Preparation and properties of 2′(or 3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate, an analog of adenosine triphosphate

An analog of adenosine triphosphate, 2′(or 3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP), was synthesized as a reporter-labeled substrate of heavy meromyosin ATPase. TNP-ATP was hydrolyzed by heavy meromyosin in the presence of CaCl₂ MgCl₂ or EDTA.TNP-ATP had absorption maxima at 259 nm, 408 nm and 470 nm at neutral pH. When bound to heavy meromyosin, TNP-ATP underwent the characteristic spectral shift. The difference spectrum resulting from the binding of TNP-ATP to heavy meromyosin at pH 8.0 had positive peaks at 415 nm and 518 nm, and a negative trough at 458 nm.The difference spectrum due to the binding of 2′(or 3′)-O-(2,4,6-trinitrophenyl)adenosine (TNP-adenosine) to heavy meromyosin had small positive peaks at 420 nm and 495 nm. This difference spectrum was similar to that of TNP-ATP or TNP-adenosine produced by 20% (v/v) ethyleneglycol perturbation. The positive peak at 495 nm in the difference spectrum due to the binding of TNP-adenosine to heavy meromyosin shifted toward 505 nm, when pyrophosphate or ATP was added to the reaction mixture.These results suggest that the difference spectrum of TNP-ATP due to the interaction with heavy meromyosin arises not only from the binding of the chromophoric portion of the TNP-ATP molecule but also from that of the phosphate portion.     

Mechanism of inhibition of skeletal muscle actomyosin by N-benzyl-p-toluenesulfonamide

N-Benzyl-p-toluenesulfonamide (BTS) is a small organic molecule that specifically inhibits the contraction of fast skeletal muscle fibers. To determine the mechanism of inhibition by BTS, we performed a kinetic analysis of its effects on the elementary steps of the actomyosin subfragment-1 ATPase cycle. BTS decreases the steady-state acto-S1 ATPase rate approximately 10-fold and increases the actin concentration for half-maximal activation. BTS primarily affects three of the elementary steps of the reaction pathway. It decreases the rate of P(i) release >20-fold in the absence of actin and >100-fold in the presence of actin. It decreases the rate of S1.ADP dissociation from 3.9 to 0.8 s(-)(1) while decreasing the S1.ADP dissociation constant from 2.3 to 0.8 microM. BTS weakens the apparent affinity of S1.ADP for actin, increasing the K(d) from 7.0 to 29.5 microM. ATP binding to S1, hydrolysis, and the affinity of nucleotide-free S1 for actin are unaffected by BTS. Kinetic modeling indicates that the binding of BTS to myosin depends on actin association/dissociation and on nucleotide state. Our results suggest that the reduction of the acto-S1 ATPase rate is due to the inhibition of P(i) release, and the suppression of tension is due to inhibition of P(i) release in conjunction with the decreased apparent affinity of S1.ADP.P(i) and S1.ADP for actin.     

Reactions of d-glyceraldehyde 3-phosphate dehydrogenase facilitated by oxidized nicotinamide–adenine dinucleotide

Transient kinetic methods have been used to study the influence of NAD(+) on the rate of elementary processes of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase. In the pH range 5-8 NAD(+) is bound to the enzyme during the following elementary processes of the mechanism: phosphorolysis of the acyl-enzyme, its formation from 1,3-diphosphoglycerate and the enzyme and the formation and breakdown of the glyceraldehyde 3-phosphate-enzyme complex. The rates of these four elementary processes only equal or exceed the turnover rate of the enzyme when NAD(+) is bound and are as much as 10(4) times the rates in the absence of NAD(+). Autocatalysis of the reductive dephosphorylation of 1,3-diphosphoglycerate occurs when glyceraldehyde 3-phosphate release is rate determining because NAD(+) is a reaction product. An important feature of the enzyme mechanism is that the negative-free-energy change of a chemical reaction, acyl-enzyme formation, is linked in a simple way to the positive-free-energy change of a dissociation reaction, NAD(+) release.     

Change in the ultraviolet absorption of an adenosine triphosphate analog, beta-napht-yl triphosphate, during its hydrolysis by heavy meromyosin.

It was found that the absorption spectrum of beta-naphthyl triphosphate is different from that of beta-naphthyl diphosphate in the range 290-335 nm. Thus, beta-naphthyl triphosphate hydrolysis by heavy meromyosin can be recorded continuously as a function of time by means of a spectrophotometer. By analyzing the time course, the apparent kinetic parameters were easily and rapidly obtained. If necessary, the true kinetic parameters, including the product dissociation constants, can be estimated spectrophotometrically. Beta-Naphthyl triphosphate hydrolysis was inhibited competitively by ATP. By analyzing the time course, it was, therefore, possible to estimate the kinetic parameters of ATP hydrolysis indirectly, and resonable values were obtained. Beta-Naphthyl triphosphate hydrolysis by heavy meromyosin was performed under various conditions. Unlike that of ATP, the hydrolysis of beta-naphthyl triphosphate was inhibited monotonously by treatment of heavy meromyosin with p-hydroxymercuribenzoate.     

Rate-determining processes and the number of simultaneously active sites of d-glyceraldehyde 3-phosphate dehydrogenase

Transient kinetic studies of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase show that all four sites of the tetrameric lobster enzyme are simultaneously active, apparently with equal reactivity. The rate-determining step of the oxidative phosphorylation is NADH release at high pH and phosphorolysis of the acyl-enzyme at low pH. For the reverse reaction the rate-determining step is a process associated with NADH binding, probably a conformation change, at high pH and d-glyceraldehyde 3-phosphate release at low pH. NADH has previously been shown to be a competitive inhibitor of the enzyme with respect to d-glyceraldehyde 3-phosphate and vice versa. This is consistent with the mechanism deduced from transient experiments given the additional proviso that 1-arseno-3-phosphoglycerate has a half-life of about 1min or longer at pH7. The dissociation constants of d-glyceraldehyde 3-phosphate and 1,3-diphosphoglycerate to the NAD(+)-bound enzyme are too large to measure but are nevertheless consistent with the low K(m) values of these substrates.     

Further studies on the interaction of actin with heavy meromyosin and subfragment 1 in the presence of ATP.

It has been postulated that, during the hydrolysis of ATP, both normal and SH1-blocked heavy meromyosin undergo a rate-limiting transition from a refractory state which cannot bind to actin to a nonrefractory state which can bind to actin. This model leads to several predictions which were studied in the present work. First, the fraction of heavy meromysin or subfragment 1 which remains unbound to actin when the ATPase equals Vmax should have the same properties as the original protein. In the present study it was determined that the unbound protein has normal ATPase activity which suggests that it is unbound to actin for a kinetic reason rather than because it is a permanently altered form of the myosin. Second, if the heavy meromyosin heads act independently half as much subfragment 1 as heavy meromyosin should bind to actin. Experiments in the ultracentrifuge demonstrate that about half as much subfragment 1 as heavy meromyosin sediments with the actin at Vmax. Third, the ATP turnover rate per actin monomer at infinite heavy meromyosin concentration should be much higher than the ATP turnover rate per heavy meromyosin head at infinite actin concentration. This was found to be the case for SH1-blocked heavy meromyosin since, even at very high concentrations of SH1-blocked heavy meromyosin, in the presence of a fixed actin concentration, the actin-activated ATPase rate remained proportional to the SH1-blocked heavy meromyosin concentration. All of these results tend to confirm the refractory state model for both SH1-blocked heavy meromyosin and unmodified heavy meromyosin and subfragment 1. However, the nature of the small amount of heavy meromyosin which does bind to actin in the presence of ATP at high actin concentration remains unclear.     

Reaction mechanism of the magnesium ion-dependent adenosine triphosphatase of frog muscle myosin and subfragment 1.

The Mg2+-dependent ATPase (adenosine 5'-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' - 1 E.ATP k' + 2 in equilibrium k' - 2 E.ADP.Pi k' + 3 in equilibrium k' - 3 E.ADP + Pi k' + 4 in equilibrium k' - 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 X 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 X 10(4) M-1.S-1 and 7.4 X 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.     

Human myosin Vc is a low duty ratio, nonprocessive molecular motor

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a V(max) of 1.8 +/- 0.3 s(-1) and a K(ATPase) of 43 +/- 11 microm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (~1.5 s(-1)), rather than the ADP release step (~12.0-16.0 s(-1)). Nevertheless, the ADP affinity of actomyosin Vc (K(d) = 0.25 +/- 0.02 microm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was approximately 10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of approximately 24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.     

Kinetic differences in cardiac myosins with identical loop 1 sequences

The kinetics of nucleotide turnover vary considerably among isoforms of vertebrate type II myosin, possibly due to differences in the rate of ADP release from the nucleotide binding pocket. Current ideas about likely mechanisms by which ADP release is regulated have focused on the hyperflexible surface loops of myosin, i.e. loop 1 (ATPase loop) and loop 2 (actin binding loop). In the present study, we investigated the kinetic properties of rat and pig beta-myosin heavy chains (beta-MHC) in which we have found the sequences of loop 1 (residues 204-216) to be virtually identical, i.e. DQSKKDSQTPKG, with a single conservative substitution (rat E210D pig). Pig myocardium normally expresses 100% beta-MHC, whereas rat myocardium was induced to express 100% beta-MHC by surgical thyroidectomy and subsequent treatment with propylthiouracil. Slack test measurements at 15 degrees C yielded unloaded shortening velocities of 1.1 +/- 0.8 muscle lengths/s in rat skinned ventricular myocytes and 0.35 +/- 0.05 muscle lengths/s in pig skinned myocytes. Similarly, solution measurements at the same temperature showed that actin-activated ATPase activity was 2.9-fold greater for rat beta-myosin than for pig beta-myosin. Stopped-flow methods were then used to assess the rates of acto-myosin dissociation by MgATP both in the presence and absence of MgADP. Although the rates of MgATP-induced dissociation of acto-heavy meromyosin (acto-HMM) were virtually identical for the two myosins, the rate of ADP dissociation was approximately 3.8-fold faster for rat beta-myosin (135 s(-)(1)) than for pig beta-myosin (35 s(-)(1)). ATP cleavage rates were nearly 30% faster for rat beta-myosin. Thus, whereas loop 1 appears from other studies to be involved in nucleotide turnover in the pocket, our results show that loop 1 does not account for large differences in turnover kinetics in these two myosin isoforms. Instead, the differences appear to be due to sequence differences in other parts of the MHC backbone.     

Two conserved lysines at the 50/20-kDa junction of myosin are necessary for triggering actin activation

Actin stimulates myosin's activity by inducing structural alterations that correlate with the transition from a weakly to a strongly bound state, during which time inorganic phosphate (P(i)) is released from myosin's active site. The surface loop at the 50/20-kDa junction of myosin (loop 2) is part of the actin interface. Here we demonstrate that elimination of two highly conserved lysines at the C-terminal end of loop 2 specifically blocks the ability of heavy meromyosin to undergo a weak to strong binding transition with actin in the presence of ATP. Removal of these lysines has no effect on strong binding in the absence of nucleotide, on the rate of ADP binding or release, or on the basal ATPase activity. We further show that the 16 amino acids of loop 2 preceding the lysine-rich region are not essential for actin activation, although they do modulate myosin's affinity for actin in the presence of ATP. We conclude that interaction of the conserved lysines with acidic residues in subdomain 1 of actin either triggers a structural change or stabilizes a conformation that is necessary for actin-activated release of P(i) and completion of the ATPase cycle.     

The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface

The time course of the extracellular reaction sequence ATP----ADP----AMP----adenosine has been examined during recirculation of substrate solutions over cultured pig aortic endothelial cells attached to polystyrene beads. This permits the study of reactions at volume to cell surface ratios approaching those of small blood vessels. When endothelial cells were presented with an initial bolus of ATP, high concentrations of the intermediates ADP and AMP developed before significant conversion of AMP to adenosine occurred. Further, the higher the initial ATP concentration, the slower the conversion of AMP to adenosine. Kinetic constants for each reaction were estimated by fitting simulated reaction curves to observed time courses. Apparent Km values estimated in this way agreed well with those reported for initial velocity measurements (ATPase = 300 microM; ADPase = 240 microM; and 5'-nucleotidase = 26 microM). The ratio of maximum velocities was ATPase:ADPase:AMPase = 6:1.5:1, with absolute values varying among cell batches. The data could only be fitted if the model incorporated inhibition of 5'-nucleotidase by ATP or ADP, and satisfactory fitting was achieved with a Ki value for ADP of 5 microM. These kinetic properties maximize the time separation of the intermediate pools. In vivo, at sites of platelet degranulation, they would create a time gap proportional to the size of the initial release between release of ADP (a proaggregatory milieu) and the appearance of adenosine (an anti-aggregatory milieu).     

Both heads of tissue-derived smooth muscle heavy meromyosin bind to actin in the presence of ADP

The effect of ADP and phosphorylation upon the actin binding properties of heavy meromyosin was investigated using three fluorescence methods that monitor the number of heavy meromyosin heads that bind to pyrene-actin: (i) amplitudes of ATP-induced dissociation, (ii) amplitudes of ADP-induced dissociation of the pyrene-actin-heavy meromyosin complex, and (iii) amplitudes of the association of heavy meromyosin with pyrene-actin. Both heads bound to pyrene-actin, irrespective of regulatory light chain phosphorylation or the presence of ADP. This behavior was found for native regulated heavy meromyosin prepared by proteolytic digestion of chicken gizzard myosin with between 5 and 95% heavy chain cleavage at the actin-binding loop, showing that two-head binding is a property of heavy meromyosin with uncleaved heavy chains. These data are in contrast to a previous study using an uncleaved expressed preparation (Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240-23245), which showed that one head of the unphosphorylated heavy meromyosin-ADP complex bound to actin and that the partner head either did not bind or bound weakly. Possible explanations for the differences between the two studies are discussed. We have shown that unphosphorylated heavy meromyosin appears to adopt a special state in the presence of ADP based upon analysis of actin-heavy meromyosin association rate constants. Data were consistent with one head binding rapidly and the second head binding more slowly in the presence of ADP. Both heads bound to actin at the same rate for all other states.     

Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles.

The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase.     

Thermodynamics of the hydrolysis of adenosine 5'-triphosphate to adenosine 5'-diphosphate

The enthalpy of hydrolysis of the enzyme-catalyzed (heavy meromyosin) conversion of adenosine 5'-triphosphate (ATP) to adenosine 5'-diphosphate (ADP) and inorganic phosphate has been investigated using heat-conduction microcalorimetry. Enthalpies of reaction were measured as a function of ionic strength (0.05-0.66 mol kg-1), pH (6.4-8.8), and temperature (25-37 degrees C) in Tris/HCl buffer. The measured enthalpies were adjusted for the effects of proton ionization and metal ion binding, protonation and interaction with the Tris buffer, and ionic strength effects to obtain a value of delta H0 = -20.5 +/- 0.4 kJ mol-1 at 25 degrees C for the process, ATP4-(aq) + H2O(l) = ADP3-(aq) + HPO2-4(aq) + H+(aq) where aq is aqueous and l is liquid. Heat measurements carried out at different temperatures lead to a value of delta C0p = -237 +/- 30 J mol-1 K-1 for the above process.     

Magnesium ion dependent adenosine triphosphatase activity of heavy meromyosin as a function of temperature between +20 and -15 degrees C.

The hydrolysis of Mg2+-adenosine 5'-triphosphate (ATP) by heavy meromyosin has been studied between +20 and -15 degrees C, especially in the low-temperature range, in a medium containing 30% (v/v) ethylene glycol by fluorometric, spectrophotometric, and potentiometric measurements. The time course of the fluorescence changes of the enzyme during the reaction depends markedly on the temperature in consequence of large differences between the activation energies of the various steps. The observed kinetics have been analyzed according to the simplified scheme of Bagshaw & Trentham [Bagshaw, C. R., & Trentham, D. R. (1974) Biochem. J. 141, 331-349]. The following results have been obtained. (1) The rate-limiting step of the reaction changes in this temperature range; at 20 degrees C M**.ADP.Pi is the predominant steady-state complex, and M*.ADP predominates at -15 degrees C, with a half-life of approximately 10 min. (2) As expected, on the basis that it is the dissociation of the M*.ADP complex which becomes rate limiting at low temperature, one observes, in the pre-steady-state below 0 degrees C, both a proton burst and a lag phase in ADP release. (3) At low temperature, the equilibrium M*.ATP in equilibrium M**.ADP.Pi is displaced to the left All the kinetic data obtained in this study are compatible with a simple pathway for the Mg2+-ATP hydrolysis by myosin and with sequential release of the reaction products.     

Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin.

Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.     

The enzymic properties of a modified ox heart myosin adenosine triphosphatase on covalent binding to an insoluble cellulose matrix

The preparation of ox heart myosin and its partial digestion with cellulose-bound papain is described. A procedure is outlined by which heavy meromyosin subfragment 1 can be covalently bound to a cellulose ion-exchange matrix. Attachment of heavy meromyosin subfragment 1 to the insoluble matrix results in a change in the ion specificity towards ATP hydrolysis. Unlike the soluble enzyme the bound form is activated by both Ca(2+) and Mg(2+). Maximal activation by Ca(2+) occurred at a lower concentration for the bound enzyme. Mg(2+) activates at a concentration which causes near-maximal inhibition of the Ca(2+)-activated adenosine triphosphatase (ATPase) of the non-bound enzyme. The Mg(2+)-activated ATPase of the bound enzyme was in turn inhibited by the presence of Ca(2+). The activation by Mg(2+) resembles the characteristic enzymic action of the actin-subfragment 1 complex.     

The mechanism of smooth muscle caldesmon-tropomyosin inhibition of the elementary steps of the actomyosin ATPase

Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%. Inhibitory caldesmon concentrations had little effect upon the rate of S1 binding to actin, actin-S1 dissociation by ATP, and dissociation of ADP from actin-S1 x ADP; however the rate of phosphate release from the actin-S1 x ADP x P(i) complex was decreased by more than 80%. In addition the transient of phosphate release displayed a lag of up to 200 ms. The presence of a lag phase indicates that a step on the pathway prior to phosphate release has become rate-limiting. Premixing the actin-tropomyosin filaments with myosin heads resulted in the disappearance of the lag phase. We conclude that caldesmon inhibition of the rate of phosphate release is caused by the thin filament being switched by caldesmon to an inactive state. The active and inactive states correspond to the open and closed states observed in skeletal muscle thin filaments with no evidence for the existence of a third, blocked state. Taken together these data suggest that at physiological concentrations, caldesmon controls the isomerization of the weak binding complex to the strong binding complex, and this causes the inhibition of the rate of phosphate release. This inhibition is sufficient to account for the inhibition of the steady state actomyosin ATPase by caldesmon and tropomyosin.     

The covalent modification of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate. Reaction at a binding site other than the active site.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfide bonds between the 6 thiol group on the purine ring and certain key cysteines on myosin, heavy meromyosin, and subfragment one. The EDTA ATPase activities of myosin and heavy meromyosin were completely inactivated when 4 mol of thiopurine nucleotide was bound. When similarly inactivated, subfragment one, depending on its method of preparation, incorporated either 1 or 2 mol of thiopurine nucleotide. Modification of a single cysteine on subfragment one resulted in an inhibition of both the Ca2+ and the EDTA ATPase activities, but the latter always to a greater extent. Modification of two cysteines per head of heavy meromyosin had the same effect suggesting that the active sites were not blocked by the thiopurine nucleotides. Direct evidence for this suggestion was provided by equilibrium dialysis experiments. Heavy meromyosin and subfragment one bound 1.9 and 0.8 mol of [8-3H]adenylyl imidodiphosphate per mol of enzyme, respectively, with an average dissociation constant of 5 X 10(-7) M. Heavy meromyosin with four thiopurine nucleotides bound or subfragment one with two thiopurine nucleotides bound retained 65-80% of these tight adenylyl imidodiphosphate binding sites confirming the above suggestion. Thus previous work assuming reaction of thiopurine nucleotide analogs at the active site of myosin must be reevaluated. Ultracentrifugation studies showed that heavy meromyosin which had incorporated four thiopurine nucleotides did not bind to F-actin while subfragment one with one thiopurine nucleotide bound interacted only very weakly with F-actin. Thus reaction of 6,6'-dithiobis(inosinyl imidodiphosphate) at nucleotide binding sites other than the active sites reduces the rate of ATP hydrolysis and inhibits actin binding. It is suggested that these second sites may function as regulatory sites on myosin.     

Pre-steady state kinetic studies on H(+)-ATPase from Candida albicans.

The mechanism of ATP hydrolysis by plasma membrane H(+)-ATPase from Candida albicans has been investigated by following the kinetics of H(+) liberation/absorption and the UV difference spectrum in a stopped flow spectrophotometer. A distinct pre-steady state phase of ATP hydrolysis could be defined. While the rapid mixing of P(i) and ATPase produced no transient pH changes, the mixing of ADP leads to the release of 1 H(+) per molecule of ATPase. Rapid mixing of ATP with ATPase releases about 2 H(+) per molecule of ATPase, of which around 1.3 H(+) are reabsorbed. The magnitudes of both H(+) release and absorption were found to be independent of ATP concentration. The rate of H(+) release (k(f)) shows ATP dependence while the rate of H(+) absorption is independent of ATP concentration. The rate of H(+) liberation with ADP, on a concentration basis, was far less as compared with ATP, indicating a low affinity of the ATPase for ADP. No change in the difference spectrum was observed with ADP. The stoichiometry of ATP binding to PM-ATPase was found to be unity from UV-difference spectrum studies. The k(f) values for H(+) release and for the appearance of a difference spectrum following the addition of ATP were found to be similar beyond a 1:1 ratio of ATP:ATPase. The results obtained lead us to propose a 4-step kinetic scheme for the mechanism of ATP hydrolysis.