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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.

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 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.

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.