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J K Sheehan

Publications and source records attributed to J K Sheehan.

At least 73 records · Page 4Linked to original sources
PubMed1984

Hydrodynamic properties of human cervical-mucus glycoproteins in 6M-guanidinium chloride.

Cervical mucins and fragments thereof were studied by sedimentation-velocity, rotatory viscometry and laser light-scattering performed as photon-correlation spectroscopy as well as low-angle total-intensity measurements. The Mr of the whole mucins is 10 X 10(6)-15 X 10(6), whereas fragments obtained after reduction of disulphide bonds ('subunits') have Mr 2.1 X 10(6)-2.9 X 10(6), depending on the method used. Subsequent trypsin digestion of subunits afforded glycopeptides with Mr approx. 0.4 X 10(6). The high frictional ratio for the whole mucins is interpreted as a large degree of expansion. The Stokes radius calculated from the diffusion coefficient is approx. 110nm for the whole mucins, which is in agreement with that estimated from the radius of gyration (130nm) by using the concept of the equivalent hydrodynamic sphere. The ratio of the concentration-dependence parameter for the reciprocal sedimentation coefficient (Ks) to the intrinsic viscosity ( [eta] ) for the whole mucins is 1.42, suggesting that the individual macromolecule occupies a spheroidal domain in solution. The relationship between [eta] and Mr for whole mucins, subunits and T-domains suggests that they are linear flexible macromolecules behaving as somewhat 'stiff' random coils. This conclusion is supported by the relationships between the sedimentation coefficients, the diffusion coefficients and the Mr. The hydrodynamic behaviour of the mucins is thus close to that expected for coiling macromolecules entrapping a lot of solvent, which is consistent with the postulated polymeric structure.

Cervix Mucus↗
PubMed1984

Macromolecular properties and polymeric structure of mucus glycoproteins.

Mucus glycoproteins (mucins) were isolated from cervical and gastric mucus as well as from chronic bronchitic sputum. The mucus gel was solubilized by slow stirring in 6M-guanidinium chloride supplemented with low-Mr proteinase inhibitors. Subsequent removal of non-mucin proteins and DNA was achieved with isopycnic density-gradient centrifugation. The cervical and the respiratory mucins were of similar size (Mr about 10 X 10(6) and 18 X 10(6)), respectively), whereas the gastric mucins were considerably larger (Mr about 45 X 10(6)). 'Subunits' isolated after disulphide bond cleavage were the same size for the three mucins, as were glycopeptides obtained after subsequent trypsin digestion of the subunits. Physical data suggest that the respiratory and gastric mucins conform to the model for the polymeric structure proposed previously for cervical mucins. The macromolecules are described as linear flexible chains behaving, in dilute solution, as random coils. We propose that mucus glycoproteins are considerably larger than hitherto recognized and that mucins of various origins are very similar in their macromolecular properties and polymeric structure.

Animals↗
PubMed1984

Macromolecular architecture and hydrodynamic properties of human cervical mucins.

Cervical mucus glycoproteins (mucins) were extracted by using slow stirring in 6M-guanidinium chloride supplemented with proteinase inhibitors. Subsequent purification was achieved by isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. The whole mucins (Mr 10 X 10(6) - 15 X 10(6)) were degraded into subunits (Mr 2 X 10(6) - 3 X 10(6)) by reduction. Trypsin digestion of subunits afforded glycopeptides (T-domains) with Mr 0.4 X 10(6). The relationship between the intrinsic viscosity and Mr for the whole mucins and the fragments suggests that cervical mucins are linear flexible macromolecules. This view is supported by hydrodynamic data.

Cervix Mucus↗
PubMed1983

Hyaluronic acid: molecular conformation and interactions in the tetragonal form of the potassium salt containing extended chains.

X-ray diffraction data were used to determine the detailed crystal structure of a tetragonal form of potassium hyaluronate containing relatively extended 4-fold helical chains (rise per disaccharide h = 0.95 nm). The polysaccharide chains are left-handed (4(3] helices. Two antiparallel chains pack in a tetragonal unit cell (a = b = 0.996 nm, c = 3.788 nm) with P4(3)2(1)2 space group symmetry. The chain conformations are stabilized intramolecularly by O4-O5 hydrogen bonds across the beta(1 leads to 3) linkage and by a pair of intermolecular hydrogen bonds per disaccharide between adjacent antiparallel chains. Fourier difference synthesis revealed one potassium ion and two water molecules per disaccharide. Six polyanion oxygen atoms from three neighboring chains together with one of these water molecules form the co-ordination polyhedra. Further stability is brought about through inter- and intrachain water bridges involving both water molecules. The probable reason for the stability of this extended allomorph is discussed in terms of the preferred co-ordination geometry of the potassium ion.

Carbohydrate Conformation↗
PubMed1983

Hyaluronic acid: molecular conformations and interactions in the orthorhombic and tetragonal forms containing sinuous chains.

The conformation, packing and probable mode of cation binding have been determined for compact potassium hyaluronate chains (average rise per disaccharide h = 0.89 nm) organized in an orthorhombic unit cell (a = 1.173 nm, b = 0.925 nm, c = 3.542 nm). The space group symmetry is P2(1)2(1)2(1) and the unit cell contains two antiparallel polysaccharide chains that are disturbed 4(3) helices. Each chain is stabilized intramolecularly by four hydrogen bonds and between adjacent antiparallel chains there are two intermolecular hydrogen bonds per crystallographic tetrasaccharide repeat. Fourier difference synthesis revealed two potassium ions and two water molecules in each asymmetric unit. Both potassium ions show octahedral co-ordination geometries and link adjacent antiparallel polysaccharide chains. The water molecules provide further intermolecular association through water bridges. A comparison of this potassium hyaluronate structure with the orthorhombic and tetragonal sodium hyaluronate structures containing similar compact hyaluronate chains revealed that the apparently isomorphous orthorhombic sodium and potassium salts did not have the same packing arrangements. The relative orientations of the sinuous hyaluronate chains in the sodium and potassium salts are about 90 degrees apart. The locations of the cations and hence the coordination schemes of K+ and Na+ are therefore very different. These analyses have delineated, for the first time, how hyaluronate chains can respond differentially to two different monovalent cations.

Carbohydrate Conformation↗
PubMed1983

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.

Amino Acids↗
PubMed1983

The macromolecular structure of human cervical-mucus glycoproteins. Studies on fragments obtained after reduction of disulphide bridges and after subsequent trypsin digestion.

Human cervical-mucus glycoproteins (mucins) were extracted with 6 M-guanidinium chloride in the presence of proteinase inhibitors and purified by isopycnic density-gradient centrifugation. The whole mucins (Mr approx. 10 X 10(6] were degraded into 'subunits' (Mr approx. 2 X 10(6] by reduction of disulphide bonds. Trypsin digestion of the 'subunits' produced glycopeptides with Mr approx. 380000, which appear to be rod-like with a length of approx. 105 nm. The relationship between the radius of gyration and the Mr value obtained by light-scattering for whole mucins, 'subunits' and 'domains' suggest that cervical-mucus glycoproteins are linear flexible macromolecules composed of, on the average, four or five 'domains'/subunit and four subunits/whole mucin macromolecule. The shape-dependent particle scattering function for the whole mucins and the 'subunits' are in accordance with that of a linear flexible chain. No evidence for a branched or a star-like structure was found. A tentative model for cervical mucins is proposed.

Amino Acids↗
PubMed1983

Isolation and characterization of human cervical-mucus glycoproteins.

Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 X 10(6) and 5.9 X 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).

Amino Acids↗
PubMed1982

Self-association of scleral proteodermatan sulfate. Evidence for interaction via the dermatan sulfate side chains.

Previous studies on scleral proteoglycans (proteodermatan sulfate) using light scattering and ultracentrifugation techniques have shown that the molecules form aggregates in 0.15 M NaCl (Cöster, L., Fransson, L.-A., Sheehan, J. K., Nieduszynski, I. A., and Phelps, C. F. (1981) Biochem. J., 197, 483-490). Aggregation was not promoted by hyaluronate but addition of free scleral dermatan sulfate chains enhanced multimerization. To investigate the possibility that scleral proteoglycans interact via their dermatan sulfate side chains, we have adopted an affinity chromatography procedure where binding of proteoglycans to various dermatan sulfate-agaroses may be studied. The evidence for an interaction between the side chains of the macromolecules and the immobilized dermatan sulfates are as follows: (a) the dermatan sulfate chains released from the proteoglycan by proteolysis display affinity for dermatan sulfate-agarose, (b) a significant proportion of the [3H]acetylated proteoglycans that were bound to the dermatan sulfate gel can be displaced by eluting with a solution of dermatan sulfate chains, (c) selective periodate oxidation of L-iduronate in the dermatan sulfate chains of the proteoglycans abolishes the affinity, (d) the core protein prepared by chondroitinase ABC digestion of the proteoglycan does not bind to dermatan sulfate-agarose, and (e) binding is retained after reduction-alkylation of the protein core. Furthermore, free [3H]dermatan sulfate chains co-elute with the proteoglycan upon gel filtration in 0.2 M NaCl.

Animals↗
PubMed1981

Self-association of heparan sulfate. Demonstration of binding by affinity chromatography of free chains on heparan sulfate-substituted agarose gels.

We have developed an affinity chromatography procedure which measures binding of heparan sulfate species to agarose gels substituted with different heparan sulfates. Three major subfractions of bovine lung heparan sulfate (HS2, HS3, and HS4) which differ in sulfate content and hexuronate composition have been used. Association-prone variants of these species (HS2-A, HS3-A, and HS4-A) were prepared by gel chromatography. Free heparan sulfate chains were applied to columns of various heparan sulfate-agaroses which were eluted with a linear guanidine gradient and binding was assessed by measuring the hexuronate content of the effluent. Associating heparan sulfate of a particular subfraction was chiefly bound to gels that were substituted with chains of the same kind, i.e. HS2-A to HS2-A-agarose, HS3-A and HS4-A to HS3-A-agarose, and HS4-A to HS4-A-agarose. N-Desulfation and N-acetylation of HS2-A markedly reduced binding to HS2-A-agarose and periodate oxidation of glucuronate in HS3-A completely abolished binding to HS3-A-agarose. Partially oxidized HS2-A was separated into bound and unbound material by affinity chromatography on HS2-A-agarose. Gel chromatography of these fractions indicated that unbound chains were significantly smaller than bound ones. It is concluded that association between heparan sulfate chains may be quite specific and that the strength of binding is dependent on co-operative interactions between a number of contact zones. The latter may correspond to the N-sulfated and iduronate- and glucuronate-containing segments.

Animals↗
PubMed1981

Isopycnic-centrifugation studies in caesium chloride and in caesium sulphate on dermatan sulphate proteoglycans from bovine sclera.

1. Two proteodermatan sulphate species from bovine sclera (fractions PG-I and PG-II) separable by gel chromatography were studied by isopycnic centrifugations in CsCl and Cs(2)SO(4) both in an analytical and a preparative mode. 2. In CsCl, fraction PG-I formed a broad band at a density rho=1.75g/ml whereas fraction PG-II banded sharply at rho=1.64g/ml. However, in Cs(2)SO(4), fraction PG-II banded at rho=1.51g/ml and fraction PG-I at rho=1.40g/ml, a reversal of the relative banding positions of the two species in CsCl. 3. Preparative isopycnic centrifugations in the two caesium salts permitted further subfractionation of fractions PG-I and PG-II. In both CsCl and Cs(2)SO(4) fraction PG-I was split into subfractions that varied greatly in uronate/protein ratios but had very similar uronate composition. In contrast, isopycnic centrifugation of fraction PG-II in Cs(2)SO(4) gave rise to subfractions with similar uronate/protein ratios but markedly different uronate composition (iduronate content, 88-42%). 4. Subfractions of fractions PG-I and PG-II obtained in preparative centrifugations in CsCl or Cs(2)SO(4) were examined in the analytical ultracentrifuge. These subfractions banded at discrete positions in the gradient. Estimations of apparent molecular weight for these subfractions from data from the analytical isopycnic centrifugations gave values that were much higher (around 1x10(6)) than were those obtained previously for their ;monomeric' states (fraction PG-I 160000-410000, and fraction PG-II 70000-130000). 5. In CsCl, fraction PG-I may be subfractionated according to the number of side chains in the molecule. Fraction PG-I increased its net solvation (i.e. lowered its buoyant density) to a larger extent than did fraction PG-II in going from CsCl to Cs(2)SO(4) (i.e. from lower to higher water activity). It is proposed that the presence of large amounts of iduronate in fraction PG-II makes these molecules relatively less solvated in Cs(2)SO(4). Thus the uronate composition may be an important factor in determining the banding position of proteodermatan sulphates in density-gradient centrifugations.

Animals↗
PubMed1980

Interaction between heparan sulphate chains. I. A gel chromatographic, light-scattering and structural study of aggregating and non-aggregating chains.

1. Heparan sulphate from bovine lung was fractionated with cetylpyridinium chloride. Solubilisation of complexes was accomplished by increasing concentrations of NaCl in a step-wise manner. Fractions I-IV, which were low-sulphated, contained more D-glucuronic acid than L-iduronic acid, fraction V contained equal proportions while fraction VI was L-iduronic acid-rich. 2. Gel chromatography of heparan sulphates II-IV in 0.5 M sodium acetate yielded extremely asymmetric profiles, while fractions V, VI and heparin did not. 3. Heparan sulphate IV was separated into aggregatable and non-aggregatable species by gel chromatography in 0.5 M sodium acetate. The particle/molecular weights of the two species were determined by light scattering. In 0.15 M NaCl or KCl the aggregatable chains yielded particle weights of 60 000-100 000 while the molecular weight was 20 000 (in 4.0 M guanidine HCl). Non-aggregatable chains afforded 'monomeric' values in 0.15 M NaCl or KCl. 4. Periodate oxidation of D-glucuronic acid residues in N-acetylated block regions followed by scission in alkali was used to fragment aggregating and non-aggregating heparan sulphate IV. The former chains yielded, on average, shorter oligosaccharides than did the latter. Reoxidation of the remaining D-glucuronic acid residues (adjacent to N-sulphated amino sugars) in the oligosaccharides followed by alkaline cleavage resulted in distinctly different fragmentation patterns in the two cases. The iduronate-containing oligosaccharides derived from aggregatable chains were markedly degraded into fragments ranging from glucosamine-L-iduronic acid-glucosamine-(C-3 fragment) to higher saccharides. Only higher saccharides were obtained from fragments of non-aggregatable chains. 5. It is concluded that self-associating heparan sulphates comprise both D-glucuronic acid- and L-iduronic acid-containing repeating units and that these units are arranged in an alternating or mixed fashion. These characteristics are analogous to those observed with self-associating dermatan sulphate species (Fransson, L.-A. and Cöster, L. (1979) Biochim. Biophys. Acta 582, 132-144).

Animals↗
PubMed1980

Equilibrium-binding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions.

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 x 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 x 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol..wt. 670 000), an average Kd of 12 x 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.

Animals↗
PubMed1978

Self-association of proteoglycan subunits from pig laryngeal cartilage.

Proteoglycans from pig laryngeal cartilage prepared by dissociative extraction in guanidine hydrochloride were studied in dilute solution by light-scattering and ultracentrifugation. In buffered 150mM-NaCl, pH7.4, the proteoglycan particle weights were about 5x10(6) daltons, but at 100mM-, 200mM- and 300mM-NaCl particle weights of 2.5x10(6)--3.0x10(6) daltons were observed. These results, together with corroborative evidence from sedimentation-velocity experiments, were interpreted in terms of proteoglycans self-associating at physiological ionic strength. The data were examined by using a proteoglycan monomer-dimer model. Proteoglycan preparations that had thiol groups partially carboxymethylated gave particle weights of 3.2x10(6)--3.5x10(6) daltons in 150mM-NaCl, which suggested that carboxymethylation inhibited multimerization and hence that the protein core is implicated in the binding site. Further studies showed that the multimers were stable to 60 degrees C, unlike the hyaluronate-proteoglycan complex.

Animals↗