Hanna Strzelecka-Golaszewska

Role of the DNase-I-binding loop in dynamic properties of actin filament.

Effects of proteolytic modifications of the DNase-I-binding loop (residues 39-51) in subdomain 2 of actin on F-actin dynamics were investigated by measuring the rates of the polymer subunit exchange with the monomer pool at steady state and of ATP hydrolysis associated with it, and by determination of relative rate constants for monomer addition to and dissociation from the polymer ends. Cleavage of actin between Gly-42 and Val-43 by protease ECP32 resulted in enhancement of the turnover rate of polymer subunits by an order of magnitude or more, in contrast to less than a threefold increase produced by subtilisin cleavage between Met-47 and Gly-48. Probing the structure of the modified actins by limited digestion with trypsin revealed a correlation between the increased F-actin dynamics and a change in the conformation of subdomain 2, indicating a more open state of the filament subunits relative to intact F-actin. The cleavage with trypsin and steady-state ATPase were cooperatively inhibited by phalloidin, with half-maximal effects at phalloidin to actin molar ratio of 1:8 and full inhibition at a 1:1 ratio. The results support F-actin models in which only the N-terminal segment of loop 39-51 is involved in monomer-monomer contacts, and suggest a possibility of regulation of actin dynamics in the cell through allosteric effects on this segment of the actin polypeptide chain.

Divalent Cation-, Nucleotide-, and Polymerization-Dependent Changes in the Conformation of Subdomain 2 of Actin

Conformational changes in subdomain 2 of actin were investigated using fluorescence probes dansyl cadaverine (DC) or dansyl ethylenediamine (DED) covalently attached to Gln41. Examination of changes in the fluorescence emission spectra as a function of time during Ca2+/Mg2+ and ATP/ADP exchange at the high-affinity site for divalent cation-nucleotide complex in G-actin confirmed a profound influence of the type of nucleotide but failed to detect a significant cation-dependent difference in the environment of Gln41. No significant difference between Ca- and Mg-actin was also seen in the magnitude of the fluorescence changes resulting from the polymerization of these two actin forms. Evidence is presented that earlier reported cation-dependent differences in the conformation of the loop 38-52 may be related to time-dependent changes in the conformation of subdomain 2 in DED- or DC-labeled G-actin, accelerated by substitution of Mg2+ for Ca2+ in CaATP-G-actin and, in particular, by conversion of MgATP- into MgADP-G-actin. These spontaneous changes are associated with a denaturation-driven release of the bound nucleotide that is promoted by two effects of DED or DC labeling: lowered affinity of actin for nucleotide and acceleration of ATP hydrolysis on MgATP-G-actin that converts it into a less stable MgADP form. Evidence is presented that the changes in the environment of Gln41 accompanying actin polymerization result in part from the release of Pi after the hydrolysis of ATP on the polymer. A similarity of this change to that accompanying replacement of the bound ATP with ADP in G-actin is discussed.

Studies on the exchange of G-actin-bound calcium with bivalent cations

Abstract 1. The possibility of the replacement of G-actin-bound calcium by various bivalent cations has been investigated. After the reaction with all cations studied, with the exception of Cu 2+ , action remains active, i.e. , contains bound ATP and polymerizes in 0.1 M KCl. 2. The amount of G-actin-bound calcium, as well as the sum of bivalent cation after replacement, not removable by short-time Dowex-50 treatment, accounts to about 1 mole per 50000 g of G-actin. 3. The rate of exchange is of the same order for bivalent cations studied, including calcium. 4. G-actin-bound Ca 2+ is fully replaced, besides free Ca 2+ , by free Mn 2+ and Cd 2+ . The replacement with Mg 2+ , Co 2+ , Ni 2+ and Zn 2+ is not complete, and there is practically no reaction with Ba 2+ and Sr 2+ . 5. Assuming the affinity constant of Ca 2+ as 1, the following affinity constants for other bivalent cations were obtained: Mn 2+ , 0.90; Cd 2+ , 1.07; Mg 2+ , 0.27; Zn 2+ , 0.22; Co 2+ , 0.18; Ni 2+ , 0.08. 6. The results obtained show that there exists a close correlation between the ionic radius of a particular bivalent cation, and its ability to replace bound Ca 2+ .

Relative affinities of divalent cations to the site of the tight calcium binding in G-actin

Abstract Replacement of G-actin-bound Ca 2+ by other divalent cations in the presence of ATP was examined at various ratios of the cation to ATP. The results are discussed from the point of view of finding proper conditions for determination of relative affinity constants of divalent cations to the site of the tight Ca 2+ binding in G-actin. The replacement of the bound Ca 2+ by other divalent cations was found to be an endothermic process. Relative apparent affinity constants of several divalent cations to G-actin at pH 7.6, at 0 °C and at room temperature were calculated taking into account binding of the cations to ATP. The results show that specificity of Ca 2+ binding to the site of the tight binding in G-actin is much less than it followed from the previously reported values, in the calculation of which the interaction of the cations with ATP was neglected.

Divalent Cations, Nucleotides, and Actin Structure

Actin has one high-affinity site for a divalent cation, with a Kd for Ca2+ and Mg2+ in the nanomolar range. This binding site is located at the bottom of the cleft between the two domains of the molecule (Fig. 1). The cation is coordinated not only by amino acid residues but also by the oxygen of the γ- and/or β- phosphate groups of the nucleotide, ADP or ATP respectively, which is bound further up in the cleft (Valentin-Ranc and Carlier 1989; Kabsch et al. 1990). The tightly bound cation of G-actin exchanges with other divalent cations by a simple competitive mechanism.

Activation of smooth muscle myosin by smooth and skeletal muscle actins.

It has been reported that actins from human uterine and from chicken gizzard smooth muscle are less effective than rabbit skeletal muscle actin in activating the Mg*+-ATPase activity of rabbit skeletal muscle myosin or heavy meromyosin (HMM, [ 1,2]). The difference has been shown to result from an -2-fold greater apparent dissociation constant (Kapp) for gizzard actin, the maximum ATPase rate at infinite actin concentration (V,,) being the same for both gizzard and skeletal muscle actins [2]. The results obtained with uterine actin [l], likewise, also seem to indicate a difference in the Kapp-value rather than in V,, [2]. The aim of this study was to establish whether this difference in the interaction of smooth and skeletal muscle actins with myosin are preserved with smooth muscle myosin. To avoid complications connected with the partial degradation and/or phosphorylation of the myosin regulatory light chain we have used mainly smooth muscle myosin heavy meromyosin subfragment 1 (SFl) whichlacks the regulatory light chains (see [3]). Such SF1 preparations are obtained by papain digestion of gizzard or arterial myosin, and, like’intact myosin in a phosphorylated state, are activated by actin in a Ca’+-insensitive manner [4-91. Chicken gizzard and pig stomach myosin were prepared as in [3]. SF1 was prepared by papain digestion as in [lo] for skeletal muscle myosin with an additional purification step that included ammonium sulfate fractionation (45-55%) and gel filtration chromatography (AcA44; LKB). Papain was 0.03 and 0.05 mg/ml for pig stomach and chicken gizzard myosin, respectively. The SF1 was stored in the form of an ammonium sulfate pellet in liquid nitrogen.

Subunit composition and some other properties of myosin from skeletal muscles of the frog rana esculenta

Abstract 1. 1. Two out of three light chains of myosin from mixed hind leg muscles of the frog (LC 1 and LC 2 ) undergo a proteolytic cleavage during preparation with procedures routinely used to obtain myosin from skeletal muscles of other vertebrates. 2. 2. The intact LC 1 and LC 3 comigrate on SDS-polyacrylamide gels with the respective light chains of rabbit fast skeletal muscle; LC 2 has lower mobility than the rabbit LC 2 . All the three light chains differ from their counterparts in the rabbit myosin in mobilities on urea-polyacrylamide gels. 3. 3. The LC 2 of frog myosin is released upon DTNB-EDTA † treatment more easily than LC 2 of rabbit fast skeletal muscle. 4. 4. Differences in the fragmentation of the heavy chains of myosin from hind leg and from rabbit fast skeletal muscles by trypsin indicate some differences in primary and/or secondary structure. 5. 5. Fast inactivation accompanied by aggregation of frog myosin was confirmed. Pyrophosphate was shown to have a protective effect. 6. 6. Myosin from mixed hind leg muscles of the frog is similar to myosin from rabbit slow skeletal muscle in its instability under mild alkaline conditions.

Paracrystalline assemblies of light meromyosins with various chain weights

SummaryParacrystals formed from well defined insoluble fragments of myosin rod: LMM-A, LMM-B, LMM-C, and LMM-D with apparent chain weights of 78 000, 72 000, 68 000, and 56 000, respectively (Nyitrayet al., 1983) were studied in the electron microscope with a negative staining technique. All fragments formed tactoids with 14.3 and 43 nm periodicities as well as aperiodic tactoids and sheets. Tactoids and sheets described earlier with a 43 nm periodicity and a pattern of alternating light bands 10 nm wide and dark bands 33 nm wide were observed in LMM-A preparations only. LMM-B and LMM-C formed tactoids with a 43 nm periodicity but without the diversified band pattern. LMM-D formed sheets and tactoids with a newly observed band pattern of alternating light bands 23 nm wide and dark bands 20 nm wide. This pattern can be explained assuming the length of LMM-D molecules to be 66 nm which is fairly consistent with the chain weight of this fragment. A model for molecular arrangement in this type of paracrystal is presented. The model involves both parallel and antiparallel interactions with a parallel axial displacement of the molecules by 43 nm as suggested by Bennett (1981) for paracrystals formed from LMM molecules 90 nm long. It is deduced from the model that LMM-D is shorter than LMM-A by 15 nm at the NH2-terminal end and by 9 nm at the COOH-terminal end.LMM-D, like the other insoluble fragments of myosin rod, is also able to form square and hexagonal nets with an approximately 40 nm distance between lattice points. The structural features of the nets obtained from LMM-D can be explained assuming the same kinds of molecular interactions within the strands of the net as those in the sheets and tactoids with a 43 nm axial repeat.It is concluded that all insoluble fragments of myosin rod are able to form paracrystalline assemblies involving the same types of parallel and antiparallel interactions.

Interaction of myosin filaments and minifilaments with actin: a comparative study

SummaryVarious aspects of actin-myosin interaction were investigated using myosin in the form of filaments and minifilaments obtained by dialysis against citrate-Tris buffer or by adding this buffer to preformed myosin filaments. Considerable similarities in the behaviour of the two systems were found. (1) Although the minifilaments are soluble structures, they form insoluble complexes with actin, which superprecipitate upon addition of MgATP. Observations in the electron microscope and from centrifugation experiments have shown that the two actomyosin systems undergo essentially similar structural changes during superprecipitation. (2) At low substrate concentrations the rate of ATP hydrolysis in both systems declines with time, which is typical of insoluble superprecipitating actomyosin. (3) In contrast to soluble myosin subfragments, both filamentous and minifilamentous myosin give biphasic actin-activation curves. (4) The Mg2+-ATPase activities of myosin minifilaments and standard myosin preparations at low KCl extrapolate to similarVmax at infinite actin concentration.Since our values ofVmax for myosin filaments and minifilaments are in the range of those reported for myosin subfragments, the results of this investigation confirm the view that the catalytic properties of myosin subfragments and intact myosin are equivalent. Moreover, the data show that the extent of myosin aggregation in the initial preparations has no appreciable effect on the characteristic features of the interaction between intact myosin and actin at pH 8.

Dependence of the length of the heavy chain of chymotryptic subfragment 1 on the temperature of myosin digestion

Limited digestion of filamentous myosin with chymotrypsin at 0°C in the absence of divalent cations generates two forms of subfragment 1 (S1), with heavy chains of 95 kDa and 98 kDa. The difference is at the C‐terminal end of the chain. The 98 kDa form prevails, in contrast to the preparations obtained by digestion at room temperature which consist of the shorter species and only traces of the longer one. The results support the idea of a temperature‐dependent conformational transition at the head‐rod junctional region of the myosin heavy chain.