Interaction of myosin subfragment-1 with F- and G-actin in equilibrium.

The structural changes of the F-actin-myosin head (S1) complex during the cross-bridge cycle are essential in muscle contraction. Although a large body of evidence has accumulated showing that the actin: S1 stoichiometry in the decorated F-actin-S1 filament is 1:1 at saturation by S1, a recent report by Andreev and Borejdo (1991, Biochem. Biophys. Res. Comm. 177, 350-356) indicated that under some conditions, the actin: S1 stoichiometry could be 2:1 at saturation by S1. Because of the important implications of this result in the mechanism of acto-myosin motility, we have re-investigated this issue. It is shown here that evidence for the 2:1 stoichiometry was circumstantial and was only observed under conditions where 50% of the actin was F-actin, i.e. at a total actin concentration twice as large as the critical concentration. The interaction of S1 with both F- and G-actin in dynamic equilibrium is studied in detail. The present data fully support the 1:1 actin: S1 stoichiometry in the decorated filament at saturation by S1.     

Kinetic analysis of F-actin depolymerization in the presence of platelet gelsolin and gelsolin-actin complexes

Platelet gelsolin (G), a 90,000-mol-wt protein, binds tightly to actin (A) and calcium at low ionic strength to form a 1:2:2 complex, GA2Ca2 (Bryan, J., and M. Kurth, 1984, J. Biol. Chem. 259:7480-7487). Chromatography of actin and gelsolin mixtures in EGTA-containing solutions isolates a stable binary complex, GA1Ca1 (Kurth, M., and J. Bryan, 1984, J. Biol. Chem. 259:7473-7479). The effects of platelet gelsolin and the binary gelsolin-actin complex on the depolymerization kinetics of rabbit skeletal muscle actin were studied by diluting pyrenyl F-actin into gelsolin or complex-containing buffers; a decrease in fluorescence represents disassembly of filaments. Dilution of F- actin to below the critical concentration required for filament assembly gave a biphasic depolymerization curve with both fast and slow components. Dilution into buffers containing gelsolin, as GCa2, increased the rate of depolymerization and gave a first order decay. The rate of decrease in fluorescence was found to be gelsolin concentration dependent. Electron microscopy of samples taken shortly after dilution into GCa2 showed a marked reduction in filament length consistent with filament severing and an increase in the number of ends. Conversely, occupancy of the EGTA-stable actin-binding site by an actin monomer eliminated the severing activity. Dilution of F-actin into the gelsolin-actin complex, either as GA1Ca1 or GA1Ca2, resulted in a decrease in the rate of depolymerization that was consistent with filament end capping. This result indicates that the EGTA-stable binding site is required and must be unoccupied for filament severing to occur. The effectiveness of gelsolin, GCa2, in causing filament depolymerization was dependent upon the ionic conditions: in KCI, actin filaments appeared to be more stable and less susceptible to gelsolin, whereas in Mg2+, actin filaments were more easily fragmented. Finally, a comparison of the number of kinetically active ends generated when filaments were diluted into gelsolin versus the number formed when gelsolin can function as a nucleation site suggests that gelsolin may sever more than once. The data are consistent with a mechanism where gelsolin, with both actin-binding sites unoccupied, can sever but not cap F-actin. Occupancy of the EGTA-stable binding site yields a gelsolin-actin complex that can no longer sever filaments, but can cap filament ends.     

Actin deoxyroboncuclease I interaction. Depolymerization and nucleotide exchange

Deoxyribonuclease I (DNase I) forms a 1:1 complex with globular actin (G-actin) and also will depolymerize filamentous actin (F-actin) to form a 1:1 complex. The effect of DNase I on the exchange of the actin nucleotide has been investigated. When DNase I is added to G-actin, the rate of nucleotide exchange is decreased from 1.16 +/- 0.25 X 10(-4) s-1 to 0.28 +/- 0.09 X 10(-4) s-1 (0 degrees C). The presence of ATP or ADP in the actin has little effect on the rate of exchange of the nucleotide for ATP. This suggests that the weaker affinity of ADP than ATP for actin is due to a slower association rate of ADP. The rate of the nucleotide exchange in the actinDNase I complex is increased by the addition of NaCl or MgCl2. When DNase I is added to F-actin, the rate of nucleotide exchange (6.2 +/- 1.6 X 10(-4) x-1, 0 degrees C) is similar to the rate of depolymerization as measured by loss of viscosity. The actinDNase I complex formed by depolymerization of F-actin exchanges nucleotide at a 4-fold faster rate than the G-actinDNase I complex in the same ionic conditions. This and other experiments suggest that DNase I binds first to F-actin before dissociating the monomer from the filament. These results are discussed in terms of possible mechanisms of action depolymerization.     

Ca2+ regulation of gelsolin activity: binding and severing of F-actin.

Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.     

Oligomeric Aip2p/Dld2p modifies the protein conformation of both properly folded and misfolded substrates in vitro

Oligomeric actin-interacting protein 2 (Aip2p) [Nat. Struct. Biol. 2 (1995) 28]/D-lactate dehydrogenase protein 2 (Dld2p) [Yeast 15 (1999) 1377, Biochem. Biophys. Res. Commun. 295 (2002) 910] exhibits the unique grapple-like structure with an ATP-dependent opening [Biochem. Biophys. Res. Commun. 320 (2004) 1271], which is required for the F-actin conformation modifying activity in vitro and in vivo [Biochem. Biophys. Res. Commun. 319 (2004) 78]. To further investigate the molecular nature of oligomeric Aip2p/Dld2p, the substrate specificity of its binding and protein conformation modifying activity was examined. In the presence of 1mM ATP or AMP-PNP, oligomeric Aip2p/Dld2p bound to all substrates so far examined, and modified the conformation of actin, DNase I, the mature form of invertase, prepro-alpha-factor, pro-alpha-factor, and mitochondrial superoxide dismutase, as determined by the trypsin susceptibility assay. Of note, the activity could modify even the conformation of pathogenic highly aggregated polypeptides, such as recombinant prion protein in beta-sheet form, alpha-synuclein, and amyloid beta (1-42) in the presence of ATP. The in vivo protein conformation modifying activity, however, depends on the growth stage; the most significant substrate modification activity was observed in yeast cells at the log phase, suggesting the presence of a cofactor/s in yeast cells, where F-actin is supposed to be a major target in vivo. These data further support our previous notion that the oligomeric Aip2p/Dld2p may belong to an unusual class of molecular chaperones [Biochem. Biophys. Res. Commun. 320 (2004) 1271], which can target both properly folded and misfolded proteins in an ATP-dependent manner in vitro.     

Cytochalasin D acts as an inhibitor of the actin-cofilin interaction

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.     

Mechanisms responsible for F-actin stabilization after lysis of polymorphonuclear leukocytes

While actin polymerization and depolymerization are both essential for cell movement, few studies have focused on actin depolymerization. In vivo, depolymerization can occur exceedingly rapidly and in a spatially defined manner: the F-actin in the lamellipodia depolymerizes in 30 s after chemoattractant removal (Cassimeris, L., H. McNeill, and S. H. Zigmond. 1990. J. Cell Biol. 110:1067-1075). To begin to understand the regulation of F-actin depolymerization, we have examined F-actin depolymerization in lysates of polymorphonuclear leukocytes (PMNs). Surprisingly, much of the cell F-actin, measured with a TRITC-phalloidin-binding assay, was stable after lysis in a physiological salt buffer (0.15 M KCl): approximately 50% of the F-actin did not depolymerize even after 18 h. This stable F-actin included lamellar F-actin which could still be visualized one hour after lysis by staining with TRITC-phalloidin and by EM. We investigated the basis for this stability. In lysates with cell concentrations greater than 10(7) cells/ml, sufficient globular actin (G-actin) was present to result in a net increase in F-actin. However, the F-actin stability was not solely because of the presence of free G-actin since addition of DNase I to the lysate did not increase the F-actin loss. Nor did it appear to be because of barbed end capping factors since cell lysates provided sites for barbed end polymerization of exogenous added actin. The stable F-actin existed in a macromolecular complex that pelleted at low gravitational forces. Increasing the salt concentration of the lysis buffer decreased the amount of F-actin that pelleted at low gravitational forces and increased the amount of F-actin that depolymerized. Various actin-binding and cross-linking proteins such as tropomyosin, alpha-actinin, and actin-binding protein pelleted with the stable F-actin. In addition, we found that alpha-actinin, a filament cross-linking protein, inhibited the rate of pyrenyl F-actin depolymerization. These results suggested that actin cross-linking proteins may contribute to the stability of cellular actin after lysis. The activity of crosslinkers may be regulated in vivo to allow rapid turnover of lamellipodia F-actin.     

Cross-linking study on skeletal muscle actin: interaction of suberimidate-treated actin with deoxyribonuclease I

We have recently reported that actin modified with dimethyl suberimidate takes a filamentous form even under depolymerizing conditions, and this phenomenon is accounted for by the conformational fixation caused by the introduction of an intramolecular cross-link (Ohara, O., Takahashi, S., Ooi, T., & Fujiyoshi, Y. (1982) J. Biochem. 91, 1999-2012). The suberimidate-treated actin (SA) is not immediately depolymerized by deoxyribonuclease I (DNase I) but is depolymerized after incubation for one day, i.e., depolymerization is much slower than that for intact F-actin. The results on circular dichroic spectra of a mixture of SA and DNase I suggest that DNase I flips the conformation of SA into a G-actin-like state from the F-actin-like one when a tight SA-DNase I complex is formed. The suberimidate cross-link introduced in an SA molecule does not completely prevent the conformational change from the F-state to the G-state but stabilizes the actin conformation very greatly in the F-state.     

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.     

Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin

Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548-1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27-3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin-Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of approximately 16 nm, a sedimentation coefficient of 17-20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions.     

Interaction of D-lactate dehydrogenase protein 2 (Dld2p) with F-actin: implication for an alternative function of Dld2p

D-Lactate dehydrogenase protein 2 [Yeast 15 (1999) 1377; Biochem. Biophys. Res. Commun. 295 (2002) 910] was initially identified as the actin interacting protein 2 (Aip2p) using a two-hybrid screen to search for proteins that interact with actin [Nat. Struct. Biol. 2 (1995) 28], but no other evidence indicating an interaction between Aip2p and actin cytoskeleton has been reported so far. During our search for the protein conformation modifying activity, we serendipitously identified Aip2p isolated from Saccharomyces cerevisiae as exhibiting an interaction with F-actin both in vitro and in vivo. Incubation with Aip2p facilitated the formation of the circular form of F-actin in vitro, which exhibited an aberrant trypsin susceptibility. Overexpression of Aip2p induced multi-buds in yeast cells, whereas reduced expression interfered with the formation of the cleavage furrow for the cell division, which was rescued by the introduction of wild-type Aip2p. While Aip2p-treated F-actin in the circular form was negligibly stained by rhodamine-labeled phalloidin (rhodamine-phalloidin) in vitro, rhodamine-phalloidin staining profiles in actin interacting protein 2 gene (AIP2)-modified cells suggested a correlation between the conformation of F-actin and the expression of Aip2p in vivo. AIP2-deleted cells became sensitive to osmotic conditions, a hallmark of actin dysfunction. Finally, immunoprecipitation of yeast cells using anti-Aip2p antibody demonstrated that Aip2p associates with actin. These properties suggest that Aip2p may interact with F-actin in vivo and play an important role in the yeast cell morphology.     

Trifluoperazine binding to calmodulin: a shift reagent 43CA NMR study

43Ca NMR experiments of Ca2+ binding to calmodulin (CaM) were performed in the presence and absence of the calmodulin antagonist trifluoperazine (TFP). By making use of the shift reagent Dy(PPP)(7-) (a 1:2 complex of DyCl3 and Na5P3O10) we have succeeded in separating the 43Ca resonances of protein-bound Ca2+ and free Ca2+ in the otherwise unresolved spectra. This experimental strategy has allowed us to demonstrate unequivocally that the affinity of CaM for Ca2+ is markedly increased in the presence of TFP. Thus Ca2+ is not liberated from the protein upon addition of TFP as had been suggested based on earlier 43Ca NMR experiments (Shimuzu, T., Hatano, M., Nagao, S. and Nozawa, Y. (1982), Biochem. Biophys. Res. Comm. 106, 1112-1118).     

Differential dynamic behavior of actin filaments containing tightly-bound Ca2+ or Mg2+ in the presence of myosin heads actively hydrolyzing ATP.

We have investigated how Ca2+ or Mg2+ bound at the high-affinity cation binding site in F-actin modulates the dynamic response of these filaments to ATP hydrolysis by attached myosin head fragments (S1). Rotational motions of the filaments were monitored using steady-state phosphorescence emission anisotropy of the triplet probe erythrosin-5-iodoacetamide covalently attached to cysteine 374 of actin. The anisotropy of filaments containing only Ca2+ increased from 0.080 to 0.137 upon binding S1 in a rigor complex and decreased to 0.065 in the presence of ATP, indicating that S1 induced additional rotational motions in the filament during ATP hydrolysis. The comparable anisotropy values for Mg(2+)-containing filaments were 0.067, 0.137, and 0.065, indicating that S1 hydrolysis did not induce measurable rotational motions in these filaments. Phalloidin, a fungal toxin which stabilizes F-actin and increases its rigidity, increased the anisotropy of F-actin containing either Ca2+ or Mg2+ but not the anisotropy of the 1:1 S1-actin complexes of these filaments. Mg(2+)-containing filaments with phalloidin bound also displayed increased rotational motions during S1 ATP hydrolysis. A strong positive correlation between the phosphorescence anisotropy of F-actin under specific conditions and the extent of the rotational motions induced by S1 during ATP hydrolysis suggested that the long axis torsional rigidity of F-actin plays a crucial role in modulating the dynamic response of the filaments to ATP hydrolysis by S1. Cooperative responses of F-actin to dynamic perturbations induced by S1 during ATP hydrolysis may thus be physically mediated by the torsional rigidity of the filament.     

Heparin increases mRNA levels of thrombospondin but not fibronectin in human vascular smooth muscle cells

The effects of heparin (180 micrograms/ml) on steady state mRNA levels for fibronectin, thrombospondin, actin and collagen types I and III were investigated in human umbilical artery smooth muscle cells. Heparin caused a 120% increase in thrombospondin mRNA levels and a 60% and 180% increase in the mRNA levels of procollagen chains alpha 2(I) and alpha 1(III), respectively. No change in fibronectin or actin mRNA levels resulted from heparin treatment. We reported earlier (Biochem. Biophys. Res. Comm. 148:1264, 1987) that heparin increases smooth muscle cell synthesis of both fibronectin and thrombospondin. These data show that heparin coordinately regulates thrombospondin mRNA and protein levels. The heparin induced increase in fibronectin biosynthesis apparently reflects control at the translational or post-translational level.     

Gamma-Actinin, a new regulatory protein from rabbit skeletal muscle. II. Action on actin.

The interaction of gamma-actinin and actin was investigated under various conditions. It has been shown that gamma-actinin affects the G-F transformation of actin, causing an increase in the number of actin monomers required to form a nucleus in the initial step of polymerization. Sonicated fragments of F-actin and heavy meromyosin caused the immediate polymerization of actin under the influence of gamma-actinin. Therefore, it can be concluded that gamma-actinin inhibits the nucleation step of G-F transformation. Actin filaments which were formed in the presence of gamma-actinin (F-actin) were shown to possess certain characteristic properties when compared with control F-actin. These were as follows: F-actin solution had a high critical concentration; F-actin showed a high rate of depolymerization; the flow birefringence of F-actin decreased with time upon incubation in the absence of free ATP; finally, F-actin was demonstrated to have ATP-splitting activity. These dynamic features of F-actin were accounted for in terms of an increase in the rate constant of depolymerization in F-actin under the influence of gamma-actinin.     

Effect of tropomyosin on the interactions of actin with actin-binding proteins isolated from pig platelets

Several non-muscle tropomyosins have been reported to lack the ability to polymerize in a head-to-tail manner [Dabrowska, R. et al. (1983) J. Muscle Res. Cell Motil. 1, 83-92; C?té, G.P. (1983) Mol. Cell. Biochem. 57, 127-146]. Unlike rabbit skeletal muscle tropomyosin, these proteins could therefore not protect the F-actin microfilaments neither from disassembly or from cross-linking by the other actin-associating factors. However, we have provided evidence that, in vitro, pig platelet tropomyosin, although shorter in molecular length, exhibits the same properties as the muscle protein: it self-associates and forms a 1:6 complex with platelet filamentous actin under physiological conditions [Prulière et al. (1984) J. Muscle Res. Cell Motil. 6, 126]. In this paper, we examine the effects of several other actin-binding proteins on the microfilaments saturated with this non-muscle tropomyosin. Since contractile proteins often vary with the cell type and may require different conditions for their interactions, we have developed a procedure which allows the parallel purification of actin-binding protein (ABP), vinculin, alpha-actinin, gelsolin as well as actin and tropomyosin from the same batch of cells. Thus, using an homogeneous system, we show by viscometry, sedimentation and densitometry, and by electron microscopy, that pig platelet tropomyosin can protect the structure of the microfilaments from the action of the modulating factors to the same extent as rabbit skeletal muscle alpha-tropomyosin. Our data suggest that interaction of ABP, vinculin or alpha-actinin can occur only with the ends of the filaments when F-actin is saturated with tropomyosin, while cross-linking takes place by interactions with sites localized along the entire length of F-actin in the absence of tropomyosin. Moreover, the presence of tropomyosin on F-actin leads to the total inhibition of gelsolin severing activity, although it did not prevent the binding of gelsolin to the F-actin--tropomyosin complex. This suggests that pig platelet as well as skeletal muscle tropomyosins have the ability to increase the strength of the interaction between actin monomers within the filament. This also suggests that the binding sites of gelsolin along the filaments are not localized in the groove of the F-actin helix.     

Influence of NG-monomethyl-L-arginine on endothelium-dependent relaxations in the perfused mesenteric vascular bed of the rat

Endothelium-dependent relaxation mediated by the formation of nitric oxide (NO) from L-arginine, is prevented by the arginine analog NG-monomethyl L-arginine (L-NMMA) (Palmer et al., Biochem. Biophys. Res. Comm. 153:1251-1256 (1988)). In the rat mesenteric arterial bed, incubation with L-NMMA did not prevent acetylcholine-induced relaxation, which, however, was reversed when L-NMMA was added during its maximum effect. A similar profile of action was observed with methylene blue, an inhibitor of guanylate cyclase. Methylene blue, but not L-NMMA, increased basal perfusion pressure. These data indicate that in the mesenteric arterial bed, NO formation via the L-NMMA-sensitive pathway occurs during stimulation with acetylcholine, but not under basal conditions.     

Proposed tertiary structure of the sodium channel.

On the basis of our recent results of the complete amino acid sequence of the squid Loligo bleekeri sodium channel deduced by cloning and sequence analysis of the complementary DNA (Sato, C. and Matsumoto, G. Biochem. Biophys. Res. Comm. 186, 1), we have proposed a tertiary structure model of the sodium channel where the transmembrane segments are octagonally aligned and the four linkers of S5-6 between segments S5 and S6 play a crucial role in the activation gate, voltage sensor and ion selective pore, which can slide, depending on membrane potentials, along inner walls consisting of segments S2 and S4 alternately. The proposed model is contrasted with that of Noda et al. (Nature 320; 188-192, 1986).     

Drebrin-induced Stabilization of Actin Filaments

Drebrin is a mammalian neuronal protein that binds to and organizes filamentous actin (F-actin) in dendritic spines, the receptive regions of most excitatory synapses that play a crucial role in higher brain functions. Here, the structural effects of drebrin on F-actin were examined in solution. Depolymerization and differential scanning calorimetry assays show that F-actin is stabilized by the binding of drebrin. Drebrin inhibits depolymerization mainly at the barbed end of F-actin. Full-length drebrin and its C-terminal truncated constructs were used to clarify the domain requirements for these effects. The actin binding domain of drebrin decreases the intrastrand disulfide cross-linking of Cys-41 (in the DNase I binding loop) to Cys-374 (C-terminal) but increases the interstrand disulfide cross-linking of Cys-265 (hydrophobic loop) to Cys-374 in the yeast mutants Q41C and S265C, respectively. We also demonstrate, using solution biochemistry methods and EM, the rescue of filament formation by drebrin in different cases of longitudinal interprotomer contact perturbation: the T203C/C374S yeast actin mutant and grimelysin-cleaved skeletal actin (between Gly-42 and Val-43). Additionally, we show that drebrin rescues the polymerization of V266G/L267G, a hydrophobic loop yeast actin mutant with an impaired lateral interface formation between the two filament strands. Overall, our data suggest that drebrin stabilizes actin filaments through its effect on their interstrand and intrastrand contacts.     

Evaluation of mechanical parameters of the mechanochemically 'active state' of actin filaments on the basis of purely chemical data

Oosawa and his collaborators (cf. F. Oosawa, Biophys. Chem. 11 (1980) 443), employing various optical techniques, have shown that the flexibility of actin filaments increases upon interacting with the enzymatically active myosin fragments, particularly heavy meromyosin (HMM). It has been reported (S. Hitchock, L. Carlsson and U. Lindberg, Cell 7 (1976) 53) that HMM can accelerate the DNase 1-induced depolymerization of F-actin, provided MgATP is also present. Since, as we have demonstrated (cf. J. Borejdo myosin, is endowed with mechanochemical capability, we made an attempt to correlate the enhanced rate of depolymerization with the decrease in rigidity of the G-G bonds in F-actin. On the basis of the chemical kinetic data of Hitchcock et al. we could derive the approximate value of the HMM-MgATP-induced change in rigidity which is a mechanical molecular parameter. Since interaction between HMM or HMM subfragment-1 and F-actin in the presence of MgATP leads to the movement of the myosin heads along the actin filaments, it is argued that the enzymic behavior of this system should not be analyzed on the basis of simple, equilibrium, complex formation.