Effect of ionic strength on the conformation of myosin subfragment 1-nucleotide complexes.

The effect of ionic strength on the conformation and stability of S1 and S1-nucleotide-phosphate analog complexes in solution was studied. It was found that increasing concentration of KCl enhances the reactivity of Cys(707) (SH1 thiol) and Lys(84) (reactive lysyl residue) and the nucleotide-induced tryptophan fluorescence increment. In contrast, high KCl concentration lowers the structural differences between the intermediate states of ATP hydrolysis in the vicinity of Cys(707), Trp(510) and the active site, possibly by increasing the flexibility of the molecule. High concentrations of neutral salts inhibit both the formation and the dissociation of the M**.ADP.Pi analog S1.ADP.Vi complex. High ionic strength profoundly affects the structure of the stable S1.ADP.BeF(x) complex, by destabilizing the M*.ATP intermediate, which is the predominant form of the complex at low ionic strength, and shifting the equilibrium to favor the M**.ADP.Pi state. The M*.ATP intermediate is destabilized by perturbation of ionic interactions possibly by disruption of salt bridges. Two salt-bridge pairs, Glu(501)-Lys(505) in the Switch II helix and Glu(776)-Lys(84) connecting the catalytic domain to the lever arm, seem most appropriate to consider for participating in the ionic strength-induced transition of the open M*.ATP to the closed M**.ADP.Pi state of S1.

Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1.

Bertrand et al. [Bertrand, R., Derancourt, J. & Kassab, R. (1995) Biochemistry 34, 9500-9507] reported that 6-[fluoresceine-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS) selectively modifies Lys553, which is part of the strong actin-binding site of myosin subfragment 1 (S1). We found that the reaction of FHS with Lys533 is accompanied by a decrease in the fluorescence intensity of the reagent. The rate of the FHS reaction increased with increasing pH implying that the unprotonated form of the epsilon-amino group of Lys553 reacts with FHS. Addition of 0.4 M KCl reduced the rate of reaction significantly, which indicates ionic strength-dependent changes in the structure of S1. Limited trypsinolysis of S1 before the FHS reaction also decreased the rate of the reaction showing that the structural integrity of S1 is needed for the reactivity of Lys553. ATP, ADP, ADP.BeF(x), ADP.AlF(4), ADP.V(i) and pyrophosphate significantly decreased the rate of Lys553 labelling, suggesting nucleotide-induced conformational changes in the environment of Lys553. The fluorescence emission spectrum of the Lys553-bound FH moiety and the quenching of its fluorescence by nitromethane was not influenced by nucleotides, implying that the chemical reactivity but not the accessibility of Lys553 was decreased by the nucleotide-induced conformational change. In the presence of ATP when the M(**)ADP.P(i) state of the ATPase cycle is predominantly populated, the reaction rate decreased more than in the case of the S1.ADP.AlF(4)(-) and S1.ADP.V(i) complexes, which are believed to mimic the M(**)ADP.P(i) state. This indicates that the conformation of the S1-ADP.AlF(4)(-) and S1.ADP.V(i) complexes in the vicinity of Lys553 does not resemble the structure of the M(**)ADP.P(i) state. The rate of Lys553 labelling decreased strongly in the presence of actin. The nitromethane quenching of the Lys553-bound FHS was not influenced by actin, which indicates that the reduced reaction rate is not due to steric hindrance caused by the bulky protein but by actin induced conformational changes in the vicinity of Lys553.

Trinitrophenylated reactive lysine residue in myosin detects lever arm movement during the consecutive steps of ATP hydrolysis.

Trinitrophenylation of the reactive lysine (Lys84) in skeletal myosin subfragment 1 (S1) introduces a chiral probe (TNP) into an interface of the catalytic and lever arm domains of S1 [Muhlrad (1977) Biochim. Biophys. Acta 493, 154-166]. Characteristics of the TNP absorption and circular dichroism (CD) spectra in TNP-modified S1 (TNP-Lys84-S1), and the Lys84 trinitrophenylation rate in native S1, indicate a one-to-one correspondence between ATPase transients and trapped phosphate analogues. Phosphate analogue-induced structures of TNP-Lys84-S1 were modeled using the crystallographic coordinates of S1 [Rayment et al. (1993) Science 261, 50-58] with swivels at Gly699 and Gly710 to approximate conformational changes during ATPase. The CD and absorption spectral characteristics of the model structures were compared to those observed for analogue-induced structures. The model calculations, first tested on a trinitrophenylated hexapeptide with known structure, were applied to TNP-Lys84-S1. They showed that ATP binding initiates swiveling at Gly699 and that swiveling at both Gly710 and Gly699 accompanied ATP splitting just prior to product release. The computed lever arm trajectory during ATPase suggests (i) a plausible mechanism for the nucleotide-induced inhibition of Lys84 trinitrophenylation, and (ii) trinitrophenylation-induced changes in S1 Mg2+- and K+-EDTA ATPase are from collision of the lever arm with TNP at Lys84. TNP is a site-specific structural perturbant of S1 and a chiral reporter group for the effect of Lys84 modification on dynamic S1 structure. As such, TNP-Lys84-S1 is equivalent to a genetically engineered mutant with intrinsic sensitivity to structure local to the modified residue.

Interaction of myosin subfragment 1 with two non-nucleotide ATP analogues.

2-[(2-Nitrophenyl)amino]ethyl triphosphate (NPhAETP) is the smallest ATP analogue that serves as a substrate for the actin-activated ATPase of myosin subfragment 1 (S1) and supports the development of active tension in skinned fibers. 2-(Phenylamino)ethyl triphosphate (PhAETP), in which the nitro group on the phenyl ring of NPhAETP is substituted by a H atom, is also a substrate of the actin-activated ATPase but does not support active tension [Wang, D., Pate, E., Cooke, R., and Yount, R. (1993) J. Muscle Res. Cell Motil. 14, 484-497]. We compared the S1-catalyzed hydrolysis of these analogues, their ability to support the formation of stable complexes with S1 and phosphate analogues, and their effect on S1 conformation. The analogues were hydrolyzed by S1 under various conditions both in the presence and in the absence of actin. In some cases, the effects of the two analogues are similar to each other and to those of ATP; they protect S1 from heat denaturation at 40 degreesC and inhibit the formation of the N-terminal 29 kDa fragment during the tryptic digestion of S1 and the modification of Lys-83 with trinitrobenzene sulfonate. However, in other cases, the effect of the two analogues is different; the effect of NPhAETP resembles that of ATP. NPhAETP and ATP decrease while PhAETP increases the rate of reaction of the SH1 thiol (Cys-707) with coumarin maleimide. The diphosphate forms of the two analogues induce a much smaller change in the near-UV CD spectrum of S1 than ADP. NPhAEDP forms stable complexes with S1 in the presence of beryllium fluoride (BeFx), aluminum fluoride (AlF4-), or vanadate (Vi) phosphate analogues, while the S1.PhAEDP complex is stable in the presence BeFx but much less stable with AlF4- and Vi. These results indicate that the S1.PhAEDP.Pi state is poorly populated during the PhAETP hydrolysis. The models of the atomic structure of S1 complexed by the two analogues show that PhAETP, unlike NPhAETP or ATP, does not form a H bond with Tyr-134 in S1, which is the probable structural reason of the lack of tension development, with PhAETP as the substrate.

Near UV circular dichroism from biomimetic model compounds define the coordination geometry of vanadate centers in MeVi- and MeADPVi-rabbit myosin subfragment 1 complexes in solution.

The circular dichroism (CD) spectrum was measured from vanadate (Vi) cyclic esters of chiral vicinal diols, hydroxycarboxylates, and cyclodextrines as a function of Vi concentration ([Vi]) and at the lowest energy transitions of the vanadium. At low [Vi] and in the presence of excess vicinal diols, hydroxycarboxylates, or cyclodextrines the CD signal intensity scales linearly with [Vi] indicating the predominance of a monomeric cyclic ester. At higher [Vi], the signal intensity in the presence of the vicinal diols and hydroxycarboxylates become nonlinear in [Vi], indicating formation of a dimeric cyclic ester. Vanadium-51 NMR (51V-NMR) indicates the coordination geometry of several of these model Vi centers in solution and identifies the CD signals characteristic to Vi trigonal bipyramidal (tbp) and octahedral (Oh) coordination geometries from monomeric and dimeric species. The CD spectra from monomeric and dimeric forms of the tbp-coordinated model compounds have two apparent transitions with amplitudes of opposite sign at wavelengths > or = 240 nm. Spectra from the monomeric and dimeric Oh coordinated species are distinct from the tbp-type spectra over the same wavelength domain because of the presence of two additional transitions with opposite sign amplitudes. These model spectra were compared to the vanadate CD spectra from Vi bound to rabbit myosin subfragment 1 (S1) in solution, in the presence of divalent metal cations (MeVi-S1) or trapped with MeADP (MeADPVi-S1). Polymeric MeVi binds to the active site of S1 and the vanadate centers in MnVi-S1 or CoVi-S1 produce a CD signal resembling that from the tbp model. The trapped ATPase transition state analog MeADPVi produces a different CD signal resembling that from the Oh model.

Effect of metal cations on the conformation of myosin subfragment-1-ADP-phosphate analog complexes: a near-UV circular dichroism study.

The interaction of myosin with actin, coupled with hydrolysis of ATP, is the molecular basis of muscle contraction. The head segment of myosin, called S1, contains the distinct binding sites for ATP and actin and is responsible for the ATPase activity. The myosin-catalyzed ATP hydrolysis consists of several intermediate steps and each step is accompanied by conformational changes in the S1 segment. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1 x ADP x Pi complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate, beryllium fluoride (BeFx) or aluminum fluoride (AlF4-), yields stable complexes which mimic the intermediates of the ATP hydrolysis. In this work, tertiary structure changes in S1 in the vicinity of aromatic residues was studied by comparing near-UV circular dichroism (CD) spectra from S1-nucleotide-phosphate analog complexes in the presence of Mg2+ and other cations. A significant difference between the MgATP and MgADP spectra indicated notable tertiary structural changes accompanying the M**ADP x Pi --> M*ADP transition. The spectra of the S1 x MgADP x BeFx and S1 x MgADP x AlF4- complexes resemble to those obtained upon addition of MgATPgammaS and MgATP to S1, and correspond to the M* x ATP and M** x ADP x Pi intermediates, respectively. We have found recently that the presence of divalent metal cations (Me2+) is essential for the formation of stable S1 x MeADP x PA complexes. Moreover, the nature of the metal cations strongly influences the stability of these complexes [Peyser, Y. M., et al. (1996) Biochemistry 35, 4409-4416]. In the present work we studied the effect of Mg2+, Mn2+, Ca2+, Ni2+, Co2+, and Fe2+ on the near-UV CD spectrum of the ATP, ADP, ADP x BeFx, and ADP x AlF4- containing S complexes. The CD spectra obtained with ADP, ATP ADP x BeFx and ADP x AlF4- were essentially identical in the presence of Co2+ and rather similar in the case of Ca2+, while they were partially different in other cases. An interesting correlation was found between actin activation and ATP versus ADP difference spectra in the presence of various metal ions. The distribution of the fractional concentration of the intermediates of ATP hydrolysis was estimated in the presence of each cation from the CD spectra with phosphate analogs. In the presence of Mg2+ the predominant intermediate is the M** x ADP x Pi state, which is in accordance with the kinetic studies. On the other hand with non-native cations the predominant intermediate is the M* x ADP state and the release of ADP is the rate limiting step in the myosin-catalyzed ATP hydrolysis. According to the results, the near-UV CD spectrum originating from aromatic residues in S1 not only can distinguish identifiable states in the ATP hydrolysis cycle but can also pinpoint to changes in the tertiary structure caused by complex formation with nucleotide or nucleotide analog and various divalent metal cations. These findings, that are correlative with actin activation, and thus with the power stroke, suggest new strategies for perturbing S1 structure in the continuous efforts directed toward the elucidation of the mechanism of muscle contraction.

Effect of complexes of ADP and phosphate analogs on the conformation of the Cys707-Cys697 region of myosin subfragment 1.

Recent crystallographic studies have suggested structural differences between the complexes of S1.Mg.ADP with the phosphate analogs aluminium fluoride (AlF4-), vanadate (VO(4)3-) and beryllium fluoride (BeFx) [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960-8972; Smith, R. & Rayment, I. (1996) Biochemistry 35, 5404-5417]. In this work, chemical modifications, namely labeling of Cys707 (the reactive SH1 thiol) and Cys707-Cys697 (SH1-SH2) cross-linking, were used to compare the S1.ADP.BeFx, S1.ADP. AlF4- and S1.ADP-VO(4)3- complexes with specific states of the myosin-ATPase pathway. Modification of Cys707 with the fluorescent monofunctional reagents 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and N-iodoacetyl-N'-(5-sulfo-1-naphtyl)ethylenediamine has shown that the reactivity of the SH1 group depends on the nucleotide bound to S1. The observed rates of Cys707 modification at 20 degrees C lead to the conclusion that S1.ADP.BeFx is similar to S1*.ATP, while S1.ADP.AlF4- and S1.ADP.VO(4)3- are more similar to S1**.ADP.Pi. The conformations of the analog states were also compared by monitoring the dissociation of the fluorescent nucleotide analog 1-N6-ethenoadenosine diphosphate (ADP[C2H2]) from the active site of Cys707-modified (by N-ethylmaleimide) and Cys707-Cys697-cross-linked (by N,N'-p-phenylene dimaleimide) S1.ADP[C2H2].AlF4- and S1.ADP[C2H2]. BeFx. Our results suggest that the conformations of the S1.ADP.AlF4-, S1.ADP.VO(4)3- and S1.ADP.BeFx complexes in the Cys707-Cys697 region are distinct from each other, with the former two at least partially resembling the S1**.ADP.Pi state, while the latter is similar to the prehydrolyzed S1*.ATP state.

Effect of divalent cations on the formation and stability of myosin subfragment 1-ADP-phosphate analog complexes.

Myosin belongs to the family of motor proteins. Its interaction with actin coupled with hydrolysis of ATP is the molecular basis of muscle contraction. The head segment of myosin, called subfragment 1 (S1), contains the distinct binding sites for ATP and actin and responsible for the ATPase activity. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1-MgADP-Pi complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate (Vi) or beryllium fluoride (BeF(x)), highly stabilizes the complex. We studied the role of the divalent cations in the ATPase activity and in the formation and decomposition of PA-containing stable complexes by substituting Mg2+ with Fe2+, Mn2+, Ni2+, Co2+, and Ca2+. These metal ions supported the actin activation of S1 ATPase and affected the obtained kinetic parameters, Km and V(max). The ATPase activity of S1 in the absence of actin increased with the increasing ionic radius of the metal (Me) ions. These ions also substituted for Mg2+ in the formation of the stable ternary S1-MeADP-PA complexes, which cannot be generated in the absence of divalent cations. Upon formation of stable ternary complexes, S1 reversibly loses its ability to catalyze the hydrolysis of ATP. The formation of the complexes can be followed by monitoring the disappearance of the ATPase activity. The rate of the complex formation depends on the divalent cation present and decreases in the order Mn > Fe > Ni > Co > Mg and Ca > Mn > Fe > Mg > Co in the Vi- and BeF(x)-containing complexes, respectively. The ATPase activity of S1 is recovered upon addition of actin, which causes the decomposition of the complex. The spontaneous decomposition of the complexes was studied in the presence of ethylenediaminetetraacetic acid (EDTA), which chelates the metal divalent cations released from the complex and prevents its reformation. The rate of decomposition was assessed by monitoring the recovery of the ATPase activity of S1 in the presence of EDTA. The rate of decomposition of the Vi- and BeF(x)-containing complexes follows the order Mn > Fe > Co > Mg > Ni and Ca >> Mn > Fe > Co > Mg, respectively. The rate of decomposition increases with the increasing ionic radius of the metal ions, similarly as observed in the case of ionic radius dependence of the ATPase activity. On the basis of this similarity, it is assumed that the decomposition of the complexes consists of two steps, the first step being the very slow release of PA followed by a rapid dissociation of MeADP from S1. The stability of the complexes has been calculated from the formation and decomposition rates. Except in the case of Mg, the stabilities of the BeF(x) complexes are higher than those containing Vi. The results indicate that the metal cations have a significant role in maintaining the proper structure of the transient state complex in the myosin-catalyzed ATP hydrolysis.

Characterization of stable beryllium fluoride, aluminum fluoride, and vanadate containing myosin subfragment 1-nucleotide complexes.

Beryllium and aluminum fluorides are good phosphate analogues. These compounds, like orthovanadate, form stable complexes with myosin subfragment 1 (S1) in the presence of MgADP. The formation of the stable S1-nucleotide complexes is characterized by the loss of ATPase activity. For the complete loss of ATPase activity there was necessary a higher concentration of aluminum than of beryllium or vanadate. In the presence of MgATP the onset of the inhibition is delayed, which indicates that stable complexes cannot form when a specific site is occupied by the gamma-phosphate of ATP or by P(i) derived from the gamma-phosphate. The half-lives of the S1-MgADP-(BeF3-), S1-MgADP-(AlF4-), and S1-MgADP-Vi complexes at 0 degrees C are 7, 2, and 4 days, respectively. In the presence of actin the rate of decomposition of all of the complexes is significantly enhanced; however, the order of decomposition is reversed, the fastest rate being observed with beryllium and the slowest with aluminum. The formation of the S1-MgADP-(BeF3-) and S1-MgADP-(AlF4-) complexes is accompanied by an increase in tryptophan fluorescence similar to that observed upon addition of MgATP to S1. The fluorescence increase develops rather slowly, by suggesting that the rate-limiting step in the formation of the stable complex is an isomerization. The rate of the fluorescence change accompanying the formation of the Be complex is faster than that for the Al complex. Addition of vanadate to S1 causes a static quenching of the tryptophan fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ions on vanadate-induced photocleavage of myosin subfragment 1.

Myosin subfragment 1 (S1) is cleaved by near-ultraviolet irradiation in the presence of vanadate at three sites located at 23, 31 and 74 kDa from the N-terminus. Since vanadate is considered to be a good structural analogue of phosphate, it is assumed that the cleavage sites participate in forming the phosphate-binding site(s) of S1. In this work, the effect of various ions on the vanadate-induced photocleavage of S1 was studied. Monovalent anions were found to inhibit photocleavage in the 50-200 mM range. The inhibition is more expressed at a site 74 kDa from the N-terminus than at the 23-kDa and 31-kDa sites. The inhibitory effect of the monovalent anions increases in the order acetate = F- less than Cl- less than Br- less than I- = SCN-. The order of the inhibitory effect is identical to the protein-structure-damaging effect of monovalent anions in the von Hippel series [von Hipel, P. H. & Wong, K. Y. (1964) Science 145, 577-581]. Therefore, it is assumed that decreased photocleavage is due to local perturbations of structure, especially at the 74-kDa site, in addition to increased ionic strength. Divalent anions, sulfate and thiosulfate, strongly inhibit photocleavage at 2 mM. The inhibition is very pronounced at the 23-kDa and 31-kDa sites, while the 74-kDa site is hardly affected. Since photocleavage at the 23-kDa and 31-kDa sites is regulated jointly and independently from cleavage at the 74-kDa site, it is assumed that S1 has two distinct phosphate-binding sites: the regions of the 23-kDa and 31-kDa cleavage sites, which are proximal to each other in the spatial structure, participate in forming the first phosphate-binding site, while the 74-kDa site is part of the second binding site. Sulfate was also found to inhibit the trapping of vanadate and to facilitate its release from the S1-MgADP-Vi (Vi, inorganic vanadate) complex. Photocleavage of S1 takes place at all three sites, both in the presence or absence of divalent cations, indicating that these, including Mg2+, are not essential for cleavage.

Effect of actin, ATP, phosphates, and pH on vanadate-induced photocleavage of myosin subfragment 1.

Near-UV irradiation in the presence of vanadate cleaves the heavy chain of myosin subfragment 1 at three specific sites located at 23, 31, and 74 kDa from the N-terminus. Increasing the pH from 6.0 to 8.5, gradually, reduces the efficiency of the cleavage and completely eliminates the 31-kDa cut. Actin specifically inhibits the photocleavage at the sites located 31 and 74 kDa from the N-terminus. ATP strongly protects from cleavage at the 23- and 31-kDa sites and less strongly from the cut at the 74-kDa site. ADP and pyrophosphate have similar, but less pronounced, effects as ATP. Orthophosphate inhibits the photocleavage at the 23- and 74-kDa sites with a similar efficiency. In the ternary actin-S-1-ATP complex, the photocleavage is inhibited at all sites, and the effects of actin and ATP are additive. Photocleavages affect the K+(EDTA)-, Ca2(+)-, and actin-activated ATPase activity of subfragment 1. Loss of all three ATPases is caused by cleavage at the 23-kDa site, while the cut at the 74-kDa site only leads to the loss of actin-activated ATPase activity. It is concluded that subfragment 1 contains at least two distinct phosphate binding sites, the first being part of the "consensus" ATP binding site wherein the 23-kDa photocleavage site is located. This site is responsible for the binding and hydrolysis of ATP. It is possible that the 31-kDa cleavage site is also associated with the "consensus" site through a loop. The 74-kDa cleavage site is a part of another phosphate binding site which may play a role in the regulation of the myosin-actin interaction.

51V NMR study of vanadate binding to myosin and its subfragment 1.

The binding of various forms of vanadate to myosin and myosin subfragment 1 (S-1) was studied by 51V NMR at increasing vanadate concentrations between 0.06 and 1.0 mM. The distribution of the various forms of vanadate in the solution depended on the total concentration of vanadate. At low concentrations, the predominant vanadate form was monomeric, while at high concentration, it was tetrameric. The presence of myosin or S-1 in the solution produced a significant broadening of the signal of each form of vanadate, indicating that all of them bind to the protein. Addition of ATP, which does not affect the 51V NMR spectra in the absence of proteins, causes their significant alteration in the presence of myosin or S-1. The changes, which include the broadening of the signal of the monomeric and the narrowing of the signal of the oligomeric vanadate forms, indicate that more monomeric and less oligomeric vanadate binds to the proteins in the presence than in the absence of ATP. Irradiation by near-UV light in the presence of vanadate cleaves S-1 at three specific sites--at 23, 31, and 74 kDa from the N-terminus. The cleavages at 23 and 31 kDa are specifically inhibited by the addition of ATP. The vanadate-associated photocleavage of S-1 also depends on the total concentration of vanadate; it is observed only when the concentration of vanadate is at least 0.2 mM. This was also the lowest concentration at which oligomeric vanadate was detected in the 51V NMR spectra. From the parallel concentration dependence of the photocleavage and the appearance of the tetrameric vanadate, it is concluded that photocleavage occurs only when tetrameric vanadate binds to S-1.

Tryptophan-130 is the most reactive tryptophan residue in rabbit skeletal myosin subfragment-1.

Rabbit skeletal muscle myosin subfragment-1 (S-1) was reacted with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) resulting in modification of 0.8 tryptophan residues per S-1. In order to assign the most reactive tryptophan of the 5 S-1 tryptophans, antibodies were raised in rabbits against bovine serum albumin modified with DHNBS. The antibodies reacted with the 27 kDa tryptic fragment of DHNBS-treated S-1, indicating that the reactive tryptophan resides on this domain. The 27 kDa fragment was isolated from DHNBS-treated S-1 and was further cleaved at a single cysteine residue by 2-nitro-5-thiocyanobenzoic acid. This cleavage resulted in two peptides, each of them containing one tryptophan. The antibodies reacted with the smaller peptide consisting of residues 122-204. The only tryptophan residing on this peptide is Trp130, and this is therefore the most reactive tryptophan of S-1.

Modification of myosin subfragment 1 tryptophans by dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide.

Modification of tryptophanyl residues (Trps) of myosin subfragments 1 (S-1) was performed with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS). Under controlled conditions, pH 6 at 0 degrees C and 10-min reaction with 10-100-fold molar excess, K+(EDTA) activity was reduced down to less than half, whereas Ca2+-ATPase activity increased and acto-S-1-ATPase was not affected. The number of modified Trps (up to 2.5) agreed well with the number of 2-hydroxy-5-nitrobenzyl moieties incorporated in S-1. The thiol groups of S-1 were not affected up to 50-fold molar excess of DHNBS, thus indicating that the modification was selective for Trps. The modification of as few as one Trp caused a blue shift of the emission spectrum, accompanied by a reduction in the fluorescence quantum yield. The accessibility of Trps to the fluorescence quencher acrylamide is drastically reduced upon modification, indicating that DHNBS-reactive Trps are more "exposed" than the DHNBS-refractive ones. DHNBS modification did not seem to affect the ATP-induced tryptophan fluorescence enhancement of S-1. The effect of DHNBS modification of the intrinsic fluorescence of S-1 indicates that the modified Trps are located in a polar environment and that they may be identical with the long-lifetime Trps of Torgerson [Torgerson, P. (1984) Biochemistry 23, 3002-3007]. The most reactive Trp is located in the N-terminal 27-kDa fragment of the S-1 heavy chain. It might also be inferred from the above data that the nonexposed and ATP-perturbed Trp(s) is (are) located in the 50-kDa fragment.

Alterations in cardiac myosin isoenzymes distribution as an adaptation to chronic environmental heat stress in the rat.

The distribution of cardiac myosin isoenzymes, using gel pyrophosphate electrophoresis, was studied in laboratory rats during acclimation to heat (34 degrees C, 0-2 month) with and without daily administration of triiodotyronin (0.3 micrograms/100 g b.wt) Thyroxin (T4) and triiodotyronin (T3) concentrations during that period were measured in the acclimated rats as well. Control rats exhibited only the V1 myosin form during the entire experimental period. In the heat acclimated rats, after three weeks of acclimation, in addition to the V1 band, bands of V2 and V3 myosin forms appeared. After the fourth week of acclimation V3 became the dominant myosin. Changes in the cardiac isoenzymes distribution occurred 1 week after a significant decrease in T3 was recorded. Administration of additional thyroxin dose didn't allow the development of V3 band in the heat acclimated rats. It was concluded that the changes observed in cardiac isoenzymes distribution could be related to an alteration in thyroid activity and that these changes could play a role in the adaptation of the cardiac muscle to chronic heat.