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.     

High affinity divalent cation exchange on actin. Association rate measurements support the simple competitive model

Each actin molecule has one high affinity site which binds a divalent cation. It has been proposed that an isomerization of the actin molecule is involved in divalent cation exchange at this site ("isomerization model," Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886); we have maintained that exchange is by a simple competitive mechanism (Estes, J. E., Selden, L. A., and Gershman, L. C. (1987) J. Biol. Chem. 262, 4952-4957). Here, using fluorescent-labeled actin, we measure the apparent rate constant for exchange (kapp) as a function of the ratio of free Ca2+ and Mg2+ concentrations, ([Ca]/[Mg]), and show that both models are consistent with the data. The major parameter controlling this relationship in the simple competitive exchange model, the ratio of the association rate constants for Ca2+ and Mg2+ to actin (kCa/kMg), is found to have a value of about 90. We have verified this parameter by direct measurements of kCa and kMg, finding that kCa = 1.9 x 10(7) M-1 s-1 and kMg = 2.3 x 10(5) M-1 s-1, consistent with the characteristics of the Ca2+ and Mg2+ aquo ions. The corresponding parameter derived from the isomerization model is not verifiable. We conclude that high affinity divalent cation exchange on actin proceeds by a simple competitive mechanism.     

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.     

Activation of heavy meromyosin adenosine triphosphatase by various states of actin.

F-actin monomer (F-monomer) is formed upon the addition of neutral salt to G-actin. Since F-monomer has a digestibility similar to that of F-actin and much lower than that of G-actin, it has been proposed that F-monomer has a conformation different from that of G-actin and similar to the conformation of the subunits in F-actin. To examine whether F-monomer will enhance the magnesium-activated myosin adenosine triphosphatase (Mg2+-ATPase) as much as F-actin, the ability of partially polymerized actin populations at equilibrium to activate the Mg2+-ATPase of heavy meromyosin was investigated. Correlations were made between ATPase activities and the polymerization state of actin as determined by measurements of viscosity and digestibility. No significant activation of the heavy meromyosin ATPase was observed under conditions where G-actin or mixtures of G-actin and F-monomer were present. As polymer formation occurred at higher actin concentrations, or with increased KCl concentrations, substantial activation characteristic of F-actin was observed. The data suggest that F-monomer may undergo a further conformational change as it forms nuclei or joins onto polymers. Alternatively, the site of actin which activates the myosin ATPase may involve the crevice between two adjacent actin subunits.     

Mobility of normal and virus-transformed cells in cellular aggregates

The mobility of embryonic chick cells and cells of four established cell lines was examined in cellular aggregates. This was done by preparing aggregates of unlabeled cells and allowing cells of the same type, but prelabeled with [3H]thymidine, to adhere to the surface of the aggregates. After 2-1/2 days in agitated liquid culture the positions of the labeled cells within the aggregates were determined by autoradiographic techniques. Since the labeled and unlabeled cells were otherwise identical, the degree of penetration of the labeled cells into the aggregates was taken as a measure of the mixing or mobility of cells in the aggregate. With this procedure, embryonic chick liver, heart, and neural retina cells were found to move an average of 2.12, 2.68, and 4.00 cell diameters inward, respectively. Mouse fibroblast BALB/c 3T3 cells moved an average of 1.13 cell diameters inward, while Simian virus 40 (SV40)-transformed BALB/c 3T3 cells moved as much as 8.80 cell diameters inward, indicating that cells of the malignant SV40-transformed line were considerably more mobile than the corresponding nonmalignant 3T3 cells. In contrast, cells of the hamster fibroblast line NIL B moved 4.17 cell diameters in 2-1/2 days, while SV40-transformed NIL B cells moved 3.00 cell diameters in the same time. It was therefore concluded that infection with oncogenic viruses does not necessarily result in increased cellular mobility.     

Opportunities for female choice in the bed bug Cimex lectularius

Bed bugs are cited as exemplars of sexual conflict because mating can only occur via traumatic insemination. However, past antagonistic coevolution between the sexes does not necessarily preclude current female choice. Here, we investigate opportunities for precopulatory female choice in bed bugs. We examined whether females seek out mating opportunities when they gain the most benefit: when females are virgin and/or have recently fed. But, we found that female mating and feeding status had little effect on female attraction to males and male odor. To determine whether females approach male harborages (home crevices) to seek matings in nature, we investigated where matings occurred among unfamiliar pairs of bed bugs. We found that, despite female attraction to male odor, matings were most likely to take place in the female's harborage rather than the male's harborage. We also examined the effect of feeding on male and female ability to mate. Whereas previous research reported that engorgement impaired female ability to refuse matings, we found that male feeding status had a larger effect on the success of mating encounters than female feeding status. Fed males had poor mating success, suggesting that males may be faced with a trade‐off between mating and feeding.