Reaktionen und relative Affinitäten zwischen verschiedenen Nucleotiden und F-Actin unter Ultraschall

Abstract

1. Like the polymerization of G-actin, the repolymerization of F-actin treated with USV occurs in the presence of every nucleosidetriphosphate (NTP) as well as nucleosiddiphosphate (NDP) that have been investigated. 2. At 4°C solutions of F-actin exchange about 9 per cent of their bound nucleotide instantaneously and about 7 percent during the next 24 hours even then when they are not mechanically treated. 3. On the other hand, every nucleotide is exchanged with considerable velocity if the F-actin solution at pH ~ 7 and 4°C is mechanically treated with USV or in the homogenisator. The rate of exchange decreases considerably if the KCl of the solution is replaced by MgCl2. Moreover, the rate of exchange decreases with the increase of the concentration of F-actin from 1,5·10-5 M to 5·10-5 M if the solution is treated with USV (but not when treated in the homogenisator). 4. The exchanged amounts of all investigated nucleotides are identical if during the mechanical treatment the nucleotides are present in a concentration of ≧ 10-3 M. Under optimal conditions (KCl, 1,6 ·10-5 M F-actin) either in a USV-period of 40′ or after a thousand movements of the teflon piston in the homogenisator, the exchange of the nucleotides reaches an average of 90 per cent. Even after mechanical treatment one, F-actin still binds one mole nucleotide. 5. The kinetics of exchange is monomolecular in any case. 6. If the nucleotide entering by exchange is a triphosphate one phosphate is split off. The kinetics of the splitting is linear. 7. At zero time the rate of exchange and that of splitting are virtually the same, but later the measured rate of exchange lags more and more behind the rate of splitting. At a given moment the ratio of split nucleotide, to exchanged nucleotide is the ratio one would expect under the following two conditions: (a) The single nucleotide ion is split only during the regeneration of a break of the F-actin filament if it has been bound to the point of breaking immediately before the regeneration, (b) The breaks between the single monomeres are statistically distributed over the lengths of the filament (i. e. the interaction of all monomers is of equal strength). 8. If the exchange occurs in the presence of two competing nucleotides the ratio of the affinities of the competing nucleotides to F-actin can be calculated. Thus it has been found that the relative affinities decrease in the following sequence: ATP>ITP>ADP>IDP>GTP>CTP. This sequence coincides with that of the relative affinity constants to G-actin; in addition the numerical values of the affinity constants to F-actin and to G-actin are similar. 9. The same rules obey the binding of the different nucleotides to G-actin and to F-actin as well as the splitting of the nucleotides during the polymerization of G-actin and during the repolymerization of broken F-actin filaments. Thus the seemingly different behaviour of G-actin nucleotide compounds and of F-actin nucleotide compounds becomes the same or nearly so if the steric hindrance of the nucleotide reactions (because of the special quaternary structure of F-actin) is removed by mechanical treatment such as USV or treatment in the homogenisator.