A bovine seminal plasma inhibitor of actin-stimulated myosin adenosine triphosphatase

During attempts to isolate bovine sperm actin, persistent low molecular weight proteinaceous (LMWP) contaminants were found. A LMWP fraction was prepared by gel filtration chromatography on Sephadex G150. The LMWP was found in extracts of washed bovine ejaculated spermatozoa and in clarified bovine seminal plasma. It was substantially reduced in amount in bovine epididymal spermatozoa, indicating that it originated from secondary sex gland secretions. The LMWP inhibited rabbit muscle actin-stimulated myosin adenosine triphosphatase (actin-myosin ATPase) activity. The LMWP:actin ratio for 50% inhibition of actin-myosin ATPase was 2.6 +/- 0.12 mg LMWP per mg actin. The LMWP interfered with actin inhibition of deoxyribonuclease, indicating that LMWP interacted with actin. The LMWP from seminal plasma had an estimated molecular weight of 8300 and consisted of several acidic components. It had negligible protease activity and its inhibition of actin-myosin ATPase was independent of divalent cations. The LMWP appears to readily aggregate with itself and other proteins, which may be related to its physiological role in semen.     

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.     

Requirement of domain-domain interaction for conformational change and functional ATP hydrolysis in myosin

Coordination between the nucleotide-binding site and the converter domain of myosin is essential for its ATP-dependent motor activities. To unveil the communication pathway between these two sites, we investigated contact between side chains of Phe-482 in the relay helix and Gly-680 in the SH1-SH2 helix. F482A myosin, in which Phe-482 was changed to alanine with a smaller side chain, was not functional in vivo. In vitro, F482A myosin did not move actin filaments and the Mg2+-ATPase activity of F482A myosin was hardly activated by actin. Phosphate burst and tryptophan fluorescence analyses, as well as fluorescence resonance energy transfer measurements to estimate the movements of the lever arm domain, indicated that the transition from the open state to the closed state, which precedes ATP hydrolysis, is very slow. In contrast, F482A/G680F doubly mutated myosin was functional in vivo and in vitro. The fact that a larger side chain at the 680th position suppresses the defects of F482A myosin suggests that the defects are caused by insufficient contact between side chains of Ala-482 and Gly-680. Thus, the contact between these two side chains appears to play an important role in the coordinated conformational changes and subsequent ATP hydrolysis.     

The effects of calcium and magnesium ions on the adenosine triphosphatase and inosine triphosphatase activities of myosin A

1. The effects of Ca(2+) and Mg(2+) on the enzymic activity of myosin were studied with myosin preparations treated by the ion-exchange resin Chelex-100. A reaction mixture containing 0.05m-potassium chloride was chosen in which the effects of univalent ions such as K(+), Na(+) and Cl(-) do not change significantly with small variations in their concentrations. 2. The relationship between the rate of hydrolysis of ATP or ITP and the concentration of Ca(2+) suggests that a relatively weak binding of Ca(2+) either to myosin or to the substrate nucleotide is responsible for the activation of the enzymic activity. According to the experiments with an ultrafiltration technique, the binding of Ca(2+) to myosin proceeds in at least two steps, the first occurring at one site on every 500000 atomic mass units of myosin with an apparent association constant, K(app.), 1.3x10(6)m(-1), and the second seeming to be so weak that its binding parameters cannot be determined by the method used. The first type of Ca(2+) binding is not observable with N-ethylmaleimide-modified myosin, yet this modified myosin shows activation by Ca(2+) of its adenosine triphosphatase and inosine triphosphatase. 3. The inhibition by Mg(2+) can be related to a binding reaction of Mg(2+) with myosin having K(app.) approximately 10(6)m(-1). Mg(2+) replaces the Ca(2+) bound tightly to myosin. The K(app.) for Mg(2+)-myosin binding calculated by assuming a competition between Ca(2+) and Mg(2+) for the same site is 2.1x10(5)-3.0x10(5)m(-1). When myosin is modified with a thiol reagent (p-mercuribenzoate) at a certain ratio to myosin, the inhibition by Mg(2+) becomes unobservable. 4. The behaviour of the hydrolytic activity of myosin on ATP or ITP in the presence of both Ca(2+) and Mg(2+) is consistent with the explanation that the inhibition by Mg(2+) is due to the tight binding of Mg(2+) to myosin, whereas the activation by Ca(2+) is caused either by a weak binding of Ca(2+) to myosin or by CaATP(2-) or by both.     

The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation

The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.     

Effect of ozone on erythrocyte membrane adenosine triphosphatase.

Human erythrocyte membrane fragments were exposed to O3 over varying lengths of time. Ozone was found to have a deleterious effect on the ouabainsensitive ATPase (EC 3.6.1.3) in the membrane fragments. After 1 min of exposure to O3, which was generated at a rate of 4.0 mumol/min, ouabain-sensitive ATPase activity decreased to 26% of the control. Ouabain-insensitive ATPase was found to be unaffected by O3 exposure under the test conditions. Additions of ascorbic acid or cysteine, prior to O3 exposure, partially protected the enzyme from inactivation. However, the inactivating effect of O3 could not be reversed by addition of either ascorbic acid or cysteine after exposure. Superoxide dismutase or catalase did not afford significant protection. The enzyme could not be protected by Ellman's reagent. The inactivating effect of O3 on the ouabain-sensitive ATPase was also demonstrated in exposure of intact erythrocytes. No detectable change was observed in glycolytic activity in the hemolysate prepared from O3-treated erythrocytes, however. It was postulated that inactivation of the membrane ATPase by O3 may be responsible for the destructive effect of O3 on the red cell.     

Transport adenosine triphosphatase: absence of ATP: p-nitrophenol phosphotransferase activity.

It is generally accepted that transport adenosine triphosphatase hydrolyzes both ATP and p-nitrophenyl phosphate, and other authors have shown that the enzyme can be phosphorylated in the same location by either substrate. We could detect no label exchange between ATP and p-nitrophenol. This finding indicates that any common phosphorylated intermediate must be formed from either substrate in a poorly reversible reaction and places constraints on models for the sodium pump.     

Analysis of the conformational change of myosin during ATP hydrolysis using fluorescence resonance energy transfer

In order to elucidate the molecular basis of energy transduction by myosin as a molecular motor, a fluorescent ribose-modified ATP analog 2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl]-ATP (NBD-ATP), was utilized to study the conformational change of the myosin motor domain during ATP hydrolysis using the fluorescence resonance energy transfer (FRET) method. The FRET efficiency from the fluorescent probe, BD- or AD-labeled at the reactive cysteine residues, SH1 (Cys 707) or SH2 (Cys697), respectively, to the NBD fluorophore in the ATP binding site was measured for several transient intermediates in the ATPase cycle. The FRET efficiency was greater than that using NBD-ADP. The FRETs for the myosin.ADP.AlF4- and myosin.ADP.BeFn ternary complexes, which mimic the M*.ADP.P(i) state and M.ATP state in the ATPase cycle, respectively, were similar to that of NBD-ATP. This suggests that both the SH1 and SH2 regions change their localized conformations to move closer to the ATPase site in the M*.ATP state and M**.ADP.P(i) state than in the M*.ADP state. Furthermore, we measured energy transfer from BD in the essential light chain to NBD in the active site. Assuming the efficiency at different states, myosin adopts a conformation such that the light chain moves closer to the active site by approximately 9 A during the hydrolysis of ATP.     

The possible relationship between a membrane conformational change and photosystem II dependent hydrogen ion accumulation and adenosine 5'-triphosphate synthesis.

Data are presented which suggest that photosystem II dependent hydrogen ion accumulation and ATP synthesis can occur only after the lamellar membranes have undergone a conformational change. This membrane conformational change is detected by the electron transport dependent incorporation of diazonium benzene[35S]sulfonate into membrane components. Previously it was established that electron flux from the photosystem II primary acceptor to plastoquinone is a necessary event for the occurrence of the diazonium-detected conformational change. These studies indicate that the release of hydrogen ions during photosystem II oxidation of the primary reductant is also a necessary event for the diazonium-detected conformational change. When iodide were substituted for water (or other proton-releasing donors) as the primary reductant of system II the conformational change did not occur even though a substantial rate of electron flow from the primary acceptor to plastoquinone occurred.     

Trypsin digestion of canine cardiac myosin. Low molecular weight fragment of myosin with high adenosine triphosphatase activity.

(1) Trypsin digestion of dog cardiac myosin leads to the formation of two dissimilar types of enzymatically active species based on the elution pattern of Sephadex G-200 columns. (2) When the digestion is performed in 0.6 m KCl the major protein peak is eluted at the exclusion limit of the column. Sodium dodecyl sulfate (SDS)-gel electrophoresis of this peak shows the heterogeneity of the heavy chain component, indicating multiple sites of cleavage by trypsin. (3) When the trypsinization is carried out in 0.15 m KCl in the presence of EDTA and β-mercaptoethanol, the major protein peak (retarded on the Sephadex G-200 column) has a high Ca²⁺-ATPase activity. On SDS-gel electrophoresis it shows only two major bands with corresponding molecular weights of 58,000 and 28,000, respectively. The 28,000-molecular-weight band apparently corresponds to cardiac light chain 1 of native myosin. (4) The results suggest that, with trypsinization of myosin in 0.15 m KCl, only a limited number of sites is exposed to trypsin. The fragment isolated under these conditions differs from a papain digestion fragment with respect to its molecular weight and the composition of the heavy chain fraction. On the basis of the molecular weight of the undissociated fragment it seems likely that the fragment retains a heavy meromyosin type (two heads) of configuration.     

G-actin conformational change and polymerization induced by paraquat.

Paraquat (1,1'-dimethyl-4,4'-bipyridilium dichloride) is a broad-spectrum herbicide that is highly toxic to animals (including man), the major lesion being in the lung. In mammalian cells, paraquat causes deep alterations in the organization of the cytoskeleton, marked decreases in cytoskeletal protein synthesis, and alterations in cytoskeletal protein composition; therefore, the involvement of the cytoskeleton in cell injury by paraquat was suggested. We previously demonstrated that monomeric actin binds paraquat; moreover, prolonged actin exposure to paraquat, in depolymerizing medium, induces the formation of actin aggregates, which are built up by F-actin. In this work we have shown that the addition of paraquat to monomeric actin results in a strong quenching of Trp-79 and Trp-86 fluorescence. Trypsin digestion experiments demonstrated that the sequence 61-69 on actin subdomain 2 undergoes paraquat-dependent conformational changes. These paraquat-induced structural changes render actin unable to completely inhibit DNase I. By using intermolecular cross-linking to characterize oligomeric species formed during paraquat-induced actin assembly, we found that the herbicide causes the formation of actin oligomers characterized by subunit-subunit contacts like those occurring in oligomers induced by polymerizing salts (i.e., between subdomain 1 on one actin subunit and subdomain 4 on the adjacent subunit). Furthermore, the oligomerization of G-actin induced by paraquat is paralleled by ATP hydrolysis.     

Catalysis of partial reactions of ATP synthesis by beef heart mitochondrial adenosine triphosphatase

We have found that when the ATP hydrolysis activity of beef heart mitochondrial adenosine triphosphatase (F1) is eliminated by either cold treatment or chemical modification, the enzyme attains the ability to catalyze the Pi in equilibrium ATP exchange reaction. The ATP hydrolysis activity of isolated F1 was lost upon chemical modification by phenyglyoxal, butanedione, or 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole. The F1 thus chemically modified was able to catalyze an ADP-dependent Pi in equilibrium ATP exchange reaction. In addition F1 that had been cold-treated to eliminate ATP hydrolysis activity, also catalyzed the Pi in equilibrium ATP exchange reaction. The Pi in equilibrium ATP exchange catalyzed by modified F1 was shown to be totally inhibited by the F1-specific antibiotic efrapeptin. We have previously shown that isolated beef heart mitochondrial ATPase will catalyze the formation of a transition state analog of the ATP synthesis reaction (Bossard, M. J., Vik, T. A., and Schuster, S. M. (1980) J. Biol. Chem. 255, 5342-5346). While the F1-catalyzed ATP hydrolysis activity was lost rapidly upon chemical modification or cold treatment, the ability of the enzyme to produce Pi . adenosine 5'-diphosphate (chromium(III) salt) from phosphate and monodentate adenosine 5'-diphosphate (chromium(III) salt) was unimpaired. The implications of these data with regard to the mechanism of ATP synthesis are discussed.