Biomedical subjects

G Drewes

Publications and source records attributed to G Drewes.

24 records · Page 2Linked to original sources

PubMed1991

A reversible conformational transition in muscle actin is caused by nucleotide exchange and uncovers cysteine in position 10.

ATP-G-actin in the absence of excess ATP and divalent metal ions was treated with ADP in amounts large enough to ensure complete formation of ADP-G-actin. Under these conditions the monomer undergoes a very slow structural transition as seen by the exposure of 2.0 +/- 0.2 thiol groups per actin molecule. Once exposed, the second thiol group reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) at a rate approximately 10-fold higher than that of cysteine 374. Labeling experiments with 2,4-dinitrophenyl [1-14C]cysteinyl disulfide followed by digestion and peptide analysis showed (besides reaction with cysteine 374) nearly exclusive labeling of cysteine 10. Since this residue is completely shielded in ATP-G-actin, exchange of ATP for ADP must have caused a partial unfolding of the protein uncovering the side chain of this cysteine. The transition is reversible, because addition of ATP or of excess divalent metal ions restored the conformation with only cysteine 374 exposed. Reversibility of the transition allowed us to directly determine the relative affinities of ATP and ADP to monomeric actin in the absence of Me2+ ions. By determination of the 50% exposure value of cysteine 10 from either side of the equilibrium we found a value of KATP/KADP = 30. The rate of uncovering of the thiol of cysteine 10 at 0 degree C was distinctly slower (t1/2 = 9 h) than its reshielding by the addition of ATP (t1/2 = 3 h). The structural change was accompanied by a decrease in polymerization rate. Relative polymerization rates were determined as ATP-G(1S)-actin:ADP-G(approximately 1S)-actin:ADP-G(2S)-actin = 1.0:0.35:0.1. From the data presented here we conclude that preparations of ADP-G-actin remain undefined unless the number of thiol groups exposed has been determined.

Actins↗

PubMed1990

The enhanced ATPase activity of glutathione-substituted actin provides a quantitative approach to filament stabilization.

[Cys374]glutathionyl-actin was prepared by isolation of the reaction product of G-actin with Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid], followed by reaction with glutathione. Filaments of this actin disulfide are susceptible to even weak shearing stress as exerted, for example, by heating to 37 degrees C. This treatment produces a 25-fold enhanced steady-state ATPase activity as compared to unsubstituted F-actin at room temperature. Monitoring the reduction of this enhanced ATPase activity is a reliable method for quantifying the effectiveness of filament-stabilizing agents and for determining their apparent dissociation constants. A detailed comparative study of filament-stabilizing agents was performed, and some hitherto unknown filament-protecting effects were revealed. Inorganic phosphate provides stabilization only to a maximum of 45% ATPase inhibition, but reaches this effect already at cytoplasmic Pi concentrations (approximately 4 mM). Arsenate seems to bind with similar affinity, but with distinctly less protective activity (maximum of 16%). High concentrations of alkali ions provide a more effective protection (maximum of 95%), Li+ being more efficient than Na+ and K+. Divalent cations (Ca2+, Mg2+) had a strong stabilizing effect on KCl-polymerized actin; we confirmed the presence of two distinct classes of binding sites for divalent metal ions with moderate and low affinities, apparent in a strong stabilizing effect on KCl-polymerized actin. The stabilizing effects of KCl and Pi are independent and additive. Correspondingly, at K2HPO4 concentrations greater than 4 mM, K+ ions contribute considerably to stabilization. In the presence of 100 mM KCl plus 4 mM Pi, conditions which mimic the physiological environment, filament protection is nearly as effective as with the mushroom toxin phalloidin. The strong stabilizing effect of phalloidin occurred at concentrations far below stoichiometric, suggesting a very high degree of cooperativity in its interaction with actin filaments.

Actin Cytoskeleton↗

PubMed1990

Glutathionyl(cysteine-374) actin forms filaments of low mechanical stability.

Rabbit muscle actin reacts with 2,4-dinitrophenylglutathionyldisulfide, forming a mixed disulfide in position 374. the product S-(cysteine-374)glutathionyl actin forms filaments which are easily disrupted under shearing stress. Even weak mechanical strain, as exerted, for example, during capillary viscometry or heating the solution to 37 degrees C, leads to considerable breakage of these filaments. Because of spontaneous repair which consumes ATP, the mechanically broken filaments exhibit an approx. 6-fold enhanced steady-state ATPase activity as compared to normal F-actin. Monomers of glutathionyl actin have a reduced affinity for their bound nucleotide and a slightly increased critical concentration. Disruption of the filaments and enhanced ATPase activity are reversed by the addition of KCl or the mushroom toxin phalloidin. By the large stabilizing effects of KCl and phalloidin on glutathionyl actin filaments we propose glutathionyl actin as a tool for detecting filament-stabilizing agents and for studying the different mechanisms of filament stabilization.

Actin Cytoskeleton↗

PubMed1990

2,4-Dinitrophenyl [14C]cysteinyl disulfide allows selective radioactive labeling of protein thiols under spectrophotometric control.

2,4-Dinitrophenyl [1-14C]cysteinyl disulfide readily introduces by disulfide exchange [14C]cysteine as a label into proteins with exposed thiols. The release of an equivalent amount of colored 2,4-dinitrothiophenolate allows the labeling reaction to be followed spectrophotometrically. In reaction with two cysteine residues of rabbit skeletal muscle actin, the thiol selectivity of the reagent corresponded to that of 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) and was superior to that of N-[14C]ethylmaleimide. Labeling of single SH groups of actin and papain proceeded faster than titration with Ellman's reagent under the same conditions. The [14C]cysteine label could be removed under mild conditions, e.g., with dithiothreitol, but proved to be stable during cyanogen bromide degradation of the protein and peptide purification. 2,4-Dinitrophenyl cysteinyl disulfide can be easily prepared within a few hours.

Actins↗

PubMed1980

[The use of contrast media in computer tomography (author's transl)].

There are a variety of applications for contrast media in CT. They can be used for better anatomic demonstration or for showing pathological processes. Nearly all available contrast media may be found useful. Intravenously injected renographic contrast media are most commonly employed. In addition to their inherent contrast, specific pharmaco-kinetic changes may be of diagnostic value. This adds some knowledge of function to the purely morphological information obtained by computer tomography. For this purpose, contrast has to be injected rapidly and a fast scanner must be used (so-called angio-CT).

Contrast Media↗

PubMedDate not supplied

Tau domains, phosphorylation, and interactions with microtubules.

We consider the interactions of tau protein with microtubules from two points of view, phosphorylation and domain structure. Tau can be phosphorylated at many sites and by several kinases, notably by proline-directed kinases (MAPK, GSK-3, cdk5) which generate Alzheimer-like antibody epitopes. Other kinases phosphorylate Ser 262, a site that has a particularly pronounced influence on the affinity of tau for microtubules. All of these sites can be cleared by phosphatases PP-2a and calcineurin. The site Ser262 lies within the repeat domain of tau. However, when probing the domains of tau for their effects on microtubule binding, nucleation, assembly, or bundling, the repeat domain has only a weak influence. Whereas the repeat domain of tau binds to microtubules with low affinity, repeat-less tau binds strongly yet unproductively in terms of microtubule assembly. Productive binding of tau to microtubules depends on the combination of (some) repeats with the flanking regions, as if the flanking regions acted as "jaws" for the proper positioning of tau on the microtubule surface.

Animals↗