scholarly journals The composition of cartilage proteoglycans. An investigation using high-and low-ionic-strength extraction procedures

1973 ◽  
Vol 131 (3) ◽  
pp. 541-553 ◽  
Author(s):  
Robert W. Mayes ◽  
Roger M. Mason ◽  
David C. Griffin
Keyword(s):  
Amino Acid ◽  
Ionic Strength ◽  
Density Gradient ◽  
Amino Acid Analysis ◽  
Chondroitin Sulphate ◽  
High Ionic Strength ◽  
Guanidinium Chloride ◽  
Equilibrium Conditions ◽  
Chondroitin Sulphate Proteoglycans ◽  
Low Ionic Strength

1. A proteoglycan fraction (the proteoglycan subunit fraction) was prepared from extracts, with 0.15m-KCl (low-ionic-strength) and 0.5m-LaCl3, 2.0m-CaCl2 and 4.0m-guanidinium chloride (high-ionic-strength), of bovine nasal cartilage by equilibrium-density-gradient centrifugation, essentially as described by Hascall & Sajdera (1969). 2. The use of different centrifugation times showed that near-equilibrium conditions were reached by 48h for the fractions prepared from the high-ionic-strength extracts. The fraction isolated from the low-ionic-strength extract required a longer centrifugation time to reach equilibrium conditions. 3. The composition of the proteoglycan fractions from the various extracts was compared by analyses of their carbohydrate and amino acid contents. Difference indices were calculated from the amino acid analysis to compare the degree of compositional relationship between the protein components of the proteoglycans. 4. Small compositional differences were found between the proteoglycans isolated from the various high-ionic-strength extracts. The protein content of the fractions from the CaCl2 extract and the guanidinium chloride extract showed the greatest difference in this respect, although their amino acid analysis was similar. 5. The proteoglycan fraction isolated from the low-ionic-strength extract shows marked differences in composition from the fractions isolated from the high-ionic-strength extracts. Its protein and glucosamine contents were lower whereas its hexuronic acid and galactosamine contents were higher than those of the latter. It also exhibits major differences in its amino acid composition. The glucosamine:galactosamine ratio of the fraction from the low-ionic-strength extract indicates that it may be an almost exclusively chondroitin sulphate–proteoglycan. Its analysis correlates closely with that of a low-molecular-weight proteoglycan isolated from pig laryngeal cartilage by Tsiganos & Muir (1969). 6. The proteoglycan fractions from both the low- and high-ionic-strength extracts migrate as a single band in zone electrophoresis carried out in a sucrose-density gradient at both pH3.0 and pH7.0, although each showed evidence of band widening during the electrophoresis. All the proteoglycan fractions migrated with the same electrophoretic mobility at pH3.0, irrespective of the differences in composition between them. 7. The differences between the proteoglycans from the low- and high-ionic-strength extracts are discussed and the view is advanced that they may be due to association between predominantly chondroitin sulphate–proteoglycans and a keratan sulphate-enriched proteoglycan species.

scholarly journals Multiple supramolecular structures formed by interaction of actin with protamine

1982 ◽  
Vol 205 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Enrico Grazi ◽  
Ermes Magri ◽  
Ivonne Pasquali-Ronchetti
Keyword(s):  
Ionic Strength ◽  
Exchange Reaction ◽  
Dead Time ◽  
High Ionic Strength ◽  
Supramolecular Structures ◽  
Molar Ratio ◽  
First Case ◽  
Atpase Reaction ◽  
Low Ionic Strength ◽  
Millipore Filters

When protamine is added to actin, different supramolecular structures are formed depending on the molar ratio of the two proteins and of the ionic strength of the medium. At low ionic strength, and going from a molar ratio of protamine to G-actin of 4:1, 2:1 and 1:1, globular aggregates are first converted into extended structures and then to long threads in which the constituent ATP–G-actin is rapidly exchangeable with the actin of the medium. At high ionic strength {Tyrode [(1910) Arch. Int. Pharmacodyn. Ther.20, 205–212] solution}, starting from G-actin and protamine in the 1:1 molar ratio, long ropes are formed that can be resolved into intertwining filaments of 4–5nm diameter. The addition of protamine in a 1:1 molar ratio to a solution of F-actin in Tyrode solution causes the breakage of the actin filaments, which is also revealed by the decrease of the viscosity of the solution and the formation of ordered latero-lateral aggregates. The structures formed by reaction of protamine with G-actin can be separated from free G-actin and protamine by filtration through 0.45μm-pore-size Millipore filters. This technique has been exploited to study the exchange reaction between free actin and the actin–protamine complexes. For these studies the 1:1 actin–protamine complex formed at low ionic strength and the 2:1 actin–protamine complex formed in the presence of 23nm-free Mg2+ have been selected. In the first case the exchange reaction is practically complete in the dead time of the experiment (20s). In the second case, where the complex operates like a true ATPase, the rate of the exchange is initially comparable with the rate of the ATP cleavage. Later on, however, the complex undergoes a change and the rate of the exchange between free actin and the actin bound to protamine becomes lower than the rate of the ATPase reaction. It is proposed that the ATP exchanges for ADP directly on the G-actin bound in the complex.

scholarly journals The Use of 2-Chloroethanol for Reversible Depolymerisation of the Protein Shell of Bacteriophage fr

1970 ◽  
Vol 25 (7) ◽  
pp. 711-713 ◽  
Author(s):  
D. Schubert ◽  
H. Frank
Keyword(s):  
Acetic Acid ◽  
Ionic Strength ◽  
Organic Solvent ◽  
High Ionic Strength ◽  
Protein Subunits ◽  
Viruslike Particles ◽  
Low Ionic Strength ◽  
Protein Shell

In mixtures of 1 volume of buffer and 2 volumes of 2-chloroethanol, the icosahedral bacteriophage fr is split into RNA and monomeric protein subunits. After removal of the RNA and after replacement of the organic solvent by water, viruslike particles can be obtained by dialysis of the protein against neutral buffers of high ionic strength, whereas multishell particles are formed in buffers of low ionic strength. All results achieved by the use of 2-chloroethanol are very similar to those obtained using acetic acid.

scholarly journals Microtubule assembly kinetics. Changes with solution conditions

1987 ◽  
Vol 247 (3) ◽  
pp. 505-511 ◽  
Author(s):  
J S Barton ◽  
D L Vandivort ◽  
D H Heacock ◽  
J A Coffman ◽  
K A Trygg
Keyword(s):  
Activation Energy ◽  
Ionic Strength ◽  
Low Temperature ◽  
Number Concentration ◽  
High Ionic Strength ◽  
Microtubule Assembly ◽  
Microtubule Protein ◽  
Assembly Kinetics ◽  
Low Ionic Strength ◽  
Kinetics Of

The assembly kinetics of microtubule protein are altered by ionic strength, temperature and Mg2+, but not by pH. High ionic strength (I0.2), low temperature (T less than 30 degrees C) and elevated Mg2+ (greater than or equal to 1.2 mM) induce a transition from biphasic to monophasic kinetics. Comparison of the activation energy obtained for the fast biphasic step at low ionic strength (I0.069) shows excellent agreement with the values obtained at high ionic strength, low temperature and elevated Mg2+. From this observation it can be implied that the tubulin-containing reactant of the fast biphasic event is also the species that elongates microtubules during monophasic assembly. Second-order rate constants for biphasic assembly are 3.82(+/- 0.72) x 10(7) M-1.s-1 and 5.19(+/- 1.25) x 10(6) M-1.s-1, and for monophasic assembly the rate constant is 2.12(+/- 0.56) x 10(7) M-1.s-1. The microtubule number concentration is constant during elongation of microtubules for biphasic and monophasic assembly.

Breakdown and reassociation of bacterial spinae

1982 ◽  
Vol 28 (7) ◽  
pp. 795-808
Author(s):  
K. B. Easterbrook ◽  
R. W. Coombs
Keyword(s):  
Ionic Strength ◽  
Protein Conformation ◽  
High Ionic Strength ◽  
Random Coil ◽  
Temperature Dependent ◽  
Calcium Effect ◽  
Α Helix ◽  
Low Ionic Strength ◽  
Β Sheet ◽  
Polymeric Structures

The tubular appendage, spina (Easterbrook and Coombs. 1976. Can. J. Microbiol. 22: 438–440), dissociates most efficiently under conditions of low ionic strength (0.01 M), high pH (10), and high temperature (95 °C). The protomer, spinin, thus produced is stable under these conditions and reassociates on cooling to give two distinct filamentous polymeric structures that differ in their stability, protein conformation, and reassociation characteristics. Under conditions of low ionic strength (0.01 M), reassociation is relatively slow and leads to a product that has significant amounts of α-helix in addition to the high β-sheet component; under conditions of high ionic strength (1 M), reassociation is rapid and the non-β-sheet component is in the random coil configuration. Since polymerization of the latter structure is "seeded" by either endogenous or exogenously supplied spina fragments, the protomers comprising it are assumed to be in the same conformation as in the spinae. High ionic strength induces folding of the protomer, multimeric association, and finally, elongation by a temperature-dependent process. Reassociation appears to be pH (6–10) independent and, apart from a possible minor calcium effect, cation nonspecific.

scholarly journals Evidence against a Simple Tethering Model for Enhancement of Herpes Simplex Virus DNA Polymerase Processivity by Accessory Protein UL42

2002 ◽  
Vol 76 (20) ◽  
pp. 10270-10281 ◽  
Author(s):  
Murari Chaudhuri ◽  
Deborah S. Parris
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Ionic Strength ◽  
Real Time ◽  
Dna Polymerase ◽  
Dissociation Rate ◽  
High Ionic Strength ◽  
Apparent Affinity ◽  
Simplex Virus ◽  
Low Ionic Strength

ABSTRACT The DNA polymerase holoenzyme of herpes simplex virus type 1 (HSV-1) is a stable heterodimer consisting of a catalytic subunit (Pol) and a processivity factor (UL42). HSV-1 UL42 differs from most DNA polymerase processivity factors in possessing an inherent ability to bind to double-stranded DNA. It has been proposed that UL42 increases the processivity of Pol by directly tethering it to the primer and template (P/T). To test this hypothesis, we took advantage of the different sensitivities of Pol and Pol/UL42 activities to ionic strength. Although the activity of Pol is inhibited by salt concentrations in excess of 50 mM KCl, the activity of the holoenzyme is relatively refractory to changes in ionic strength from 50 to 125 mM KCl. We used nitrocellulose filter-binding assays and real-time biosensor technology to measure binding affinities and dissociation rate constants of the individual subunits and holoenzyme for a short model P/T as a function of the ionic strength of the buffer. We found that as observed for activity, the binding affinity and dissociation rate constant of the Pol/UL42 holoenzyme for P/T were not altered substantially in high- versus low-ionic-strength buffer. In 50 mM KCl, the apparent affinity with which UL42 bound the P/T did not differ by more than twofold compared to that observed for Pol or Pol/UL42 in the same low-ionic-strength buffer. However, increasing the ionic strength dramatically decreased the affinity of UL42 for P/T, such that it was reduced more than 3 orders of magnitude from that of Pol/UL42 in 125 mM KCl. Real-time binding kinetics revealed that much of the reduced affinity could be attributable to an extremely rapid dissociation of UL42 from the P/T in high-ionic-strength buffer. The resistance of the activity, binding affinity, and stability of the holoenzyme for the model P/T to increases in ionic strength, despite the low apparent affinity and poor stability with which UL42 binds the model P/T in high concentrations of salt, suggests that UL42 does not simply tether the Pol to DNA. Instead, it is likely that conformational alterations induced by interaction of UL42 with Pol allow for high-affinity and high-stability binding of the holoenzyme to the P/T even under high-ionic-strength conditions.

Activation and inactivation of acetylcholinesterase by metal ions

1981 ◽  
Vol 59 (9) ◽  
pp. 728-735 ◽  
Author(s):  
George Tomlinson ◽  
Bulent Mutus ◽  
Ian McLennan
Keyword(s):  
Ionic Strength ◽  
Metal Ions ◽  
Metal Ion ◽  
Prolonged Exposure ◽  
Chelating Agent ◽  
High Ionic Strength ◽  
Metal Ion Binding ◽  
Peripheral Site ◽  
Metal Ion Effects ◽  
Low Ionic Strength

The kinetic consequences of acetylcholinesterase peripheral site occupation by metal ions were examined using three substrates; acetylthiocholine, p-nitrophenylacetate, and 7-(dimethylcarbamoyloxy)-N-methylquinolinium iodide. Two classes of metal ion effects were noted: activation by a group including Mg2+, Ca2+, Mn2+, and Na+, and inactivation by a second group which to date includes Zn2+, Cd2+, Hg2+, Ni2+, Cu2+, and Pb2+. Activation is demonstrable only in solutions of low ionic strength whereas inactivation can be readily observed in solutions of both low and high ionic strength. Activation appears to be due to a combination of metal ion binding and ionic strength effects and involves binding to peripheral sites which are distinct from those which bind organic cationic activators such as gallamine, propidium, and 7-(dimethylcarbamoyloxy)-N-methylquinolinium. The principal activating effect is on the deacylation phase of the enzyme–substrate reaction. Inactivators effect a slow conversion of the enzyme to an unreactive form. The kinetics of inactivation are biphasic at low ionic strength but become essentially monophasic at high ionic strength. More than 80% of the enzyme activity can be recovered upon addition of EDTA provided the chelating agent is added immediately following completion of the inactivation process. Prolonged exposure to inactivators results in a progressive decrease in the amount of recoverable activity. Although peripheral ligand interactions may result in a variety of catalytic site conformations, the macroscopic properties can be accounted for in terms of three ligand-dependent states of the enzyme in which catalytic ability (actual or potential) is retained, and a fourth denatured state.

scholarly journals Subunit composition and structure of subcomponent C1q of the first component of human complement

1976 ◽  
Vol 155 (1) ◽  
pp. 19-23 ◽  
Author(s):  
K B M Reid ◽  
R R Porter
Keyword(s):  
Ionic Strength ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Helical Structure ◽  
Guanidinium Chloride ◽  
Molecular Weights ◽  
Composition And Structure ◽  
Covalently Linked ◽  
Low Ionic Strength ◽  
Expected Position

1. Unreduced human subcomponent C1q was shown by electrophoresis on polyacrylamide gels run in the presence of sodium dodecyl sulphate to be composed of two types of non-covalently linked subunits of apparent mol.wts. 69 000 and 54 000. The ratio of the two subunits was markedly affected by the ionic strength of the applied sample. At a low ionic strength of applied sample, which gave the optimum value for the 54 000-apparent mol.wt. subunit, a ratio of 1.99:1.00 was obtained for the ratio of the 69 000-apparent mol.wt. subunit to the 5400-apparent-mol.wt. subunit. The amount of the 54 000-apparent-mol.wt. subunit detected in the expected position on the gel was found to be inversely proportional to increases in the ionic strength of the applled sample. 2. Human subcomponent C1q on reduction and alkylation, or oxidation, yields equimolar amounts of three chains designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. The results obtained by Yonemasu & Stroud [(1972) Immunochemistry 9, 545-554], which showed that the 69 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the A and B chains and that the 54 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the C chain, were confirmed. 3. Gel filtration on Sephadex G-200 in 6.0M-guanidinium chloride showed that both types of unreduced subunit were eluted together as a single symmetrical peak of apparent mol.wt. 49 000-50 000 when globular proteins were used as markers. The molecular weights of the oxidized or reduced A, B and C chains have been shown previously to be very similar all being in the range 23 000-24 000 [Reid et al. (1972) Biochem. J. 130, 749-763; Reid (1974) Biochem. J. 141, 189-203]. 4. It is proposed that subcomponent C1q (mol.wt. 410000) is composed of nine non-covalently linked subunits, i.e. six A-B dimers and three C-C dimers. 5. A structure for subcomponent C1q is proposed and is based on the assumption that the collagen-like regions of 78 residues in each of the A, B and C chains are combined to form a triple-helical structure of the same type as is found in collagens.

Aromatic sulphonates and the hydrolysis of 2-(p-nitrophenoxy)tetrahydropyran

1984 ◽  
Vol 62 (7) ◽  
pp. 1320-1324 ◽  
Author(s):  
Stella O'Leary
Keyword(s):  
Ionic Strength ◽  
Rate Constant ◽  
Polyacrylic Acid ◽  
Order Rate Constant ◽  
High Ionic Strength ◽  
Buffer Concentration ◽  
Salt Effects ◽  
Low Ionic Strength ◽  
Hydrolysis Of ◽  
Aromatic Sulphonates

The rate of hydrolyis of 2-(p-nitrophenoxy)tetrahydropyran was measured in a variety of buffers in water at 30 °C. At low ionic strength (μ = 0.05), 3,6-disulphonaphthoxyacetic acid catalysed the reaction. The second-order rate constant was 20 times faster than predicted from pKa. At high ionic strength (μ = 0.5), plots of kobs vs. total buffer concentration for both 3,6-disulphonaphthoxyacetic acid and 6,8-disulphonaphthyoxyacetic acid go through a maximum. Polyacrylic acid catalysed the reaction. The results are discussed in terms of aggregation and salt effects.

scholarly journals Adhesion of Pseudomonas fluorescens(ATCC 17552) to Nonpolarized and Polarized Thin Films of Gold

2001 ◽  
Vol 67 (7) ◽  
pp. 3188-3194 ◽  
Author(s):  
J. P. Busalmen ◽  
S. R. de S�nchez
Keyword(s):  
Thin Films ◽  
Ionic Strength ◽  
Pseudomonas Fluorescens ◽  
Theoretical Calculations ◽  
Gold Surface ◽  
High Ionic Strength ◽  
Reversible Adhesion ◽  
Controlled Flow ◽  
Low Ionic Strength

ABSTRACT The adhesion of Pseudomonas fluorescens (ATCC 17552) to nonpolarized and negatively polarized thin films of gold was studied in situ by contrast microscopy using a thin-film electrochemical flow cell. The influence of the electrochemical potential was evaluated at two different ionic strengths (0.01 and 0.1 M NaCl; pH 7) under controlled flow. Adhesion to nonpolarized gold surfaces readily increased with the time of exposition at both ionic-strength values. At negative potentials (−0.2 and −0.5 V [Ag/AgCl-KCl saturated {sat.}]), on the other hand, bacterial adhesion was strongly inhibited. At 0.01 M NaCl, the inhibition was almost total at both negative potentials, whereas at 0.1 M NaCl the inhibition was proportional to the magnitude of the potential, being almost total at −0.5 V. The existence of reversible adhesion was investigated by carrying out experiments under stagnant conditions. Reversible adhesion was observed only at potential values very close to the potential of zero charge of the gold surface (0.0 V [Ag/AgCl-KCl sat.]) at a high ionic strength (0.1 M NaCl). Theoretical calculations of the Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction energy for the bacteria-gold interaction were in good agreement with experimental results at low ionic strength (0.01 M). At high ionic strength (0.1 M), deviations from DLVO behavior related to the participation of specific interactions were observed, when surfaces were polarized to negative potentials.

scholarly journals Parallel modulation of brush border myosin conformation and enzyme activity induced by monoclonal antibodies.

1989 ◽  
Vol 109 (2) ◽  
pp. 549-556 ◽  
Author(s):  
S Citi ◽  
R A Cross ◽  
C R Bagshaw ◽  
J Kendrick-Jones
Keyword(s):  
Enzyme Activity ◽  
Monoclonal Antibodies ◽  
Ionic Strength ◽  
Brush Border ◽  
Active Sites ◽  
High Ionic Strength ◽  
Enzymatic Activities ◽  
Filament Assembly ◽  
Order Of Magnitude ◽  
Low Ionic Strength

Monoclonal antibodies binding to distinct epitopes on the tail of brush border myosin were used to modulate the conformation and state of assembly of this myosin. BM1 binds 1:3 of the distance from the tip of the tail to the head and prevents the extended-tail (6S) monomer from folding into the assembly-incompetent folded-tail (10S) state, whereas BM4 binds to the tip of the myosin tail, and induces the myosin to fold into the 10S state. Thus, at physiological ionic strength BM1 promotes and BM4 blocks the assembly of the myosin into filaments. Using BM1 and BM4 together, we were able to prevent both folding and filament assembly, thus locking myosin molecules in the extended-tail 6S monomer conformation at low ionic strength where they normally assemble into filaments. Using these myosin-antibody complexes, we were able to investigate independently the effects of folding of the myosin tail and assembly into filaments on the myosin MgATPase. The enzymatic activities were measured from the fluorescent profiles during the turnover of the ATP analogue formycin triphosphate (FTP). Extended-tail (6S) myosin molecules had an FTPase activity of 1-5 X 10(-3) s-1, either at high ionic strength as a monomer alone or when complexed with antibody, or at low ionic strength as filaments or when maintained as extended-tail monomers by the binding of BM1 and BM4. Folding of the molecules into the 10S state reduced this rate by an order of magnitude, effectively trapping the products of FTP hydrolysis in the active sites.

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