scholarly journals Self-association of α-chymotrypsin at low ionic strength in the vicinity of its pH optimum

1977 ◽  
Vol 161 (3) ◽  
pp. 687-694 ◽  
Author(s):  
R Tellam ◽  
D J Winzor
Keyword(s):  
Ionic Strength ◽  
Electrostatic Interactions ◽  
Sedimentation Equilibrium ◽  
Velocity Sedimentation ◽  
Gel Chromatography ◽  
Ph Optimum ◽  
Equilibrium Sedimentation ◽  
Equilibrium Distributions ◽  
Self Association ◽  
Low Ionic Strength

The self-association of alpha-chymotrypsin and its di-isopropyl phosphoryl derivative in in I0.03 sodium phophate buffer, pH7,9, was investigated by velocity sedimentation, equilibrium sedimentation and difference gel chromatography. No differences between the native and chemically modified enzyme were observed in the ultracentrifuge studies, and only a marginal (0.6%) difference in weight-average elution volume was detected by difference gel chromatography of 5g/litre solutions on Sephadex G-75. From quantitative analyses of sedimentation velocity and sedimentation-equilibrium distributions obtained with iPr2P (di-isopropylphosphoryl)-chymotrypsin, the polymerizing system is postulated to involve an indefinite association of dimer (with an isodesmic association constant of 0.68 litre/g) that is formed by a discrete dimerization step with equilibrium constant 0.25 litre/g. In addition to providing the best fit of the experimental results, this model of chymotrypsin polymerization at low ionic strength is also consistent with an earlier observation that dimer formation is a symmetrical head-to-head phenomenon under conditions of higher ionic strength (I0.29, pH7.9) where association is restricted to a monomer-dimer equilibrium. It is proposed that the dimerization process is essentially unchanged by variation in ionic strength at pH7.9, and that higher polymers are formed by an entirely different mechanism involving largely electrostatic interactions between dimeric species.

Physicochemical studies on the aggregation of bovine cardiac tropomyosin with ionic strength

1969 ◽  
Vol 47 (4) ◽  
pp. 411-413 ◽  
Author(s):  
William D. McCubbin ◽  
Cyril M. Kay
Keyword(s):  
Ionic Strength ◽  
High Ionic Strength ◽  
Optical Rotatory Dispersion ◽  
Rotatory Dispersion ◽  
Sedimentation Equilibrium ◽  
Velocity Sedimentation ◽  
Optical Rotatory ◽  
Dispersion Parameters ◽  
Tertiary Structures ◽  
The Individual

The aggregation of bovine cardiac tropomyosin as a function of ionic strength has been studied by the techniques of sedimentation velocity, sedimentation equilibrium, osmometry, viscometry, and optical rotatory dispersion. The measurements indicate that in aqueous buffers at neutral pH and at ionic strengths below 0.6, cardiac tropomyosin is heterogeneous and consists of a monomer in equilibrium with its aggregates. Dissociation of the aggregates occurs on dilution to yield a molecular weight species of approximately 70 000, in very good agreement with the value obtained at high ionic strength. These observations essentially parallel similar findings noted for the polymerization of skeletal tropomyosin, with the exception that the cardiac protein shows no tendency to polymerize at high ionic strength. The virtual constancy of all the optical rotatory dispersion parameters with polymerization suggests that the association is probably linear rather than lateral, with no accompanying changes in secondary and tertiary structures of the individual monomers.

Quasielastische Lichtstreuung von isoliertem Chromatin / Quasielastic Light Scattering of Isolated Chromatin

1983 ◽  
Vol 38 (1-2) ◽  
pp. 126-134 ◽  
Author(s):  
Bernd Meuel ◽  
Holger Notbohm
Keyword(s):  
Light Scattering ◽  
Ionic Strength ◽  
Structural Changes ◽  
Gel Chromatography ◽  
Quasielastic Light Scattering ◽  
Self Assembling ◽  
X Ray Scattering ◽  
Monovalent Salt ◽  
Quaternary Structures ◽  
Low Ionic Strength

Chromatin undergoes structural changes in dependence on the ionic strength of monovalent cations. At low ionic strength an extended chain of nucleosomes is apparent while with increasing ionic strength more compact structures are formed. Soluble chromatin was prepared from rat liver and fractionated by gel chromatography. Quasielastic light scattering experiments on chromatin were done with monovalent salt concentrations ranging from 3-150 mм. Using this method translational diffusion coefficients have been derived. These appeared to be nearly independent of monovalent salt concentrations, indicating that the hydrodynamic radius of chromatin molecules did not change. On the other hand, sedimentation coefficients were increasing according to an exponential relation. Taken together, both findings reveal a rising of the molar mass with increasing ionic strength. On the contrary, chromatin prepared in physiological salt apparently disintegrates by lowering the ionic strength. Furthermore, it could be demonstrated by earlier small angle X-ray scattering studies that the diameter of the higher order chromatin fiber remained constant at approximately 32 nm even if these structures had been reconstituted from smaller pieces starting at low ionic strength. Thus, isolated chromatin fibers are capable of self-assembling to regular quaternary structures, even if the DNA does not form a continuous strand.

Fc domain mediated self-association of an IgG1 monoclonal antibody under a low ionic strength condition

2011 ◽  
Vol 112 (4) ◽  
pp. 326-332 ◽  
Author(s):  
Hirotaka Nishi ◽  
Makoto Miyajima ◽  
Naoki Wakiyama ◽  
Kei Kubota ◽  
Jun Hasegawa ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Ionic Strength ◽  
Strength Condition ◽  
Igg1 Monoclonal Antibody ◽  
Self Association ◽  
Low Ionic Strength

Influence of pH and ionic strength on the lysis of Micrococcus luteus cells by hen lysozyme at low (20°C) and high (physiological, 40°C) temperature*

1981 ◽  
Vol 1 (2) ◽  
pp. 119-123 ◽  
Author(s):  
J. Saint-Blancard ◽  
J. P. Maurel ◽  
J. F. Constant ◽  
J. Berthou ◽  
P. Jolles
Keyword(s):  
High Temperature ◽  
Ionic Strength ◽  
Low Temperature ◽  
Micrococcus Luteus ◽  
Ph Domain ◽  
Ph Optimum ◽  
Temperature Form ◽  
Low Ionic Strength ◽  
Influence Of Ph ◽  
High Temperature Form

From isoactivity curves (showing activity as a function of pH and ionic strength) it was found that in the pH domain 6.7–8.6 frequently used in experiments involving hen lysozyme, the pH optimum of lysis of Micrococcus luteus ceils at low ionic strength (0.02–0.05) by the high-temperature form (40°C physiological temperature) was one to two pH units lower than that by the low-temperature form (20°C).

scholarly journals Structure and interactions of cartilage proteoglycan binding region and link protein

1985 ◽  
Vol 228 (1) ◽  
pp. 77-85 ◽  
Author(s):  
F Bonnet ◽  
D G Dunham ◽  
T E Hardingham
Keyword(s):  
Polyclonal Antibodies ◽  
Sedimentation Equilibrium ◽  
Antigenic Determinants ◽  
Gel Chromatography ◽  
Binding Region ◽  
Keratan Sulphate ◽  
Link Protein ◽  
Lysine Residues ◽  
Proteoglycan Aggregates ◽  
Self Association

Binding region and link protein were prepared from pig laryngeal cartilage proteoglycans after chondroitinase ABC and trypsin digestion. Experiments on gel chromatography showed the purified binding region to interact reversibly with hyaluronate (HA), and this binding was also shown to be stabilized by native link protein. The trypsin-prepared link protein showed properties of self-association in solution that were partially inhibited by oligosaccharides (HA10-16) and abolished by modification of free amino groups (lysine residues) with 2-methylmaleic anhydride. The Mr (sedimentation equilibrium) of the modified link protein was 41 700. Analysis of binding region showed it to contain 25% (w/w) carbohydrate, mainly in galactose, glucosamine, mannose and galactosamine. It contained some keratan sulphate, as digestion with endo-beta-D-galactosidase (keratanase) removed 28% galactose and 25% glucosamine and the Mr (sedimentation equilibrium) decreased from 66 500 to 60 800. After keratanase digestion the interaction with polyclonal antibodies specific for binding region was unaffected, but the response in a radioimmunoassay with a monoclonal antibody to keratan sulphate was decreased by 47%. Preparation of a complex between binding region, link protein and HA approximately 34 showed a single component (5.5S) of Mr (sedimentation equilibrium) 133 500. In this complex the antigenic determinants of link protein appeared masked, as previously found with proteoglycan aggregates. The isolated binding region and link protein were thus shown to retain properties comparable with those involved in the structure and organization of proteoglycan aggregates.

Further characterization of the second oestrogen-binding species of the rat granulosa cell

1984 ◽  
Vol 102 (1) ◽  
pp. 93-102 ◽  
Author(s):  
G. B. Kudolo ◽  
M. G. Elder ◽  
L. Myatt
Keyword(s):  
Ionic Strength ◽  
Granulosa Cell ◽  
Oestrogen Receptor ◽  
Sucrose Density Gradient ◽  
Gel Chromatography ◽  
Dissociation Kinetics ◽  
Receptor Kinetics ◽  
Stokes Radius ◽  
Cell Cytosol ◽  
Low Ionic Strength

ABSTRACT Rat granulosa cell cytosol contains a second oestrogen-binding species (SOB) distinguished from the classical oestrogen receptor by its lower dissociation constant (approx. 45 nmol/l) and the ability to bind oestrogens, antioestrogens, androgens and progesterone but not diethylstilboestrol. The SOB and the oestrogen receptor can be further distinguished by their differential adsorption to spheroidal hydroxylapatite and Concanavalin A–Sepharose. Addition of chaotropic salts or molybdate to granulosa cell cytosol did not alter the concentration of SOB or oestrogen receptor measured, indicating that there are no 'masked' binding sites in the two species caused by aggregation phenomena. The association rate of oestradiol with SOB at 4°C (1·72 ± 0·27(s.e.m.) × 108 mol/h) and 25°C (4·50 ± 0·36 × 108 mol/h) was faster than with the oestrogen receptor (7·20 ± 0·15 × 107 mol/h and 1·23 ± 0·15 × 108 mol/h respectively). The biphasic dissociation kinetics of [3H]oestradiol from the oestrogen receptor at 25°C (rate constants k−1 = 0·30±0·07/min and k−2 = 3·73±0·57 × 10−3/min) were similar to those reported in other target tissues but the dissociation of [3H]oestradiol from SOB appeared to be much more rapid and could not be measured by the Sephadex LH-20 separation method employed for determining receptor kinetics. Using sucrose density-gradient (SDG) analysis and Sephacryl S-200 gel chromatography the oestrogen receptor fractionated in an aggregated form (10·3S, Stokes radius >5·2 nm) in low ionic strength buffers and as a small species (4·4S, Stokes radius 3·5 nm) in buffers containing 0·4 m-KCl. However, the SOB fractionated as 2–3S, Stokes radius 3·7–4·0 nm at low ionic strength and as 5·8S, Stokes radius 3·5 nm in 0·4 m-KCl. In contrast to the receptor from other target tissues the granulosa cell oestrogen receptor did not bind to the artificial acceptor matrix oligo(dT)-cellulose and heat activation did not promote a 4S to 5S conversion when analysed on SDG. The salt-extracted form of nuclear receptor sedimented at 4·6S, mol. wt 69–72 000 on SDG. J. Endocr. (1984) 102, 93–102

scholarly journals Self-association of troponin

1977 ◽  
Vol 167 (1) ◽  
pp. 131-136 ◽  
Author(s):  
S J Lovell ◽  
D J Winzor
Keyword(s):  
Ionic Strength ◽  
Thin Filament ◽  
Velocity Sedimentation ◽  
Troponin Complex ◽  
Self Association

Ox muscle troponin was shown by equilibrium- and velocity-sedimentation studies to undergo concentration-dependent dissociation into its constituent subunits as well as self-association in imidazole buffers, pH 6.9. The extent of troponin association was found to be strongly dependent on ionic strength and also to exhibit a dependence on pH and temperature; under conditions physiological in regard to pH, temperature and ionic strength the extent of polymerization of troponin is considerable in 2 mg/ml solutions. The ability of polymeric troponin to bind to tropomyosin has been inferred from studies of mixtures containing actin-tropomyosin and an excess of troponin over the amount required for the normal 7:1:1 actin-tropomyosin-troponin complex. These findings should be relevant to studies of reconstituted actin-tropomyosin-troponin preparations, since they signify possible chemical as well as physical differences between the gel, paracrystalline and filamentous states of the complex that result from adoption of different preparative procedures for analogues of the native thin filament.

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