Mechanism of the Interaction of Superoxide Ion and Ascorbate with Anthracycline Antibiotics

The interaction of superoxide ion and ascorbate anion with anthracycline antibiotics (adriamycin and aclacinimycin A) as well as with their Fe³⁺ complexes has been studied in aprotic and protic media. It was found that both superoxide and ascorbate reduce anthracyclines to deoxyaglycons via a one-electron transfer mechanism under all conditions studied. The reaction of ascorbate anion with adriamycin and aclacinomycin A in aqueous solution proceeded only in the presence of Fe³⁺ ions; it is supposed that an active catalytic species was Fe³⁺ adriamycin. It is also supposed that the reduction of anthracycline antibiotics by O,⁷ and ascorbate in cells may increase their anticancer effect.     

Novel Mode of Cooperative Binding between Myosin and Mg-actin Filaments in the Presence of Low Concentrations of ATP

Cooperative interaction between myosin and actin filaments has been detected by a number of different methods, and has been suggested to have some role in force generation by the actomyosin motor. In this study, we observed the binding of myosin to actin filaments directly using fluorescence microscopy to analyze the mechanism of the cooperative interaction in more detail. For this purpose, we prepared fluorescently labeled heavy meromyosin (HMM) of rabbit skeletal muscle myosin and Dictyostelium myosin II. Both types of HMMs formed fluorescent clusters along actin filaments when added at substoichiometric amounts. Quantitative analysis of the fluorescence intensity of the HMM clusters revealed that there are two distinct types of cooperative binding. The stronger form was observed along Ca²⁺-actin filaments with substoichiometric amounts of bound phalloidin, in which the density of HMM molecules in the clusters was comparable to full decoration. The novel, weaker form was observed along Mg²⁺-actin filaments with and without stoichiometric amounts of phalloidin. HMM density in the clusters of the weaker form was several-fold lower than full decoration. The weak cooperative binding required sub-micromolar ATP, and did not occur in the absence of nucleotides or in the presence of ADP and ADP-Vi. The G680V mutant of Dictyostelium HMM, which over-occupies the ADP-Pi bound state in the presence of actin filaments and ATP, also formed clusters along Mg²⁺-actin filaments, suggesting that the weak cooperative binding of HMM to actin filaments occurs or initiates at an intermediate state of the actomyosin-ADP-Pi complex other than that attained by adding ADP-Vi.     

Conformational change in actin filament induced by the interaction with heavy meromyosin: effects of pH, tropomyosin and deoxy-ATP.

Conformational changes in pure and tropomyosin-containing F-actin during interaction with heavy meromyosin in the absence and presence of deoxy-ATP, were studied by measurements of the changes in fluorescence intensity of e-ADP² incorporated into the F-actin instead of ADP. The actin filaments were found to be stabilized by tropomyosin and were more stable at pH 7 than at pH 8. The rigor binding of HMM to F-actin caused an increase in the fluorescence intensity. The increase with F-actin containing TM was higher than that with pure F-actin at each HMM concentration. A linear relation between the fluoresence change and moles of HMM per actin was found regardless of the presence of TM, with a maximum value of 0.5 moles of HMM per actin. In the presence of deoxy-ATP, (which is a substrate for acto-HMM but cannot bind to actin) no changes in fluorescence intensity of e-ADP bound to pure F-actin were observed. In the case of F-actin containing TM, the fluorescence intensity increased with increasing HMM concentration, although the light scattering intensity of the acto-HMM solutions indicated that almost all the HMM was dissociated from the F-actin. This suggests that the conformational change in F-actin-TM induced by the interaction with HMM in the presence of deoxy-ATP has a long lifetime which continues for some time even after the detachment of the HMM.     

Comparison of the reaction of N-ethylmaleimide with myosin and heavy meromyosin subfragment 1

Myosin reacted at low ionic strength with NEM forms an actomyosin which is Ca⁺⁺ insensitive. With HMM S-1 the reaction with NEM causes a marked loss of the actin activated ATPase activity and the Ca⁺⁺ sensitivity is reduced but not eliminated. The presence of actin during the sulfhydryl reaction does not significantly alter this result. HMM S-1 prepared from myosin previously desensitized by NEM regains Ca⁺⁺ sensitivity. These results indicate that the conformations of myosin and HMM S-1 are different and could reflect a difference between insoluble (filamentous) myosin and myosin, or its fragments, in solution.     

Actin detected in Mouse Neuroblastoma Cells by Binding of Heavy Meromyosin

HEAVY meromyosin (HMM) fragments of myosin from striated muscle specifically bind with actin filaments to form complexes that are readily observed by electron microscopy¹ in both negatively-stained preparations and sectioned material. The composite or “decorated filaments” appear like a line of arrowheads. The existence of such decorated filaments in cells or some cell fraction after treatment with HMM indicates that actin is present. Ishikawa et al.² used this to demonstrate actin in a number of cultured cell types. More recently, other workers have similarly demonstrated actin filaments in slime mould³, amoebae^4,5, blood platelets⁶, microvilli⁷, macrophages⁸ and, less convincingly, in sperm tails⁹ and the mitotic spindle¹⁰. We prove here that filaments from the cortical region of mouse neuroblastoma cells bind HMM and therefore contain actin.     

Rotational dynamics of spin-labeled F-actin in the sub-millisecond time range.

The rotational motions of F-actin filaments and myosin heads attached to them have been measured by saturation transfer electron paramagnetic resonance spectroscopy using spin-labels rigidly bound to actin, or to the myosin head region in intact myosin molecules, heavy meromyosin, and subfragment-1. The spin-label attached to F-actin undergoes rotational motion having an effective correlation time of the order of 10^?4 seconds. This cannot be interpreted as rotation of the entire F-actin filament or local rotation of the spin-label, but must represent an internal rotational mode of F-actin, possibly a bending or flexing motion, or a rotation of an actin monomer or a segment of it. The rate of this rotational motion is reduced approximately fourfold by myosin, HMM or S-1; HMM and S-1 are equally effective, on a molar basis, in slowing this rotation and both produce their maximal effect at a ratio of about one molecule of HMM or S-1 per ten actin monomers. With chymotryptic S-1, the effect is partially reversed at higher concentrations. With S-1 prepared with papain in the presence of Mg²⁺, the reversal is smaller, while with HMM or myosin there is no reversal at higher concentrations. Tropomyosin slightly decreases the actin rotational mobility, and the addition of HMM to the actin-tropomyosin complex produces a further slowing. The rotational correlation time for acto-HMM is the same whether the spin-label is on actin or HMM, indicating that the rotation of the head region of HMM when bound to F-actin is controlled by a mode of rotation within the F-actin filaments.     

Induction of contractile activity by rigor complexes in myofibrils

The relation between ATPase rate and substrate concentration was investigated for myofibrils with varying amounts of added HMM. There was a biphasic, 3 to 5-fold increase in ATPase in the absence of Ca⁺⁺. In the absence of added HMM, the peak activity occurred at ≤ 0.1 mM MgATP. With increasing concentrations of HMM, the position and magnitude of the ATPase peak shifted to larger substrate concentrations and higher rates. The cofactor activity of regulated actin in myofibrils is activated to a similar degree by Ca⁺⁺ as by HMM (rigor links). SDS gel electrophoretic patterns of myofibrils mixed with HMM indicated the soluble HMM binds to myofibrils at 0.1 mM MgATP and is dissociated at higher MgATP concentrations. Thus, in well-regulated myofibrils in the absence of Ca⁺⁺ actin cofactor activity can be activated by rigor complexes.     

Tension development in skinned glycerinated rabbit psoas fiber segments irrigated with soluble myosin fragments

Single glycerinated rabbit psoas muscle fibers were skinned by splitting them lengthwise. The fiber segments thus obtained were more easily accessible to solutes in the surrounding medium than the intact fibers. Using such segments, active tension could be fully abolished by adding N-ethylmaleimide under conditions which lead to inhibition of actin activation of the ATPase activity of myosin. Such muscles could, however, develop tension after irrigation with myosin or with the water-soluble active myosin fragments heavy meromyosin (HMM) or its subfragment 1 (HMM-S1). The induced tensions increased with increasing protein concentration in the irrigating solution. At any given protein concentration, the tension generated by myosin was larger than that produced by HMM which was, in turn, greater than that induced by HMM-S1 e.g. at 15 mg/ml protein the tensions produced by these three myosin moieties were 44.0, 14.0 and 2.8 g/cm², respectively. The tension was found to be intimately associated with ATP splitting; thus, HMM and HMM-S1 which have been treated with reagents abolishing actin-activated ATPase failed to induce tension development. A contractile force may thus be generated through the interaction with actin of the water-soluble, enzymatically active, myosin subfragments involving the splitting of ATP.     

Two pathways for oxygen exchange by heavy meromyosin and their dependence on actin

In the hydrolysis of MgATP by acto heavy meromyosin (HMM) there are two enzymatic pathways that differ in the properties of their intermediate oxygen exchange; one of these is designated the low exchange pathway (P1); the other is designated the high exchange pathway (P2). A plot of the P1 flux versus the actin concentration gives a sigmoid curve, whereas the corresponding curve for the P2 flux rises in an approximately hyperbolic manner. At low concentrations of actin, where the sigmoid curve of the P1 flux is in a lag phase, the major flux is along P2; but at higher concentrations of actin, as the P1 curve rises sharply, the flux along P1 comes to predominate. Even at the highest levels of actin, at saturating levels for both pathways, the kinetics of exchange along P1 and P2 are significantly different. In addition to these differences in the actin dependence, the flux of P1 relative to P2 is markedly inhibited by KCl. Therefore, which of the two pathways dominates during the hydrolysis of MgATP by HMM is strongly dependent on experimental conditions. The findings suggest that P1 involves the interaction of HMM with two actin units whereas P2 involves the interaction of HMM with one actin unit. The results are discussed in relation to a kinetic scheme based on this proposal.     

Motion of myosin fragments during actin-activated ATPase: fluorescence correlation spectroscopy study.

The rates of the translational motion of myosin fragments, heavy meromyosin (HMM), and heavy meromyosin subfragment-1 (HMM S-1) were measured during actin-activated ATPase reaction by the method of fluorescence correlation spectroscopy. This technique monitors the random fluctuations in the concentration of fluorescent molecules in an open volume which result from the translational diffusion of the molecular species under observation. The statistical behavior of the fluctuations is represented in the form of the autocorrelation function, which is related to the translational diffusion coefficient of the fluorescent molecules. The translational motion of fluorescently labeled myosin fragments was progressively slowed down after additions of increasing amounts of actin in the presence of excess MgATP. When these results are interpreted according to a simple binding scheme, the extent of the retardation can be used to obtain the apparent association constant for binding of S-1 and HMM to actin in the presence of MgATP. In 0.1M KCl and at 23°C, the apparent association constants were determined as K_app^HMM = 2.2 × 10⁴M^?1 and K_app^S-1 = 8.8 × 10³ for HMM and S-1, respectively.     

Inhibition of sliding movement of F-actin by crosslinking emphasizes the role of actin structure in the mechanism of motility

The effects of crosslinking of monomeric and polymeric actin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), disuccinimidyl suberate (DSS) and glutaraldehyde on the interaction with heavy meromyosin (HMM) in solution and on the sliding movement on glass-attached HMM were examined. The Vmax values of actin-activated HMM ATPase decreased in the following order: intact actin = EDC F-actin greater than DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than EDC G-actin. The affinity of actin for HMM in the presence of ATP decreased in the following order: DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than intact actin greater than EDC F-actin greater than EDC G-actin. However, sliding movement was inhibited only in the case of glutaraldehyde-crosslinked F and G-actin and EDC-crosslinked G-actin. Interestingly, after copolymerization of "non-motile" glutaraldehyde or EDC-crosslinked monomers with "motile" monomers of intact actin sliding of the copolymers was observed and its rate was independent of the type of crosslinked monomer, i.e. of the manner of their interaction with HMM. These data strongly indicate that inhibition of the sliding of actin by crosslinking cannot be explained entirely by changes in the Vmax value or affinity for myosin heads. We conclude that movement is generated by interaction of myosin with segments of F-actin containing a number of intact monomers, and the mechanism of inhibition involves an effect of the crosslinkers on the structure of F-actin itself.     

Mechanism of the Interaction of Superoxide Ion and Ascorbate with Anthracycline Antibiotics

The interaction of superoxide ion and ascorbate anion with anthracycline antibiotics (adriamycin and aclacinimycin A) as well as with their Fe³⁺ complexes has been studied in aprotic and protic media. It was found that both superoxide and ascorbate reduce anthracyclines to deoxyaglycons via a one-electron transfer mechanism under all conditions studied. The reaction of ascorbate anion with adriamycin and aclacinomycin A in aqueous solution proceeded only in the presence of Fe³⁺ ions; it is supposed that an active catalytic species was Fe³⁺ adriamycin. It is also supposed that the reduction of anthracycline antibiotics by O,⁷ and ascorbate in cells may increase their anticancer effect.     

NADH-dependent cinerulose reductase in rat liver microsomes.

In the course of studies on the metabolism of a new antitumor anthracycline antibiotic, aclacinomycin A, the new keto reductase which catalyzes the reduction of keto group of L-cinerulose of aclacinomycin A to L-rhodinose was found in rat liver microsomal membrane. The enzyme requires NADH for the reduction and showed optimum pH at 7.0. Km value for aclacinomycin A, 2.1 × 10^?5 M and the concentration of NADH need to half maximal activity, 6.2 × 10^?5 M were obtained. The activity was potently inhibited by detergents, such as Triton X-100, sodium deoxycholate and sodium dodecyl sulfate.     

Inorganic polyphosphate hydrolysis catalyzed by skeletal muscular actomyosin complexes is uncoupled with motility

Hydrolysis of the triphosphate moiety of ATP, catalyzed by myosin, induces alterations in the affinity of the myosin heads for actin filaments via conformational changes, thereby causing motility of the actomyosin complexes. To elucidate the contribution of the triphosphate group attached to adenosine, we examined the enzymatic activity of heavy meromyosin (HMM) with actin filaments for inorganic tripolyphosphate (3PP) using a Malachite green method and evaluated using fluorescence microscopy the effects of 3PP on actin filament motility on HMM-coated glass slides. In the presence of MgCl₂, HMM hydrolyzed 3PP at a maximum rate of 0.016 s⁻¹ HMM⁻¹, which was four times lower than the hydrolysis rate of ATP. Tetrapolyphosphate (4PP) was hydrolyzed at a rate similar to that of 3PP hydrolysis. The hydrolysis rates of 3PP and 4PP were enhanced by roughly 10-fold in the presence of actin filaments. In motility assays, the presence of polyphosphates did not lead to the sliding movement of actin filaments. Moreover, in the presence of ATP at low concentrations, the sliding velocity of actin filaments decreased as the concentration of added polyphosphate increased, indicating a competitive binding of polyphosphate to myosin heads with ATP. These results suggested that the energy produced by standalone triphosphate hydrolysis did not induce the unidirectional motion of actomyosin and that the link between triphosphate and adenosine was crucial for motility.     

Regulatory light chains modulate in vitro actin motility driven by skeletal heavy meromyosin

Phosphorylation and Ca²⁺-Mg²⁺ exchange on the regulatory light chains (RLCs) of skeletal myosin modulate muscle contraction. However, the relation between the mechanisms for the effects of phosphorylation and metal ion exchange are not clear. We propose that modulation of skeletal muscle contraction by phosphorylation of the myosin regulatory light chains (RLCs) is mediated by altered electrostatic interactions between myosin heads/necks and the negatively charged thick filament backbone. Our study, using the in vitro motility assay, showed actin motility on hydrophilic negatively charged surfaces only over the HMM with phosphorylated RLCs both in the presence and absence of Ca²⁺. In contrast, good actin motility was observed on silanized surfaces (low charge density), independent of RLC phosphorylation status but with markedly lower velocity in the presence of Ca²⁺. The data suggest that Ca²⁺-binding to, and phosphorylation of, the RLCs affect the actomyosin interaction by independent molecular mechanisms. The phosphorylation effects depend on hydrophobicity and charge density of the underlying surface. Such findings might be exploited for control of actomyosin based transportation of cargoes in lab-on-a chip applications, e.g. local and temporary stopping of actin sliding on hydrophilic areas along a nanosized track.     

Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.     

Evidence of multifactorial mechanisms in an adriamycin-resistant HL-60 promyelocytic leukemia cell line

HL-60/AR leukemia cells, which were 60-fold resistant to the growth inhibitory activity of adriamycin, remained sensitive to the antiproliferative and differentiation-inducing activities of aclacinomycin A. The replication of HL-60/AR and of adriamycin sensitive parental HL-60 cells was inhibited by greater than 80% by 30 nM aclacinomycin A and the majority of cells (about 60 to 70%) of each line underwent granulocytic differentiation when treated with this agent, as assessed by the reduction of nitroblue tetrazolium. Measurement of the initial rates of uptake of daunorubicin and steady-state levels of adriamycin in sensitive and resistant lines indicated that transport differences do not fully account for the insensitivity of HL-60/AR cells to these anthracyclines. Furthermore, 30-fold greater levels of cell-associated adriamycin were required in HL-60/AR cells for toxic effects equivalent to those occurring in parental HL-60 cells. Analysis of DNA histograms of adriamycin treated HL-60 cells indicated that cell-cycle progression was blocked in G2-M, while this antibiotic blocked progression of resistant HL-60/AR cells in the S phase. These results suggest that, in addition to alterations in membrane permeability, differential sensitivity of multiple biochemical targets may be important in the toxicity and the development of resistance to anthracyclines. Furthermore, the finding that HL-60/AR cells do not exhibit cross-resistance to aclacinomycin A indicates that this oligosaccharide-containing anthracycline may have utility in the treatment of adriamycin resistant neoplasms.     

Crosslinking of trypsin digested acto-heavy meromyosin as a probe of the affinity of the two myosin heads to actin

The interaction of the two heads of the myosin molecule with actin was studied by tryptic digestion of HMM in the presence of actin, followed by crosslinking the two nicked heavy chains with Nbs2 at the S2 region. In view of the protection by actin of the 50/60 kDa junction against proteolysis, the percentage of the heads interacting with actin was estimated from the proportion of the 110 kDa to the 60 kDa digestion product. Under conditions such that about 50% of HMM heads were protected by actin (at an actin to HMM head molar ratio of 1:1 in the absence of nucleotide, or 3:1 in the presence of 5 mM ADP), the crosslinking of the digestion products yielded a 230 kDa (110 + 110 kDa), 125 kDa (60 + 60 kDa) and 175 kDa (60 + 110 kDa) species. Since the latter should be the only crosslinking product when only one head of HMM molecule is protected by actin, it is concluded that there is no preferential binding of one of the two HMM heads to actin in the presence of ADP or at equimolar actin to myosin heads ratio.     

Heavy meromyosin binds actin with negative cooperativity.

The association of fluorescently labeled heavy meromyosin (HMM) and F-actin was measured by time-resolved fluorescence depolarization. The effects of varying the protein concentrations, temperature, KCl concentration, and pH were determined. Measurements of HMM mobility supported a model of no interaction between the two heads in the absence of actin. Measurements of actin binding, when compared with results for myosin subfragment I, indicated that the two heads of HMM do not bind independently in the rigor complex. This could result from actin-transmitted negative cooperativity or from steric inhibition due to the structure of HMM. For HMM and actin in 0.15 7 kcl at 25 degrees C: Ka = 3.9 X 10(7) M-1, deltaHco' = 36 +/- 2 J M-1, deltaSco' = 0.26 +/- 0.02 kJ M-1 K-1; the slope of ln Ka vs. [KCl]1/2 = -3.88 and the pH of maximum association was 6.9.     

Ultrastructural Localization of Filamentous Actin within Neuronal Interphase Nuclei in Situ

Previous biochemical studies utilizing isolated nuclei and nuclear matrices have shown actin to be a constituent of the interphase nucleus. In addition, recent ultrastructural work has shown the presence of actin and myosin within nuclei of interphase cells in situ. It was unclear, however, whether this intranuclear actin is present in the unpolymerized globular actin or the filamentous (F)-actin form. The present work, using confocal microscopy and ultrastructural cytochemical techniques, demonstrates the presence of F-actin within interphase nuclei of intact, uncompromised, dorsal root ganglion neurons in vitro and in vivo. Labeling by FITC-phalloidin detected the presence of intranuclear F-actin adjacent to the nucleolar periphery in a small fraction of cells in vitro, an observation confirmed by three-dimensional reconstruction. Ultrastructural analyses of cells exposed to heavy meromyosin (HMM), showed the presence of typical "arrowhead" complexes. The observation that these complexes were associated with nucleoli confirms that the intranuclear ligand detected by FITC-phalloidin indeed represents F-actin. Postembedding labeling with HMM conjugated to 20-nm colloidal gold (HMM-Au₂₀) resulted in labeling similar to that obtained with HMM. However, HMM-Au₂₀ was found to label a much larger fraction of cells, both in vitro and in vivo, than did FITC-phalloidin or HMM. This finding indicates that labeling with HMM-Au₂₀ more accurately reflects the extent of actin polymerization in nuclei. Results from double labeling with HMM-Au₂₀ and an antibody to α-sarcomeric actin confirmed that only a small amount of nuclear actin is in the F-form. Together, these results represent a first ultrastructural demonstration of the presence of F-actin in nuclei of neurons. While the role of nuclear F-actin has yet to be defined, the results suggest that F-actin may represent a component of the molecular motor responsible for the dynamic positioning of specific chromatin domains into the tissue-specific, nonrandom patterns observed in many cell types.