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We have shown that F-actin, polymerized in 50 mM-KCl at 20 degrees C and pH 8.0, can be resolved by centrifugation into two polymer populations, which differ morphologically as well as kinetically. The first population represents about 10% of the overall polymer and is composed of small amorphous aggregates. It rapidly exchanges the bound nucleotide with free ATP in the medium, either directly or through the monomers. The second population is composed of long actin filaments. These are labelled by free ATP in the medium only through condensation with labelled small amorphous aggregates.

When the temperature is lowered from 20 to 4 degrees C, the specific viscosity of actin polymerized in the presence of either 4 mM-CaCl2 or 2 mM-MgCl2, but not of actin polymerized in the presence of 90 mM-KCl, is decreased by 50% in the absence of free ATP. Addition of ATP restores the viscosity of the actin polymerized by Mg2+, but not that of actin polymerized by Ca2+, to the original value. The effect of temperature on actin polymerized in the presence of Ca2+ is due to (a) polymer-into-monomer conversion, (b) latero-lateral aggregation of filaments, and (c) fragmentation of the filaments. Fragmentation, as demonstrated by fractional centrifugation and electron microscopy, was the most important of these.

Mg2+, at submicromolar concentrations, is needed for the nucleation of actin [Maruyama (1981) J. Biol. Chem. 256, 1060-1062]. I show here that Mn2+ fulfils the same function. It binds to oligomers present in the ATP-G-actin solutions with a ratio of 2-3 Mn2+ ions per 100 actin monomers and with an association constant of 0.66 × 10(10) M-1 at pH 8.2 at 25 degrees C. The time course of the binding of Mn2+ to polymerizing actin is not affected by the initial concentration of the protein. Analysis of the distribution of the binding shows that, both in the large oligomeric species and in the polymers, 1 Mn2+ ion is bound for every 14-25 actin monomers, whereas in the smaller oligomeric species 1 Mn2+ ion is bound for every 4 actin monomers. The proposal is made that Mn2+ stabilizes actin nuclei and decreases the concentration of the monomers at the steady state. It is also proposed that, at least in some experimental conditions, the direct condensation of oligomers of intermediate length is an effective mechanism of F-actin formation.

We compared the properties of the nuclei that accumulate in 7.5 mM-KCl in ATP-G-actin solutions and of the oligomers that are formed by sonication of either G-actin or F-actin. We found that the ability of the above species to prime the polymerization of actin decays with different rates. The nuclei are stable in 7.5 mM-KCl (they decay with a rate constant of 1.5 X 10(-3) s -1 at pH 7.8 at 22 degrees C in the absence of KCl). The oligomers formed by sonication of either G-actin or F-actin, once the sonication is stopped, revert to simpler structures or evolve into F-actin, depending on the KCl concentration in which they are kept. In 10.5 mM-KCl at pH 7.8 at 22 degrees C their priming ability decays with a rate constant of 6 X 10(-3) s -1. We propose that the nuclei that form spontaneously in 7.5 mM-KCl are not directly susceptible to elongation. They must first be converted into activated nuclei, which exist in very low concentration at the steady state. The activated nuclei are directly susceptible to elongation, they have a short life and they decay rapidly into the ground state unless the elongation reaction occurs. Sonication displaces the steady-state concentration in favour of the activated state.

Mechanical stimulation (forced circulation in narrow tubing) accelerates as much as 10-fold the rate of polymerization of actin. The increase in the rate is proportional to the intensity of the stimulation for flow rates between 0 and 3 cm/s. This supports the hypothesis that a statistical factor (the orientation of the flowing particles) is influenced by the flow. Comparison of the kinetics of the polymerization of resting and of mechanically stimulated actin solutions shows that both the nucleation and the elongation steps are accelerated. It is thus concluded that flow orients not only the oligomeric structures but also the actin monomers. The elongation reaction, also in the flow-stimulated samples, occurs always by the addition of ATP-G-actin (or ATP-containing oligomers) and not by the fusion of ADP-containing oligomeric structures.

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° in F-actin and about 90° 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.

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.

The rate of oxidation of ferricyanide of the aldolase-dihydroxyacetone phosphate complex was measured under different conditions. The following conclusions are drawn. 1. In the cleavage of fructose diphosphate, catalysed by native aldolase, the steady-state concentration of the enzyme-dihydroxyacetone phosphate carbanion intermediate represents less than 6% of the total enzyme-substrate intermediates. 2. Fructose diphosphate and dihydroxyacetone phosphate compete for the four catalytic sites on aldolase, the binding of fructose diphosphate being about twice as tight. 3. The equilibrium concentration of the carbanion intermediate formed by reaction of carboxypeptidase-treated aldolase with dihydroxyacetone phosphate is independent of pH between 5.0 and 9.0. The rates of fromation of the carbanion intermediate and of the reverse reaction are, however, concomitantly increased by increasing pH between 5.0 and 6.5.

The osmotic behavior of myosin subfragment 1 was studied at 22°C and pH 7.45 in 0.1 m KCl, 2 mm MgCl₂, and 10 mm triethanolamine or in 25 mm phosphate, 2 mm MgCl₂, and 2 mm MgADP. It was found that, in 0.1 m KCl, myosin subfragment 1 behaved as a spheroidal particle, with an average diameter of 8.09 nm, composed of two myosin subfragment 1 molecules. The lower limit of the thermodynamic dimerization constant was estimated to be 3.5 ×104 M−1. Above 5 mm as monomer, myosin subfragment 1 departed from the behavior expected of a dimeric spheroidal model because of the onset of a "hydration force." This force measured at the contact distance between particles equals 2.18 × 107 dynes/cm2 and falls off exponentially with a decay distance of 0.27 nm. In 25 mm orthophosphate, myosin subfragment 1, with an increase in the protein osmotic pressure, shifted from the behavior of a sphere to that of a cylinder. Between 1 × 105 and 4 × 105 dynes/cm2, the behavior of myosin subfragment 1 was different in the presence and in the absence of MgADP. In particular, at 1.8 × 105 dynes/cm2, the protein osmotic pressure in frog muscle, myosin subfragment 1 behaved as a sphere of 3.21-nm radius in the presence of MgADP and as a cylinder with a length to diameter ratio of 2.07 in the absence of MgADP. Under the solution conditions used in this work, S1 never behaved as a fully extended particle.Copyright 1995, 1999 Academic Press, Inc.

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.

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.

The ATP-G-actin aggregates formed by incubation of ATP-G-actin in 7.5 mM-KCl were characterized by electron-microscopical observation, by high-pressure liquid chromatography and by the study of the 1,N6-etheno-ATP-ATP exchange reaction between the free and the actin-bound nucleotide. In 30 mM-KCl the initial rate of the reduced-viscosity increase is found to be directly related to the amount of the aggregates formed in the course of the preincubation in 7.5 mM-KCl.

We have compared the osmotic properties of the hydrated, native actin filament and of hydrated phalloidin-F-actin. We have found that phalloidin-F-actin interacts much more strongly with water than native F-actin. It is therefore very likely that the interaction with myosin (that requires the expulsion of the protein solvation water) is more problematic for phalloidin-F-actin that for native F-actin. We conclude that phalloidin-F-actin is not a bona fide substitute for native F-actin in the "in vitro motility assay".Copyright 1995, 1999 Academic Press, Inc.

The diameter of the actin filament is influenced by osmotic stress, being 9.0 nm at 1×105dynes/cm2and 6.8 nm at 9.00×106dynes/cm2. At 1.81×105dynes/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 2×105dynes/cm2), the decorated filament is significantly more resistant to compression than the undecorated actin filament.

Fructose 1,6-bisphosphate aldolase from rabbit muscle forms by reaction with dihydroxyacetone phosphate a pyruvaldehyde-aldolase-orthophosphate complex that is in equilibrium with the eneamine intermediate. The new intermediate accumulates in two phases. The first one is practically complete in 40ms, and the second occurs with an apparent first-order rate constant of 4.6 +/- 0.5s-1. The new intermediate breaks down slowly with the release into the medium of pyruvaldehyde and Pi. The rate of the spontaneous release is higher at acidic than at neutral pH.

The formation and dissociation of the aldolase-dihydroxyacetone phosphate complex were studied by following changes in A240 [Topper, Mehler & Bloom (1957), Science 126, 1287-1289]. It was shown that the enzyme-substrate complex (ES) slowly isomerizes according to the following reaction: (formula: see text) the two first-order rate constants for the isomerization step being k+2 = 1.3s-1 and k-2 = 0.7s-1 at 20 degrees C and pH 7.5. The dissociation of the ES complex was provoked by the addition of the competitive inhibitor hexitol 1,6-bisphosphate. At 20 degrees C and pH 7.5, k+1 was 4.7 X 10(6)M-1-S-1 and k-1 was 30s-1. Both the ES and the ES* complexes react rapidly with 1.7 mM-glyceraldehyde 3-phosphate, the reaction being practically complete in 40 ms. This shows that the ES* complex is not a dead-end complex. Evidence was also provided that aldolase binds and utilizes only the keto form of dihydroxyacetone phosphate.

Chicken kidney contains two arginases with different sedimentation coefficients and substrate specificity. The ligher of these arginases, which hydrolyses only L-arginine, has been purified about 3000-fold. Like the “ureotelic” arginase, developed in chicken liver after starvation, it displays many of the properties of the arginase of the “ureotelic” species. This seems to exclude the possibility that ureotelism and uricotelism are characterized by a specific type of arginases. Both liver and kidney arginases are located in the mitochondrial matrix. The rate of hydrolysis of arginine thus not only depends on the arginase activity but also on the rate of transport of arginine into the matrix. This last process therefore is of regulatory significance.

A purification procedure for the preparation of chicken liver arginase in a homogeneous form is presented. The enzyme hydrolyses both arginine and argininic acid. Kinetic analysis reveals that the enzyme binds arginine when the amino group is protonated or unprotonated; however, the unprotonated form seems to be hydrolysed more rapidly. The enzyme exchanges with the medium approx. 1.6Mn2+ions per molecule.

[gamma-32P]ATP-G-actin was polymerized in 4 mM-CaCl2, and the distribution of the radioactive nucleotide among the oligomeric and the polymeric species was studied. The results obtained are best explained by assuming spontaneous fragmentation.

The release of Pi from the Pi-G-actin-ADP complex is the rate-limiting step in the ATPase activity that is shown by ATP-G-actin in the presence of protamine.