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E Grazi

Publications and source records attributed to E Grazi.

At least 37 records · Page 2Linked to original sources
PubMed1994

Actin may contribute to the power stroke in the binary actomyosin system.

At the physiological protein osmotic pressure, the angle formed between the long axis of the actin monomer and the pointed end of the filament axis is roughly 61 degrees in F-actin and about 90 degrees in the myosin subfragment 1--decorated F-actin. This implies that, in the course of the contractile cycle, actin itself contributes, by about 4 nm, to the displacement of the actin filament toward the center of the sarcomer.

Actins↗
PubMed1994

alpha-Actinin from chicken gizzard: at low temperature, the onset of actin-gelling activity correlates with actin bundling.

The effect of alpha-actinin from chicken gizzard on the properties of F-actin solutions at 37 degrees C and at 4 degrees C was investigated. Beside the well-known increase of the gelling activity of alpha-actinin, it was found that lowering temperature to 4 degrees C: (a) modifies the shape of the alpha-actinin-F-actin binding isotherm; (b) increases the light scattering of the alpha-actinin-F-actin mixtures; (c) induces the formation of ribbons and bundles of F-actin. It was also observed that, by warming to 37 degrees C, the bundles of F-actin formed at 4 degrees C were dissociated into quasi-parallel actin filaments running at a distance of 25-42 nm from each other. On subsequent cooling to 4 degrees C, these parallel filaments were rapidly assembled into bundles. As at 37 degrees C, alpha-actinin displays a potent gelling activity on bundles but not on filaments of actin, and as the gelling activity at 4 degrees C is accompanied by the formation of actin bundles, it is concluded that actin bundling is a necessary condition to promote the actin-gelling activity.

Actinin↗
PubMed1993

Osmotic stress is the main determinant of the diameter of the actin filament.

The diameter of the actin filament is influenced by osmotic stress, being 9.0 nm at 1 x 10(5) dynes/cm2 and 6.8 nm at 9.00 x 10(6) dynes/cm2. At 1.81 x 10(5) dynes/cm2, the protein osmotic pressure in frog muscle, the diameter is 7.95 nm. The diameter of the tropomyosin-decorated actin filament is also influenced by osmotic stress even though, at the low pressures (up to 2 x 10(5) dynes/cm2), the decorated filament is significantly more resistant to compression than the undecorated actin filament.

Actins↗
PubMed1993

Diffusion hindrance and geometry of filament crossings account for the complex interactions of F-actin with alpha-actinin from chicken gizzard.

The interaction of alpha-actinin from chicken gizzard with F-actin is quite complex. The apparent dissociation constant, C, increases with the increase of actin concentration according to the following expression: C = Ko + a[actin] - c[actin]5/2. At pH 7.5 and 37 degrees C, in the presence of 0.1 M KCl and 2 mM MgCl2, the dissociation constant at infinite actin dilution, Ko, is 2.17 microM. The binding of alpha-actinin to actin is related by the term a[actin] to the diffusion of actin filaments and by the term c[actin]5/2 to the crossing number concentration of the F-actin network. Especially at low actin concentration, the binding of alpha-actinin to actin is increased by gelsolin, which fragments actin filaments and increases their diffusion. The different binding isotherms of alpha-actinin to actin filaments and to actin bundles are discussed.

Actinin↗
PubMed1992

Preferential binding of alpha-actinin to actin bundles.

At 37 degrees C, the alpha-actin-F-actin binding isotherm is anomalous. In 6.7% polyethylene glycol 6000, concomitantly with the formation of actin bundles, the binding isotherm becomes hyperbolic (Kdiss. = 11.3 microM). alpha-Actinin increases the rigidity of the networks formed by actin bundles in polyethylene glycol and by paracrystalline actin in 16 mM MgCl2 but not by F-actin. It is proposed that in the cell alpha-actinin functions are mostly carried on by interaction with actin bundles.

Actinin↗
PubMed1992

The control of cellular shape and motility. Mg2+ and tropomyosin regulate the formation and the dissociation of microfilament bundles.

At pH 7.14 and 37 degrees C, in 7.2% (w/v) poly(ethylene glycol) 6000, tropomyosin-regulated actin filaments are converted into filament bundles by increasing the free Mg2+ concentration to 1.7-2.0 mM. When free Mg2+ concentration is decreased below 1.7 mM, bundles dissociate back into tropomyosin-regulated actin filaments. Pure actin filaments are insensitive to this mechanism of control and are found as filament bundles in all the range of free Mg2+ concentrations tested (1.37-2.2 mM). The mechanism of regulation described above is likely to operate in the cell, where the concentration of free Mg2+ is linked to the energy charge of the adenine nucleotide system.

Actin Cytoskeleton↗
PubMed1992

'Macromolecular crowding' is a primary factor in the organization of the cytoskeleton.

We propose that, in the cell, the reversible conversion of actin filaments into actin bundles is controlled by the concentration of the macromolecules [we have employed poly(ethylene glycol) 6000 to mimic the macromolecules of the cell] as well as by the nature of the ancillary cytoskeletal proteins that decorate actin filaments. The proposal is based on the following evidence. (1) Under our experimental conditions the transition from filaments into bundles occurs at increasing concentrations of poly(ethylene glycol), with the following sequence: caldesmon-actin, 3%; filamin-actin, 4-5%; caldesmon-tropomyosin-actin, 5-7%; actin, 6-7%; tropomyosin-actin, 9-10%. (2) Under conditions of low osmoelastic stress [3% poly(ethylene glycol)], preformed caldesmon-actin bundles are dissociated by the addition of either tropomyosin or tropomyosin-decorated actin. The dissociation of the bundles promoted by the addition of tropomyosin-decorated actin is faster than that promoted by the addition of tropomyosin.

Actins↗
PubMed1991

The control of cellular motility and the role of gelsolin.

Solation of actin gel by gelsolin is much less efficient in the presence of a high concentration of macromolecular solutes. The rigidity of the gel formed by 12 microM actin is lowered from 4 to 0.33 dynes/cm2 by 15 nM gelsolin, while in 6% (w/v) polyethylene glycol, rigidity is lowered only from 20 to 11 dynes/cm2 by 64 nM gelsolin. Owing to the large concentration of protein, transitions in the fluid- and gel-like properties of the cytoplasm are expected to be problematic when promoted by gelsolin alone.

Actins↗
PubMed1991

Binding of alpha-actinin to F-actin or to tropomyosin F-actin is a function of both alpha-actinin concentration and gel structure.

We have studied by electron microscopy as well as by measurements of low shear viscosity, rigidity and binding, the effect of alpha-actinin on the gel formed at 37 degrees C with F-actin and with tropomyosin-decorated F-actin. Contrary to previous reports in the literature, alpha-actinin at nanomolar concentrations is an efficient actin gelling protein, even at 37 degrees C, provided that the concentration of actin (or of tropomyosin-decorated F-actin) is low (1.2-2.4 microM). The binding of alpha-actinin to F-actin, as a function of actin concentration, is anomalous. The amount of bound alpha-actinin increases when actin concentration increases from 0 to 1.2 microM but does not change significantly when actin concentration is further increased up to 48 microM. A similar result is obtained with tropomyosin-decorated F-actin. These observations can be explained by an hypothesis that binding is a function of the alpha-actinin - F-actin association constant as well as of the rigidity of the gel. When the concentration of actin increases, the rigidity of the gel also increases and more work is required to bring two actin filaments to the reaction distance with alpha-actinin and, consequently, a larger alpha-actinin concentration is required to attain the same ratio of bound alpha-actinin to actin monomers in the filaments.

Actinin↗
PubMed1990

The actin gelling activity of chicken gizzard alpha-actinin at physiological temperature is triggered by water sequestration.

At 37 degrees C, in the presence of 6% (w/v) polyethylene glycol 6000, 30 nM alpha-actinin from chicken gizzard induces the gelation of 12 microM actin. Static measurement shows that the addition of 30 nM alpha-actinin increases the rigidity of the system from 23.5 to 54 dynes/cm2. According to the theory of osmoelastic coupling, also large additives, such as the proteins of the cell sap, are able to cause an osmotic stress equivalent to that caused by polyethylene glycol. We thus conclude that, in vivo, alpha-actinin acts as an actin gelling protein.

Actinin↗
PubMed1990

Divergent effects of filamin and tropomyosin on actin filaments bundling.

Filamin increases and tropomyosin decreases the susceptibility of F-actin to form bundles of filaments in the presence of polyethylene glycol 6000. The two proteins, which are located in the leading edge and in the internal part of the cell, respectively, are thus likely to display divergent effects on the microfilaments into bundles transition in these two areas of the cell.

Actin Cytoskeleton↗
PubMed1990

Microfilament gel rigidity cooperates negatively with the binding of actin gelling proteins.

At 37 degrees C, in the presence of 0.1 M KC1 and 2 mM MgCl2, the binding of alpha-actinin to F-actin increases with the concentration of alpha-actinin but not with the concentration of F-actin. This implies that binding is determined by additional factors, beside the alpha-actinin - F-actin association constant. We propose that one of these factors is the rigidity of the gel, which cooperates negatively to the binding by increasing the work needed to bring two actin filaments at the reaction distance with alpha-actinin.

Actin Cytoskeleton↗
PubMed1989

The influence of substoichiometric concentrations of myosin subfragment 1 on the state of aggregation of actin under depolymerizing conditions.

In 3 mM KCl, 2 mM Tris/HCl pH 7.5, 22 degrees C, 0.38 microM myosin subfragment 1 delays the depolymerization of F-actin (7.2 microM measured as monomer). The depolymerization proceeds rapidly for a few minutes and then slows down suddenly when the ratio between the monomers in the actin filaments and myosin subfragment 1 reaches the value of 11. At this time myosin subfragment 1 is substantially all bound to the actin polymers which form an irregular and discontinuous network of filaments running in doublets and in triplets, perhaps cross-linked by myosin subfragment 1. Depolymerization proceeds then for several hours, apparently ending up with the formation of the 1:1 actin-S1 heteropolymer. The ratio between the monomers in the actin filaments and myosin subfragment 1 at the end of the rapid depolymerization process is different for different protein preparations and may be as low as 5.5. In 2 mM Tris/HCl pH 7.5, 25 degrees C, 1 microM myosin subfragment 1 is able to induce the formation of undecorated actin filaments from 12 microM ATP--G-actin. These filaments probably originate by redistribution of myosin subfragment 1 between the newly formed 1/1 actin-S1 heteropolymer and G-actin in the medium, a process which allows the transient formation of undecorated actin filaments.

Actin Depolymerizing Factors↗
PubMed1989

Substoichiometric concentrations of ATP-G-actin are required to anneal actin polymerized by calcium ions.

At 3 degrees C and pH 7.0, the addition of 40 nM ATP-G-actin to F-actin (12 microM as the monomer), polymerized in the presence of 4 mM CaCl2, determines a substantial and rapid increase of the viscosity of the solution, which is accompanied by the incorporation of the ATP-G-actin added into the polymer. The hypothesis that the presence of ATP-actin at the filament end(s) promotes the annealing reaction is substantiated by the finding that, after the addition of ATP-G-actin, the average filament length is increased. This finding is relevant, not only because it provides evidences in favour of the existence of annealing but also because it shows that the concentration of ATP-G-actin influences the filaments length distribution through a mechanism different from the elongation reaction.

Actins↗
PubMed1989

Opposite effects of alfa-actinin and of fructose 1,6-bisphosphate aldolase on the microfilament network. The role of orthophosphate revisited.

At pH 7.5, in the presence of 0.1 M KCl, 2 mM MgCl2 and 15 mM phosphate, the binding of 1 molecule of alfa-actinin for each strand of 1000 actin monomers doubles the apparent viscosity of an F-actin solution (12 microM as the monomer). Further binding of one molecule of aldolase for each strand of 280 actin monomers halves the apparent viscosity of the alfa-actinin-F-actin system without any desorption of alfa-actinin. The effect of aldolase is not hindered by the addition of 0.1 mM fructose 1,6-bisphosphate. It is shown that orthophosphate acts as a damper of the regulatory effect of fructose bisphosphate on the interaction between aldolase and microfilaments.

Actin Cytoskeleton↗