Characteristics of affinity modification of myosin ATPase under the action of monoaldehyde derivatives of ADP]

It was shown that the highly purified monoaldehyde derivative of ADP obtained by partial reduction of the dialdehyde derivative of ADP causes strong irreversible inhibition of the Ca-ATPase activity of myosin subfragment I, the inhibiting effect being of the affinity modification type. The addition to the reaction medium of Mg2+ (but not Ca2+) during the subfragment I interaction with the inhibitor fully prevents the inhibiting effect at all substrates used (Ca-, Mg- or K, EDTA-ATPases). Contrariwise, the subfragment I modified in the absence of Mg2+ exhibits the same degree of inhibition for all the three types of the ATPase activity. An unexpected result that was previously unobserved for other affinity modifiers of myosin ATPase is the maintenance of activity in 50% of active centers, when "two-head" forms of the enzyme (the myosin proper and heavy meromyosin, HMM) are modified. Noteworthy that the affinity modification reaction is characterized by the same values of inhibition constants as in the case of myosin subfragment I (Ki = 3.3-3.5 X 10(-4) M; ki = 0.03-0.04 min-1). This finding provides additional evidence in favour of functional asymmetry of myosin heads in the myosin molecule which seems to be due to the screening of the active center of one head by the other one.     

Coupling of proteolysis with ATP hydrolysis by Escherichia coli Lon proteinase. I. Kinetic aspects of ATP hydrolysis

Some aspects of the ATPase function of the Escherichia coli Lon protease were studied around the optimum pH value. It was revealed that, in the absence of the protein substrate, the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg2+ ions in the area of their millimolar concentrations. Free components of the substrate complex (ATP-Mg)2- inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg2+ ions is caused by the formation of the "ADP-Mg-form" of the ATPase centers. It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)2- complex to the enzyme, by the elimination of the inhibiting action of free Mg2+ ions without altering the efficiency of catalysis of ATP hydrolysis (based on the kcat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)- complex (without changing the Ki value). Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.     

Counteracting effects of urea and methylamines in function and structure of skeletal muscle myosin

Myosin is an asymmetric protein that comprises two globular heads (S1) and a double-stranded alpha-helical rod. We have investigated the effects of urea and the methylamines trimethylamine oxide (TMA-O) and glycine betaine (betaine) on activity and structure of skeletal muscle myosin. K(+) EDTA ATPase activity of myosin was almost completely inhibited by urea (2M); TMA-O stimulated myosin activity, whereas betaine had no effect. When combined with urea (0-2M), TMA-O or betaine (1 M) effectively protected the ATPase activity of myosin against inhibition. Intrinsic fluorescence measurements showed that in urea or TMA-O (0-2M), there were no shifts in the center of mass of the fluorescence spectrum of myosin, despite a decrease in fluorescence intensity. However, these osmolytes at concentrations above 2M produced a red shift in the emission spectrum. Betaine alone did not alter the center of mass at any concentration tested up to 5.2M. Thus, modifications in ATPase activity induced by low concentrations of solutes (<2M) are not directly correlated with the modifications in myosin structure detected by fluorescence. Both methylamines (>or=1M) were also able to protect myosin structure against urea-induced effects (2-8M). Protection was not observed for S1, supporting the hypothesis that these osmolytes have a biphasic effect on myosin: at lower concentrations there is an effect on the globular portion (S1), and at higher concentrations there is an effect on the coiled-coil (rod) portion of myosin.     

Dialdehyde derivatives of purine mononucleotides: substrate properties and affinity modification of myosin ATPase

It was demonstrated that the dialdehyde derivative of ATP is a good substrate for Ca-ATPase of heavy meromyosin (Km = (1.2-1.4) X 10(-4) M; V = VATP). At the same time, this compound can induce irreversible inhibition of the enzyme. Since oxo-ATP is rapidly hydrolyzed by myosin to form oxo-ADP, this inhibition is the result of the enzyme interaction with oxo-ADP. It was found that the kinetics of heavy meromyosin inhibition by oxo-ADP are typical of affinity modification; in this case ATP fully protects heavy meromyosin from the activity loss. Similar results on the irreversible inhibition of the ATPase activity under the action of oxo-ADP were obtained in the presence of myosin, heavy meromyosin, subfragment I and natural actomyosin and in the absence of bivalent cations, thus suggesting the modification of the active center of myosin ATPase.     

Effect of staphylococcus active substances on ATPase activity of smooth muscle actomyosin and myosin

The effect of staphylococcus active substances--protein A (PA) and peptidoglican (PG) at concentrations 10(-6)-10(-2) mg/ml on the ATPase activity of pig stomach natural actomyosin and myosin was studied. It was shown that PA and PG at direct contact with smooth muscle contractile proteins caused the activation and inhibition of ATPase activity, respectively. On the basis of this investigation it was assumed that staphylococcal active substances were able to modify of the ATPase activity smooth muscle contractile proteins perhaps via direct action on the myosin molecule, which could be accompanied by conformational changes of the active center of myosin ATPase.     

Modification of myosin subfragment 1 by carbodiimide in the presence of a nucleophile. Effect on adenosinetriphosphatase activities

The modification of myosin subfragment 1 by N-cyclohexyl-N'-[2-(4-morpholinyl)ethyl]carbodiimide methyl p-toluenesulfonate in the presence of the nucleophile nitrotyrosine ethyl ester was investigated. For elimination of interference of the thiol groups, the two most reactive thiols were protected by cyanylation with 2-nitro-5-(thiocyanato)benzoic acid. The ATPase activity of the cyanylated myosin subfragment 1 was not lost, but had changed. At pH 5.9, carbodiimide in the presence of the nucleophile rapidly inactivated the cyanylated enzyme. The inactivation followed first-order kinetics. The K+(EDTA)--, Ca2+--, and Mg2+--ATPase activities decreased at the same rate. Inactivation and incorporation of nucleophile occurred simultaneously. A full loss of activity resulted from the incorporation of 1 mol of nitrotyrosine per mol of myosin subfragment 1. Pyrophosphate, ITP, ADP, and ATP protected against inactivation, and the efficiency of the protection was parallel to the ligand binding strength. These results suggested that one carboxyl group was essential for the active conformation of myosin.     

Coupling of proteolysis to ATP hydrolysis uponEscherichia coli lon protease functioning: I. kinetic aspects of ATP hydrolysis

Some aspects of theEscherichia coli Lon protease ATPase function were studied around the optimum pH value. It was revealed that in the absence of the protein substrate the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg²⁺ ions in the area of their millimolar concentrations. Free components of the substrate complex (ATP-Mg)²⁻ inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg²⁺ ions is caused by the formation of the “ADP-Mg-form” of ATPase centers. It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)²⁻ complex to the enzyme, by an elimination of the inhibiting action of free Mg²⁺ ions without altering the efficiency of catalysis of ATP hydrolysis (based on thek _cat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)⁻ complex (without changing theK _i value). Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.     

Distinct structures of ATP and GTP complexes in the myosin ATPase

The active site of the myosin subfragment-1 ATPase was affinity-labeled with ribose-modified fluorescent analogs of ADP, dADP, CDP, UDP, IDP, and GDP in combination with vanadate, forming a stable myosin-nucleoside diphosphate-vanadate complex that is analogous to the normal myosin-ADP-Pi intermediate [Hiratsuka, T. (1984) J. Biochem. 96, 147-154]. Labeled enzyme was isolated free of unbound analog and vanadate, and fluorescent properties of the fluorophore at the active site were examined. Fluorescence emission and acrylamide quenching studies revealed that the hydrophobicity of environment around the fluorophore and the degree of its burial in the protein vary with the base structure of NDP. It was found that the fluorophore of ADP analog is most buried into the protein, while that of the GDP analog is least buried. The results suggest that the deep burial of ATP into the myosin active site is essential for muscle contraction.     

Fluorescence decay of sarcoplasmic reticulum ATPase. Ligand binding and hydration effects

Multifrequency phase-modulation lifetime data were acquired for sarcoplasmic reticulum Ca2+-ATPase. The intrinsic tryptophan fluorescence decay was complex and was fitted either with three exponentials or with bimodal Lorentzian distributions of lifetimes. Ca2+ binding to the high affinity sites in the ATPase produced an increase of 11% in the center of the main component of the bimodal distribution, shifting the lifetime from 4.04 to 4.50 ns. The effects of solvent on the ATPase were studied with the enzyme dissolved in reverse micelles of detergent bis-(2-ethylhexyl)sulfosuccinate in hexane. Increasing amounts of water up to a water/bis-(2-ethylhexyl)sulfosuccinate molar ratio of 4 produced marked changes in the fluorescence emission of the protein. Comparison of data obtained for micellar solutions of tryptophan or ATPase indicated that the tryptophan residues in the protein are protected from exposure to water. Correlation of water effects on emission intensity and lifetimes suggested that interaction with solvent may result in structural changes that cause a mixture of dynamic and static quenching of ATPase intrinsic fluorescence. Evidence for an effect of hydration on the structure of the active site was obtained by measurements of the fluorescence properties of fluorescein isothiocianate-labeled ATPase in reverse micelles.     

Effects of mercury on myosin ATPase in the ventricular myocardium of the rat

Mercury reduces twitch and tetanic force development in isolated rat papillary muscles, and a putative toxic effect on the contractile machinery has been suggested. Based on that, the actions of HgCl2 on the myosin ATPase activity of the left ventricular myocardium were investigated. Samples for assay of myosin ATPase activity were obtained from rats' left ventricles. Increasing concentrations of HgCl2 reduced dose-dependently the activity of the myosin ATPase. This reduction was observed even at very small concentrations, 50 nM HgCl2. This effect was dependent on the presence of SH groups in the myosin molecule since DTT and glutathione protected the myosin ATPase against toxic effects of mercury; full activity being restored by using 500 nM DTT or 500 nM glutathione. Results also suggested that the metal acts as an uncompetitive inhibitor with a Ki of 200 nM HgCl2. Our results suggest that mercury reduces the activity of the myosin ATPase by an uncompetitive mechanism at a very low dose that does not depress force. DTT and glutathione are effective for protection against the actions of mercury suggesting that SH groups might be the sites of action of the metal on the myosin molecule.     

Electrochemical studies of arsenite oxidase: an unusual example of a highly cooperative two-electron molybdenum center

Arsenite oxidase from Alcaligenes faecalis, an unusual molybdoenzyme that does not exhibit a Mo(V) EPR signal during oxidative-reductive titrations, has been investigated by protein film voltammetry. A film of the enzyme on a pyrolytic graphite edge electrode produces a sharp two-electron signal associated with reversible reduction of the oxidized Mo(VI) molybdenum center to Mo(IV). That reduction or oxidation of the active site occurs without accumulation of Mo(V) is consistent with the failure to observe a Mo(V) EPR signal for the enzyme under a variety of conditions and is indicative of an obligate two-electron center. The reduction potential for the molybdenum center, 292 mV (vs SHE) at pH 5.9 and 0 degrees C, exhibits a linear pH dependence for pH 5-10, consistent with a two-electron reduction strongly coupled to the uptake of two protons without a pK in this range. This suggests that the oxidized enzyme is best characterized as having an L(2)MoO(2) rather than L(2)MoO(OH) center in the oxidized state and that arsenite oxidase uses a "spectator oxo" effect to facilitate the oxo transfer reaction. The onset of the catalytic wave observed in the presence of substrate correlates well with the Mo(VI/IV) potential, consistent with catalytic electron transport that is limited only by turnover at the active site. The one-electron peaks for the iron-sulfur centers are difficult to observe by protein film voltammetry, but spectrophotometric titrations have been carried out to measure their reduction potentials: at pH 6.0 and 20 degrees C, that of the [3Fe-4S] center is approximately 260 mV and that of the Rieske center is approximately 130 mV.     

Influence of heavy metal ions on the ATPase activity of actomyosin complex and myosin subfragment-1 from smooth muscle of the uterus

The effect of divalent cations--Co2+, Cu2+, Mn2+ and Ni2+ (5 mM) on the activity of actomyosin complex ATPase and ATPase of subfragment-1 (S1,head) of myosin from smooth muscle of the uterus was studied. It has been shown that Co2+, Mn2+ and Ni2+ inhibited, while Cu2+ activates the enzyme activity of both actomyosin and myosin S1. Mg and Mn ions had practically no effect on the emission intensity of eosin Y associated with actomyosin, while one could observe the most marked suppression of emission of related fluorescent probe in the presence of Cu cations and less pronounced suppression in the presence of Co2+. In the presence of Mn, Co and Ni cations the average hydrodynamic diameter (HD) of actomyosin complex and of subfragment-1 of the smooth muscle of the uterus is virtually identical to the HD in the presence of Mg2+. In the presence of Cu cations there is a considerable (ten-fold) increase in the size of the protein particles that may be a result of their aggregation. The results obtained evidence for the significant changes in the structure and function of the actomyosin complex of the myometrium in the presence of heavy metals and allow us to assume that the target of the effect of these metals on the contractile proteins is a subfragment-1 of myosin, where the active site of ATPase and actin-binding sites are localized.     

Magnesium ion dependent rabbit skeletal muscle myosin guanosine and thioguanosine triphosphatase mechanism and a novel guanosine diphosphatase reaction.

The mechanism of the Mg2+-dependent myosin subfragment 1 catalyzed hydrolysis of GTP and 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate (thioGTP) has been investigated by rapid-reaction techniques. The myosin was isolated from rabbit skeletal muscle. The steady-state intermediate of these reactions consists pre-dominantly of a protein-substrate complex unlike the myosin subfragment 1 ATPase reaction which has a protein-products complex as the principal steady-state component. The mechanism of GTP hydrolysis catalyzed by subfragment 1 has other marked differences from the ATPase mechanism. The second-order rate constant of binding of GTP to subfragment 1 is tenfold greater than that for GDP binding. The dissociation rate constant of GDP from subfragment 1 is 0.06 s-1 compared with the subfragment 1 catalytic center activity for GTP hydrolysis of 0.5 s-1 at pH 8.0 and 20 degrees C. This shows that GDP bound to subfragment 1 forms a complex which is not kinetically competent to be an intermediate of the GTPase mechanism. GDP is hydrolyzed in the presence of subfragment 1 to GMP and Pi. The subfragment 1 GTPase mechanism has a nuber if features in common with that of the elongation factor Tu GTPase of the protein biosynthetic system of Escherichia coli.     

Carboxyl groups at the two active centers of sucrase-isomaltase from rabbit small intestine.

1. Seveal selective reagents were employed to identify the amino acid residues essential for the catalytic activity of sucrase-isomaltase. 2. Modification of histidine, lysine and carboxyl residues resulted in a partial inactivation of the enzyme. Substrates or competitive inhibitors provided protection against inactivation only in the reaction of carboxyl groups with carbodiimide (+lycine ethyl ester) or with diazoacetic ethyl ester. This indicated the occurrence of carboxyl groups at the two active centers of the enzyme complex. 3. Protection against inactivation of the enzyme by carbodiimide was provided also by the presence of alkali and alkaline earth metal ions, which are non-essential activators of sucrase-isomaltase. The presence of Na+ and Ba2+ protected approximately one carboxyl group per active center from reacting with carbodiimide plus glycine ethyl ester. 4. The carbodiimide-reactive groups were not identical with the two carboxylate groups recently found to react with conduritol-B-epoxide, an active-site-directed inhibitor of sucrase-isomaltase (Quaroni, A. and Semenza, G., 1976, J. Biol. Chem 251,3250--3253). A possible role for the carbodiimide-reactive carboxyl groups at the active centers of sucrase-isomaltase is discussed.     

ATPase activity of myosin in hair bundles of the bullfrog''s sacculus.

Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor.     

A comparative study of myosin and its subunits in adult and neonatal-rat hearts and in rat heart cells from young and old cultures.

A possible explanation for the decrease in myosin Ca2+-dependent ATPase activity as rat heart cells age in culture is presented. The subunit structure and enzyme kinetics of myosin from adult and neonatal rat hearts and from rat heart cells of young and old cultures are compared. These studies indicate that the loss in Ca-ATPase activity of myosin from older cultures was an intrinsic property of the myosin itself. Myofibrillar fractions from the indicated four sources showed no qualitative or quantitative differences in electrophoretic patterns. Myosin from older cultures was more sensitive to alkaline denaturation than was myosin from younger cultures, as indicated by its more accelerated loss of K+(EDTA)-dependent ATPase activity after 10 min of incubation at pH 10. Furthermore, myosin from older cultures was more temperature-sensitive, as indicted by a more rapid loss of Ca-ATPase with decrease in assay temperature. It is suggested that there is either a change in conformation of myosin molecules at or near the active site of the enzyme or alternatively there is a change in light chain 1-light chain 2 and/or light-chain-heavy-chain interaction(s) in the myosin molecules under study.     

Demonstration of a direct role for myosin light chain kinase in fibroblast-populated collagen lattice contraction.

Mixing feed fibroblasts with soluble collagen and serum-supplemented culture medium at 37 degrees C results in the entrapment of cells within the polymerizing collagen matrix. This cellular-collagen complex is referred to as a fibroblast-populated collagen lattice (FPCL). In time, this FPCL undergoes a reduction in size called lattice contraction. The proposed mechanism for lattice contraction is cellular force produced by cytoplasmic microfilaments which organize collagen fibrils compacting the matrix. When the regulatory subunits of myosin, myosin light chains, are phosphorylated by myosin light chain kinase (MLCK), myosin ATPase activity is increased and actin-myosin dynamic filament sliding occurs. Elevated levels of myosin ATPase are required for maximal lattice contraction. Cholera toxin inhibits lattice contraction by increasing intracellular levels of cAMP. It is proposed that increased cytoplasmic concentrations of cAMP promote phosphorylation of MLCK, the enzyme important for maximizing myosin ATPase activity. Phosphorylating MLCK in vitro inhibits activity by decreasing its sensitivity to calcium-calmodulin complex. A decrease in MLCK activity would result in lower levels of myosin ATPase activity. MLCK, purified from turkey gizzard, was subjected to limited proteolytic digestion to produce calmodulin-independent-MLCK. The partially digested kinase does not require calcium-calmodulin for activation. Independent-MLCK is not subject to inhibition by phosphorylation. The electroporetic inoculation of independent-MLCK into fibroblasts before FPCL manufacture produced enhanced lattice contraction. Lattice contraction, in the presence of cholera toxin, was restored to normal levels by the prior electroporetic introduction of independent-MLCK. These findings support the hypothesis that increases in cAMP hinder lattice contraction by a mechanism involving inhibition of MLCK and myosin ATPase.     

The contractile and regulatory proteins of insect flight muscle

1. Myosin, actin and the regulatory proteins were prepared from insect flight muscle. 2. The light subunit composition of the myosin differed from that of vertebrate muscle myosin. The ionic strength and pH dependence of the myosin adenosine triphosphatase (ATPase) were measured. 3. Actin was associated with a protein of subunit molecular weight 55000 and was purified by gel filtration. Impure actin had protein bound at a periodicity of about 40nm. 4. Regulatory protein extracts had tropomyosin and troponin components of subunit molecular weight 18000, 27000 and 30000. Crude extracts of regulatory proteins inhibited the ATPase activity of desensitized or synthetic actomyosin; this inhibition was relatively insensitive to high Ca(2+) concentrations. Purified insect regulatory protein produced as much sensitivity to Ca(2+) as did the rabbit troponin-tropomyosin complex. 5. Synthetic actomyosins were made from rabbit and insect proteins. Actomyosins containing insect myosin had a low ATPase activity that was activated by tropomyosin. The Ca(2+) sensitivity of actomyosins containing insect myosin or actin, with added troponin-tropomyosin complex from rabbit, was comparable with that of rabbit actomyosin.     

Relation between the thermostability of the tetrameric molecule of lactate dehydrogenase from swine muscles and the degree of occupancy of its active sites with ligands

Using differential scanning microcalorimetry and measurements of protein fluorescence, the thermal denaturation of lactate dehydrogenase (LDH) from porcine muscle (in the apo-form as well as in the form of the enzyme-pyruvate, enzyme-NAD+ and enzyme-NAD-pyruvate-adduct complexes) was studied. Pyruvate binding did not affect the thermal stability of LDH. NAD+ exerted a stabilizing effect on the enzyme, the value of which was proportional to the number of ligand molecules bound per LDH tetramer. The formation of the abortive LDH-NAD-pyruvate complex in one, two or three active centers of the enzyme tetramer did not influence the values of calorimetric parameters of thermal denaturation in comparison with those for the apoenzyme. The occupancy of all four active centers of LDH by the adduct resulted in a sharp increase of the enzyme thermal stability and tightness of the LDH adduct complex as compared with complexes formed upon partial saturation. The experimental results are suggestive of the existence of a concerted conformational transition of the LDH tetramer induced by the formation of the LDH-NAD-pyruvate complex in the last active center of the tetramer.     

Role of bivalent and monovalent cations in the functioning of myosin ATPase

The effects of bivalent (Mg2+, Ca2+, Sr2+) and monovalent (K+, Na+, NH4+) cations on the ATPase activity of subfragment 1 of myosin (SI) with a decreased Mg2+ content (EDTA-SI) were studied. Mg2+ activate the EDTA-SI ATPase, but only in the absence of other activating cations. K+, NH4+, a2+ and Sr2+ have a much stronger activating effect on EDTA-SI ATPase than on Mg-SI (SI enriched with Mg2+) ATPase. Monovalent cations inhibit Mg2+-ATPase and Ca2+-ATPase of EDTA-SI, while K+ and NH4+ activate Sr2+-ATPase of EDTA-SI. Based on experimental results and literary data, a hypothesis on the participation of the cations in the functioning of myosin ATPase was postulated. This hypothesis entails the existence of two closely interconnected cation-binding sites in the vicinity of the myosin active center (one for bivalent and one for monovalent cations); the ATPase activity of myosin is at any moment dependent on the nature of cations present in these two sites. An attempt to explain the role of the cations in the accomplishment of the ATPase reaction by myosin was made.