A new method to precipitate myosin V from rat brain soluble fraction

Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg(2+)-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg(2+)-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg(2+)-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide.     

The action of ATP on natural actomyosin.

1. When ATP is added to Limulus myosin B and the mixture is centrifuged at 50,000 rev/min for 3 hr to dissociate it into a supernatant of myosin and a pellet of actin, the results are contrary to expectation. The pellet does contain actin but no more than one obtains if the ATP is omitted. The supernatant also contains actin. 2. If this is done in the Model E centrifuge, the major peak disappears upon ATP addition and a new peak, sedimenting at a rate typical of myosin, appears. When only this slow moving peak is run on SDS gels, actin is clearly seen. 3. It is concluded that ATP does not dissociate this actomyosin but rather causes a conformational change.     

Reversible changes in size of cell nuclei isolated from Amoeba proteus: role of the cytoskeleton.

Micrurgically isolated interphasal nuclei of Amoeba proteus, which preserve F-actin cytoskeletal shells on their surface, shrink after perfusion with imidazole buffer without ATP, and expand to about 200% of their cross-sectional area upon addition of pyrophosphate. These changes in size may be reproduced several times with the same nucleus. The shrunken nuclei are insensitive to the osmotic effects of sugars and distilled water, whereas the expanded ones react only to the distilled water, showing further swelling. The shrinking-expansion cycles are partially inhibited by cytochalasins. They are attributed to the state of actomyosin complex in the perinuclear cytoskeleton, which is supposed to be in the rigor state in the imidazole buffer without ATP, and to dissociate in the presence of pyrophosphate. Inflow of external medium to the nuclei during dissociation of the myosin from the perinuclear F-actin may be due to colloidal osmosis depending on other macromolecular components of the karyoplasm.     

Purification and partial characterization of myosin II from rat testis

The intent, in this work, was to isolate rat testis myosin II. Testis 40,000 x g x 40' supernatant was frozen at -20 degrees C for 48 h and, after it was thawed and centrifuged. The precipitate, after washed twice, was enriched in three polypeptides bands: p205, p43 and one that migrated together with the front of the gel. These polypeptides were solubilized in pH 10.8 at 27 degrees C and separated in Sephacryl S-400 column. Three low weight polypeptides co-eluted together with p205. The p205 was marked with anti-myosin II, possess actin-stimulated Mg-ATPase activity and co-sedimented with F-actin in the absence, but not in the presence, of ATP. In the present study, we have been developing a method for purification of myosin II from rat testis.     

Myosin from molluscan abalone, Haliotis discus. Isolation and enzymatic properties.

Actomyosin was extracted from smooth muscle of molluscan abalone with 0.1 M PPit pH 6.4. Myosin was separated from the actomyosin by centrifugation at 100,000 X g in the presence of 5 mM ATP and 10 mM MgCl2. Myosin in the supernatant was further purified by gel filtration on a Sepharose 4B column. Paramyosin contamination of the actomyosin preparation interfered with the isolation of myosin and complete removal of actin and paramyosin from the myosin has not been accomplished. The myosin appeared to consist of a single f-chain and a single g-chain, as examined by SDS-disc electrophoresis in 8 or 13.7% acrylamide gel. The ATPase [EC 3.6.1.3] activity of this myosin in 0.5 M KCL at neutral pH and at 0 degrees was rather unstable and decreased by 10-20% per day. The effects of rho-chloromercuribenzoate and EDTA on the ATPase activity were similar to those observed with other smooth muscle myosin but the dependence upon pH or KCL concentration was different.     

Control of filament length by the regulatory light chains in skeletal and cardiac myosins

The possible role of the regulatory light chains (LC2) in in vitro assembly of rabbit skeletal and dog cardiac myosins was examined by formation of minifilaments and synthetic thick filaments. After LC2 was removed, the resulting myosin preparations exhibited little aggregation in 0.5 M KCl and 0.05 M potassium phosphate (pH 6.5). Minifilaments migrated as a single, hypersharp peak during sedimentation velocity, but electron microscopic analysis revealed a more destabilized structure for LC2-deficient minifilaments. Thick filaments were formed in buffers containing 0.15 M KCl and the following: 20 mM imidazole; 20 mM imidazole, 5 mM ATP; or 20 mM imidazole, 5 mM ATP, and 5 mM MgCl2, all at pH 7.0. Skeletal and cardiac myosin filaments formed in imidazole buffer alone were bipolar, tapered at both ends, and about 1.6 micron long. Removal of LC2 resulted in the formation of shorter thick filaments (1.2 micron long). This effect could be reversed by reassociation with LC2. Inclusion of ATP in the buffer disrupted the filament structure, resulting in irregular, short filaments (less than 0.6 micron); addition of both ATP and MgCl2 largely reversed the effects of ATP alone. In cardiac myosin filaments, the bare zone diameter increased from 16 nm as measured in control and LC2-recombined samples to 20 nm in LC2-deficient myosin assemblies. These results implicate LC2 in an active role in controlling synthetic thick filament length in both skeletal and cardiac muscles.     

The myosin cardiac loop participates functionally in the actomyosin interaction

The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on beta-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in beta-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using site-directed mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.     

C-terminal sites of caldesmon drive ATP hydrolysis cycle by shifting actomyosin itermediates from strong to weak binding of myosin and actin

Polarized fluorimetry technique and ghost muscle fibers containing tropomyosin were used to study effects of caldesmon (CaD) and recombinant peptides CaDH1 (residues 506-793), CaDH2 (residues 683-767), CaDH12 (residues 506-708) and 658C (residues 658-793) on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to Cys-707 of myosin subfragment-1 (S1) in the absence of nucleotide, and in the presence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. It was shown that at modelling different intermediates of actomyosin ATPase, the orientation and mobility of dye dipoles changed discretely, suggesting a multi-step changing of the myosin head structural state in ATP hydrolysis cycle. The maximum difference in orientation and mobility of the oscillator (4 degrees and 30%, respectively) was observed between actomyosin in the presence of MgATP, and actomyosin in the presence of MgADP. Caldesmon actin-binding sites C and B' inhibit formation of actomyosin strong binding states, while site B activates it. It is suggested that actin-myosin interaction in ATP hydrolysis cycle initiates nucleotide-dependent rotation of myosin motor domain, or that of its site for dye binding as well as the change in myosin head mobility. Caldesmon drives ATP hydrolysis cycle by shifting the equilibrium between strong and weak forms of actin-myosin binding.     

Differences between smooth and skeletal muscle myosins in their interactions with F-actin

Myosin and F-actin were prepared from bovine carotid arterial smooth muscle and the properties of the binding of myosin to F-actin were compared with those of the binding of skeletal muscle myosin to F-actin. The following differences were observed between skeletal and smooth muscle myosins. 1. The rate of ATP-induced dissociation of arterial actomyosin was equal to that of hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin, but was much lower than those of skeletal muscle actomyosin and of hybrid actomyosin reconstituted from skeletal muscle myosin and arterial F-actin. 2. The amount of ATP necessary for complete dissociation of arterial actomyosin was 2 mol/mol of myosin, although it is well known that skeletal muscle actomyosin is dissociated completely by the addition of 1 mol ATP per mol of myosin. 3. Arterial actomyosin and hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin did not dissociate upon addition of 0.1 mM PPi, while skeletal muscle actomyosin dissociated completely. 4. In the absence of Mg2+, neither dissociation by ATP nor ATPase [EC 3.6.1.3] activity was observed with arterial actomyosin and hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin. On the other hand, skeletal muscle actomyosin dissociated almost completely upon addition of ATP and showed a considerably high ATPase activity. These observations reveal marked differences between myosins from skeletal and smooth muscles in their binding properties to F-actin.     

Reaction of two heads of gizzard myosin with ATP

The reaction intermediates formed by the two heads of smooth muscle myosin were studied. The amount of myosin-phosphate-ADP complex, MPADP, formed was measured from the Pi-burst size over a wide range of ATP concentrations. At low concentrations of ATP, the Pi-burst size was 0.5 mol/mol myosin head, and the apparent Kd value was about 0.15 microM. However, at high ATP concentrations, the Pi burst size increased from 0.5 to 0.75 mol/mol myosin head with an observed Kd value of 15 microM. The binding of nucleotides to gizzard myosin during the ATPase reaction was directly measured by a centrifugation method. Myosin bound 0.5 mol of nucleotides (ATP and ADP) with high affinity (Kd congruent to 1 microM) and 0.35 mol of nucleotides with low affinity (Kd = 24 microM) for ATP. These results indicate that gizzard myosin has two kinds of nucleotide binding sites, one of which forms MPADP with high affinity for ATP while the other forms MPADP and MATP with low affinity for ATP. We studied the correlation between the formation of MPADP and the dissociation of actomyosin. The amount of Pi-burst size was not affected by the existence of F-actin, and when 0.5 mol of ATP per mol of myosin head was added to actomyosin (1 mg/ml F-actin, 5 microM myosin at 0 degrees C) most (93%) of the added ATP was hydrolyzed in the Pi-burst phase. All gizzard actomyosin dissociated when 1 mol of ATP per mol myosin head was added to actomyosin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-sensitive modulation of the actomyosin ATPase by fodrin

Fodrin, a spectrin-like protein isolated from brain, is a long flexible molecule which binds calmodulin and cross-links F-actin. The effects of fodrin on the actin-activated ATPase of myosin have been examined. When added after ATP, fodrin inhibited the actomyosin ATPase. Two to three times as much fodrin was required for inhibition in the presence of Ca2+ as in its absence. Complete inhibition in the absence of Ca2+ occurred at about one fodrin to 200 actins. Inhibition does not appear to result from fodrin cross-linking F-actin, and, thereby, preventing the myosin filaments from reaching the actin filaments; but cross-linking may promote inhibition by trapping the myosin filaments within the cross-linked F-actin. When added before ATP, fodrin stimulated the actomyosin ATPase almost 3-fold in the presence of Ca2+ and by less than 50% in the absence of Ca2+. Stimulation is thought to result from fodrin cross-linking F-actin. After several minutes the stimulations in Ca2+ were greatly reduced, and in the absence of Ca2+ the actomyosin ATPases were substantially inhibited. Whether added before or after ATP, fodrin inhibited the actin-activated ATPase of myosin subfragment 1. This inhibition was also slightly Ca2+ sensitive.     

Comparison of kinetic properties of the ATPase reaction of arterial smooth muscle myosin with skeletal muscle myosin

Myosin was prepared from arterial smooth muscle, and a hybrid actomyosin was formed from arterial myosin and rabbit skeletal muscle F-actin. We performed kinetics on the ATPase reaction [EC 3.6.1.3] of arterial myosin and the hybrid actomyosin at high ionic strength, and compared the kinetic properties of arterial myosin ATPase with those of skeletal muscle myosin ATPase. No significant difference was found between these two myosins in the size of the initial Pi burst, the amount of bound nucleotides, and the rates of various elementary steps in the ATPase reaction. On the other hand, two important differences were observed between the hybrid actomyosin and skeletal muscle actomyosin: (i) The amounts of ATP necessary for complete dissociation of the hybrid and skeletal muscle actomyosins were 2 and 1 mol/mol of myosin, respectively. (ii) The rate of dissociation of the hybrid actomyosin induced by ATP was much lower than that of skeletal muscle actomyosin and also was lower than that of fluorescence enhancement.     

A simple method for the isolation of actin from myxomycete plasmodia.

A new, simple method for the isolation of actin from myxomycete plasmodia has been developed. Plasmodium myosin B was incubated at 55 degrees C for 15 min in the presence of ATP or was treated with 90% acetone. By this treatment myosin was denatured completely. Actin was then extracted with a dilute ATP and cysteine solution from the heat- or acetone-treated myosin B. The method is simple and almost pure actin was obtained in high yield. The purified G-actin polymerized to F-actin on addition of 0.1 M KCl or 2 mM MgCl2. The viscosity of the purified F-actin was 8-10 dl/g. The F-actin activated muscle myosin ATPase, and actomyosin synthesized from the F-actin and muscle myosin showed superprecipitation on addition of ATP.     

The phosphorylation-dephosphorylation process as a myosin-linked regulation of superprecipitation of fast skeletal muscle actomyosin

The dependence of the onset and course of turbidity changes ( superprecipitation) induced by ATP were studied in a natural actomyosin suspension with the dephosphorylated and phosphorylated forms of light chains (LC2) of myosin. It was found that the onset and time course of the changes in turbidity of the natural actomyosin suspension are strongly dependent on the (phosphorylated and dephosphorylated) form of these chains of myosin. The ATPase activity of actomyosin with phosphorylated LC2 was lower and the half-time for achieving maximal turbidity of actomyosin suspension after addition of ATP was higher than that of actomyosin with dephosphorylated LC2. Natural actomyosin preparations contain endogenous light-chain kinase and phosphatase. The changes of turbidity induced by ATP in the natural actomyosin suspension are greatly diminished in the presence of phosphate. Thiophosphorylation of LC2 of myosin leads to a decrease of the extent of superprecipitation of natural actomyosin. The release of [32P]phosphate from actomyosin containing [32P]ATP-phosphorylated LC2 of myosin increases with increased turbidity of actomyosin suspension. The change of the form LC2 as a kind of additional myosin-linked regulation of superprecipitation is discussed.     

Competitive and uncompetitive effects of 2,4-dinitrophenol on ATPase activities of rabbit skeletal actomyosin and myosin.

A kinetic study of the ATPase reactions catalyzed by myosin and actomyosin was carried out by varying the concentrations of ATP and 2,4-dinitrophenol (DNP). Mg-ATPase of myosin in the initial burst and that of actomyosin were both inhibited competitively by DNP. The dissociation contants for the DNP-myosin interaction (Ki) were estimated to be very similar, that is, 4.2 mM in the initial burst of ATP splitting, and 3.3 mM for the actomyosin ATPase. It is therefore suggested that DNP acts at the same site when it inhibits the burst splitting of ATP and the actomyosin ATPase. In contrast, Mg,-Ca-, and EDTA-ATPase activities of myosin in the steady state were all affected uncompetitively by DNP. Moreover, the Ki value for Mg-ATPase of myosin in the steady state was found to be 31 mM, which is much higher than those mentioned above for the initial burst and actomyosin ATPase. It is therefore suggested that the site at which DNP acts to inhibit the burst splitting of ATP is different from the site at which DNP acts to affect Mg-, Ca-, and EDTA-ATPases in the steady state.     

Factors influencing interaction of phosphorylated and dephosphorylated myosin with actin

The influence of various factors on the interaction of phosphorylated and dephosphorylated myosin with actin was examined. It was found that the difference between the values of specific activity of the two myosin forms of actin-stimulated Mg2+-ATPase is affected by changes in KCl, MgATP and actin concentration. The effect of increased pH on the differences in the rate of ATP hydrolysis by actomyosin containing phosphorylated myosin as compared with that of the dephosphorylated one, observed in the presence of EGTA, is abolished by addition of Ca2+. Tropomyosin strongly inhibits the actin-stimulated Mg2+-ATPase of phosphorylated myosin (by about 60%). The tropomyosin-troponin complex and native tropomyosin lowered the rate of ATP hydrolysis by actomyosin containing both phosphorylated and dephosphorylated myosin by about of 60% of the value obtained in the absence of those proteins. These results indicate that the change of negative charge on the myosin head due to phosphorylation and dephosphorylation of myosin light chains modulates the actin-myosin interaction at different steps of the ATP hydrolysis cycle. Phosphorylation of myosin seems to be a factor decreasing the rate of ATP hydrolysis by actomyosin under physiological conditions.     

Interactions between actin, myosin, and an actin-binding protein from rabbit alveolar macrophages. Alveolar macrophage myosin Mg-2+-adenosine triphosphatase requires a cofactor for activation by actin.

The interactions were analyzed between actin, myosin, and a recently discovered high molecular weight actin-binding protein (Hartwig, J. H., and Stossel, T. P. (1975) J. Biol Chem.250,5696-5705) of rabbit alveolar macrophages. Purified rabbit alveolar macrophage or rabbit skeletal muscle F-actins did not activate the Mg2+ATPase activity of purified rabbit alveolar macrophage myosin unless an additional cofactor, partially purified from macrophage extracts, was added. The Mg2+ATPase activity of cofactor-activated macrophage actomyosin was as high as 0.6 mumol of Pi/mg of myosin protein/min at 37 degrees. The macrophage cofactor increased the Mg2+ATPase activity of rabbit skeletal muscle actomyosin, and calcium regulated the Mg2+ATPase activity of cofactor-activited muscle actomyosin in the presence of muscle troponins and tropomyosin. However, the Mg2+ATPase activity of macrophage actomyosin in the presence of the cofactor was inhibited by muscle control proteins, both in the presence and absence of calcium. The Mg2+ATPase activity of the macrophage actomyosin plus cofactor, whether assembled from purified components or studied in a complex collected from crude macrophage extracts, was not influenced by the presence of absence of calcium ions. Therefore, as described for Acanthamoeba castellanii myosin (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697), rabbit alveolar macrophage myosin requires a cofactor for activation of its Mg2+ATPase activity by F-actin; and no evidence was found for participation of calcium ions in the regulation of this activity.In macrophage extracts containing 0.34 M sucrose, 0.5 mM ATP, and 0.05 M KCl at pH 7.0,the actin-binding protein bound F-actin into bundles with interconnecting bridges. Purified macrophage actin-binding protein in 0.1 M KCl at pH 7.0 also bound purified macrophage F-actin into filament bundles. Macrophage myosin bound to F-actin in the absence but not the presence of Mg2+ATP, but the actin-binding protein did not bind to macrophage myosin in either the presence or absence of Mg2+ATP.     

Studies on the isolation and chemical properties of porcine follicle-stimulating hormone.

Porcine follicle-stimulating hormone (pFSH) has been prepared from acetone dried pituitary glands. The acetone powder was initially extracted in pH 5 ethanol-acetate buffer (40% ethanol and acetate at 0.5 ionic strength) containing 0.02% PMSF. The gonadotropin rich fraction, obtained in the 80% ethanol precipitate, was dissolved in 0.12 M NH4HCO3, heated at 60 degrees for 3 min, cooled, and centrifuged. The supernatant was lyophilized for chromatography on QAE-Sephadex, then Sephadex G-100. The pFSH obtained has a biological activity of 15 X NIH-FSH-P-1 and 2.1% sialic acid. The amino acid analysis is characterized by high lysine, aspartic acid, and glutamic acid with notable amounts of threonine, proline, and half-cystine.     

Steady-state studies of the actin-activated adenosine triphosphatase activity of myosin.

Reconstituted actomyosin (ATP phosphohydrolase, EC 3.6.1.3) (0.400 mg F-actin/mg myosin) in 10.0 muM ATP loses 96% of its specific ATPase activity when its reaction concentration is decreased from 42.0 mug/ml down to 0.700 mug/ml. The loss of specific activity at the very low enzyme concentrations is prevented by the addition of more F-actin to 17.6 mug/ml. It is concluded that at low actomyosin concentrations the complex dissociates into free myosin with a very low specific ATPase activity and free F-actin with no ATPase. The dissociation of the essential low molecular weight subunits of myosin from the heavy chains at very low actomyosin concentrations may be a contributing factor. Actomyosin has its maximum specific activity at pH 7.8-8.2. The Km for ATP is 9.4 muM, which is at least 20-fold greater than myosin's Km for ATP. The actin-activated ATPase of myosin follows hyperbolic kinetics with varying F-actin concentrations. The Km values for F-actin are 0.110 muM (4.95 mug/ml) at pH 7.4 and 0.241 muM (10.8 mug/ml) at pH 7.8. The actin-activated maximum turnover numbers for myosin are 9.3 s-1 at pH 7.4 and 11.6 s-1 at pH 7.8. The actomyosin ATPase is inhibited by KCl. This KCl inhibition is not competitive with respect to F-actin, and it is not a simple form of non-competitive inhibition.     

Superprecipitation of actomyosin with p-chloromercuribenzoate-modified myosin reconstituted from rabbit skeletal muscle

Myosin head modified with p-chloromercuribenzoate (CMB) forms rigor-like complex with actin in the presence of ATP. Actomyosins with CMB-modified myosin were reconstituted to study the effect of rigor-like complexes on superprecipitation. As native myosin was increasingly replaced by CMB-modified myosin, superprecipitation of the actomyosin was strongly suppressed. Further, the suppression of superprecipitation occurred in a different fashion depending on how CMB-modified myosin was incorporated in myosin filaments of the reconstituted actomyosin. The present results indicate that superprecipitation requires the dissociation of actin and myosin head to take place (i.e., the presence of molecular rearrangements of actomyosin network), and further suggest that superprecipitation is associated with dynamic rearrangements of actomyosin network along myosin filaments.