Model connective tissue systems. A physical study of gelatin gels containing proteoglycans

The swelling behavior of gelatin gels containing proteoglycans (sulphated proteoglycans from bovine intervertebral discs and a hyaluronate proteoglycan from bovine synovial fluid) when immersed in osmotically active solutions of dextran have been measured. The presence of the proteoglycans markedly affects the internal osmotic contribution to the swelling pressure of the gel. These internal osmotic pressures are considerably in excess of the sum of the osmotic activities of the individual components. This behavior is understood in terms of an entropic interaction between the gelatin and the proteoglycan molecules. By use of the “dilute solution” treatment of Flory, the osmotic pressure excesses are related to the volumes and hence dimensions of the interact acting species. A comparison of these values with those calculated by other means shows good agreement. The osmotic behavior of the complex gels can be understood on a mechanistic basis, if we regard the gelatin and sulphated proteoglycans as spheres and the hyaluronate proteoglycan as a rod.     

Extraction and purification of proteoglycans from various types of connective tissue

A new procedure for the isolation of proteoglycans has been described. Tissues are extracted with 4 M guanidinium chloride. the extracting solven is then exchanged for 7 M urea and the extract is chromatographed on a DEAE-cellulose column previously equilibrated with 7 M urea. Non-proteoglycan proteins were eluted with urea in weak salt solutions. Subsequently proteoglycans were eluted with strong salt solutions. By the procedure proteoglycans from tissues containing only small amounts of proteoglycans can be obtained virtually free from collagen in a form suitable for further fractionation.     

Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans

Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential distribution of 4- and 6-sulfated and unsulfated proteoglycans in tissue sections of cartilage and other noncartilaginous tissues. Digestion with chondroitinase ABC or ACII can be used to differentiate between chondroitin sulfate and dermatan sulfate proteoglycan in different connective tissues. In addition, the presence of a 6-sulfated chondroitin sulfate proteoglycan that is associated with membranes surrounding nerve and muscle fiber bundles is described. Monoclonal antibodies were also raised against the link protein(s) of cartilage proteoglycan aggregate. They have been used in peptide map analyses of link protein and in demonstrating the presence of a high-mannose oligosaccharide chain of the link proteins. The presence of high-mannose oligosaccharide structures on the link protein(s) accounts for the microheterogeneity of the link proteins (link proteins 1, 2, or 3) that is observed on sodium dodecyl sulfate-polyacrylamide gels.     

Interaction of connective tissue structural glycoprotein with proteoglycans and glycosaminoglycans]

It was shown that structural glycoprotein (SGP) of connective tissue isolated from be cattle heart valves and nasal septum cartilage formed water-soluble complexes with protein-chondroitin-4-sulphate and different heparin fractions. SGP formed no mentioned complexes with hyaluronic acid. Probably this plays an important role in the formation of collagen and elastin fibers.     

NMR and molecular modeling studies on two glycopeptides from the carbohydrate-protein linkage region of connective tissue proteoglycans.

Complete 1H and 13C NMR assignments are reported for two glycopeptides representing the carbohydrate-protein linkage region of connective tissue proteoglycans. These glycopeptides are the octasaccharide hexapeptide, Ser(GlcpAbeta(1-->3) Galpbeta(1-->3)Galpbeta(1-->4)Xylpbeta)-Gly-Ser-Gly-Se r (GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)Xylp beta)-Gly (1), and the tetrasaccharide dipeptide, Ser(GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)X ylpbeta)-Gly (2). The vicinal coupling constant data show that the monosaccharide residues adopt4 C 1 chair conformations. Distance geometry/simulated annealing calculations using 2D NOESY derived distance constraints yielded a single family of structures for the tetrasaccharide moiety, with well defined interglycosidic linkage conformations. The straight phi torsion angles of the glycosidic C1'-O1 bonds showed a strict preference for the -sc range whereas the psi torsion angles (O1-Cn) exhibited dependence upon the interglycosidic linkage position (-ac for beta(1-->3) linkage, +ac for beta(1-->4) linkage). The predominant conformation about the glycopeptide bond is straight phi = -sc and psi = +ac. The presence of strong daN (i, i+1) NOE contacts, and the general absence of dNN (i, i+1) contacts (except for a weak Ser-5/Gly-6 dNN contact) and the dbN (i, i+1) contacts (except for Ser-1/Gly-2) in the ROESY spectrum, suggest that the backbone for 1 is predominantly in an extended conformation. A comparison of the ROESY data for 1 with those obtained from the unglycosylated hexapeptide (3) of the same sequence suggests that glycosylation has only a marginal influence on the backbone conformation of the hexapeptide.     

The core proteins of large and small interstitial proteoglycans from various connective tissues form distinct subgroups.

Large and small proteoglycans were separately isolated from a number of connective tissues and compared to determine the extent of structural similarity. This was studied by enzyme-linked immunosorbent assays and by the peptide patterns obtained when 125I-labelled proteoglycans were digested with trypsin. All the large proteoglycans, i.e. from tendon, sclera, cartilage and aorta, appear to contain the structure typical for the hyaluronic acid-binding region, both shown by enzyme-linked immunosorbent assay and by content of peptides unique for this region. These proteoglycans also share other structural features of the protein core, as indicated by immunological cross-reactivity and similar peptide patterns. The large proteoglycans from aorta in addition show the presence of unique structures both upon immunoassay and with regard to peptide pattern. Among the small proteoglycans two groups can be identified, although amino acid composition and protein core sizes are grossly similar. One group consists of the small proteoglycans from aorta and cartilage having similar peptide maps and showing immunological cross-reactivity in enzyme-linked immunosorbent assay. The other distinctly different group consists of the small proteoglycans from bone, cornea, sclera and tendon, which among them show identity in enzyme-linked immunosorbent assay and similar peptide patterns. Proteoglycans from the two groups, however, show partial immunological cross-reactivity.     

The effect of trypsin on membrane transport of non-electrolytes

Trypsinization of alveolar macrophages depresses lysine transport but has no effect on nucleoside transport. The depression of the initial rate of lysine transport is due to a decreased rate of exchange diffusion presumably secondary to lowered intracellular pools of amino acids. It is shown directly that trypsinization accelerates the efflux of amino acids but not certain other metabolites, and reduces the capacity of the cell to concentrate amino acids.     

Plasma concentrations of connective tissue compounds: I. the effect of uterine involution.

The information obtained in a prospective study of plasma concentrations of connective tissue components during the third trimester of pregnancy, 4 days, and again at 6 weeks postpartum, suggests that the increased concentrations of plasma protein-hydroxyproline (hypro-protein), free proline, and GAG are associated with increased rate of hydrolysis of extraskeletal connective tissue components.     

Interaction between sodium ion and non-electrolytes in the countercurrent systems of the kidney

A change in the electrolyte concentration in a multicomponent system will in general change the activity of non-electrolyte species, a particular example being the salting-out of dissolved gases (although for some solutes a salting-in effect occurs). It follows that countercurrent multiplication of sodium ions in the renal medulla will inevitably be associated with change in activity of non-electrolytes since this process is characterized by two orthogonal [Na ⁺] gradients in the multiplier loop, the one longitudinal, the other transverse. Using a single loop model we show that the two [Na+] gradients act independently to produce opposing effects on activity so that given, say, a salting-out effect, the longitudinal gradient effects an increase in activity and the transverse a decrease, both being dependent on the magnitude of the interaction. In the latter case, since attenuation results from direct involvement of the solute in the countercurrent multiplication process, the diffusion coefficient of necessity also has a positive influence. In the non-steady state we show that a measure of the time taken to reach equilibrium varies with the square of the transit time of tubular fluid. This means that there are likely to be transient activity differences between the two countercurrent systems, active and passive, in the medulla. Urine appears to be in equilibrium with the inner medulla and so will reflect these activity changes in the steady and non-steady state. This analysis is consistent with previous experimental findings on urinary inert gases. These results could suggest an indirect method of monitoring countercurrent multiplication of sodium. There are also implications for clinical studies since it is often assumed that urine reflects blood concentrations of biologically inert solutes.