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↗

PubMedDate not supplied

Binding of calcium to myoplasmic buffers contributes to the frequency-dependent inotropy in heart ventricular cells.

In guinea-pig ventricular cells, the Ca2+ buffer capacity of the myoplasm was estimated from the ratio of ionized calcium (from Indo-1 fluorescence) through total calcium (ionized plus bound calcium, from x-ray microprobe analysis). During post-rest potentiation (1 Hz paired-pulses in voltage-clamp), where diastolic sarcomere length remained nearly constant, Ca2+ buffer capacity slowly fell from 5500:1 to 700:1 suggesting that slow Ca2+ binding sites became saturated. We discuss that frequency-inotropy depends not only on the replenishment of intracellular stores with Ca2+, but also on binding of Ca2+ to these slow sites; the slow Ca2+ sites could complete with the fast activator sites on troponin C for systolic Ca2+, or they could enhance the Ca2+ affinity of the fast Ca2+ sites on troponin C by cooperative interaction.

Action Potentials↗

PubMedDate not supplied

Disruption of microfilament organization after injection of F-actin capping proteins into living tissue culture cells.

Capping proteins are F-actin binding proteins which interfere with the in vitro growth of an actin filament by blocking one of its ends (for recent reviews see refs 1-3). The majority of such proteins described so far "cap' the fast-growing (positive) end of the polar filament, thus reducing the velocity of filament growth while increasing the number of filaments being formed de novo from a monomer pool. We have studied the effects of capping proteins on the organization of actin filaments in living tissue culture cells by microinjection in conjunction with fluorescence, reflection contrast and electron microscopy. Our results, reported here, indicate that capping proteins from different sources disrupt microfilament bundles in a variety of cell types causing their disintegration from the distal end towards the centre of the cell.

Animals↗

PubMedDate not supplied

Regulation of actin polymerization by non-polymerizable actin-like proteins.

Three functionally distinct actin-capping proteins from the slime mould Physarum are structurally closely related to actin itself. In Physarum, actin polymerization is regulated by a set of non-polymerizable actin-like proteins. It remains to be established whether these proteins and actin are each encoded by separate genes.

Actins↗

PubMedDate not supplied

Ca entry and contraction as studied in isolated bovine ventricular myocytes.

Single bovine ventricular myocytes were superfused with Tyrode solution containing 1.8 mM CaCl2. The cells did not bear external load and contracted isotonically. Contraction and relaxation were characterized by the shortening and relengthening of the sarcomeres which resembled in their time course the isometric twitches of bovine papillary muscles. Resemblance was also found in regard to positive inotropic interventions as increase in the stimulation frequency, exposure to elevated [Ca]0 or to adrenaline. A two-microelectrode voltage-clamp technique was applied to the single myocyte. The transmembrane Ca inward current ICa was defined as difference current sensitive to 5 mM Ni or to 2 microM D600. During a voltage step from -45 to +5 mV, ICa peaked within 3 ms to -6 nA, afterwards it decayed to 15% of peak amplitude (incomplete inactivation with a 2 exponential time course). Experiments in Na-free media suggested that Na entry does not significantly contaminate ICa. Therefore, Ca entry could be calculated from ICa. The increment in total intracellular Ca concentration (delta[Ca]Ti) was estimated by referring Ca entry to the cell volume (50 pl). Within 100 ms delta[Ca]Ti came to 25 microM at control conditions, to 55 microM at [Ca]0 = 3.6 mM and to 88 microM when 0.1 microM adrenaline were present. The delta[Ca]Ti values were sufficient to activate contraction without the necessity of Ca-release from SR. Despite the new data, the relationship between Ca entry and activation of contraction was complex: during the "positive Herztreppe" ICa slightly attenuated but contractility doubled. Therefore, the old EC-model (M. Morad and Y. Goldman, Progr. Biophys. Mol. Biol. 27, 257 (1973)) was adapted. The Ca-entry's capability to load and to overload the intracellular Ca store (SR) is discussed.

Animals↗

PubMedDate not supplied

Prevention of ischemic renal damage with prostacyclin.

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Animals↗