Nucleotide-induced states of myosin subfragment 1 cross-linked to actin

Actomyosin interactions and the properties of weakly bound states in carbodiimide-cross-linked complexes of actin and myosin subfragment 1 (S-1) were probed in tryptic digestion, fluorescence, and thiol modification experiments. Limited proteolysis showed that the 50/20K junction on S-1 was protected in cross-linked acto-S-1 from trypsin even under high-salt conditions in the presence of MgADP, MgAMPPNP, and MgPPi (mu = 0.5 M). The same junction was exposed to trypsin by MgATP and MgATP gamma S but mainly on S-1 cross-linked via its 50K fragment to actin. p-Phenylenedimaleimide-bridged S-1, when cross-linked to actin, yielded similar tryptic cleavage patterns to those of cross-linked S-1 in the presence of MgATP. By using p-nitrophenylenemaleimide, it was found that the essential thiols of cross-linked S-1 were exposed to labeling in the presence of MgATP and MgATP gamma S in a state-specific manner. In contrast to this, the reactive thiols were protected from modification in the presence of MgADP, MgAMPPNP, and MgPPi at mu = 0.5 M. These modifications were compared with similar reactions on isolated S-1. Experiments with pyrene-actin cross-linked to S-1 showed enhancement of fluorescence intensity upon additions of MgATP and MgATP gamma S, indicating the release of the pyrene probe on actin from the sphere of S-1 influence. The results of this study contrast the "open" structure of weakly bound actomyosin states to the "tight" conformation of rigor complexes.     

Cross-linking of actin to myosin subfragment 1: course of reaction and stoichiometry of products

The cross-linking of actin to myosin subfragment 1 (S-1) with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide was reexamined by using two cross-linking procedures [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306; Sutoh, K. (1983) Biochemistry 22, 1579-1585] and two independent methods for quantitating the reaction products. In the first approach, the cross-linked acto-S-1 complexes were cleaved with elastase at the 25K/50K and 50K/22K junctions in S-1. This enabled direct measurements of the cross-linked and un-cross-linked fractions of the 50K and 22K fragments of S-1. We found that in all cases actin was preferentially cross-linked to the 22K fragment and that the overall stoichiometry of the main cross-linked products was that of a 1:1 complex of actin and S-1. In the second approach, actin was cross-linked to tryptically cleaved S-1, and the course of these reactions was monitored by measuring the decay of the free 50K and 20K fragments and the formation of cross-linked products. After selecting the optimal cross-linking procedure and conditions, we determined that the rate of actin cross-linking to the 20K fragment of S-1 was 3-fold faster than the reaction with the 50K peptide. The overall rate of cross-linking actin to S-1 corresponded to the sum of the individual reactions of the 50K and 20K fragments, indicating their mutually exclusive cross-linking to actin. Thus, the reactions with tryptically cleaved S-1 were consistent with the 1:1 stoichiometry of actin and S-1 in the main cross-linked products and verified the preferential cross-linking of actin to the 20K fragment of S-1. These results are discussed in the context of the binding of actin to S-1.     

Tryptic cleavage and substructure of bovine cardiac myosin subfragment 1

The method of limited tryptic proteolysis has been used to compare and contrast the substructure of bovine cardiac myosin subfragment 1 (S-1) to that of skeletal myosin S-1. While tryptic cleavage of cardiac S-1, like that of skeletal S-1, yields three fragments, the 25K, 50K, and 20K peptides, the digestion of cardiac S-1 proceeds at a 2-fold faster rate. The increased rate of cleavage is due entirely to an order of magnitude faster rate of cleavage at the 25K/50K junction of cardiac S-1 compared to that of skeletal, with approximately equal rates of cleavage at the 50K/20K junctions. Actin inhibits the tryptic attack at this latter junction, but its effect is an order of magnitude smaller for the cardiac than for the skeletal S-1. Furthermore, the tryptic susceptibility of the 50K/20K junction of cardiac S-1 in the acto-S-1 complex is increased in the presence of 2 mM MgADP. This effect is not due to partial dissociation of the cardiac acto-S-1 complex by MgADP. Our results indicate that in analogy to skeletal S-1, the cardiac myosin head is organized into three protease-resistant fragments connected by open linker peptides. However, the much faster rate of tryptic cleavage of the 25K/50K junction and also the greater accessibility of the 50K/20K junction in the cardiac acto-S-1 complex indicate substructural differences between cardiac and skeletal S-1.     

Tryptic digestion of rabbit skeletal myofibrils: an enzymatic probe of myosin cross-bridges

Tryptic digestion of rabbit skeletal myofibrils under physiological ionic strength and pH conditions was used as a probe of cross-bridge interaction with actin in the presence of nucleotides and pyrophosphate. Under rigor conditions, digestion of myofibrils at 24 degrees C results in the formation of 25K, 110K [heavy meromyosin (HMM)], and light meromyosin (LMM) fragments as the main reaction products. Very little if any 50K peptide is generated in such digestions. In the presence of magnesium pyrophosphate, magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and MgATP, the main cleavage proceeds at two positions, 25K and 75K from the N-terminal portion of myosin, yielding the 25K, 50K, and 150K species. The relative amounts of the 50K, 110K, and 150K peptides and the rates of myosin heavy-chain digestion in the presence of pyrophosphate and AMPPNP indicate partial dissociation of myosin from actin. Direct centrifugation measurements of the binding of HMM and subfragment 1 (S-1) to actin in myofibrils confirm that cross-bridges partition between attached and detached states in the presence of these ligands. In the presence of MgADP, HMM and S-1 remain attached to actin at 24 degrees C. However, tryptic digestion of myofibrils containing MgADP is consistent with the existence of a mixed population of attached and detached cross-bridges, suggesting that only one head on each myosin molecule is attached to actin. As shown by tryptic digestion of myofibrils and the measurements of HMM and S-1 binding to actin, nucleotide- and pyrophosphate-induced dissociation of cross-bridges is more pronounced at 4 than at 24 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural aspects of myosin subfragment 1 as studied by subtilisin digestion of trypsin-split S-1 (27 kDa-50 kDa-20 kDa)

Limited subtilisin digestion of myosin subfragment 1 (S-1) was carried out, varying the enzyme: substrate weight ratio from 1:200 to 1:10, and changes in structure, and in the MgATPase activities of S-1 and acto-S-1 after proteolysis, were followed. When the starting material--tryptically-cleaved S-1 (27 kDa-50 kDa-20 kDa) ("split S-1")--was subjected to further subtilisin digestion, it was found that with increasing enzyme concentration, the 50 kDa fragment degraded into an 18 kDa fragment via a 33 kDa peptide (50----33----18 kDa), which was not cross-linked with F-actin. On the other hand, the 27 and 20 kDa fragments were rather stable at lower subtilisin concentrations and started to degrade only at higher subtilisin concentrations. These degradations lowered the MgATPase activities of S-1 and acto-S-1. The losses of MgATPase activities of S-1 and of acto-S-1 were mainly due to the degradations of the 27 and 20 kDa fragments, respectively. Addition of EDTA did not affect the subtilisin cleavage pattern of split S-1 but the breakdown of the 50 kDa fragment was extremely depressed, suggesting that some conformational change of the 50 kDa fragment is induced by the binding of divalent cation. The binding of MgADP to split S-1 accelerated the degradation of the 27 kDa fragment and produced a new cut in the 27 kDa fragment (27----20 kDa), resulting in a further loss of the S-1 MgATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of nitrite reductase from a denitrifier, Alcaligenes sp. NCIB 11015. A novel copper protein

Covalent cross-linking reaction between SH1 and SH2 groups in myosin subfragment-1 (S-1) by N,N'-p-phenylenedimaleimide (pPDM) was followed by the degree of inactivation of NH4+-EDTA ATPase activity. The rate of the cross-linking reaction decreased to less than a 20th in the presence of F-actin. The inhibitory effect of F-actin was not observed in the presence of MgATP. Binding of F-actin to S-1 was measured using ultracentrifugation. S-1 whose SH1 and SH2 were covalently cross-linked by pPDM or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) did not bind F-actin. After the DTNB-cross-linked S-1 is reduced by dithiothreitol, the ability to bind F-actin is recovered. These results suggest that S-1 has a binding site for F-actin in the region between SH1 and SH2. This site appears to determine the high affinity of acto-S-1 complex at the rigor while decreasing the affinity more than 10(2) times in the presence of MgATP.     

Cross-linking of the skeletal myosin subfragment 1 heavy chain to the N-terminal actin segment of residues 40-113

Glutaraldehyde (GA) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), a hydrophobic, carboxyl group directed, zero-length protein cross-linker, were employed for the chemical cross-linking of the rigor complex between F-actin and the skeletal myosin S-1. The enzymatic properties and structure of the new covalent complexes obtained with both reagents were determined and compared to those known for the EDC-acto-S-1 complex. The GA- or EEDQ-catalyzed covalent attachment of F-actin to the S-1 heavy chain induced an elevated Mg2+-ATPase activity. The turnover rates of the isolated cross-linked complexes were similar to those for EDC-acto-S-1 (30 s-1). The solution stability of the new complexes is also comparable to that exhibited by EDC-acto-S-1. The proteolytic digestion of the isolated AEDANS-labeled covalent complexes and direct cross-linking experiments between actin and various preformed proteolytic S-1 derivatives indicated that, as observed with EDC, the COOH-terminal 20K and the central 50K heavy chain fragments are involved in the cross-linking reactions of GA and EEDQ. KI-depolymerized acto-S-1 complexes cross-linked by EDC, GA, or EEDQ were digested by thrombin which cuts only actin, releasing S-1 heavy chain-actin peptide cross-linked complexes migrating on acrylamide gels with Mr 100K (EDC), 110K and 105K (GA), and 102K (EEDQ); these were fluorescent only when fluorescent S-1 was used. They were identified by immunostaining with specific antibodies directed against selected parts of he NH2-terminal actin segment of residues 1-113.(ABSTRACT TRUNCATED AT 250 WORDS)     

MgADP-induced changes in the structure of myosin S1 near the ATPase-related thiol SH1 probed by cross-linking

The structural consequences of MgADP binding at the vicinity of the ATPase-related thiol SH1 (Cys-707) have been examined by subjecting myosin subfragment 1, premodified at SH2 (Cys-697) with N-ethylmaleimide (NEM), to reaction with the bifunctional reagent p-phenylenedimaleimide (pPDM) in the presence and absence of MgADP. By monitoring the changes in the Ca2(+)-ATPase activity as a function of reaction time, it appears that the reagent rapidly modifies SH1 irrespective of whether MgADP is present or not. In the absence of nucleotide, only extremely low levels of cross-linking to the 50-kDa middle segment of S1 can be detected, while in the presence of MgADP substantial cross-linking to this segment is observed. A similar cross-link is also formed if MgADP is added subsequent to the reaction of the SH2-NEM-pre-modified S1 with pPDM in the absence of nucleotide. Isolation of the labeled tryptic peptide from the cross-linked adduct formed with [14C]pPDM, and subsequent partial sequence analyses, indicates that the cross-link is made from SH1 to Cys-522. Moreover, it appears that this cross-link results in the trapping of MgADP in this S1 species. These data suggest that the binding of MgADP results in a change in the structure of S1 in the vicinity of the SH1 thiol relative to the 50-kDa "domain" which enables Cys-522 to adopt the appropriate configuration to enable it to be cross-linked to SH1 by pPDM.     

Effect of nucleotide on the binding of N,N'-p-phenylenedimaleimide-modified S-1 to unregulated and regulated actin

In our previous study [Chalovich, J. M., Greene, L. E., & Eisenberg, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4909-4913], myosin subfragment 1 that was modified by having its two reactive thiol groups cross-linked by N,N'-p-phenylenedimaleimide (pPDM) was found to resemble the myosin subfragment 1-adenosine 5'-triphosphate (S-1.ATP) complex in its interaction with actin. In the present study, we examined the effect of actin on adenosine 5'-diphosphate (ADP) trapped at the active site of pPDM.S-1. Our results indicate first that, in the presence of actin, ADP is no longer trapped at the active site but exchanges rapidly with free nucleotide. Different pPDM.S-1.nucleotide complexes were then formed by exchanging nucleotide into the active site of pPDM.S-1 in the presence of actin. The binding of pPDM.S-1.ATP or pPDM.S-1.PPi to actin is virtually identical with that of unmodified S-1 in the presence of ATP. Specifically, at mu = 18 mM, 25 degrees C, pPDM.S-1.ATP or pPDM.S-1.PPi binds to unregulated actin with the same affinity as does S-1.ATP, and this binding does not appear to be affected by troponin-tropomyosin. On the other hand, pPDM.S-1.ADP and pPDM.S-1 with no bound nucleotide both show a small, but significant, difference between their binding to actin and the binding of S-1.ATP; pPDM.S-1 and pPDM.S-1.ADP both bind about 2- to 3-fold more strongly to unregulated actin than does S-1.ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthetic peptide of the sequence 632-642 on myosin subfragment 1 inhibits actomyosin ATPase activity.

A synthetic peptide corresponding to a sequence 632-642 (S632-642) on the myosin subfragment 1 (S-1) heavy chain and spanning the 50/20 kDa junction of S-1 binds to actin in the presence and absence of S-1. The binding of 1.0 mole of peptide per actin causes almost complete inhibition of actomyosin ATPase activity and only partial inhibition of S-1 binding to actin. The binding of S632-642 to the N-terminal segment of actin is supported by competitive carbodiimide cross-linking of S-1 and S632-642 to actin and the catalytic properties of cross-linked acto-S-1 and actin-peptide complexes. These results show that the sequence 632-642 on S-1 is an autonomous binding site for actin and confirm the catalytic importance of its interactions with the N-terminal segment of actin.     

New states of actomyosin

Unstained frozen hydrated samples of myosin subfragment 1 (S-1) cross-linked to actin with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide have been examined by electron microscopy in an effort to probe structural states of the attached cross-bridge. The cross-linked complex in the absence of ATP has a rigor-like appearance. In contrast, both in the presence of ATP and after the N, N'-p-phenylenedimaleimide (pPDM) bridging of the reactive thiols of S-1, the covalently attached cross-bridges of the acto X S-1 complex appear more disordered and no longer assume the characteristic rigor 45 degrees angle with the actin filaments. The images both in the presence and absence of ATP bear a striking resemblance to those obtained by negative staining of the cross-linked acto X S-1 complex (Craig, R., Greene, L. E. & Eisenberg, E. (1985) Proc. Natl. Acad. Sci. U.S. A. 82, 3247-3251). The actin-bound pPDM S-1 complex, formed by treating the cross-linked complex with pPDM in the presence of ATP, is an expected analog of the weakly bound cross-bridge state. The disordered appearance of S-1 molecules of the cross-linked complex in the presence of ATP and after pPDM treatment may reflect the structural state of the weakly bound cross-bridge.     

Functional, chymotryptically split actin and its interaction with myosin subfragment 1

We have prepared chymotryptically split actin that retains the characteristic properties of intact actin. Chymotryptic digestion of G-actin produces an intermediate 35-kilodalton (kDa) fragment and from this a final product of 33 kDa known as the C-terminal "core". These fragments remain attached to an N-terminal 10-kDa fragment. The 35-kDa-10-kDa complex is able to polymerize upon addition of KCl and MgCl2, like intact actin, whereas the 33-kDa-10-kDa complex is not. The 35-kDa-10-kDa complex is here termed "split actin". In the rigor state, split actin binds to myosin subfragment 1 (S-1) strongly, with the same stoichiometry as intact actin. In the rigor state, split actin forms a carbodiimide-induced cross-linked product with S-1; the cross-linking sites on the split actin and on S-1 were proved to be the N-terminal 10-kDa fragment of split actin and the 20-kDa domain of S-1. There was no cross-linking between the 50-kDa domain of S-1 and the 10 kDa of actin. Therefore, the structure of the split actin-S-1 complex differs somewhat from that of the complex with intact actin. The cross-linking of split actin to S-1 causes superactivation of S-1 ATPase to approximately the same extent as does cross-linking of intact actin, whereas non-cross-linked split actin activates S-1 ATPase to a lesser extent. The N-terminus of the 35-kDa fragment was found to be residue 45 (Val-45) by amino acid sequence analysis; so there is no residue missing in split actin.     

Potentiated state of the tropomyosin actin filament and nucleotide-containing myosin subfragment 1

The main purpose of this study was to determine whether potentiation of acto-S-1 ATPase activity (activity higher than that obtained with tropomyosin-free actin) could be caused by nucleotide-containing acto-S-1 complexes. In addition, we wanted to know whether these complexes also have a positive cooperative effect on their own apparent binding constant under conditions where nucleotide-free acto-S-1 complexes cause potentiation of ATPase activity. Using calcium-saturated troponin-tropomyosin actin filaments, we observed potentiation of ATPase activity in the presence of 5.0 mM magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP) and calculated that the ability of acto-S-1-AMPPNP complexes to cause potentiation must have been very similar to that of nucleotide-free acto-S-1 complexes. In extension of earlier studies, potentiated acto-S-1 ATPase activity was characterized by an increase in Vmax and, as observed before, a lowering of the apparent Km for subfragment 1 (S-1). Under conditions similar to those that produce the potentiation of acto-S-1 ATPase activity, the apparent actin binding constant of nucleotide-free S-1 was increased about 3-5 fold while the apparent binding constant of AMPPNP to actin-bound S-1 was reduced to (2.5-10) x 10(2) M-1 compared to that of about (1-5) x 10(3) M-1 for S-1 bound to tropomyosin-free actin. Under the same conditions, the apparent binding constant of S-1-AMPPNP to actin was not increased. We suggest that a potentiated state of the tropomyosin actin filament is produced by the cooperative action of acto-S-1 or acto-S-1-AMPPNP complexes. The potentiated state is characterized by an increase in the Vmax of the acto-S-1 ATPase activity, increased binding constants for S-1 and S-1-ADP, and increased binding of tropomyosin to actin.     

Nucleotide-induced changes in the proteolytically sensitive regions of myosin subfragment 1

Limited proteolytic digestions of myosin subfragment 1 (S-1) with elastase, subtilisin, papain, and thermolysin yield fragments that correspond within 1-2K daltons to the 25K, 50K, and 20K fragments produced by trypsin. While papain and thermolysin cut preferentially at the 26K/70K junction, elastase and subtilisin cleave both the 26K/70K and the 75K/22K junctions in S-1. Using the above proteases as conformational probes, we have previously demonstrated that the binding of actin is sensed at both the 26K/50K and the 50K/22K junctions [Applegate, D., & Reisler, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7109-7112]. We report here that the binding of nucleotides at the active site is also sensed at both junctions. Both 2 mM MgADP and 5 mM MgATP slow the rate of elastase and subtilisin cleavage of the 95K heavy chain. With elastase, the 3-fold decrease in the rate of cleavage induced by nucleotides is evidenced at both the 26K/50K and the 50K/22K junctions. The analysis of subtilisin digestions is complicated by Mg nucleotide induced cleavage at a new site to produce a 91K fragment. Using N-methyl-6-anilinonaphthalene-2-sulfonyl chloride (MnsCl) to fluorescently label the 26K peptide, we demonstrate that the additional cleavage site is approximately 4K daltons from the N-terminal portion of the 95K heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of nucleotide on interaction of the 567-578 segment of myosin heavy chain with actin

To probe the effect of nucleotide on the formation of ionic contacts between actin and the 567-578 residue loop of the heavy chain of rabbit skeletal muscle myosin subfragment 1 (S1), the complexes between F-actin and proteolytic derivatives of S1 were submitted to chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. We have shown that in the absence of nucleotide both 45 kDa and 5 kDa tryptic derivatives of the central 50 kDa heavy chain fragment of S1 can be cross-linked to actin, whereas in the presence of MgADP.AlF4, only the 5 kDa fragment is involved in cross-linking reaction. By the identification of the N-terminal sequence of the 5-kDa fragment, we have found that trypsin splits the 50 kDa heavy chain fragment between Lys-572 and Gly-573, the residues located within the 567-578 loop. Using S1 preparations cleaved with elastase, we could show that the residue of 567-578 loop that can be cross-linked to actin in the presence of MgADP.AlF4 is Lys-574. The observed nucleotide-dependent changes of the actin-subfragment 1 interface indicate that the 567-578 residue loop of skeletal muscle myosin participates in the communication between the nucleotide and actin binding sites.     

Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head

We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.     

The conformation of cross-linked actin.S-1 in the presence and absence of ATP

Electron microscopy studies have shown that the structure of the complex of myosin subfragment 1 (S-1) cross-linked to actin with 1-ethyl-3-[3-(dimethyl-amino) propyl] carbodiimide is very different in the presence and absence of ATP (Craig, R., Greene, L. E., and Eisenberg, E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3247-3251). More recent studies have found that the structure of the cross-linked complex between S-1 modified extensively with N-ethylmaleimide (NEM.S-1) and actin resembles that of the rigor complex both in the presence and absence of ATP, whereas the structure of the cross-linked complex between S-1 modified with N',N'-p-phenylenedimaleimide (pPDM.S-1) and actin resembles that of the cross-linked actin.S-1 complex in the presence of ATP. In the present study, we have obtained biochemical evidence supporting these results. The conformation of the different cross-linked actin.S-1 complexes was determined by studying their effect on the troponin-tropomyosin-actin complex (regulated actin). The basis of this probe for conformation is that S-1.ATP, which is in the weak-binding conformation, interacts very differently with regulated actin than S-1 or S-1.ADP, which are in the strong-binding conformation. We find that both in the presence and absence of ATP, cross-linked NEM.S-1 appears to be in the strong-binding conformation, whereas cross-linked pPDM.S-1 appears to be shifted toward the weak-binding conformation. In contrast, cross-linked unmodified S-1 appears to be in the strong-binding conformation in the presence of ADP and the weak-binding conformation in the presence of ATP. Therefore, in agreement with electron microscopy studies, the cross-linked actin.S-1 complex appears to be able to alternate between the weak-binding and strong-binding conformation during the cross-bridge cycle.     

Interaction of myosin subfragment-1 with actin. II. Location of the actin binding site in a fragment of subfragment-1 heavy chain

The heavy chain of subfragment-1 prepared by chymotrypsin treatment had a molecular weight of about 96K. The heavy chain was split into 26 K, 50 K, and 21 K fragments by trypsin. When the trypsin-treated subfragment-1 was cross-linked with dimethyl suberimidate, cross-linked products of 26 K, 50 K, and 21 K fragments and of 50 K and 21 K fragments appeared, but there was little cross-linked product of 26 K and 50 K fragments or of 26 K and 21 K fragments. When the cross-linking experiments were carried out in the presence of actin, a new band appeared and the amount of cross-linked product of 26 K, 50 K, and 21 K fragments decreased by about 50%. The molecular weight of the new band was lower than that of the cross-linked product of 26 K, 50 K, and 21 K fragments, and higher than that of the dimer of actin. Based on this and some other results, we suggest that this band represented a cross-linked product of actin and the 50 K fragment. We also suggest that the decrease in the amount of cross-linked product of 26 K, 50 K, and 21 K fragments reflected the conformational change in subfragment-1 due to the binding of actin.     

Propagation of Acto-S-1 ATPase reaction-coupled conformational change in actin along the filament

F-Actin was partially cross-linked to myosin subfragment-1 (S-1) at various molar ratios (r = S-1/actin) with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The cross-linked acto-S-1 ATPase showed so called "super-activation," Vx. S-1 was added further to the cross-linked acto-S-1 and the ATPase activity, Vy, was measured. Since the added S-1 can interact only with the bare actin protomers within the cross-linked actin filament, the difference, delta V = Vy - Vx - Vs (where Vs is the ATPase activity of the additional S-1 alone), can indicate the state of the bare actin protomers while the cross-linked acto-S-1 is hydrolyzing ATP. With increasing r, delta V decreased much more rapidly than delta Vo(1 - r) (where delta Vo is delta V at r = 0) and reached a minimum around r = 0.15. As r increased further, delta V approached the level of delta Vo(1 - r). When SH1/SH2-blocked S-1 was cross-linked to F-actin, delta V decreased according to delta Vo(1 - r). Therefore, the large reduction of delta V, observed when intact S-1 was cross-linked, was coupled to the high ATPase activity of the cross-linked acto-S-1. Combining these data with other kinetic data, we could deduce that structural distortion in a cross-linked actin induced by the ATPase reaction of the S-1 partner propagated over several bare actin protomers along the filament and reduced their affinity for the S-1-ADP-Pi complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular movements in the actomyosin complex: F-actin-promoted internal cross-linking of the 25- and 20-kDa heavy chain fragments of skeletal myosin subfragment.

We describe, for the first time, the F-actin-promoted changes in the spatial relationship of strands in the NH2-terminal 25-kDa and COOH-terminal 20-kDa heavy chain fragments of the skeletal myosin subfragment 1 (S-1), detected by their exclusive chemical cross-linking in the rigor F-actin-S-1 complex with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS). Quantitative electrophoretic analysis of the reaction products showed extensive conversion of the 95-kDa heavy chain of the actin-bound S-1 into a new species with an apparent mass of 135 kDa (yield = 50-60%), whereas the heavy chain mobility remained unaffected when actin was omitted. The 135-kDa entity retained the fluorescence of AEDANS-S-1 but not of AEDANS-actin, indicating that it was not a cross-linked acto-heavy chain adduct. Its extent of production depended markedly on the S-1: actin molar ratio and was maximum near a ratio of 1:4. The MBS treatment of acto-S-1 led also to some covalent actin-actin oligomers which could be suppressed by using trypsin-truncated F-actin lacking Cys-374, without altering the generation of the 135-kDa heavy chain derivative.(ABSTRACT TRUNCATED AT 250 WORDS)