The kinetics of actin unfolding induced by guanidine hydrochloride has been studied. On the basis of obtained experimental data a new kinetic pathway of actin unfolding was proposed. We have shown that the transition from native to inactivated actin induced by guanidine hydrochloride (GdnHCl) passes through essential unfolding of the protein. This means that inactivated actin should be considered as the off-pathway species rather than an intermediate conformation between native and completely unfolded states of actin, as has been assumed earlier. The rate constants of the transitions that give rise to the inactivated actin were determined. At 1.0-2.0 M GdnHCl the value of the rate constant of the transition from native to essentially unfolded actin exceeds that of the following step of inactivated actin formation. It leads to the accumulation of essentially unfolded macromolecules early in the unfolding process, which in turn causes the minimum in the time dependencies of tryptophan fluorescence intensity, parameter A, characterizing the intrinsic fluorescence spectrum position, and tryptophan fluorescence anisotropy. 相似文献
A series of linear peptides with the general formula H‐Glu(R1)‐Glu(R2)‐OH was subjected to cyclization under standard conditions. Formation of respective 2,5‐diketopiperazines was accompanied by transformation of the N‐terminal Glu(R1) to pyroglutamic acid residue. Even in the case R1 is an amino acid residue attached to the N‐terminal γ‐carboxyl group, lactamization leads to its elimination. The observed reaction has not been reported so far in the literature. Correspondingly, an alternative route to Glu(R1)‐Glu(R2)‐containing 2,5‐diketopiperazines was applied to improve the overall yields. 相似文献
Summary The influence of the internal dynamics of two polypeptides comprising transmembrane -helix A or two -helices A and B of bacterioopsin on experimentally accessible 15N NMR relaxation rates was investigated by molecular dynamics (MD) simulations, combined with more simple mechanic considerations. Model-free order parameters and correlation times of internal motions [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559] were calculated for these models. It was found that both peptides exhibit two types of internal motions of the amide bonds, on the pico- and nanosecond time scales, affecting 15N NMR relaxation. The fast fluctuations are local and correspond to the librational motions of the individual N–H vectors in an effective potential of atoms of the surrounding matrix. In contrast, the motions on the nanosecond time scale imply concerted collective vibrations of a large number of atoms and could be represented as bending oscillation of -helices, strongly overdamped by the ambient solvent. A few other molecular mechanisms of slow internal motion were found, such as local distortions of the -helices (e.g., -aneurysm), delocalized distortions of the -helical backbone, as well as oscillations of the tilt angle between the axes of the -helices A and B. The results are compared with 15N NMR relaxation data measured for the (1–36)bacterioopsin and (1–71)bacterioopsin polypeptides in chloroform-methanol (1:1) and in SDS micelles [Orekhov, V.Yu., Pervushin, K.V. and Arseniev, A.S. (1994) Eur. J. Biochem., 219, 887–896].Abbreviations C2
baeterioopsin-(7–63)-peptide
- sA
bacterioopsin-(7–32)-peptide
- CPMG
Carr-Purcell-Meiboom-Gill
- MD
molecular dynamics
- rmsd
root-mean-square deviation 相似文献
Summary Two NAD-dependent dehydrogenases exhibiting activity with L-alanine were detected in crude extracts ofStreptomyces aureofaciens. The dominant enzyme was found to be an alanine dehydrogenase with 6 or 4 subunits, each ofM, 45,000. The minor enzyme exhibited high activities with branched-chain amino acids, suggesting it is a valine dehydrogenase. The results demonstrate thatS. aureofaciens has only one alanine dehydrogenase, which is catalytically active in a six- or four-subunit structure. 相似文献
Abstract Immunisation with outer membranes of Francisella tularensis induced an efficient protection in guinea pigs against challenge with the virulent strains 503 or 144/713 (type B biovar holarctica ), both clinical isolates, and prevented the development of typical signs of infection in hamadryads (baboons), challenged with the virulent strain Schu (type A, biovar tularensis ) of F. tularensis . Immunisation with a lipopolysaccharide protein complex isolated from the outer membranes afforded protection in CBA mice against challenge with strain 503. Another LPS-protein complex obtained by the simple mixture of LPS preparations from strain 503 and a 17-kDa membrane protein from the avirulent R-variant of the vaccine strain 15 also demonstrated protective properties against experimental tularemia in mice. 相似文献
Brillouin microspectroscopy is a powerful technique for noninvasive optical imaging. In particular, Brillouin microspectroscopy uniquely allows assessing a sample's mechanical properties with microscopic spatial resolution. Recent advances in background‐free Brillouin microspectroscopy make it possible to image scattering samples without substantial degradation of the data quality. However, measurements at the cellular‐ and subcellular‐level have never been performed to date due to the limited signal strength. In this report, by adopting our recently optimized VIPA‐based Brillouin spectrometer, we probed the microscopic viscoelasticity of individual red blood cells. These measurements were supplemented by chemically specific measurements using Raman microspectroscopy.
Rat C6 glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na+,K+-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K+ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K+. The specific activity of Na+,K+-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10–4 or 10–5 M noradrenaline (NA) remained without any effect on NA+,K+-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na+,K+-ATPase of C6 glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na+,K+-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10–7 M concentration. In spite of the fact that Na+,K+-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na+,K+-ATPase may contribute to the previously reported inhibition by vanadate of Na+,K+-ATPase of the whole brain tissue. 相似文献