Fluorescent coumarin-labeled nucleotides to measure ADP release from actomyosin

Several coumarin-labeled nucleotides have been synthesized, based on 2'(3')-O-(2-aminoethyl)carbamoyl-ATP (edaATP). The fluorescent coumarins coupled with the free amino group are 7-diethylaminocoumarin-3-carboxylic acid (to give deac-edaATP), coumarin 343 (but-edaATP) and 7-ethylamino-8-bromocoumarin-3-carboxylic acid (mbc-edaATP). The carbamoyl linkage of these nucleotide analogs undergoes interconversion between 2'- and 3'-hydroxyl attachment very slowly, so that the 2'- and 3'-isomers were separated and stored with minimal equilibration. 3'-Deac-edaADP had fluorescence excitation and emission maxima at 430 nm and 477 nm, with a fluorescence quantum yield of 0.012. The equivalent data for 3'-but-edaADP are 445 nm, 494 nm, and 0.51, respectively, and for 3'-mbc-edaADP, 405 nm, 464 nm, and 0.62. The interaction with skeletal myosin subfragment 1 was measured in the absence and presence of actin. In each case the fluorescence was decreased when bound to subfragment 1, 3-fold for 3'-deac-edaADP, 7-fold for 3'-but-edaADP, and 11-fold for 3'-mbc-edaADP. Steady-state ATPase measurements and the kinetics of binding and release of nucleotides were similar to those reported for the natural nucleotide. Large fluorescence changes could be observed for the release of these analogs from actomyosin subfragment 1, enabling a direct measurement of the kinetics of this process. In the case of 3'-deac-edaADP a rate constant of 474 s(-1) was measured (at pH 7.0, 20 degrees C, and low ionic strength).     

Intrinsic tryptophan fluorescence identifies specific conformational changes at the actomyosin interface upon actin binding and ADP release.

The helix-loop-helix (A-site) and myopathy loop (R-site) are located on opposite sides of the cleft that separates the proposed actin-binding interface of myosin. To investigate the structural features of the A- and R-sites, we engineered two mutants of the smooth muscle myosin motor domain with the essential light chain (MDE), containing a single tryptophan located either in the A-site (W546-MDE) or in the R-site (V413W MDE). W546- and V413W-MDE display actin-activated ATPase and actin-binding properties similar to those of wild-type MDE. The steady-state fluorescence properties of W546-MDE [emission peak (lambda(max)) = 344, quantum yield = 0.20, and acrylamide bimolecular quenching constant (k(q)) = 6.4 M(-)(1). ns(-)(1)] and V413W-MDE [lambda(max) = 338, quantum yield = 0.27, and k(q) = 3.6 M(-)(1).ns(-)(1)] demonstrate that Trp-546 and Trp-413 are nearly fully exposed to solvent, in agreement with the crystallographic data on these residues. In the presence of actin, Trp-546 shifts to a more buried environment in both the ADP-bound and nucleotide-free (rigor) actomyosin complexes, as indicated by an average lambda(max) of 337 or 336 nm, respectively, and protection from dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) oxidation. In contrast, Trp-413 has a single conformation with an average lambda(max) of 338 nm in the ADP-bound complex, but in the rigor complex it is 50% more accessible to DHNBS oxidation and can adopt a range of possible conformations (lambda(max) = 341-347 nm). Our results suggest a structural model in which the A-site remains tightly bound to actin and the R-site adopts a more flexible and solvent-exposed conformation upon ADP release.     

Kinetic and spectroscopic evidence for three actomyosin:ADP states in smooth muscle

Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin II. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to an identifiable state change. These studies indicate that for this myosin:ADP, both the catalytic site and the actin-binding site can each assume one of two conformations. Relatively loose coupling between these two binding sites leads to three discrete actin-associated ADP states. Following an initial, weakly bound state, binding of myosin:ADP to actin shifts the equilibrium toward a mixture of two states that each bind actin strongly but differ in the conformation of their catalytic sites. By contrast, fast myosins, including Dictyostelium myosin II, have reciprocal coupling between the actin- and ADP-binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly bound actomyosin ADP state in smooth muscle, would prolong the fraction of the ATPase cycle time that this actomyosin spends in a force-generating conformation and may be central to explaining the physiologic differences between this and other myosins.     

Myosin cleft movement and its coupling to actomyosin dissociation

It has long been known that binding of actin and binding of nucleotides to myosin are antagonistic, an observation that led to the biochemical basis for the crossbridge cycle of muscle contraction. Thus ATP binding to actomyosin causes actin dissociation, whereas actin binding to the myosin accelerates ADP and phosphate release. Structural studies have indicated that communication between the actin- and nucleotide-binding sites involves the opening and closing of the cleft between the upper and lower 50K domains of the myosin head. Here we test the proposal that the cleft responds to actin and nucleotide binding in a reciprocal manner and show that cleft movement is coupled to actin binding and dissociation. We monitored cleft movement using pyrene excimer fluorescence from probes engineered across the cleft.     

Optimal timing of cleft palate closure

Treatment objectives for the cleft palate patient--normal speech, normal maxillofacial growth, and normal hearing--are closely related. Controversy about the timing of cleft palate surgery is directed at the need for early palatoplasty for improved speech and hearing versus delayed hard palate repair for undisturbed facial growth. This controversy as to the value of early versus delayed closure continues into the present. The authors present an updated argument regarding this controversy along with a comprehensive literature review. They also present a logical algorithm based on the literature and their personal experience.     

Role of ADP in the control of actomyosin superprecipitation and ATPase activity.

ATP, in the presence of 0.05–0.15 m KCl and greater than 50 μm Mg²⁺, induces dissociation (clearing) followed by superprecipitation of skeletal muscle actomyosin. Superprecipitation has been studied as a model of muscle contraction, and ATP depletion has been associated with the onset of superprecipitation. Recent studies [Puszkin and Rubin (1975) Science188, 1319–1320] indicate that ADP stimulates superprecipitation without increasing the rate of ATP hydrolysis. We confirm that ADP stimulates superprecipitation; however, contrary to the experience of these investigators, ADP does stimulate ATP hydrolysis in the system studied here. We present evidence that superprecipitation is associated with generation of a critical ADP:ATP ratio but it appears that this ratio is an indirect measure of an associated but uncharacterized phenomenon which signals the onset of superprecipitation. Added ADP decreased the extent and duration of clearing, increased the rate of ATP hydrolysis, and increased the extent of superprecipitation of rat skeletal muscle actomyosin in the presence of excess Mg²⁺. The ADP effect was not mimicked by EDTA or AMP. The duration of clearing was related not to the time required to attain a specific level of any nucleotide phosphate, but to the time required to generate an ADP:ATP ratio of approximately 3.6. Apparently only that ADP generated in the system by ATP hydrolysis was involved in the critical ADP:ATP ratio. Added ADP stimulated myosin ATPase activity in 1.6 or 3.2 mm Mg²⁺. This effect was not mimicked by EDTA or AMP. The results are used to relate studies by others of myosin sulfhydryl modification to a recent model [Burke et al. (1973) Proc. Nat. Acad. Sci. USA70, 3793–3796] in which myosin MgATPase activity is inhibited by formation of a stable cyclic complex of MgATP and the S₁ and S₂ sites of heavy meromyosin.     

Microtubules accelerate ADP release by dynein

The effects of microtubules on the phosphate-water oxygen exchange reactions catalyzed by dynein were examined in order to determine the mechanism by which microtubules activate the ATPase. Microtubules inhibited the rate of medium exchange observed during net ATP hydrolysis. Inhibition of the exchange reaction was proportional to the extent of microtubule activation of ATP turnover with no effect on the partition coefficient. These data argue that microtubules do not increase the rate of release of phosphate from dynein; rather, they increase the rate of ADP release. Microtubules markedly inhibited medium phosphate-water exchange reactions observed in the presence of ADP and Pi. With increasing concentrations of ADP, the rate of exchange increased in parallel to the dissociation of dynein from the microtubules, suggesting that only free dynein and not the microtubule-dynein complex catalyzes the exchange reaction. The rates of dynein binding to microtubules in the absence and presence of saturating ADP were 1.6 X 10(6) and 9.8 X 10(5) M-1 s-1, respectively. ADP inhibited the rate of the ATP-induced dissociation of the microtubule-dynein complex with an apparent Kd = 0.37 mM for the binding of ADP to the microtubule-dynein complex. However, the rate of dissociation of ADP from the M.D.ADP complex was quite fast (approximately 1000 s-1). These data support the postulate of a high-energy dynein-ADP intermediate and indicate that microtubules activate the dynein ATPase by enhancing the rate of ADP release.     

Evidence for the dependence of arterial haemostasis on ADP

The possible involvement of adenosine diphosphate (ADP) in haemostatic platelet aggregation was investigated by determining the duration of primary haemorrhage as standardized bleeding times from punctures of small mesenteric arteries in anaesthetized rats. The bleeding times were highly significantly increased by infusing into the mesenteric arterial blood flowing towards the punctures either the nucleotide-dephosphorylating enzyme apyrase or the ADP-receptor antagonists ATP, adenosine 5'-(beta,gamma-methylene)triphosphonate (AMP-PCP) or 2-methylthioadenosine 5'-(beta,gamma-methylene)triphosphonate (2-MeS-AMP-PCP). The increases in bleeding times could not be accounted for by local vasodilator effects of the agents. It is concluded that the presence of ADP through local release and/or formation at sites of vascular injury contributes significantly to haemostasis, presumably by accelerating platelet aggregation.     

Correlation between steady-state ATP hydrolysis and vanadate-induced ADP trapping in Human P-glycoprotein. Evidence for ADP release as the rate-limiting step in the catalytic cycle and its modulation by substrates

P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resistance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis. The objective of this study was to understand how substrates affect the catalytic cycle of ATP hydrolysis by Pgp. The ATPase activity of purified and reconstituted recombinant human Pgp was measured using a continuous cycling assay. Pgp hydrolyzes ATP in the absence of drug at a basal rate of 0.5 micromol x min x mg(-1) with a K(m) for ATP of 0.33 mm. This basal rate can be either increased or decreased depending on the Pgp substrate used, without an effect on the K(m) for ATP or 8-azidoATP and K(i) for ADP, suggesting that substrates do not affect nucleotide binding to Pgp. Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal rate, they do not completely inhibit the activity. Therefore, these drugs can be classified as substrates. Vanadate (Vi)-induced trapping of [alpha-(32)P]8-azidoADP was used to probe the effect of substrates on the transition state of the ATP hydrolysis reaction. The K(m) for [alpha-(32)P]8-azidoATP (20 microm) is decreased in the presence of Vi; however, it is not changed by drugs such as verapamil or cyclosporin A. Strikingly, the extent of Vi-induced [alpha-(32)P]8-azidoADP trapping correlates directly with the fold stimulation of ATPase activity at steady state. Furthermore, P(i) exhibits very low affinity for Pgp (K(i) approximately 30 mm for Vi-induced 8-azidoADP trapping). In aggregate, these data demonstrate that the release of Vi trapped [alpha-(32)P]8-azidoADP from Pgp is the rate-limiting step in the steady-state reaction. We suggest that substrates modulate the rate of ATPase activity of Pgp by controlling the rate of dissociation of ADP following ATP hydrolysis and that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp.     

Coupling between phosphate release and force generation in muscle actomyosin

Energetic, kinetic and oxygen exchange experiments in the mid-1980s and early 1990s suggested that phosphate (Pi) release from actomyosin-adenosine diphosphate Pi (AM.ADP.Pi) in muscle fibres is linked to force generation and that Pi release is reversible. The transition leading to the force-generating state and subsequent Pi release were hypothesized to be separate, but closely linked steps. Pi shortens single force-generating actomyosin interactions in an isometric optical clamp only if the conditions enable them to last 20-40 ms, enough time for Pi to dissociate. Until 2003, the available crystal forms of myosin suggested a rigid coupling between movement of switch II and tilting of the lever arm to generate force, but they did not explain the reciprocal affinity myosin has for actin and nucleotides. Newer crystal forms and other structural data suggest that closing of the actin-binding cleft opens switch I (presumably decreasing nucleotide affinity). These data are all consistent with the order of events suggested before: myosin.ADP.Pi binds weakly, then strongly to actin, generating force. Then Pi dissociates, possibly further increasing force or sliding.     

Evidence against essential roles for subdomain 1 of actin in actomyosin sliding movements

We have engineered acto-S1chimera proteins carrying the entire actin inserted in loop 2 of the motor domain of Dictyostelium myosin II with 24 or 18 residue-linkers (CP24 and CP18, respectively). These proteins were capable of self-polymerization as well as copolymerization with skeletal actin and exhibited rigor-like structures. The MgATPase rate of CP24-skeletal actin copolymer was 1.06 s(-1), which is slightly less than the V(max) of Dictyostelium S1. Homopolymer filaments of skeletal actin, CP24, and CP18 moved at 4.7+/-0.6, 2.9+/-0.6, and 4.1+/-0.8 microm/s (mean+/-SD), respectively, on coverslips coated with skeletal myosin at 27 degrees C. Statistically thermodynamic considerations suggest that the S1 portion of chimera protein mostly resides on subdomain 1 (SD-1) of the actin portion even in the presence of ATP. This and the fact that filaments of CP18 with shorter linkers moved faster than CP24 filaments suggest that SD-1 might not be as essential as conventionally presumed for actomyosin sliding interactions.     

Evidence for a Complex between Myosin and ADP in Relaxed Muscle Fibres

TAYLOR and his co-workers have shown that myosin, when mixed with its substrate ATP, rapidly forms a stoichiometric complex in which the terminal phosphate bond of the ATP is already broken¹; this complex is relatively stable and lasts for nearly all of the time between successive ATP hydrolyses². The aim of the present work was to find out whether such a complex is formed in relaxed muscle.     

Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes.

BACKGROUND: Both single cells and multicellular systems rapidly heal physical insults but are thought to do so by distinctly different mechanisms. Wounds in single cells heal by calcium-dependent membrane fusion, whereas multicellular wounds heal by a variety of different mechanisms, including circumferential contraction of an actomyosin 'purse string' that assembles around wound borders and is dependent upon the small GTPase Rho. RESULTS: We investigated healing of puncture wounds made in Xenopus oocytes, a single-cell system. Oocyte wounds rapidly assumed a circular morphology and constricted circumferentially, coincident with the recruitment of filamentous actin (F-actin) and myosin-II to the wound borders. Surprisingly, recruitment of myosin-II to wound borders occurred before that of F-actin. Further, experimental disruption of F-actin prevented healing but did not prevent myosin-II recruitment. Actomyosin purse-string assembly and closure was dependent on Rho GTPases and extracellular calcium. Wounding resulted in reorganization of microtubules into an array similar to that which forms during cytokinesis in Xenopus embryos. Experimental perturbation of oocyte microtubules before wounding inhibited actomyosin recruitment and wound closure, whereas depolymerization of microtubules after wounding accelerated wound closure. CONCLUSIONS: We conclude the following: actomyosin purse strings can close single-cell wounds; myosin-II is recruited to wound borders independently of F-actin; purse-string assembly is dependent on a Rho GTPase; and purse-string assembly and closure are controlled by microtubules. More generally, the results indicate that actomyosin purse strings have been co-opted through evolution to dispatch a broad variety of single-cell and multicellular processes, including wound healing, cytokinesis and morphogenesis.