The Reactivity of the N-terminal Isoleucine of α-Chymotrypsin as a Function of Ionic Strength

1971 ◽  
Vol 49 (5) ◽  
pp. 529-534 ◽  
Author(s):  
A. Kurosky ◽  
J. E. S. Graham ◽  
Joan W. Dixon ◽  
T. Hofmann
Keyword(s):  
Ionic Strength ◽  
Nitrous Acid ◽  
High Ionic Strength ◽  
Amino Group ◽  
Model Compounds ◽  
Third Order ◽  
X Ray ◽  
A Value ◽  
Group A ◽  
Low Ionic Strength

The reactivity of the α-amino group of isoleucine-16 of α-chymotrypsin towards nitrous acid at pH 4.0 and 0° is strongly dependent on ionic strength. Third-order deamination rate constants at low ionic strength (μ = 0.1 M) are 500–1000 times higher than those at high ionic strength (μ = 5.0 and 6.0 M) and are independent of the nature of the ions and the chymotrypsin concentration. Extrapolation to zero ionic strength of a plot of the logarithms of the constants against ionic strength leads to a value which is the same as that for the exposed α-amino group of the model compounds isoleucylvaline and valylvaline, and of the N-terminal isoleucine of pepsin. The deamination rate constant of the dipeptide valylvaline varies only two- to three-fold between ionic strength of 0.1 M and 6 M. The results suggest that the concept of a "buried" N-terminal as shown by X-ray analysis (carried out at pH 4.2 and ionic strength 9–11 M) requires modification; at low ionic strength (0.1 M) the reactivity of the N-terminal is only little below that of an exposed amino group, a fact which suggests that the amino group is much more available than shown by the X-ray analysis. The results are interpreted in terms of an effect of the ionic strength on the equilibrium between two conformational states of the enzyme.

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.

Kinetics of the Reaction of Nitrous Acid with Model Compounds and Proteins, and the Conformational State of N-terminal Groups in the Chymotrypsin Family

1972 ◽  
Vol 50 (12) ◽  
pp. 1282-1296 ◽  
Author(s):  
A. Kurosky ◽  
T. Hofmann
Keyword(s):  
Nitrous Acid ◽  
Conformational State ◽  
Serine Proteinases ◽  
Amino Groups ◽  
Model Compounds ◽  
Third Order ◽  
Tyrosine Residues ◽  
Reactive Groups ◽  
Primary Amino ◽  
Kinetics Of

The kinetics of the reaction of nitrous acid at 4° and pH 4.0 with various amino acids, peptides, and proteins were studied. The reaction with isoleucine methyl ester was found to have a linear dependence on the square of the HONO concentration showing that N2O3 was the reactive species. Third order nitrosation rate constants of primary amino groups showed a correlation with their pK values. They were calculated for the concentration of the unprotonated species to give intrinsic reactivities. The rate of nitrosation of acetyltryptophan to give N-nitrosoacetyltryptophan was found to be a linear function of the nitrous acid concentration. This nitrosation therefore follows a different mechanism. The reaction of nitrous acid with tyrosine residues was examined by spectrophotometry. The reaction was negligible compared to that of other groups. Acetylhistidine and imidazole did not react. Reactivities for α-amino groups, ε-amino groups, and other residues in proteins were compared. The conformational state of the N-terminal residues in serine proteinases, as revealed from their reactivities, is discussed in detail. It is concluded that nitrous acid reacts preferentially with "surface" residues and is a useful tool for exploring conformational states of reactive groups in proteins, especially α-amino groups and indole rings.

scholarly journals Actin oligomers at the initial stage of polymerization induced by increasing temperature at low ionic strength: Study with small-angle X-ray scattering

BIOPHYSICS ◽  
2010 ◽  
Vol 6 ◽  
pp. 1-11 ◽  
Author(s):  
Takaaki Sato ◽  
Togo Shimozawa ◽  
Toshiko Fukasawa ◽  
Masako Ohtaki ◽  
Kenji Aramaki ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Small Angle ◽  
X Ray ◽  
X Ray Scattering ◽  
Initial Stage ◽  
Increasing Temperature ◽  
Low Ionic Strength ◽  
Ray Scattering

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 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.

Ionic strength-dependent alterations of membrane structure of red blood cells

1986 ◽  
Vol 6 (11) ◽  
pp. 1007-1015 ◽  
Author(s):  
Andreas Herrmann ◽  
Peter Müller
Keyword(s):  
Ionic Strength ◽  
Membrane Structure ◽  
Spin Labels ◽  
Head Group ◽  
Polar Head Group ◽  
Paramagnetic Resonance ◽  
Group A ◽  
Hydrophobic Tail ◽  
Low Ionic Strength ◽  
Electron Paramagnetic

Electron paramagnetic resonance (EPR) measurements using various fatty acid spin labels were performed on membranes of intact human erythrocytes at physiological, and at low ionic strength. In the case of spin probes bearing the nitroxide near the polar head group, a less restricted motion at low ionic strength was seen than with those labels with a nitroxide deeper within the hydrophobic tail of the membrane. Although these data clearly show an influence of ionic strength on membrane structure, and possibly a modified protein-lipid interaction, they cannot be simply discussed in terms of an altered membrane fluidity.

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.

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