Effect of pressure and ionic strength on the self-association of Apo-A-I from the human high density lipoprotein complex.

The self-association of apo-A-I isolated from the human high density lipoprotein complex has been investigated by gel permeation chromatography and sedimentation equilibrium. The apparent weight average molecular weight (MWapp) versus Apo-A-I concentration profile was found to be sensitive to ionic strength and pressure; MWapp increased with increasing ionic strength and decreasing rotor speed. The data were consistent with a monomer-dimer-tetrameroctamer association shceme over all conditions investigated if a change in the partial specific volume of apo-A-I upon association of 5.5 x 10(-2) ml/g is postulated.     

The effects of ionic strength on the self-association of human spectrin.

The self-association of human spectrin has been studied by means of sedimentation equilibrium in the analytical ultracentrifuge at pH 7.5 and over a range of ionic strength from 0.009 to 1.0 M. Increasing ionic strength above 0.1 M reduces the equilibrium constants for all of the measurable steps in the self-association reaction. These results support the concept of charge-charge interactions stabilizing the tetramer and higher oligomers with respect to the heterodimer. In addition, increasing ionic strength brought about a dissociation of the heterodimer to component polypeptide chains. Dissociation to the heterodimers is also enhanced with a decrease in ionic strength below 0.05 M. This low ionic strength-dependent dissociation is consistent with generalised electrostatic repulsion; however, this effect also correlates with some loss of alpha-helical content as revealed by circular dichroism. The secondary, tertiary and quaternary structures may all be partially disrupted by electrostatic free energy at low ionic strength.     

Effect of ionic strength on the conformation of myosin subfragment 1-nucleotide complexes.

The effect of ionic strength on the conformation and stability of S1 and S1-nucleotide-phosphate analog complexes in solution was studied. It was found that increasing concentration of KCl enhances the reactivity of Cys(707) (SH1 thiol) and Lys(84) (reactive lysyl residue) and the nucleotide-induced tryptophan fluorescence increment. In contrast, high KCl concentration lowers the structural differences between the intermediate states of ATP hydrolysis in the vicinity of Cys(707), Trp(510) and the active site, possibly by increasing the flexibility of the molecule. High concentrations of neutral salts inhibit both the formation and the dissociation of the M**.ADP.Pi analog S1.ADP.Vi complex. High ionic strength profoundly affects the structure of the stable S1.ADP.BeF(x) complex, by destabilizing the M*.ATP intermediate, which is the predominant form of the complex at low ionic strength, and shifting the equilibrium to favor the M**.ADP.Pi state. The M*.ATP intermediate is destabilized by perturbation of ionic interactions possibly by disruption of salt bridges. Two salt-bridge pairs, Glu(501)-Lys(505) in the Switch II helix and Glu(776)-Lys(84) connecting the catalytic domain to the lever arm, seem most appropriate to consider for participating in the ionic strength-induced transition of the open M*.ATP to the closed M**.ADP.Pi state of S1.     

Immobilization of polyribonucleotides in agarose gels at high ionic strength

Purine polyribonucleotides poly(A), poly(G), and poly(I) associate reversibly with agarose gels at high NaCl molarities over the pH range 6–10, at 20°?40°C. Pyrimidine polyribonucleotides poly (C) and poly(U) could not be immobilized in agarose gels under the above conditions. However, poly(C) could be immobilized in agarose without precipitation between pH 3.2 and 4.0. Association of poly(G) and poly(I) with agarose appears to decrease progressively with deprotonation of their purine residues, and both polymers interact with the gel very weakly above pH 10 regardless of NaCl concentration. The binding to agarose of these polymers at pH 7.5 is also strongly influenced by temperature in the range 20°?40°C. The association of single-stranded poly(A) is only shifted toward higher NaCl molarities by increased pH; its binding is also little affected by temperature in the above range. At NaCl molarities effecting the saturating retention in agarose and at neutral pH, the immobilization of several polynucleotides could be prevented by urea in a concentration-dependent manner. The corresponding profiles of urea molarity appear to disclose a number of hydrophobic interactions between polynucleotides and agarose, some of which could be relatively strong, especially in the case of poly(A).     

Effect of ADP and ionic strength on the kinetic and motile properties of recombinant mouse myosin V

Mouse myosin V is a two-headed unconventional myosin with an extended neck that binds six calmodulins. Double-headed (heavy meromyosin-like) and single-headed (subfragment 1-like) fragments of mouse myosin V were expressed in Sf9 cells, and intact myosin V was purified from mouse brain. The actin-activated MgATPase of the tissue-purified myosin V, and its expressed fragments had a high V(max) and a low K(ATPase). Calcium regulated the MgATPase of intact myosin V but not of the fragments. Both the MgATPase activity and the in vitro motility were remarkably insensitive to ionic strength. Myosin V and its fragments translocated actin at very low myosin surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 s(-1) under conditions where the V(max) was 3.3 s(-1), indicating that, although not totally rate-limiting, ADP dissociation was close to the rate-limiting step. The high affinity for actin and the slow rate of ADP release helps the myosin head to remain attached to actin for a large fraction of each ATPase cycle and allows actin filaments to be moved by only a few myosin V molecules in vitro.     

Predominant attached state of myosin cross-bridges during contraction and relaxation at low ionic strength

The chemical states of a cross-bridge--nucleotide complex were studied using a fluorescent ATP analogue, 1-N6-etheno-2-aza-ATP(epsilon-2-aza-ATP). The fluorescence of epsilon-2-aza-ATP at specific emission wavelengths was enhanced by 12.5 times upon binding to myosin in a relaxed muscle and the fluorescence from the resultant myosin(M)-epsilon-2-aza-ADP-Pi intermediate was 2.5 times greater than that from a M-epsilon-2-aza-ADP complex. Similar enhancements of the fluorescence of epsilon-2-aza-ATP and epsilon-2-aza-ADP were observed upon binding to heavy meromyosin in solution. Binding of F-actin did not change the fluorescence of epsilon-2-aza-ATP or epsilon-2-aza-ADP bound to heavy meromyosin. When a muscle went from a relaxed state to a state of isometric contraction or contraction with shortening, the fluorescence intensity decreased only slightly or not at all, i.e. the fluorescence of nucleotides bound to most of the myosin heads during contraction is the same as that of the M-epsilon-2-aza-ADP-Pi intermediate. These results suggest that an actomyosin(AM)-epsilon-2-aza-ADP-Pi intermediate is the predominant attached state during contraction. When the ionic strength of the relaxing solution was decreased, cross-bridges formed at 6 degrees C without tension generation. At 20 degrees C, a large tension was produced although the shortening velocity was negligibly small or zero. The fluorescence intensity decreased by 15% at 20 degrees C but only a small decrease of 3% was observed at 6 degrees C, suggesting that the predominant complexes in the attached state were AM-epsilon-2-aza-ATP and/or AM-2-aza-ADP-Pi at 6 degrees C and AM-epsilon-2-aza-ADP at 20 degrees C. Thus, the identification of the actomyosin-nucleotide complexes existing before and after the force-generating step lent further support to the conclusion that the sliding force is generated by conformational changes in actomyosin when the (epsilon-2-aza-)ADP-Pi complex is bound to it.     

Archaea-based microbial fuel cell operating at high ionic strength conditions

In this work, two archaea microorganisms (Haloferax volcanii and Natrialba magadii) used as biocatalyst at a microbial fuel cell (MFC) anode were evaluated. Both archaea are able to grow at high salt concentrations. By increasing the media conductivity, the internal resistance was diminished, improving the MFC’s performance. Without any added redox mediator, maximum power (P _max) and current at P _max were 11.87/4.57/0.12 μW cm⁻² and 49.67/22.03/0.59 μA cm⁻² for H. volcanii, N. magadii and E. coli, respectively. When neutral red was used as the redox mediator, P _max was 50.98 and 5.39 μW cm⁻² for H. volcanii and N. magadii, respectively. In this paper, an archaea MFC is described and compared with other MFC systems; the high salt concentration assayed here, comparable with that used in Pt-catalyzed alkaline hydrogen fuel cells, will open new options when MFC scaling up is the objective necessary for practical applications.     

Spectroscopic study of the self-association of myosin

Homodyne and heterodyne measurements have been made of the spectrum and intensity of laser light scattered from solutions of skeletal muscle myosin at high salt concentrations. The spectral broadening and intensity measurements are consistent with the hypothesis that a myosin monomer-dimer equilibrium exists in these solutions. Values have been calculated for molecular weight and radius of gyration of the myosin monomer, virial coefficients, and diffusion constants of both monomer and dimer, and equilibrium constant of the reaction to form dimer. Diffusion constant measurements yield an approximate length of 1481 Å for the monomer and 2121 Å for the dimer. The significance of these lengths is discussed in terms of the models of Huxley and Pepe for the structure of the myofilament.