ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin

The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.     

Human T cell L-plastin bundles actin filaments in a calcium-dependent manner.

The amino acid sequences deduced from cDNA analyses revealed that human leucocyte L-plastin phosphorylated in response to interleukin 1, 2 closely resembles a chicken intestinal microvilli protein, fimbrin, that bundles actin filaments [de Arruda et al. (1990) J. Cell Biol. 111, 1069-1079]. In the present work, it was observed that unphosphorylated L-plastin isolated from human T cells bundled F-actin just as fimbrin does. L-Plastin acted on T cell beta-actin, but hardly acted on muscle alpha-actin or chicken gizzard gamma-actin, whereas fimbrin bundled muscle alpha-actin. Unlike fimbrin, L-plastin's actin-bundling action was strictly calcium-dependent: the bundles were formed at pCa 7, but not at pCa 6. Under suitable conditions, approximately one molecule of L-plastin bound to 8 molecules of actin monomer in the actin filament.     

Localization of an endogenous ADP-ribose acceptor, p33, in polymorphonuclear cell granules in chicken liver interlobular connective tissue

We investigated immunohistochemically the localization of p33, an endogenous substrate protein for an arginine-specific ADP-ribosyltransferase in chicken liver. Polymorphonuclear-pseudo-eosinophilic granulocytes (heterophils) in interlobular connective tissues of the liver were exclusively and strongly stained with the antibody against p33. Strong reactivity was associated with granules in cytoplasm of the heterophils. When the chicken liver nuclear fraction was washed, the transferase activity was released into the 600 x g supernatant fraction while a nuclear enzyme poly(ADP-ribose) synthetase was retained in the pellet fraction. These results indicate that p33 and probably also ADP-ribosyltransferase, found in the liver nuclear fraction [Tanigawa et al. (1984) J. Biol. Chem. 259, 2022-2029, Mishima et al. (1988) Eur. J. Biochem. 179, 267-273], originate from interlobular heterophils of the chicken liver.     

ADP ribosylation of Arg41 of Rap by ExoS inhibits the ability of Rap to interact with its guanine nucleotide exchange factor, C3G

ExoS is a bifunctional type III cytotoxin that is secreted by Pseudomonas aeruginosa. The N-terminal domain comprises a RhoGAP activity, while the C-terminal domain comprises a ADP-ribosyltransferase activity. Previous studies showed that ExoS ADP ribosylated Ras at Arg41 which interfered with the ability of Ras to interact with its guanine nucleotide exchange factor. Rap and Ras share considerable primary amino acid homology, including Arg41. In this study, we report that ExoS ADP ribosylates Rap1b at Arg41 and that ADP ribosylation of Arg41 inhibits the ability of C3G to stimulate guanine nucleotide exchange. The mechanism responsible for this inhibition is one in which ADP-ribosylated Rap binds inefficiently to C3G, relative to wild type Rap. This identifies a second member of the Ras GTPase subfamily that can be ADP ribosylated by ExoS and indicates that ExoS can inhibit both Ras and Rap signaling pathways in eukaryotic cells.     

Isolation and characterization of covalently cross-linked actin dimer

Covalently cross-linked actin dimer was isolated from rabbit skeletal muscle F-actin reacted with phenylenebismaleimide (Knight, P., and Offer, G. (1978) Biochem. J. 175, 1023-1032). The UV spectrum of the purified cross-linked actin dimer, in a nonpolymerizing buffer, was very similar to that of native F-actin and not to the spectrum of G-actin. Cross-linked actin dimer polymerized to filaments that were indistinguishable in the electron microscope from F-actin made from native G-actin and that were similar to native F-actin in their ability to activate the Mg2+-ATPase of myosin subfragment-1. The critical concentrations of polymerization of cross-linked actin dimer in 0.5 mM and 2.0 mM MgCl2, 2 to 4 microM, and 1 to 2 microM, respectively, were similar to the values for native G-actin. Cross-linked actin dimer contained 2 mol of bound nucleotide/mol of dimer. One bound nucleotide exchanged with ATP in solution with a t 1/2 of 55 min and with ADP with a t 1/2 of 5 h. The second bound nucleotide exchanged much more slowly. The more rapidly exchangeable site contained 10 to 15% bound ADP.Pi and 85 to 90% bound ATP while the second site contained much less, if any, bound ADP.Pi. Cross-linked actin dimer had an ATPase activity in 0.5 mM MgCl2 that was 7 times greater than the ATPase activity of native G-actin and that was also stimulated by cytochalasin D. These data are discussed in relation to the possible role of ATP in actin polymerization and function with the speculation that the cross-linked actin dimer may serve simultaneously as a useful model for each of the two different ends of native F-actin.     

Evidence that F-actin can hydrolyze ATP independent of monomer-polymer end interactions

The rate of ATP hydrolysis in solutions of F-actin at steady state in 50 mM KC1, 0.1 mM CaC12 was inhibited by AMP and ADP. The inhibition was competitive with ATP (Km of about 600 microM) with Ki values of 9 microM for AMP and 44 microM for ADP. ATP hydrolysis was inhibited greater than 95% by 1 mM AMP. AMP had no effect on the time course of actin polymerization, ATP hydrolysis during polymerization, or the critical actin concentration. Simultaneous measurements of G-actin/F-actin subunit exchange and nucleotide exchange showed that nucleotide exchange occurred much more rapidly than subunit exchange; during the experiment over 50% of the F-actin-bound nucleotide was replaced when less than 1% of the F-actin subunits had exchanged. When AMP was present it was incorporated into the polymer, preventing incorporation of ADP from ATP in solution. F-actin with bound Mg2+ was much less sensitive to AMP than F-actin with bound Ca2+. These data provide evidence for an ATP hydrolysis cycle associated with direct exchange of F-actin-bound ADP for ATP free in solution independent of monomer-polymer end interactions. This exchange and hydrolysis of nucleotide may be enhanced when Ca2+ is bound to the F-actin protomers.     

Calcium-dependent regulation of actin filament bundling by lipocortin-85

Lipocortin-85 (L-85, calpactin-I/lipocortin-II heterotetramer) binds to F-actin in the presence of calcium with high affinity and in a cooperative manner. Quantitative analysis of binding curves indicate an apparent Kd (L-85) of 0.226 microM +/- 0.153 (2 S.D., n = 3), a stoichiometry of L-85/actin of 1:1.9 and a Hill coefficient of 1.37 +/- 0.14 (2 S.D., n = 3). Large anisotropic bundles were visualized by electron microscopy under these conditions, and quantitation of bundling by both low speed sedimentation and light scattering yielded apparent Kd values between 0.12 and 0.27 microM L-85. Filament bundling was dependent upon calcium, and the calcium sensitivity was increased by raising the molar ratio of lipocortin-85/F-actin. At saturating levels of L-85, apparent K0.5 values of 0.1-2 microM Ca2+f were obtained. The monomeric heavy chain, lipocortin-II, bundled F-actin to a much lesser extent and at much higher concentrations than for lipocortin-85. Bundling of F-actin by lipocortin-I was not detected at molar ratios of lipocortin-I to actin as high as 2.5 mol/mol (lipocortin-I/actin). At 5-10 microM Ca2+f and saturating levels of L-85, F-actin bundling progressed very rapidly with a t0.5 of 6 s. The process was quickly reversed by the addition of excess EGTA, and bundles could be reformed by the addition of a second burst of 5-10 microM Ca2+f. Thus, our data suggest that lipocortin-85 can rapidly regulate F-actin bundling in a calcium-dependent manner at physiologically relevant calcium levels.     

Inactivation of glutamine synthetases by an NAD:arginine ADP-ribosyltransferase

Glutamine synthetase from ovine brain has a critical arginine residue at the catalytic site (Powers, S. G., and Riordan, J.F. (1975) Proc. Natl. Acad. Sci. U.S. A. 72, 2616-2620). This enzyme is now shown to be a substrate for a purified NAD:arginine ADP-ribosyltransferase from turkey erythrocyte cytosol that catalyzes the transfer of ADP-ribose from NAD to arginine and purified proteins. The transferase catalyzed the inactivation of the synthetase in an NAD-dependent reaction; ADP-ribose and nicotinamide did not substitute for NAD. Agmatine, an alternate ADP-ribose acceptor in the transferase-catalyzed reaction, prevented inactivation of glutamine synthetase. MgATP, a substrate for the synthetase which was previously shown to protect that enzyme from chemical inactivation, also decreased the rate of inactivation in the presence of NAD and ADP-ribosyltransferase. Using [32P]NAD, it was observed that approximately 90% inactivation occurred following the transfer of 0.89 mol of [32P]ADP-ribose/mol of synthetase. The erythrocyte transferase also catalyzed the NAD-dependent inactivation of glutamine synthetase purified from chicken heart; 0.60 mol of ADP-ribose was transferred per mol of enzyme, resulting in a 95% inactivation. As noted with the ovine brain enzyme, agmatine and MgATP protected the chicken synthetase from inactivation and decreased the extent of [32P]ADP-ribosylation of the synthetase. These observations are consistent with the conclusion that the NAD:arginine ADP-ribosyltransferase modifies specifically an arginine residue involved in the catalytic site of glutamine synthetase. Although the transferase can use numerous proteins as ADP-ribose acceptors, some characteristics of this particular arginine, perhaps the same characteristics that are involved in its function in the catalytic site, make it a favored ADP-ribose acceptor site for the transferase.     

Salmonella enterica SpvB ADP-ribosylates actin at position arginine-177-characterization of the catalytic domain within the SpvB protein and a comparison to binary clostridial actin-ADP-ribosylating toxins

The SpvB protein from Salmonella enterica was recently discovered as an actin-ADP-ribosylating toxin. SpvB is most likely delivered via a type-III secretion system into eukaryotic cells and does not have a binding/translocation component. This is in contrast to the family of binary actin-ADP-ribosylating toxins from various Bacillus and Clostridium species. However, there are homologies in amino acid sequences between the C-terminal domain of SpvB and the catalytic domains of the actin-ADP-ribosylating toxins such as C2 toxin from Clostridium botulinum and iota toxin from Clostridium perfringens. We compared the biochemical properties of the catalytic C-terminal domain of SpvB (C/SpvB) with the enzyme components of C2 toxin and iota toxin. The specificity of C/SpvB concerning the modification of G- or F-actin was comparable to the C2 and iota toxins, although there were distinct differences regarding the recognition of actin isoforms. C/SpvB and iota toxin modify both muscle alpha-actin and nonmuscle beta/gamma-actin, whereas C2 toxin only modifies beta/gamma-actin. In contrast to the iota and C2 toxins, C/SpvB possessed no detectable glycohydrolase activity in the absence of a protein substrate. The maximal reaction rates were comparable for all toxins, whereas variable K(m) values for NAD were evident. We identified arginine-177 as the modification site for C/SpvB with the actin homologue protein Act88F from Drosophila.     

Arginine-specific ADP-ribosyltransferase and its acceptor protein p33 in chicken polymorphonuclear cells: co-localization in the cell granules, partial characterization, and in situ mono(ADP-ribosyl)ation.

We have reported the purification and characterization of arginine-specific ADP-ribosyltransferase from hen liver nuclei [Tanigawa, Y. et al. (1984) J. Biol. Chem. 259, 2022-2029] and the DNA-dependent mono(ADP-ribosyl)ation of p33, an acceptor protein in the nuclei [Mishima, K. et al. (1989) Eur. J. Biochem. 179, 267-273]. In the present study, we obtained evidence that among various tissues and cells from chicken, polymorphonuclear cells, so-called heterophils, possess both the ADP-ribosyltransferase and p33 at high levels. Percoll density gradient centrifugation of the postnuclear fraction of the heterophils revealed the co-localization of ADP-ribosyltransferase with p33 in the granule fraction. The enzyme and p33 were purified approximately 219- and 3.77-fold, respectively, from postnuclear pellet fraction to apparent homogeneity. The properties of heterophil ADP-ribosyltransferase and p33 were compared with those of the liver enzyme and p33. The molecular mass of the heterophil enzyme was estimated by SDS-polyacrylamide gel electrophoresis to be 27.5 kDa. The enzyme activity was stimulated by a sulfhydryl agent and inhibited by lysolecithin, NaCl, and inorganic phosphate. The mono(ADP-ribosyl)ation of p33 was markedly enhanced by polyanion, such as DNA, RNA, or poly(L-glutamate). SDS-polyacrylamide gel electrophoretic analysis after limited trypsin proteolysis of p33s, purified from chicken heterophils and liver, showed much the same pattern. Thus, it appears that ADP-ribosyltransferase and p33 present in heterophils are identical to those in the liver, respectively. p33 is considered to be an in situ substrate for ADP-ribosyltransferase, since it was specifically mono(ADP-ribosyl)ated in permeabilized heterophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bundling of actin filaments by alpha-actinin depends on its molecular length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.     

Ca2+ ionophore A23187 and thrombin inhibit the pertussis-toxin-induced ADP-ribosylation of the alpha-subunit of the inhibitory guanine-nucleotide-binding protein and other proteins in human platelets

Inhibitory guanine-nucleotide-binding proteins (Gi proteins) are substrates for pertussis toxin and the decreased pertussis-toxin-dependent ADP ribosylation of Gi proteins upon prior specific hormonal stimulation of cells is thought to reflect the receptor-mediated activation of Gi proteins, leading to their subsequent dissociation into alpha i and beta/gamma subunits. In the present study, the effect of various platelet stimuli on the subsequent pertussis-toxin-dependent ADP ribosylation of the alpha subunit of Gi (Gi alpha) in saponized platelets and platelet membranes were studied. Stimulation of intact platelets with the Ca(2+)-ionophore A23187 or thrombin, but not phorbol 12,13-dibutyrate, decreased the subsequent pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponin-permeabilized platelets in a time-dependent and dose-dependent manner. Thrombin was more effective than A23187. Parallel measurements of Ca2+ mobilization and pertussis-toxin-dependent ADP ribosylation of Gi alpha in platelets showed that Ca2+ mobilization could only partly account for the decrease in pertussis-toxin-dependent ADP ribosylation in platelets stimulated by thrombin. When the ADP-ribosylation reaction was carried out in platelet membranes, a decrease in ADP ribosylation was still observed after stimulation of platelets with thrombin, but not with A23187. In addition to Gi alpha, two other proteins were found to be ADP ribosylated by pertussis toxin; their ADP ribosylation was also decreased after A23187 and thrombin stimulation of platelets. The results indicate that Ca2+ mobilization can decrease the pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponized platelets; the decrease of pertussis-toxin-dependent ADP ribosylation of Gi alpha after thrombin stimulation of platelets can only, in part, be explained by Ca2+ mobilization and involves additional mechanisms; the decrease in pertussis-toxin-dependent ADP ribosylation after A23187 and thrombin stimulation is not confined to G1 alpha and involves other proteins. We conclude that the decrease in pertussis-toxin-dependent ADP ribosylation of Gi in thrombin-stimulated platelets might not be solely caused by a specific structural change, such as dissociation of Gi. It is likely that A23187 and thrombin stimulation of platelets generates substances which interfere with the ADP-ribosylating activity of pertussis toxin.     

Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes.

A protein purified from cytoskeletal fractions of Dictyostelium discoideum proved to be a member of the fimbrin/plastin family of actin-bundling proteins. Like other family members, this Ca(2+)-inhibited 67-kDa protein contains two EF hands followed by two actin-binding sites of the alpha-actinin/beta-spectrin type. Dd plastin interacted selectively with actin isoforms: it bound to D. discoideum actin and to beta/gamma-actin from bovine spleen but not to alpha-actin from rabbit skeletal muscle. Immunofluorescence labeling of growth phase cells showed accumulation of Dd plastin in cortical structures associated with cell surface extensions. In the elongated, streaming cells of the early aggregation stage, Dd plastin was enriched in the front regions. To examine how the bundled actin filaments behave in myosin II-driven motility, complexes of F-actin and Dd plastin were bound to immobilized heavy meromyosin, and motility was started by photoactivating caged ATP. Actin filaments were immediately propelled out of bundles or even larger aggregates and moved on the myosin as separate filaments. This result shows that myosin can disperse an actin network when it acts as a motor and sheds light on the dynamics of protein-protein interactions in the cortex of a motile cell where myosin II and Dd plastin are simultaneously present.     

Microcalorimetric measurement of the enthalpy of binding of rabbit skeletal myosin subfragment 1 and heavy meromyosin to F-actin

The heat of binding of rabbit skeletal myosin subfragment 1 (myosin-S1) and heavy meromyosin (HMM) to F-actin has been measured by batch calorimetry. Proton release measurements in unbuffered solutions indicate that less than 0.1 mol of protons is absorbed or released per mol of myosin head bound to actin. Hence, the measured heats are approximately equal to the enthalpy of myosin-S1 and HMM binding to actin. The enthalpy of binding of myosin-S1 to actin was +22 +/- 3 and +27 +/- 5 kJ/mol of myosin-S1 in two series of experiments at 12 degrees C and +26 +/- 5 kJ/mol of myosin-S1 at 0 degrees C, indicating that delta Cp for this reaction in the range of 0-12 degrees C is small (-80 J/mol/K). The enthalpy of binding of HMM to actin at 12 degrees C was found to be +26 +/- 1 kJ/mol of myosin head. The enthalpies determined here and the equilibrium constants obtained from the literature for measurements at 20 degrees C under identical solvent conditions were used to estimate the entropy of the association of myosin S1 and HMM with F-actin: +235 J/mol/K for myosin-S1 and +190 J/mol of myosin head/K for HMM. Thermodynamic parameters of the interaction of myosin-S1 with actin and ADP or AMP-PNP can be evaluated using the enthalpy of association of myosin-S1 with actin determined here, together with literature values for the equilibrium constants and enthalpies of binding of these nucleotides to myosin-S1. The calculated enthalpies of binding of ADP or AMP-PNP to actomyosin-S1 are small and negative.     

Effect of F-actin upon the binding of ADP to myosin and its fragments.

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.     

ADP-ribosylation of platelet actin by botulinum C2 toxin

Botulinum C2 toxin is a microbial toxin which possesses ADP-ribosyltransferase activity. In human platelet cytosol a 43-kDa protein was ADP-ribosylated by botulinum C2 toxin. Labelling of the 43-kDa protein using [32P]NAD as substrate was reduced by unlabelled NAD and nicotinamide. The label was removed by treatment with snake venom phosphodiesterase. Half-maximal and maximal ADP-ribosylation occurred at 0.1 microgram/ml and 3 micrograms/ml botulinum C2 toxin, respectively. The Km value of the ADP-ribosylation reaction for NAD was about 1 microM. The peptide map of the ADP-ribosylated 43-kDa protein was almost identical with platelet actin. The ADP-ribosylated 43-kDa substrate protein bound to and was eluted from immobilized DNase I in a manner similar to G-actin. Trypsin treatment of platelet cytosol decreased subsequent ADP-ribosylation of the 43-kDa protein without occurrence of smaller labelled polypeptides. Purified platelet actin was also ADP-ribosylated by botulinum C2 toxin with similar characteristics found with actin in platelet cytosol. Phalloidin decreased the ADP-ribosylation of actin in platelet cytosol and of isolated platelet actin. Half-maximal and maximal, about 90%, reduction of actin ADP-ribosylation was observed at 0.4 microM and 10 microM phalloidin, respectively. ADP-ribosylation of purified actin, induced by botulinum C2I toxin, abolished the formation of the typical microfilament network. The data indicate that platelet G-actin but not F-actin is a substrate of botulinum C2 toxin and that this covalent modification largely affects the functional properties of actin.     

Actin filament barbed end elongation with nonmuscle MgATP-actin and MgADP-actin in the presence of profilin

We have quantitated the in vitro interactions of profilin and the profilin-actin complex (PA) with the actin filament barbed end using profilin and nonmuscle beta,gamma-actin prepared from bovine spleen. Actin filament barbed end elongation was initiated from spectrin seeds in the presence of varying profilin concentrations and followed by light scattering. We find that profilin inhibits actin polymerization and that this effect is much more pronounced for beta,gamma-actin than for alpha-skeletal muscle actin. Profilin binds to beta,gamma-actin filament barbed ends with an equilibrium constant of 20 microM, decreases the filament elongation rate by blocking addition of actin monomers, and increases the dissociation rate of actin monomers from the filament end. PA containing bound MgADP supports elongation of the actin filament barbed end, indicating that ATP hydrolysis is not necessary for PA elongation of filaments. Initial analysis of the energetics for these reactions suggested an apparent greater negative free energy change for actin filament elongation from PA than elongation from monomeric actin. However, we calculate that the free energy changes for the two elongation pathways are equal if the profilin-induced weakening of nucleotide binding to actin is taken into consideration.     

Endogenous ADP ribosylation of high mobility group proteins 1 and 2 and histone H1 following DNA damage in intact cells

Endogenous ADP ribosylation of nonhistone high-mobility group (HMG) proteins and histone H1 was studied in cultured mouse mammary tumor cells following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MNNG treatment of cells caused a rapid and transient increase in ADP ribosylation of histone H1 and HMG 1 and 2, whereas (ADP-ribose)n on HMG 14 and 17 was not affected. 3-Aminobenzamide, an inhibitor of (ADP-ribose)n synthetase, prevented the increase in ADP ribosylation of histone H1 and HMG 1 and 2. This inhibitor enhanced the cell-killing effect of MNNG, but had no significant effect on the removal of methylated purines. The preferential increase in ADP ribosylation of HMG 1 and 2 and histone H1 may be necessary for cell recovery from DNA damage.     

Reannealing of phalloidin-treated actin filaments during recovery after sonication and its inhibition by beta-actinin

Phalloidin (2 mol per mol actin)-treated pyrenyl F-actin showed a critical concentration of 1.8 microM in the presence of 10 mM KCl, 0.2 mM ADP, and 5 mM Tris-HCl buffer, pH 8.0 at 25 degrees C. The filament weight concentration did not change at all during and after sonication, yet degrees of flow birefringence increased and the filament number concentration decreased after the termination of sonication. The latter changes were not affected by EDTA, but inhibited by beta-actinin. These observations suggest that reannealing of short pieces of phalloidin-treated actin filaments fragmented during sonication takes place during recovery after sonication.     

Ca(2+) activation and tension cost in myofilaments from mouse hearts ectopically expressing enteric gamma-actin

To determine the significance of actin isoforms in chemomechanical coupling, we compared tension and ATPase rate in heart myofilaments from nontransgenic (NTG) and transgenic (TG) mice in which enteric gamma-actin replaced >95% of the cardiac alpha-actin. Enteric gamma-actin was expressed against three backgrounds: mice expressing cardiac alpha-actin, heterozygous null cardiac alpha-actin mice, and homozygous null cardiac alpha-actin mice. There were no differences in maximum Ca(2+) activated tension or maximum rate of tension redevelopment after a quick release and rapid restretch protocol between TG and NTG skinned fiber bundles. However, compared with NTG controls, Ca(2+) sensitivity of tension was significantly decreased and economy of tension development was significantly increased in myofilaments from all TG hearts. Shifts in myosin isoform population could not fully account for this increase in the economy of force production of TG myofilaments. Our results indicate that an exchange of cardiac alpha-actin with an actin isoform differing in only five amino acids has a significant impact on both Ca(2+) regulation of cardiac myofilaments and the cross-bridge cycling rate.