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W H Goldmann

Publications and source records attributed to W H Goldmann.

48 records · Page 3Linked to original sources
PubMed1992

Talin anchors and nucleates actin filaments at lipid membranes. A direct demonstration.

Platelet talin nucleates actin assembly as we show here directly by using rhodamine-phalloidin labelling of actin filaments. Nucleation by talin still occurs after reconstitution into liposomal bilayers. This is also demonstrated directly after protein-lipid double labelling and light microscopic imaging. Talin, thus, is the first actin binding protein for which anchoring and nucleation of actin filament growth at lipid interfaces have been visualized.

Actin Cytoskeleton↗
PubMed1992

Probing actin and liposome interaction of talin and talin-vinculin complexes: a kinetic, thermodynamic and lipid labeling study.

Talin purified from human platelets and chicken gizzard smooth muscle is an actin and lipid binding protein. Here, we have investigated the effect of vinculin on (a) talin-nucleated actin polymerization and (b) insertion of talin into lipid bilayers. Calorimetric data show ternary complex formation between talin, vinculin, and actin. Actin-talin, actin-vinculin and actin-(talin-vinculin) binding and rate constants as well as actin polymerization rates for all three protein species have been determined by steady state titration, stopped-flow, and fluorescence assay. In contrast to an increase of the polymerization rate by a factor of less than 2 for actin-talin and actin-(talin-vinculin) when lowering the temperature, we measured a decrease in rates for actin alone and actin-vinculin. The overall equilibrium constants (Keq) in the van't Hoff plot proved linear and were of one-step reactions. Thermodynamic data exhibited signs of van der Waal's binding forces. Using the photoactivatable lipid analogue [3H]PTPC/11, which selectively labels membrane-embedded hydrophobic domains of proteins, we also show that talin partially inserts into the hydrophobic bilayer of liposomes. This insertion occurs in a similar manner irrespective of preincubation with vinculin.

Actins↗
PubMed1991

Kinetic determination of talin-actin binding.

Smooth muscle talin prepared from chicken gizzard binds to skeletal muscle actin in vitro. The stoichiometry of 1:3 for talin:fluorescent labelled G-actin was confirmed by steady state titration and viscosity measurements under non-polymerizing conditions. The binding constant (Kd) of talin and G-actin was determined by continuous fluorescence titration and gave a value of approx 0.3 microM. The association rate constant of talin and fluorescent labelled G-actin of approx 7 x 10(6) M-1 x s-1 was ascertained by the stopped flow method; the dissociation rate constant was calculated at approx 2-3 s-1.

Actins↗
PubMed1991

Talin binds to actin and promotes filament nucleation.

Platelet talin binds to actin in vitro and hence is an actin binding protein. By four different non-interfering assay conditions (fluorescence, fluorescence recovery after photobleaching, (FRAP), dynamic light scattering and DNase-I inhibition) we show that talin promotes filament nucleation, raises the filament number concentration and increases the net rate of actin polymerization but has no inhibitory effect on filament elongation. Binding of talin to actin occurs at a maximal molar ratio of 1:3 as determined by fluorescencetitration under G-buffer conditions. The overall binding constant was approximately 0.25 microM.

Actins↗
PubMed1991

A "slow" temperature jump apparatus built from a stopped-flow machine.

A simple modification to a standard thermostated stopped-flow machine is described which allows it to be used as a temperature jump machine. Temperature jumps larger than 10 degrees C can be achieved in less than 150 ms which makes it useful for the range of times where conventional rapid temperature jumps are not applicable. The apparatus has a sample size of 300 microliters and can produce temperature jumps both above and below the initial temperature.

Actins↗
PubMedDate not supplied

Native talin is a dumbbell-shaped homodimer when it interacts with actin.

Electron microscopy of glycerol-sprayed and rotary metal-shadowed talin from human platelets reveals a dumbbell-shaped molecule with an average length of approximately 51 nm. Analytical ultracentrifugation of native talin yields a single molecular species with an apparent molecular mass of 412 (+/- 28.6) kDa and a sedimentation coefficient of S20w = 11.2. Chemical cross-linking with glutaraldehyde (GA) and corresponding SDS-PAGE analysis show that the monomer band of talin can be quantitatively converted to a dimer band at GA concentrations > or = 0.45%, indicating that there is no significant amount of monomer present in solution. These structural and biophysical data are compatible with native talin being an antiparallel homodimer. Length measurements and viscometric and fluorescent assays of actin filaments polymerized in the presence of native talin and of covalently cross-linked talin dimers all yield similar effects: namely, increased nucleation and polymerization rates and an overall reduction of actin filament length. Hence, we conclude that talin in its native biological state is a dimer when promoting nucleation of actin filaments.

Actins↗