Myosin subfragment-1 interacts with two G-actin molecules in the absence of ATP

The interaction between G-actin and myosin subfragment-1 (S1) has been monitored by pyrenyl-actin fluorescence and light scattering. In low ionic strength buffer and in the absence of ATP the polymerization of G-actin induced by myosin subfragment-1 is preceded by the formation of binary GS and ternary G2S complexes in which S1 interacts tightly in rapid equilibrium (K greater than 10(7) M-1) with one and two G-actin molecules, respectively. Pyrenyl fluorescence of G-actin is enhanced 4-fold in GS and 3-fold in G2S. At concentrations of G-actin and S1 in the micromolar range and above, G2S is the predominant species at G-actin/S1 ratios equal to or greater than 1. The isomer of myosin subfragment-1 carrying the A1 light chain, S1(A1), forms a tighter ternary complex than the isomer S1(A2). Actin-bound ATP is not hydrolyzed upon formation of GS and G2S. In the presence of one molar equivalent or more of myosin subfragment-1/mol of G-actin, in low ionic strength buffer containing no nucleotides, G-actin polymerizes faster in the presence of S1(A1) than in the presence of S1(A2). The interaction of S1 with G-actin is inhibited by the binding of ATP or ADP to S1, ATP having a higher affinity for S1 than ADP. The possible structural similarity of the G2S complex to the F-acto-S1 complex in the rigor state and the potential significance of a ternary (actin)2-myosin interaction for actomyosin-based motility are discussed.     

Transmission of ADP.vanadate-induced conformational changes to three peptide segments of myosin subfragment-1

In order to study the conformational changes associated with formation of the stable ternary complex of myosin subfragment-1 (S-1) with ADP and orthovanadate (Vi), S-1 was fluorescently labeled with 9-anthroylnitrile, 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole, and 5-(iodoacetamido) fluorescein at the 23-, 50-, and 20-kDa peptide segments of S-1, respectively (Hiratsuka, T. (1989) J. Biol. Chem. 264, 18188-18194; Hiratsuka, T. (1986) J. Biol. Chem. 261, 7294-7299; Takashi, R. (1979) Biochemistry 18, 5164-5169). The extrinsic fluorescence of these S-1 derivatives was sensitive not only to binding of ADP but to formation of the stable ternary complex with ADP and Vi. By using these fluorescent properties, the kinetics of formation of the stable ternary complexes of these S-1 derivatives with ADP and Vi, M. ADP.Vi, were analyzed according to the scheme proposed by Goodno (Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2620-2624). [Formula; see text] The values obtained for KVi )0.2-0.4 mM) and k (0.03-0.05 s-1) of these S-1 derivatives were similar regardless of the peptide segments of S-1 where the fluorophore had been covalently labeled. These results suggest that the conformational changes, which are induced by formation of the stable ternary complex of S-1 with ADP and Vi, are transmitted to all three peptide segments of S-1 at a similar rate. The present results also encourage us to confirm that the ATPase site of S-1 resides at or near the region where all three peptide segments of S-1 are contiguous.     

X-ray structures of the Dictyostelium discoideum myosin motor domain with six non-nucleotide analogs

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with dinitrophenylaminoethyl-, dinitrophenylaminopropyl-, o-nitrophenylaminoethyl-, m-nitrophenylaminoethyl-, p-nitrophenylaminoethyl-, and o-nitrophenyl-N-methyl-aminoethyl-diphosphate.beryllium fluoride have been determined to better than 2.3-A resolution. The structure of the protein and nucleotide binding pocket in these complexes is very similar to that of S1dC.ADP.BeF(x) (Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960-8972). The position of the triphosphate-like moiety is essentially identical in all complexes. Furthermore, the alkyl-amino group plays the same role as the ribose by linking the triphosphate to the adenine binding pocket; however, none of the phenyl groups lie in the same position as adenine in S1dC.MgADP.BeF(x), even though several of these nucleotide analogs are functionally equivalent to ATP. Rather the former location of adenine is occupied by water in the nanolog complexes, and the phenyl groups are organized in a manner that attempts to optimize their hydrogen bonding interactions with this constellation of solvent molecules. A comparison of the kinetic and structural properties of the nanologs relative to ATP suggests that the ability of a substrate to sustain tension and to generate movement correlates with a well defined interaction with the active site water structure observed in S1dC.MgADP.BeF(x).     

Formation and characterization of kinesin.ADP.fluorometal complexes

Recent crystallographic studies of motor proteins showed that the structure of the motor domains of myosin and kinesin are highly conserved. Thus, these motor proteins, which are important for motility, may share a common mechanism for generating energy from ATP hydrolysis. We have previously demonstrated that, in the presence of ADP, myosin forms stable ternary complexes with new phosphate analogues of aluminum fluoride (AlF(4)(-)) and beryllium fluoride (BeF(n)), and these stable complexes mimic the transient state along the ATPase kinetic pathway [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. In this study, we examined the formation of kinesin.ADP.fluorometals ternary complexes and analyzed their characteristics using the fluorescent ATP analogue NBD-ATP (2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl]-ADP). Our results suggest that these ternary complexes may mimic transient state intermediates in the kinesin ATPase cycle. Thus, the kinesin.ADP.AlF(4)(-) complex resembles the kinesin.ADP state, and the kinesin.ADP.BeF(n) complex mimics the kinesin.ADP.P(i) state.     

Interaction of myosin subfragment 1 with forms of monomeric actin

The ability of myosin subfragment 1 to interact with monomeric actin complexed to sequestering proteins was tested by a number of different techniques such as affinity absorption, chemical cross-linking, fluorescence titration, and competition procedures. For affinity absorption, actin was attached to agarose immobilized DNase I. Both chymotryptic subfragment 1 isoforms (S1A1 and S1A2) were retained by this affinity matrix. Fluorescence titration employing pyrenyl-actin in complex with deoxyribonuclease I (DNase I) or thymosin beta4 demonstrated S1 binding to these actin complexes. A K(D) of 5 x 10(-8) M for S1A1 binding to the actin-DNase I complex was determined. Fluorescence titration did not indicate binding of S1 to actin in complex with gelsolin segment 1 (G1) or vitamin D-binding protein (DBP). However, fluorescence competition experiments and analysis of tryptic cleavage patterns of S1 indicated its interaction with actin in complex with DBP or G1. Formation of the ternary DNase I-acto-S1 complex was directly demonstrated by sucrose density sedimentation. S1 binding to G-actin was found to be sensitive to ATP and an increase in ionic strength. Actin fixed in its monomeric state by DNase I was unable to significantly stimulate the Mg2+-dependent S1-ATPase activity. Both wild-type and a mutant of Dictyostelium discoideum myosin II subfragment 1 containing 12 additional lysine residues within an insertion of 20 residues into loop 2 (K12/20-Q532E) were found to also interact with actin-DNase I complex. Binding of the K12/20-Q532E mutant to the actin-DNase I complex occurred with higher affinity than wild-type S1 and was less sensitive to mono- and divalent cations.     

Interaction of myosin.ADP.fluorometal complexes with fluorescent probes and direct observation using quick-freeze deep-etch electron microscopy

Myosin forms stable ternary complexes with ADP and phosphate analogues of fluorometals that mimic different ATPase reaction intermediates corresponding to each step of the cross-bridge cycle. In the present study, we monitored the formation of ternary complexes of myosin.ADP.fluorometal using the fluorescence probe prodan. It has been reported that the fluorescence changes of the probe reflect the formation of intermediates in the ATPase reaction [Hiratsuka (1998) Biochemistry 37, 7167-7176]. Prodan bound to skeletal muscle heavy-mero-myosin (HMM).ADP.fluorometal, with each complex showing different fluorescence spectra. Prodan bound to the HMM.ADP.BeFn complex showed a slightly smaller red-shift than other complexes in the presence of ATP, suggesting a difference in the localized conformation or a difference in the population of BeFn species of global shape. We also examined directly the global structure of the HMM.ADP.fluorometal complexes using quick-freeze deep-etch replica electron microscopy. The HMM heads in the absence of nucleotides were mostly straight and elongated. In contrast, the HMM heads of ternary complexes showed sharply kinked or rounded configurations as seen in the presence of ATP. This is the first report of the direct observation of myosin-ADP-fluorometal ternary complexes, and the results suggest that these complexes indeed mimic the shape of the myosin head during ATP hydrolysis.     

Interaction of a new fluorescent ATP analogue with skeletal muscle myosin subfragment-1

A new fluorescent ribose-modified ATP analogue, 2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic]-ATP (NBD-ATP), was synthesized and its interaction with skeletal muscle myosin subfragment-1 (S-1) was studied. NBD-ATP was hydrolysed by S-1 at a rate and with divalent cation-dependence similar to those in the case of regular ATP. Skeletal HMM supported actin translocation using NBD-ATP and the velocity was slightly higher than that in the case of regular ATP. The addition of S1 to NBD-ATP resulted in quenching of NBD fluorescence. Recovery of the fluorescence intensity was noted after complete hydrolysis of NBD-ATP to NBD-ADP. The quenching of NBD-ATP fluorescence was accompanied by enhancement of intrinsic tryptophan fluorescence. These results suggested that the quenching of NBD-ATP fluorescence reflected the formation of transient states of ATPase. The formation of S-1.NBD-ADP.BeF(n) and S-1.NBD-ADP.AlF(4)(-) complexes was monitored by following changes in NBD fluorescence. The time-course of the formation fitted an exponential profile yielding rate constants of 7.38 x 10(-2) s(-1) for BeF(n) and 1.1 x 10(-3) s(-1) for AlF(4)(-). These values were similar to those estimated from the intrinsic fluorescence enhancement of trp due to the formation of S-1.ADP.BeF(n) or AlF(4)(-) reported previously by our group. Our novel ATP analogue seems to be applicable to kinetic studies on myosin.     

Two-headed binding of the unphosphorylated nonmuscle heavy meromyosin.ADP complex to actin

The enzymatic and motor function of smooth muscle and nonmuscle myosin II is activated by phosphorylation of the regulatory light chains located in the head portion of myosin. Dimerization of the heads, which is brought about by the coiled-coil tail region, is essential for regulation since single-headed fragments are active regardless of the state of phosphorylation. Utilizing the fluorescence signal on binding of myosin to pyrene-labeled actin filaments, we investigated the interplay of actin and nucleotide binding to thiophosphorylated and unphosphorylated recombinant nonmuscle IIA heavy meromyosin constructs. We show that both heads of either thiophosphorylated or unphosphorylated heavy meromyosin bind very strongly to actin (K(d) < 10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (K(d) around 1 microM) when bound to actin. These findings are in line with the previously observed unusually loose coupling between nucleotide and actin binding to nonmuscle myosin IIA subfragment-1 (Kovács et al. (2003) J. Biol. Chem. 278, 38132.). Furthermore, they imply that the structure of the two heads in the ternary actomyosin-ADP complex is symmetrical and that the asymmetrical structure observed in the presence of ATP and the absence of actin in previous investigations (Wendt et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361) is likely to represent an ATPase intermediate that precedes the actomyosin-ADP state.     

The effects of substrate concentration on the Mg-adenosine triphosphatase activity of myosin.

Using myosin, heavy meromyosin, and subfragment-1 the steady state rate of Mg-modified adenosine triphosphatase (Mg-ATPase) was determined over a range of substrate concentrations between 10(-8) M and 5 X 10(-3)M, at 0.5 M and 0.05 M KC1 (pH 7.4 at 20 degrees C). At the substrate concentrations below 10(-5) M, myosin Mg-ATPase was observed to show that two active sites interact, as suggested by the analysis of transient kinetic studies (Walz, F. G., Jr.: J. Theor. Biol. 41, 357-373 (1973)). The increase in the activity at Mg-ATP concentrations higher than 10(-4) M corresponds to the binding of Mg-ATP to myosin sites not responsible for the catalytic action. With heavy meromyosin and subfragment-1, the activity was best expressed by the Michaelis equation. With heavy meromyonsin, the activation at high ATP concentrations is detectable, though not as pronounced as with myosin, but not with subfragment-1.     

Fluorescence energey transfer in myosin subfragment-1

Fluorescent probes have been selectively introduced into skeletal muscle myosin subfragment-1 and the fluorescence emission characteristics of the labeled products studied. The fluorophores employed were the thiol-specific reagents N-[[(iodoacetyl)aminolethyl-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, the spectral properties of which render them a particularly effective donor-acceptor pair in F?rster energy-transfer studies. Alkali 1 light chain, labeled at a single cysteine with either of these probes, was incorporated into chymotryptic subfragment-1 by the exchange procedure of Wagner & Weeds [Wagner, P.D., & Weeds, A.G. (1977) J. Mol. Biol. 109, 455-473]. The resultant, fluorescently labeled subfragment-1 was isolated by ion-exchange chromatography. Determination of the extent of incorporation by extinction and fluorescence indicated that greater than 80% of the subfragment-1 population possessed a fluorescently labeled alkali 1 light chain. The introduction of labeled alkali 1 did not perturb the K+-, Ca2+-, or actin-activated adenosine triphosphatases of subfragment-1. The addition of adenosine triphosphate (ATP), liganded by various cations, to this singly labeled subfragment-1 induced a 6-10% decrease in the fluorescence intensity of the extrinsic chromophore. An intensity decrease of approximately 4% was obtained when the hydrolysis of ATP was complete, and also upon direct addition of adenosine diphosphate. The ATP analogue adenylyl imidodiphosphate induced a decrease of approximately 7% in intensity. The addition of F-actin to the subfragment-1 in the presence of MgATP elicited no further fluorescence intensity change. A second, appropriate fluorophore was introduced into the singly labeled subfragment-1 at the SH1 thiol on the heavy chain. F?rster energy transfer was observed between this labeled site and the fluorophore previously introduced on the alkali 1 light chain. The measured efficiency of energy transfer indicated that the two fluorophores were approximately 40 A apart. The same value was obtained upon reversal of the donor and acceptor attachment sites, suggesting that the uncertainty in the calculated distance introduced by the choice of orientation factor is probably less than 20%. Steady-state observations did not reveal any obvious change in this distance upon the addition of MgATP and then F-actin to the doubly labeled subfragment-1.     

The 2'-O- and 3'-O-Cy3-EDA-ATP(ADP) complexes with myosin subfragment-1 are spectroscopically distinct

Ribose-modified highly-fluorescent sulfoindocyanine ATP and ADP analogs, 2'(3')-O-Cy3-EDA-AT(D)P, with kinetics similar to AT(D)P, enable myosin and actomyosin ATPase enzymology with single substrate molecules. Stopped-flow studies recording both fluorescence and anisotropy during binding to skeletal muscle myosin subfragment-1 (S1) and subsequent single-turnover decay of steady-state intermediates showed that on complex formation, 2'-O- isomer fluorescence quenched by 5%, anisotropy increased from 0.208 to 0.357, and then decayed with turnover rate k(cat) 0.07 s(-1); however, 3'-O- isomer fluorescence increased 77%, and anisotropy from 0.202 to 0.389, but k(cat) was 0.03 s(-1). Cy3-EDA-ADP.S1 complexes with vanadate (V(i)) were studied kinetically and by time-resolved fluorometry as stable analogs of the steady-state intermediates. Upon formation of the 3'-O-Cy3-EDA-ADP.S1.V(i) complex fluorescence doubled and anisotropy increased to 0.372; for the 2'-O- isomer, anisotropy increased to 0.343 but fluorescence only 6%. Average fluorescent lifetimes of 2'-O- and 3'-O-Cy3-EDA-ADP.S1.V(i) complexes, 0.9 and 1.85 ns, compare with approximately 0.7 ns for free analogs. Dynamic polarization shows rotational correlation times higher than 100 ns for both Cy3-EDA-ADP.S1.V(i) complexes, but the 2'-O-isomer only has also a 0.2-ns component. Thus, when bound, 3'-O-Cy3-EDA-ADP's fluorescence is twofold brighter with motion more restricted and turnover slower than the 2'-O-isomer; these data are relevant for applications of these analogs in single molecule studies.     

Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states

Fluorescence resonance energy transfer measurements have been made between Cys-374 on actin and Cys-177 on the alkali light chain of myosin subfragment 1 (S1) using several pairs of donor-acceptor chromophores. The labeled light chain was exchanged into subfragment 1 and the resulting fluorescently labeled subfragment 1 isolated by ion-exchange chromatography on SP-Trisacryl. The efficiency of energy transfer was measured by steady-state fluorescence in a strong binding complex of acto-S1 and found to represent a spatial separation between the two probes of 5.6-6.3 nm. The same measurements were then made with weak binding acto-S1 complexes generated in two ways. First, actin was complexed with p-phenylenedimaleimide-S1, a stable analogue of S1-adenosine 5'-triphosphate (ATP), obtained by cross-linking the SH1 and SH2 heavy-chain thiols of subfragment 1 [Greene, L. E., Chalovich, J. M., & Eisenberg, E. (1986) Biochemistry 25, 704-709]. Large increases in transfer efficiency indicated that the two probes had moved closer together by some 3 nm. Second, weak binding complexes were formed between subfragment 1 and actin in the presence of the regulatory proteins troponin and tropomyosin, the absence of calcium, and the presence of ATP [Chalovich, J. M., & Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437]. The measured efficiency of energy transfer again indicated that the distance between the two labeled sites had moved closer by about 3 nm. These data support the idea that there is a considerable difference in the structure of the acto-S1 complex between the weakly and strongly bound states.     

Photolabeling of the 6 and 10 S conformations of gizzard myosin with 3'(2')-O-(4-Benzoyl)benzoyl-ATP. Proline 324 is near the active site

3'(2')-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP) was used as a photoaffinity label of the ATP binding site of unphosphorylated chicken gizzard myosin. Specific photolabeling of the active site of 6 S myosin was assured by forming a stable myosin.Co(II)Bz2ADP.orthovanadate complex (termed trapping) prior to irradiation. Co2+ was used in place of Mg2+ to prevent the known photoreaction of vanadate with myosin which destabilizes the trapped complex. [3H] Bz2ADP.Pi was also stably trapped on gizzard myosin by forming the 10 S folded conformation of the protein in the presence of [3H]Bz2ATP and Mg2+. Irradiation of 6 S myosin containing orthovanadate trapped [3H] Bz2ADP or 10 S trapped [3H]Bz2ADP.Pi gave 32 and 30% covalent incorporation, respectively. The 50-kDa and precursor 68-kDa tryptic peptides of the subfragment-1 heavy chain derived from both forms of myosin were found to contain essentially all of the covalently attached [3H]Bz2ADP. Parallel experiments with untrapped [3H]Bz2ADP showed extensive nonspecific labeling of all of the major tryptic peptides and the light chains. Eight labeled peptides, isolated from 6 and 10 S photolabeled myosin, contained the sequence G319-H-V-P-I-X-A-Q326, where X corresponds to labeled proline 324. [14C]Bz2ADP was previously shown to label serine 324 in skeletal subfragment-1 (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995), which corresponds to alanine 325 in the gizzard sequence. Thus, this region of the 50-kDa tryptic fragment, near the nucleotide binding site, in both skeletal and smooth muscle myosins, must fold in essentially the same manner.     

Interaction of F-actin-AMPPNP with myosin subfragment-1 and troponin-tropomyosin: influence of an extra phosphate at the nucleotide binding site in F-actin on its function

An unsplitable analogue of ATP (adenylyl imidodiphosphate; AMPPNP) was incorporated into F-actin [Cooke, R. (1975) Biochemistry 14, 3250-3256]. The resulting polymers (F-actin-AMPPNP) activated the ATPase activity of myosin subfragment-1 (S1) as efficiently as normal F-actin; neither the maximum velocity at infinite actin concentration (Vmax) nor the affinity of actin to S1 in the presence of ATP (1/KATPase) changed, which indicates that the terminal phosphate of the bound nucleotide at the cleft region between the two domains of the actin molecule [Kabsch, W., Mannherz, H.G., & Suck, D. (1985) EMBO J. 4, 2113-2118] is not directly involved in a myosin binding site. However, the interaction of F-actin with troponin-tropomyosin was strongly modulated by the replacement of ADP with AMPPNP. The troponin-tropomyosin complex strongly enhanced the activation of S1-ATPase activity by F-actin-AMPPNP in the presence of Ca2+, although it has no effect on the activation by normal F-actin-ADP. KATPase was enhanced about threefold by troponin-tropomyosin in the presence of Ca2+, while Vmax was not markedly changed. F-actin-AMPPNP is highly potentiated by troponin-tropomyosin even with low S1 to actin ratios and at high ATP conditions. In the absence of Ca2+, the activation by F-actin-AMPPNP was inhibited normally by troponin-tropomyosin. The results suggest that the terminal beta-phosphate of the bound nucleotide in F-actin is located in a region which is important for regulation of the interaction with myosin.     

Comparative structural and enzymatic properties of skeletal muscle myosin from neonatal and adult rabbits

Several structural and enzymatic properties of myosin from skeletal muscles of neonatal and adult rabbits were compared. Electrophoretic analyses and proteolysis experiments indicated that differences between the two myosin types could be attributed to their heavy subunits. Circular dichroism measurements of subfragment-1 species, and trypsin-digested derivatives showed that the neonatal protein contained less alpha-helices than the adult form. The Mg2(+)-ATPase activity of neonatal myosin was lower than that of adult myosin, especially in the presence of actin. In comparison with adult subfragment-1, it was found that the binding of ATP analogues such as adenosine 5'-[beta, gamma-imino]triphosphate and PPi, or that of ATP (as deduced from the apparent KmATP) to neonatal subfragment-1 in the presence of actin was enhanced, while that of ADP was decreased. On the other hand, the association of actin with the ADP - neonatal-subfragment-1 complex was weaker. These features must be expressed in the cyclical actin-myosin association/dissociation steps occurring in ATP hydrolysis, and more particularly in the reassociation of actin with the ATP-hydrolysis-products - myosin complex.     

X-ray structures of the apo and MgATP-bound states of Dictyostelium discoideum myosin motor domain

Myosin is the most comprehensively studied molecular motor that converts energy from the hydrolysis of MgATP into directed movement. Its motile cycle consists of a sequential series of interactions between myosin, actin, MgATP, and the products of hydrolysis, where the affinity of myosin for actin is modulated by the nature of the nucleotide bound in the active site. The first step in the contractile cycle occurs when ATP binds to actomyosin and releases myosin from the complex. We report here the structure of the motor domain of Dictyostelium discoideum myosin II both in its nucleotide-free state and complexed with MgATP. The structure with MgATP was obtained by soaking the crystals in substrate. These structures reveal that both the apo form and the MgATP complex are very similar to those previously seen with MgATPgammaS and MgAMP-PNP. Moreover, these structures are similar to that of chicken skeletal myosin subfragment-1. The crystallized protein is enzymatically active in solution, indicating that the conformation of myosin observed in chicken skeletal myosin subfragment-1 is unable to hydrolyze ATP and most likely represents the pre-hydrolysis structure for the myosin head that occurs after release from actin.     

Transient kinetics of the interaction of 1,N6-ethenoadenosine 5'-triphosphate with myosin subfragment 1 under normal and cryoenzymic conditions: a comparison with adenosine 5'-triphosphate

The kinetics of the interaction of the fluorescent analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP) with myosin subfragment 1 (S1) were studied at 15 and -7.5 degrees C with 40% ethylene glycol as cryosolvent. Two techniques were used: fluorescence stopped flow and rapid flow-quench. When S1 is mixed with epsilon-ATP in a stopped-flow apparatus, biphasic fluorescence transients are obtained which are difficult to assign. Chemical sampling by the rapid-flow-quench method led to the chemical identity and the kinetics of interconversion of key intermediates, and by this method the optical signals were assigned and information about the cleavage and release of products was obtained. The data were interpreted by a shortened form of the Bagshaw-Trentham scheme for myosin adenosinetriphosphatase: M + ATP K1 in equilibrium M.ATP k2----M*.ATP k3 in equilibrium k3 M**.ADP.Pi k4----M + ADP + Pi The constants obtained were compared with those for ATP under identical conditions. In agreement with Rosenfeld and Taylor [Rosenfeld, S. S., & Taylor, E. W. (1984) J. Biol. Chem. 259, 11920-11929] we find that epsilon-ATP is bound tightly to S1 and that the chemical step is slower than with ATP. We show that the fast fluorescence transient is due to the tight binding of epsilon-ATP with K1 = 32 microM and k2 = 58 s-1 at 15 degrees C. With ATP these values are 8 microM and 16 s-1, respectively. There is a large difference in the delta H for k2: 50 kJ.mol-1 for epsilon-ATP and 119 kJ.mol-1 for ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the ATPase active site in myosin subfragment-1 with the use of vanadate plus ADP as a reversible "affinity-labeling" reagent: evidence for heterogeneity in the active sites

Our previous work showed that the active site heterogeneity in heavy meromyosin (HMM) becomes evident when highly reactive SH-groups in HMM are modified by thimerosal (Kawamura, Higuchi, Emoto, & Tawada (1985) J. Biochem. 97, 1583-1593). The heterogeneity was revealed by "affinity-labeling" analysis with vanadate plus ADP, which was developed in the previous paper. To see whether this heterogeneity is due to the head-head interaction or two different alkali light chains present in HMM, we carried out similar studies with myosin subfragment-1 (S1) and one of the isozymes, S1(A1), which contains only the alkali light chain 1, and obtained essentially the same results as those previously obtained with HMM. The S1 results are easily explained by the same hypothesis previously used for explaining the HMM results: SH-modified S1 or S1(A1) contains two kinds of active site in a 1:1 ratio with almost the same ATPase activity: one hydrolyzes ATP by a mechanism giving a protein Trp fluorescence enhancement, whereas the other hydrolyzes ATP by another mechanism giving no fluorescence enhancement.     

Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine.

The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the interaction of rabbit skeletal muscle adenylate deaminase with myosin subfragments. A kinetically regulated system

The interaction of rabbit skeletal muscle adenylate deaminase with myosin fragments (heavy meromyosin and subfragment-2) has been studied by analytical centrifugation, gel chromatography, and stopped flow light scattering. Formation of the complex is highly cooperative with respect to addition of two molecules of adenylate deaminase/molecule of myosin fragment to form a ternary complex. Ternary complex formation is also highly pH-dependent with less complex formed at higher pH values, and the pH dependence is steeper with heavy meromyosin than with subfragment-2. At pH 6.5, the dissociation constant for the heavy meromyosin-deaminase complex is approximately 1.2 X 10(-15) M2. Over the pH range 6.5-7.0, rate constants for the formation and dissociation of both the ternary and binary complexes of adenylate deaminase with heavy meromyosin have been determined. From analysis of the time course of stopped flow light scattering, the association steps are found to be extremely rapid, while the rate constant for dissociation of the first molecule of adenylate deaminase from the ternary complex is quite slow. This rate constant increases as the pH increased, but is sufficiently low that the interacting system does not equilibrate on the time scale of mass transport experiments (sedimentation velocity and gel chromatography), and thus displays apparent "slow" behavior. The kinetic regulatory properties of adenylate deaminase are influenced by heavy meromyosin and subfragment-2, particularly with respect to inhibition by GTP. The association and dissociation of adenylate deaminase and myosin fragments and the resultant changes in kinetic properties of the adenylate deaminase can markedly alter the time course of the enzymatic reaction. The time scale over which this interaction is modulated by changes in pH may have significance in the metabolism of exercising muscle.