Involvement of an arginyl residue in the catalytic activity of myosin heads.

1. Phenylglyoxal reacts rapidly with isolated myosin heads (subfragment 1) and induces two successive and distinguishable effects on their enzymic properties: first, a twofold activation of the Ca2+ and Mg2+-dependent ATPases with no effect onthe K+-ATPase followed by inhibition of the K+, Ca2+ and actin-activated Mg2+-ATPases. A specific protein-reagent reagent complex is formed during the second phase of the modification reaction (Ki approximately 5 x 10(-3) M). 2. ADP and ATP with or without cations provide efficient protection only against the loss of ATPase activities, suggesting that the second inhibitory process is occurring at or close to the active site. 3. On the basis of [14C]phenylglyoxal-labelling experiments and the composition of modified subfragment-1 derivatives, it is demonstrated that the sequential modification of two reactive arginyl residues is responsible for the observed activation-inhibition phenomena. Blocking of the first reactive residue produces a shift in the pH/activity curves related to the Ca2+ and Mg2+-dependent ATPases with an apparent activation effect. Modification of the second guanidino group does not destroy the affinity of the protein for the nucleotide substrates but does alter the nucleotide binding site as reflected in the inability of Mg2+. ATP to dissociate the modified subfragment-1--actin complex. It is concluded that electrostatic interactions between this positively charged group and the negatively charged ATP and ADP molecules may be critical for the hydrolytic efficiency of myosin heads. 4. After dissociation and separation of the polypeptide constituents of the protein in acetic acid medium, both labelled sites are found to reside in the heavy chain.     

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

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

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

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

Location of the sites of reaction of N-ethylmaleimide in papain and chymotryptic fragments of the gizzard myosin heavy chain

The thiol of the gizzard myosin heavy chain, which reacts most rapidly with N-ethylmaleimide (MalNEt), has been located in the subfragment 2 region of myosin rod by fragmentation of [14C]-MalNEt-labeled myosin with papain and chymotrypsin. MalNEt reacts more slowly with thiols present in the 70- and 25-kilodalton (kDa) papain fragments of subfragment 1. The reaction of MalNEt with thiols present in these regions is increased on addition of ATP by factors of 2 and 10, respectively, when myosin is modified in 0.45 M NaCl where it is present in the extended, 6S conformation. The rate of increase of Mg2+-activated adenosinetriphosphatase (ATPase) activity, which reflects the loss of ability of myosin to assume the folded, 10S conformation, and the rate of loss of K+-EDTA-activated activity produced by MalNEt are both accelerated 5- to 10-fold on addition of ATP. The rates at which ATPase activities change agree closely to the reaction rates of MalNEt with the 25-kDa region of subfragment 1; therefore, the changes in these activities can be attributed to modification of a thiol of the 25-kDa segment. An increase in actin-activated ATPase activity produced by reaction of myosin with MalNEt in 0.45 M NaCl is accelerated by ATP by a factor of at least 4. Reaction with [14C]MalNEt in the presence of MgATP and 0.2 M NaCl, where myosin is in the 10S form, inhibits the incorporation of radioactive MalNEt into the 25-kDa papain fragment of subfragment 1. It also prevents the increase in actin-activated ATPase activity and preserves the ability of myosin to assume the 10S form.     

Steady-state magnesium ion-adenosine triphosphatase activities of myosin and its subfragment 1 derivative

The Mg2+-ATPase activity of myosin and its subfragment 1 (ATP phosphohydrolase, EC 3.6.1.3) always followed normal Michaelis-Menten kinetics for ATP concentrations less than 10 microM. The average Km values at pH 7.4 and 25 degrees C are 0.33 +/- 0.04 microM for myosin and 0.43 +/- 0.11 microM for subfragment 1. At low salt concentration myosin yields a second hyperbolic increase in Mg2+-ATPase activity as the ATP rises from 10.2 microM to 153 microM: V doubles with a Km of 11 +/- 5 microM. This second low-salt-dependent increase in Mg2+-ATPase activity occurred between pH 6.8 and pH 8.7. It was not affected by the presence of 0.10 M EGTA to remove Ca2+ contamination. Solubilization of the catalytic sites by assaying myosin for ATPase activity in the presence of 0.60 M NaCl or by conversion of myosin to subfragment 1 eliminated the secondary hyperbolic increase. Subfragment 1 has a significantly different pH-activity curve from that of myosin. Subfragment 1 has an activity peak at pH 6.0, a rising activity as the pH goes from 8.7 to 9.8, and a deep activity valley between pH 6.8 and pH 8.4. Myosin has a very shallow trough of activity at pH 6.8 to 8.4, and in 1.0 mM ATP its activity drops as the pH decreases from 6.8 to 6.0. NaCl is a noncompetitive inhibitor of the Mg2+-ATPase activity of myosin and subfragment 1. Myosin has a greater affinity for NaCl (Ki = 0.101 +/- 0.004 M) than does subfragment 1 (Ki = 0.194 +/- 0.009 M).     

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.     

Reaction of phenylglyoxal with chicken gizzard myosin

Modification of chicken gizzard myosin with phenyl[2-14C]-glyoxal inhibited the K+-ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity as a function of time. During the 2.5 and 15 min interval 3.2 mol of the reagent were incorporated per 4.7 X 10(5) g protein and the K+-ATPase activity was 50% inhibited. Phenylglyoxal reacted with arginine residues of gizzard myosin in a mol ratio of two to one, phenylglyoxal to arginine as determined spectrophotometrically. The modification was limited to the subfragment 1 heavy chain and rod-like regions and none of the light chains were lost. The inhibition of the ATPase activity occurred when the subfragment 1 region was modified predominantly. The same results were obtained when the myosin was phosphorylated and then incubated with phenylglyoxal. Substrate MgATP2- or MgADP enhanced the inactivation of gizzard myosin; there was an increase in the incorporation of the reagent and a change in the distribution into the heavy chains. Approx. 0.5 mol of the nucleotide was bound to 4.7 X 10(5) g of phenylglyoxal myosin. Conformational changes, induced by these modifications, were responsible for the inhibition of enzymic activity. Arginine residues of gizzard myosin are necessary for the maintenance of the ATPase activity of this contractile protein.     

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

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

Identical behavior of the two active sites of myosin with respect to trinitrophenylation.

Myosin and its active subfragments were trinitrophenylated under conditions in which mainly the active site(s) was modified. Proteins modified at the active site(s) could be separated by affinity chromatography on agarose-ATP columns. By two independent methods, ATPase activity measurements and analysis of elution patterns on agarose-ATP columns, it was shown that the introduction of two trinitrophenyl groups per myosin or one per heavy meromyosin subfragment 1 molecule is responsible for the remarkable change in the ATPase activities. Heavy meromyosin subfragment 1 prepared from trinitrophenylated myosin retained the original degree of trinitrophenylation per "active head." The kinetic constant of trinitrophenylation of the epsilon-amino group of lysine at the active site was found to be 2000 S-1-M-1, whereas a much smaller constant of 2.2 S-1-M-1 was obtained for the trinitrophenylation of the unessential lysyl residues of myosin. By using affinity chromatography, we could follow the formation of mono- and ditrinitrophenyl myosin. The amounts of these myosin derivatives at various extents of the reaction corresponded approximately to the calculated amounts, assuming a random and independent trinitrophenylation of the two myosin "heads." It is concluded that in each of the two heads of myosin there is one ATPase active site and these two sites behave in an identical manner with respect to trinitrophenylation.     

The binding of divalent cations to myosin.

Centrifuge transport, equilibrium dialysis, and electron paramagnetic resonance studies on the binding of Mn2+ to myosin revealed two sets of noninteracting binding sites which are characterized at low ionic strength (0.016 M KCl) by affinity constants of 10(6) M-1 (Class I) and 10(3) M-1 (Class II), respectively. At 0.6 M KCl concentration, the affinity of Mn2+ for both sets of sites is reduced. The maximum number of binding sites is 2 for the high affinity and 20 to 25 for the low affinity set. Other divalent metal ions displace Mn2+ from the high affinity sites in the following order of effectiveness: Ca greater than Mg = Zn = Co greater than Sr greater than Ni. The inhibitory effects of Mg2+ and Ca2+ upon the Mn2+ binding are competitive with inhibitor constants of 0.75 to 1 mM which is similar to that of the low affinity divalent metal ion binding sites. Exposure of myosin to 37 degrees partially inhibits Mn2+ binding to Class I parallel with inhibition of ATPase activity. The binding of Mn2+ to the high affinity binding sites is not significantly influenced by ADP or PPi, although Mn2+ increases the affinity of ADP binding to myosin at high ionic strength.     

Modification of Lys-237 on actin by 2,4-pentanedione. Alteration of the interaction of actin with tropomyosin

It has been possible to specifically label rabbit skeletal muscle actin at Lys-237 with 2,4-pentanedione, producing an enamine. This reaction can be reversed with hydroxylamine. The modification can be carried out with actin in either the G- or F-forms and does not affect polymerization-depolymerization. The modification does affect, however, the interaction of tropomyosin (Tm) with the modified F-actin. In the absence of Ca2+ and Mg2+ (mu = 0.12), Tm failed to bind to the modified F-actin whereas it did bind to unmodified F-actin (1 Tm:7 actins). Tm binding could be restored under these conditions by the addition of either troponin (Tn), Mg2+, or Mg2+ and Ca2+. Under certain conditions, Tm alone has been shown to inhibit actin-activated heavy meromyosin (HMM)-Mg2+-ATPase. This inhibition did not occur with the modified F-actin even though Tm was bound (approximately 1 Tm:7 actins). Even when Tn was added to this system (in the absence of Ca2+), no inhibition of ATPase could be observed. Thus, this modification appears to prevent F-actin X Tm from assuming the "blocking" inhibitory position (conformation). In addition, Tn appears to enhance the activation of heavy meromyosin-Mg2+-ATPase by the modified F-actin X Tm complex whether Ca2+ is present or not. This state may be analogous to the potentiated state (Murray, J. M., Knox, M. K., Trueblood, C. E., and Weber, A. (1982) Biochemistry 27, 906-915) seen with myosin subfragment 1-saturated actin at low ATP levels. Thus, using modified and unmodified F-actin, it is possible to produce three Tm X actin states: off (F-actin X Tm), on (modified F-actin X Tm), and "potentiated" (modified F-actin X Tm X Tn).     

Incorporation of 6-carboxyfluorescein into myosin subfragment 1

We describe for the first time the introduction of a label into the "50K" domain of myosin subfragment 1 (S-1), and we investigate the properties of this fluorescent modification in relation to the ATPase and actin-binding activities, both residing in the myosin head. The labeling consists of a major incorporation of 6-carboxyfluorescein into the "50K" domain of S-1. Using different conditions for tryptic digestion that allowed a fragmentation of the "50K" domain with a loss of 5 kilodaltons (kDa) leading to a final product of 45 kDa, we have shown that the fluorescent dye remains in the 45-kDa final product. By studying cross-linking as a function of time, we have demonstrated that the "50K" domain and the 45-kDa fluorescent peptide are equally cross-linkable to actin. We have also investigated the K+EDTA-, Ca2+-, Mg2+-, and actin-activated ATPase activities of this modified S-1 and after purification observed no enzymatic changes.     

Involvement of the 50-kDa peptide of myosin heads in the ATPase activity revealed by fluorescent modification with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole

The fluorescent reagent 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F) reacted specifically with 1.9 lysyl residues/mol of the myosin subfragment-1 (S-1) ATPase. When 1.9 lysyl residues were modified, the K+- and Ca2+-ATPase activities were almost completely inhibited, whereas the Mg2+-ATPase activity was increased to 180% of original activity. The actin-activated Mg2+-ATPase activity was decreased to 30% of original activity by this modification. However, affinity of S-1 for actin in the presence of ATP was unchanged. The NBD fluorescence of the modified S-1 was quenched on addition of ATP, suggesting that ATP induced conformational changes around the NBD groups attached to S-1. Tryptic digestion of the modified S-1 revealed that the NBD groups are attached mainly to the 50-kDa peptide of S-1, more precisely the 45-kDa peptide. These results confirm the recent reports that the 50-kDa peptide of S-1 is involved in the myosin ATPase reaction (K?rner, M., Thiem, N. V., Cardinaud, R., and Lacombe, G. (1983) Biochemistry 22, 5843-5847; Hiratsuka, T. (1986) Biochemistry 25, in press).     

Cardiac myosin subfragment 1 modification by carbodiimide in the presence of a nucleophile

The subfragment 1 from dog cardiac myosin was modified by N-cyclohexyl-N′-(2-(4-morpholinyl) ethyl) carbodiimide methyl p-toluenesulfonate in the presence of the nucleophile nitrotyrosine ethyl ester. At pH 5.9, the inactivation of ATPase activity was very rapid and followed first-order kinetics. K⁺ (EDTA) - and Ca⁺⁺-ATPase activities decreased at the same rate, and the initial phosphate burst was lost. Inactivation and incorporation of the nucleophile occurred simultaneously. Complete inactivation was accompanied by the incorporation of 1 mol of (¹⁴C) nitrotyrosine per mol of myosin subfragment 1. Inactivation and incorporation of the label were essentially equal, either with the native subfragment 1, or with the subfragment 1 in which the reactive thiols were protected by cyanylation prior to modification. No protection by nucleotides was observed. These data suggest that one carboxyl group is essential for the active conformation of cardiac myosin. This finding is in general agreement with that previously obtained with skeletal subfragment 1 (Lacombe et al. (1981) Biochemistry 20, 3648–3653) except that inactivation of cardiac subfragment 1 was not prevented by nucleotides.     

Inhibition of myosin ATPase by metal fluoride complexes

Magnesium (Mg2+) is the physiological divalent cation stabilizing nucleotide or nucleotide analog in the active site of myosin subfragment 1 (S1). In the presence of fluoride, Mg2+ and MgADP form a complex that traps the active site of S1 and inhibits myosin ATPase. The ATPase inactivation rate of the magnesium trapped S1 is comparable but smaller than the other known gamma-phosphate analogs at 1.2 M-1 s-1 with 1 mM MgCl2. The observed molar ratio of Mg/S1 in this complex of 1.58 suggests that magnesium occupies the gamma-phosphate position in the ATP binding site of S1 (S1-MgADP-MgFx). The stability of S1-MgADP-MgFx at 4 degrees C was studied by EDTA chase experiments but decomposition was not observed. However, removal of excess fluoride causes full recovery of the K+-EDTA ATPase activity indicating that free fluoride is necessary for maintaining a stable trap and suggesting that the magnesium fluoride complex is bonded to the bridging oxygen of beta-phosphate more loosely than the other known phosphate analogs. The structure of S1 in S1-MgADP-MgFx was studied with near ultraviolet circular dichroism, total tryptophan fluorescence, and tryptophan residue 510 quenching measurements. These data suggest that S1-MgADP-MgFx resembles the M**.ADP.Pi steady-state intermediate of myosin ATPase. Gallium fluoride was found to compete with MgFx for the gamma-phosphate site in S1-MgADP-MgFx. The ionic radius and coordination geometry of magnesium, gallium and other known gamma-phosphate analogs were compared and identified as important in determining which myosin ATPase intermediate the analog mimics.     

Interaction of myosin subfragment 1 with Cibacron Blue F3GA

Cibacron Blue F3GA and its immobilized derivatives have been shown before to bind and inhibit nucleotide-dependent enzymes and, among them, myosin subfragment 1. Experiments have been carried out to examine the mechanism of the subfragment 1--dye interaction. Binding of subfragment 1 to immobilized dye (Affi-Gel Blue) does not involve the ATP binding site on myosin. Subfragment 1 hydrolyzes MgATP and CaATP while bound to the Affi-Gel Blue column. Inactivated subfragment 1, which contains [3H]ADP noncovalently trapped at the active site, binds and elutes from the Affi-Gel Blue column in the same manner as unmodified, active protein. Free Cibacron Blue inhibits the ATPase activity of subfragment 1. The inhibition is pH, salt, and time dependent. Complete inhibition correlates with the noncovalent binding of four to five dye molecules per mole of subfragment 1. Three to four of these dye molecules can be preferentially removed from subfragment 1 in the presence of 1 M KCl without relieving the inhibition. This inhibition, which can be traced to one dye molecule per subfragment 1, is reversible and is facilitated in the presence of MgADP and MgATP, suggesting that the dye does not bind at the active site of subfragment 1. Our observations are explained in terms of hydrophobic and electrostatic protein--dye interactions.     

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.     

Reaction intermediates of myosin ATPase from scallop adductor muscles: nonidentical two-headed structure of striated adductor muscle myosin

To determine whether or not the two heads of myosin from striated adductor muscles of scallop are nonidentical and the main intermediate of the ATPase reaction, MADPP, is produced only on one of the two heads, the Pi-burst size, the amount of total bound nucleotides and the amount of bound ADP during the ATPase reaction were measured in this study. The Pi-burst size was 1 mol per mol in the presence of 0.1-5 mM Mg2+ ions. The amount of total nucleotides bound to myosin was 2 mol per mol. Both the amounts of bound ADP and ATP at sufficiently high ATP concentrations were 1 mol per mol of striated adductor myosin, and the affinity for ADP binding was higher than that for ATP binding. These findings indicate that MADPP or MATP is produced on each of the two heads of striated adductor myosin on its interaction with ATP. The fluorescence intensity at 340 nm of striated adductor myosin was enhanced by about 7% upon addition of ATP. The time for the half maximum fluorescence enhancement, tau 1/2, at 5 microM ATP was 0.25 s, which was almost equal to the tau 1/2 values for the Pi-burst and for the dissociation of actomyosin reconstituted from striated adductor myosin and skeletal muscle F-actin. The dependences on ATP concentration of the extent of the fluorescence enhancement and the dissociation of actomyosin could be explained by assuming that these changes are associated with the formation of MADPP on one of the two heads of myosin. The Pi-burst size and the amount of bound ADP of smooth adductor myosin were slightly but significantly larger than 1 mol per mol. Both ATPase reactions of striated and smooth adductor myofibrils showed the substrate inhibition. The extent of substrate inhibition of ATPase of smooth adductor myofibrils was less than that of striated adductor myofibrils. All the present findings support the view that the nonidentical two-headed structure is required for substrate inhibition of the actomyosin ATPase reaction.     

Characterization of two types of binding sites of substrate-like inhibitors in the heavy meromyosin molecule

The effects of several phosphorylating and alkylating analogs of the substrate on the ATPase activity of myosin and heavy meromyosin were compared. The data obtained confirmed the previously made assumption on the existence of two types of substrate-like inhibitor binding sites in the enzyme molecule. In one of the sites, presumably in the active one, there occurs a reversible competitive inhibition characterized by a high affinity for the inhibitors, which are mixed anhydrides of various mononucleotides and mesitylcarboxylic acid or its derivatives. An enhancement of hydrophobicity of these compounds causes an increase in their affinity for this site. At much higher concentrations of the inhibitors an irreversible inhibition takes place, the rate of inhibition being decreased with an increase in the phosphorylating capacity of the compound. This site possesses a far lower affinity for the inhibitors and reveals a certain specificity with respect to the analog mononucleotide moiety structure, i.e. a replacement of the 6-NH2-group by the 6-OH-group or an increase in the number of the phosphate residues result in a decrease of the efficiency of inhibition. No correlation between the analog capacity to cause irreversible inhibition and to act as an effective competitive inhibitor of reversible type has been shown to exist, thus allowing to use inhibitors of preferable action in one of the two types of the binding sites. No irreversible inhibition site was revealed when the ATPase activity of myosin subfragment I with and without the DTNB chains was investigated. Actin protects myosin against the inhibiting action of the analogs tested.     

Inhibition of actin-tropomyosin activation of myosin MgATPase activity by the smooth muscle regulatory protein caldesmon.

Caldesmon inhibition of actin-tropomyosin activation of myosin MgATPase activity was investigated. greater than 90% inhibition of ATPase activation correlated with 0.035-0.1 caldesmon bound per actin monomer over a wide range of conditions. Caldesmon inhibited sheep aorta actin-tropomyosin activation of skeletal muscle heavy meromyosin (HMM) by 85%, but had no effect on the binding affinity of HMM.ADP.Pi to actin. At ratios of 2 and 0.12 subfragment 1 (S1):1 actin, addition of caldesmon inhibited the ATPase activation by up to 95%, but did not alter the fraction of S1.ADP.Pi associated with actin-tropomyosin. We concluded that caldesmon inhibited actomyosin ATPase by slowing the rate-limiting step of the activation pathway. At concentrations comparable to the ATPase measurements, S1 displaced caldesmon from native thin filaments both in the absence (rigor) and the presence of MgATP. We therefore concluded that caldesmon could displace S1.ADP.Pi from actin-tropomyosin only under exceptional circumstances. An expressed mutant of caldesmon comprising just the C-terminal 99 amino acids bound actin 10 times weaker than whole caldesmon but otherwise inhibited actin-tropomyosin activation with the same potency and same mechanism as intact caldesmon. Thus, the entire inhibitory function of caldesmon resides in its extreme C terminus.