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L A Selden

Publications and source records attributed to L A Selden.

25 records · Page 2Linked to original sources
PubMed1986

High affinity binding of divalent cation to actin monomer is much stronger than previously reported.

Monomeric actin is known to bind tightly one divalent cation per molecule. We have quantitatively reinvestigated the affinity of actin for Ca++ and Mg++ using the fluorescent Ca++ chelator Quin2 to induce and measure the dissociation of Ca++ from Ca-actin, supporting these studies with measurements using 45Ca. We found that the KD for Ca-actin is actually 1.9 +/- 0.7 nM. Kinetic analysis supported this result and demonstrated a dissociation rate constant (k-) of 0.013 s-1 and an association rate constant (k+) of 6.8 X 10(6)M-1 s-1 for Ca-actin. Competitive binding studies indicated that the binding affinity of actin for Ca++ is 5.4 times that for Mg++, yielding a calculated KD for Mg-actin of about 10 nM. Thus, the tight-binding of divalent cations to actin is 3-4 orders of magnitude stronger than previously thought.

Actins↗
PubMed1986

A kinetic comparison between Mg-actin and Ca-actin.

The kinetics of the elongation reaction in the polymerization of actin containing tightly-bound Mg2+ (Mg-actin) or tightly-bound Ca2+ (Ca-actin) have been studied. The reaction was monitored using the increase in fluorescence intensity of N-(1-pyrenyl)iodoacetamide-labelled monomeric actin as a measure of polymer formation. The actin nucleation reaction was circumvented by the addition of phalloidin-stabilized actin nuclei. Elongation rates were obtained at various actin concentrations and at various temperatures for polymerization induced by the presence of different monovalent and divalent salt concentrations. Values for the relative rate constant of forward polymerization (mk+) for Mg-actin were found to be larger than those for Ca-actin under similar conditions (m = number of polymer ends). The critical actin concentration (Cc) of Mg-actin is lower than the Cc for Ca-actin, as were estimates of the relative rate constant of depolymerization (mk-). The temperature dependence of Cc, mk+ and mk- for Mg-actin was different from that for Ca-actin, further suggesting a difference in monomeric properties due to the type of divalent cation tightly bound to actin. Estimates of the activation enthalpy change for the forward reaction in the G in equilibrium F transformation were similar for both types of actin, but the activation enthalpy change for the depolymerization of Mg-actin was significantly larger than that for Ca-actin.

Actins↗
PubMed1984

Bound-cation exchange affects the lag phase in actin polymerization.

The delay or lag phase at the onset of polymerization of actin by neutral salt is generally attributed to an actin nucleation reaction. However, when nucleation is circumvented by the use of phalloidin-stabilized nuclei, a lag phase persists when Ca2+-containing actin is polymerized with MgCl2. Pretreatment of actin with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and/or Mg2+ shortens or eliminates this lag phase, suggesting that exchange of the actin-bound divalent cation occurs during this nucleation-independent lag phase. Measurement of the actin-bound cation initially and after brief incubation with EGTA/Mg2+ directly verifies that Mg2+ has replaced Ca2+ as the actin-bound cation, producing a highly polymerizable Mg2+-actin species. Bound-cation exchange prolongs the lag phase in actin polymerization and probably explains what has been termed the monomer activation step in actin polymerization.

Actins↗
PubMed1983

The tightly bound divalent cation regulates actin polymerization.

The polymerization characteristics of Ca++-actin and Mg++-actin were studied by measuring initial rates of polymerization upon addition of phalloidin-stabilized nuclei and neutral salt. Under conditions where the effects of divalent cation exchange were minimized, CaCl2 and MgCl2 were found to be equally effective in polymerizing actin. Mg++-actin was found to nucleate and polymerize more readily than Ca++-actin, having a forward rate constant about twice that of Ca++-actin under a variety of polymerizing conditions. The critical concentration for Ca++-actin is approximately 20 times that for Mg++-actin under equivalent conditions. These data imply that the polymer of Mg++-actin must be more stable than that of Ca++-actin, having a depolymerization rate constant about 10 fold lower. Since Mg++ is probably the tightly-bound cation in vivo, whereas Ca++-actin has been more widely studied in vitro, it would appear that actin in its physiological state is probably more polymerizable and more stable in the polymer form than previously considered.

Actins↗
PubMed1981

Mechanism of action of phalloidin on the polymerization of muscle actin.

Under conditions where muscle actin only partially polymerizes, or where it does not polymerize at all, a significant enhancement of polymerization was observed if equimolar phalloidin was also present. The increased extent of polymerization in the the presence of phalloidin can be explained by the reduced critical actin concentration of partially polymerized populations at equilibrium. Under such conditions, the rate of polymerization, as judged by the length of time to reach half the viscosity plateau, was found to be essentially independent of the phalloidin concentration. Moreover, the initial rate of polymerization of actin was also found to be independent of phalloidin concentration. However, phalloidin apparently causes a reduction in the magnitude of the reverse rates in the polymerization reaction, as was demonstrated by the lack of depolymerization of phalloidin-treated actin polymers. This effect of phalloidin is also supported by the identification of actin nuclei and short polymers in populations of G-actin incubated with phalloidin in the absence of added KCl. Our conclusion, then, is that phalloidin influences the polymerization of actin by stabilizing nuclei and polymers as they are formed.

Actins↗