groups in aggrecan blocked apatite growth by linking via Ca2+ bridges to growth sites on the crystal surfaces. Similar Ca-mediated interactions may well have a critical regulatory role in cartilage mineralization.     

Darcy permeability of agarose-glycosaminoglycan gels analyzed using fiber-mixture and donnan models

Agarose-glycosaminoglycan (GAG) membranes were synthesized to provide a model system in which the factors controlling the Darcy (or hydraulic) permeability could be assessed in composite gels of biological relevance. The membranes contained a GAG (chondroitin sulfate) that was covalently bound to agarose via terminal amine groups, and the variables examined were GAG concentration and solution ionic strength. The addition of even small amounts of GAG (0.4 vol/vol %) resulted in a twofold reduction in the Darcy permeability of 3 vol/vol % agarose gels. Electrokinetic coupling, caused by the negative charge of the GAG, resulted in an additional twofold reduction in the open-circuit permeability when the ionic strength was decreased from 1 M to 0.01 M. A microstructural hydrodynamic model was developed, based on a mixture of neutral, coarse fibers (agarose fibrils), and fine, charged fibers (GAG chains). Heterogeneity within agarose gels was modeled by assuming that fiber-rich, spherical inclusions were distributed throughout a fiber-poor matrix. That model accurately predicted the Darcy permeability when the ionic strength was high enough to suppress the effects of charge, but underestimated the influence of ionic strength. A more macroscopic approach, based on Donnan equilibria, better captured the reductions in Darcy permeability caused by GAG charge.     

Ca2+-sensitive cross-bridge dissociation in the presence of magnesium pyrophosphate in skinned rabbit psoas fibers.

We find that at 6 degrees C in the presence of 4 mM MgPPi, at low or moderate ionic strength, skinned rabbit psoas fibers exhibit a stiffness and an equatorial x-ray diffraction pattern similar to that of rigor fibers. As the ionic strength is increased in the absence of Ca2+, both the stiffness and the equatorial x-ray diffraction pattern approach those of the relaxed state. This suggests that, as in solution, increasing ionic strength weakens the affinity of myosin cross-bridges for actin, which results in a decrease in the number of cross-bridges attached. The effect is Ca2+-sensitive. Assuming that stiffness is a measure of the number of cross-bridge heads attached, in the absence of Ca2+, the fraction of attached cross-bridge heads varies from approximately 75% to approximately 25% over an ionic strength range where ionic strength in solution weakens the binding constant for myosin subfragment-1 binding to unregulated actin by less than a factor of 3. Therefore, this phenomenon appears similar to the cooperative Ca2+-sensitive binding of S1 to regulated actin in solution (Greene, L. E., and E. Eisenberg, 1980, Proc. Natl. Acad. Sci. USA, 77:2616). By comparing the binding constants in solution and in the fiber under similar conditions, we find that the "effective actin concentration," that is, the concentration that gives the same fraction of S1 molecules bound to actin in solution as cross-bridge heads are bound to actin in a fiber, is in the millimolar range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hyperosmotic solutions on the filament lattice of intact frog skeletal muscle.

The effect of increasing the osmotic strength of the extracellular solution on the fifament lattice of living frog sartorius and semitendinosus muscle has been studied using low-angle x-ray diffraction to measure the lattice spacing. As the extracellular osmotic strength is increased, the filament lattice shrinks like an osmometer until a minimal spacing between the thick filaments is reached. This minimal spacing varies from 20 to 31 nm, depending on the sarcomere length. Further increase in the osmotic strength produces little further shrinkage. The osmotic shrinkage curve indicates, for both muscles, an osmotically-inactive volume of approximately 30% of the volume in normal Ringer's solution. Shrinkage appears to be independent of temperature and the type of particle used to increase the osmotic strength (glucose, sucrose, small ions). The rate at which osmotic equilibruim is reached depends on muscle size, being slower for greater muscle diameters. Equilibrium spacings are approached exponentially with time constants ranging from 20 to 60 min. Independent of osmotic equilibrium, the lattice tends to shrink slowly by approximately 3% over the first few hours after dissection, probably because of a leakage of K+ ions from inside the muscle cells. This can be partly prevented by using an extracellular solution which contains a higher concentration of K+ ions or which is hypoosmotic. The volume of the muscle filament lattice (1.155d10(2) . S) is constant over a very wide range of sarcomere lengths, and is equal to approximately 3.6 x 10(6) nm3 for a range of amphibian muscle types.