Ermes Magri

Phosphorylation of actin and removal of its inhibitory activity on pancreatic DNAase I by liver plasma membranes

It was recently reported that the 1: 1 actinDNAase I complex is dissociated by 5’-nucleotidase and the DNAase activity is restored [ 13; the same result is obtained by incubating the actin-DNAase I complex with liver plasma membranes [2]. The a~umption, in this case, is that actin is bound to the 5’-nucleotida~ embedded in the plasma membr~es and DNAase I is released free in the solution. We have confirmed the results of these experiments and describe here a new, independent mechanism of protection of DNAase I against the inactivation by Gactin. Both G-actin and F-actin, when incubated with liver plasma membranes, are phosphorylated and lose their Inhibitory activity on DNAase I. The inactivated G-actin does not polymerize. The inactivated F-&in still activates myosin.

G-actin modified by plasma membrane interaction polymerizes only in the presence of filamentous myosin

In non-muscle cells actin can occur in different forms depending on the immediate and local needs of the cell. This suggests the presence of regulatory mechanisms capable to allow an easy interconversion between polymerization states of actin ranging from functional microfilaments to oligomeric or even monomeric actin. We describe here a potential regulatory mechanism mediated by the interaction of G-actin with plasma membranes. After the interaction G-actin is modified insuch a way that it does not polymerize in the presence of 2 mM MgCl, but only in the presence ‘of myosin with the formation of contractile units.

The interaction of histone and protamine with actin. Possible involvement in the formation of the mitotic spindle.

Abstract At low ionic strength 1–2 μM protamine or 1–2 μM H1 histone induce the nucleation of G-ATP actin. At high ionic strength 1–2 μM protamine or 0.1 to 0.2 μM H1 histone accelerate by 3 to 4 times the rate of polymerisation of G-ATP actin. It is suggested that histone may trigger the formation of actin fibers running from the kinetochore of the chromosome to the pole of the mitotic spindle.

Active subunits of rabbit liver fructose diphosphatase

Abstract Fructose diphosphatase, bound to a matrix of Sepharose, retains most of the catalytic activity but becomes half desensitized to AMP. The dimers, obtained by acid dissociation of the enzyme bound to the matrix, possess half of the specific activity of the tetramers and are almost completely desensitized to AMP. The monomers are inactive.

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 μM 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.

Studies on the polymerisation of actin: a rapid method for the separation of the monomeric from the polymeric species.

A main difficulty in the study of the kinetics of actin polymerisation is the time consuming procedure available for the separation of the monomeric from the polymeric species [ 11. We have now found that G-actin and F-actin can be separated by filtration through 0.45 I.tm millipore filters. The procedure is very rapid and suitable to handle samples of very small volume. We expect this observation to provide a large impulse on the studies of the mechanism of actin polymerisation.

The stiffness of the crossbridge is a function of the intrinsic protein osmotic pressure generated by the crossbridge itself

A model is presented that makes it possible to determine the stiffness of the crossbridge from protein osmotic stress experiments. The model was elaborated while studying the osmotic properties of F-actin and of myosin subfragment-1 F-actin. These studies showed that the elastic modulus by bending of the monomer is directly related to the intrinsic protein osmotic pressure of the system. At a protein osmotic pressure of 1.8 x 10(5) dynes/cm2, the physiological protein osmotic pressure of frog skeletal muscle, it was found that the elastic moduli by bending of the monomer in F-actin and in the myosin subfragment-1 decorated F-actin are 6.5 X 10(7) and 3.3 X 10(8) dynes/cm2, respectively. The value of the elastic modulus by bending of the monomer in the myosin subfragment-1 decorated F-actin compares favorably with the values of the elastic modulus by stretching determined in skeletal muscle fibres.

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

At 37°C, in the presence of 6% ( ) polyethylene glycol 6000, 30 nM α‐actinin from chicken gizzard induces the gelation of 12 μM actin. Static measurement shows that the addition of 30 nM α‐actinin increases the rigidity of the system from 23.5 to 54 . 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, α‐actinin acts as an actin gelling protein.

Protein cross talking through osmotic work: the free energy of formation of the MgADP-myosin complexes at the muscle protein osmotic pressure

A method is presented to determine the energy of formation of the myosin-ADP complexes at the muscle protein osmotic pressure. It is found that, at 18 kP, the putative protein osmotic pressure in skeletal muscle, the increase of MgADP from 0.05 to 2 mmolal, increases the free energy of myosin-ADP and of myosin-(ADP)2 by 0. 756 and by 9.85 kJ/mol, respectively, and decreases the free energy of myosin by 8.34 kJ erg/mol. It is pointed out that the local changes of water chemical potential, induced by the binding of MgADP to myosin, can be sensed by other structures of the contractile machinery, which per se may even be insensitive to MgADP. Cross talking between macromolecules can thus be achieved by changes of the water chemical potential.

Preferential binding of α-actinin to actin bundles

At 37°C, the α‐actinin‐F‐actin binding isotherm is anomalous. In 6.7% polyethylene glycol 6000, concomitantly with the formation of actin bundles, the binding isotherm becomes hyperbolic (K diss. = 11.3 μM). α‐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 α‐actinin functions are mostly carried on by interaction with actin bundles.