scholarly journals Self-association of proteoglycan subunits from pig laryngeal cartilage

1978 ◽  
Vol 171 (1) ◽  
pp. 109-114 ◽  
Author(s):  
J K Sheehan ◽  
I A Nieduszynski ◽  
C F Phelps
Keyword(s):  
Light Scattering ◽  
Ionic Strength ◽  
Guanidine Hydrochloride ◽  
Sedimentation Velocity ◽  
Dilute Solution ◽  
Thiol Groups ◽  
Dimer Model ◽  
Laryngeal Cartilage ◽  
Self Association ◽  
Corroborative Evidence

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 5×10(6) daltons, but at 100mM-, 200mM- and 300mM-NaCl particle weights of 2.5×10(6)–3.0×10(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 3.2×10(6)–3.5×10(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.

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

1980 ◽  
Vol 185 (1) ◽  
pp. 107-114 ◽  
Author(s):  
I A Nieduszynski ◽  
J K Sheehan ◽  
C F Phelps ◽  
T E Hardingham ◽  
H Muir
Keyword(s):  
Dissociation Constant ◽  
Equilibrium Dialysis ◽  
Guanidine Hydrochloride ◽  
Dilute Solution ◽  
Binding Studies ◽  
Laryngeal Cartilage ◽  
Equilibrium Binding ◽  
Cartilage Proteoglycans ◽  
Dissociation Constant Kd

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 × 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 × 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 × 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.

scholarly journals Hydrolysable ATP is a requirement for the correct interaction of molecular chaperonins cpn60 and cpn10

2002 ◽  
Vol 364 (3) ◽  
pp. 849-855 ◽  
Author(s):  
Chris WALTERS ◽  
Neil ERRINGTON ◽  
Arther J. ROWE ◽  
Stephen E. HARDING
Keyword(s):  
Light Scattering ◽  
Molecular Chaperone ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Velocity ◽  
Dilute Solution ◽  
Binding Ability ◽  
Absorption Spectrophotometry ◽  
Molecular Hydrodynamic ◽  
Atp Analogues ◽  
Bead Modelling

Over recent years the binding ability of the molecular chaperone cpn60 (GroEL14) and its co-chaperone cpn10 (GroES7) has been reported to occur under an assortment of specific conditions from the use of non-hydrolysable ATP analogues (namely adenosine 5′-[γ-thio]triphosphate) to requiring hydrolysable ATP for any interaction to occur. We have investigated this further using the molecular hydrodynamic methods (hydrodynamic bead modelling, sedimentation-velocity analytical ultracentrifugation and dynamic light-scattering), allowing the process to be followed under physiologically relevant dilute solution conditions, combined with absorption spectrophotometry to determine GroES7—GroEL14 interaction through the rate inhibition of the cpn60's ATPase activity by GroES7. The results found here indicate that the presence of hydrolysable ATP is required to facilitate correct GroES7 interaction with GroEL14 in solution.

scholarly journals The effect of heavy chain phosphorylation and solution conditions on the assembly of Acanthamoeba myosin-II.

1989 ◽  
Vol 109 (4) ◽  
pp. 1529-1535 ◽  
Author(s):  
J H Sinard ◽  
T D Pollard
Keyword(s):  
Light Scattering ◽  
Ionic Strength ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Critical Concentration ◽  
Myosin Ii ◽  
Acidic Ph ◽  
Thick Filaments ◽  
Low Ionic Strength

At low ionic strength, Acanthamoeba myosin-II polymerizes into bipolar minifilaments, consisting of eight molecules, that scatter about three times as much light as monomers. With this light scattering assay, we show that the critical concentration for assembly in 50-mM KCl is less than 5 nM. Phosphorylation of the myosin heavy chain over the range of 0.7 to 3.7 P per molecule has no effect on its KCl dependent assembly properties: the structure of the filaments, the extent of assembly, and the critical concentration for assembly are the same. Sucrose at a concentration above a few percent inhibits polymerization. Millimolar concentrations of MgCl2 induce the lateral aggregation of fully formed minifilaments into thick filaments. Compared with dephosphorylated minifilaments, minifilaments of phosphorylated myosin have a lower tendency to aggregate laterally and require higher concentrations of MgCl2 for maximal light scattering. Acidic pH also induces lateral aggregation, whereas basic pH leads to depolymerization of the myosin-II minifilaments. Under polymerizing conditions, millimolar concentrations of ATP only slightly decrease the light scattering of either phosphorylated or dephosphorylated myosin-II. Barring further modulation of assembly by unknown proteins, both phosphorylated and dephosphorylated myosin-II are expected to be in the form of minifilaments under the ionic conditions existing within Acanthamoeba.

scholarly journals Macromolecular properties and polymeric structure of canine tracheal mucins

1991 ◽  
Vol 276 (2) ◽  
pp. 525-532 ◽  
Author(s):  
V Shankar ◽  
A K Virmani ◽  
B Naziruddin ◽  
G P Sachdev
Keyword(s):  
Gel Electrophoresis ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Hydrophobic Properties ◽  
Experimental Conditions ◽  
Protein Core ◽  
Polymeric Structure ◽  
Composite Gel ◽  
Self Association ◽  
Static Laser Light Scattering

Two high-Mr mucus glycoproteins (mucins), CTM-A and CTM-B, were highly purified from canine tracheal pouch secretions, and their macromolecular properties as well as polymeric structure were investigated. On SDS/composite-gel electrophoresis, a diffuse band was observed for each mucin. Polyacrylamide-gel electrophoresis using 6% gels also showed the absence of low-Mr contaminants in the mucins. Comparison of chemical and amino acid compositions revealed significant differences between the two mucins. Using a static-laser-light-scattering technique, CTM-A and CTM-B were found to have weight-average Mr values of about 11.0 x 10(6) and 1.4 x 10(6) respectively. Both mucins showed concentration-dependent aggregation in buffer containing 6 M-guanidine hydrochloride. Under similar experimental conditions, reduced-alkylated CTM-A had an Mr of 5.48 x 10(6) and showed no concentration-dependent aggregation. Hydrophobic properties of the mucins, investigated by the fluorescent probe technique using mansylphenylalanine as the probe, showed the presence of a large number of low-affinity (KD approx. 10(5) M) binding sites. These sites appeared to be located on the non-glycosylated regions of the protein core, since Pronase digestion of the mucins almost completely eliminated probe binding. Reduction of disulphide bonds of CTM-A and CTM-B did not significantly alter the probe-binding properties. Also, addition of increasing NaCl concentrations (0.03-1.0 M) to the buffer caused only a small change in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable degradation, using a combination of chemical and enzymic methods. On SDS/PAGE the protein core was estimated to have an Mr of approx. 60,000. On the basis of the protein and carbohydrate contents of the major mucin CTM-A, the mucin monomer was calculated to have an Mr of approx. 140,000. The high Mr (11 x 10(6] observed by physical methods is therefore due to self-association of the mucin monomer subunits.

Aggregation behaviour of stereoregular poly(methyl methacrylates) in dilute solution: light scattering and proton n.m.r. study

Polymer ◽  
1983 ◽  
Vol 24 (6) ◽  
pp. 700-706 ◽  
Author(s):  
L. Mrkvičková ◽  
J. Stêjskal ◽  
J. Spěváček ◽  
J. Horská ◽  
O. Quadrat
Keyword(s):  
Light Scattering ◽  
Dilute Solution ◽  
Aggregation Behaviour ◽  
Methyl Methacrylates

scholarly journals The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps.

1989 ◽  
Vol 109 (4) ◽  
pp. 1537-1547 ◽  
Author(s):  
J H Sinard ◽  
W F Stafford ◽  
T D Pollard
Keyword(s):  
Electron Microscopy ◽  
Light Scattering ◽  
Ionic Strength ◽  
Analytical Ultracentrifugation ◽  
Myosin Ii ◽  
Alkaline Ph ◽  
Predominant Species ◽  
Assembly Mechanism ◽  
Rapid Equilibrium ◽  
Low Ionic Strength

We used 90 degrees light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCl, Acanthamoeba myosin-II is monomeric. At low ionic strength (less than 100 mM KCl), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCl, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCl. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that intermediate structures exist at these salt concentrations, and that they are in rapid equilibrium with each other. We believe these structures represent assembly intermediates and have used equilibrium analytical ultracentrifugation and electron microscopy to identify them. Polymerization begins with the formation of antiparallel dimers, with the two tails overlapping by approximately 15 nm. Two antiparallel dimers then associated with a 15-nm stagger to form an antiparallel tetramer. Finally, two tetramers associate with a 30-nm stagger to form the completed minifilament. At very low ionic strengths, the last step in the assembly mechanism is largely reversed and antiparallel tetramers are the predominant species. Alkaline pH, which can also induce minifilament disassembly, produces the same assembly intermediates as are found for salt induced disassembly.

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