Localisation of light chain and actin binding sites on myosin

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.     

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.     

Interactions of myosin subfragment 1 isozymes with G-actin

The polymerization of G-actin by myosin subfragment 1 (S-1) isozymes, S-1(A1) and S-1(A2), and their proteolytically cleaved forms was studied by light-scattering, fluorescence, and analytical ultracentrifugation techniques. As reported previously, S-1(A1) polymerized G-actin rapidly while S-1(A2) could hardly promote the assembly reaction (Chaussepied & Kasprzak, 1989a; Chen and Reisler, 1990). This difference between the isozymes of S-1 was traced to the very poor, if any, ability of G-actin-S-1(A2) complexes to nucleate the assembly of actin filaments. The formation of G-actin-S-1(A2) complexes was verified in sedimentation velocity experiments and by fluorescence measurements using pyrene-labeled actin. The G-actin-S-1(A2) complexes supported the growth of actin filaments and accelerated the polymerization of actin in solutions seeded with MgCl2-, KCl-, and S-1(A1)-generated nuclei. The growth rates of actin-S-1(A2) filaments were markedly slower than those for actin-S-1(A1) filaments. Proteolytic cleavage of S-1 isozymes at the 50/20-kDa junction of the heavy chain greatly decreased their binding to G-actin and thus inhibited the polymerization of actin by S-1(A1). These results are discussed in the context of G-actin-S-1 interactions.     

Isolation and characterization of the N-terminal 23-kilodalton fragment of myosin subfragment 1

The 23-kDa N-terminal tryptic fragment was isolated from the heavy chain of rabbit skeletal myosin subfragment 1 (S-1). The heavy-chain fragments were dissociated by guanidine hydrochloride following limited trypsinolysis, and the 23-kDa fragment was isolated by gel filtration and ion-exchange chromatography. Finally, the fragment was renatured by removing the denaturants. The CD spectrum of the renatured fragment shows the presence of ordered structure. The tryptophan fluorescence emission spectrum of the fragment is considerably shifted to the red upon adding guanidine hydrochloride which indicates that the tryptophans are located in relatively hydrophobic environments. The two 23-kDa tryptophans, unlike the rest of the S-1 tryptophans, are fully accessible to acrylamide as indicated by fluorescence quenching. The isolated 23-kDa fragment cosediments with F-actin in the ultracentrifuge and significantly increases the light scattering of actin in solution which indicates actin binding. The binding is rather tight (Kd = 0.1 microM) and ionic strength dependent (decreasing with increasing ionic strength). ATP, pyrophosphate, and ADP dissociate the 23-kDa-actin complex with decreasing effectiveness. The isolated 23-kDa fragment does not have ATPase activity; however, it inhibits the actin-activated ATPase activity of S-1 by competing presumably with S-1 for binding sites on actin. F-Actin binds to the 23-kDa fragment immobilized on the nitrocellulose membrane. The fragment was further cleaved, and one of the resulting peptides, containing the 130-204 stretch of residues, was found to bind actin on the nitrocellulose membrane, indicating that this region of the 23-kDa fragment participates in forming an actin binding site.     

Localization and characterization of a 7.3-kDa region of caldesmon which reversibly inhibits actomyosin ATPase activity.

Cleavage of caldesmon with chymotrypsin yields a series of fragments which bind both calmodulin and actin and inhibit the binding of myosin subfragments to actin and the subsequent stimulation of ATPase activity. Several of these fragments have been purified by cation exchange chromatography and their amino-terminal sequences determined. The smallest fragment has a molecular mass of about 7.3 kDa and extends from Leu597 to Phe665. This polypeptide inhibits the actin-activated ATPase of myosin S-1; this inhibition is augmented by smooth muscle tropomyosin and relieved by Ca(2+)-calmodulin. The binding of the 7.3-kDa fragment to actin is competitive with the binding of S-1 to actin. Thus, this polypeptide has several of the important features characteristic of intact caldesmon. However, although an intact caldesmon molecule covers between six and nine actin monomers, the 7.3-kDa fragment binds to actin in a 1:1 complex. Comparison of this fragment with others suggests that a small region of caldesmon is responsible for at least part of the interaction with both calmodulin and actin.     

Superprecipitation induced by soluble myosin fragments.

Superprecipitation (s.p.) took place when $\text{both}$ an active myosin fragment [heavy meromyosin (HMM) or HMM subfragment-1 (S-1)] and an inactivated myosin were added to actin. The duration of the “clearing phase” decreased, while the rate and extent of s.p. increased up to a constant value when the myosin fragment concentration was raised. The extent and rate were higher while the delay time shorter for HMM, as compared to S-1 at the same concentration, No s.p. could be detected when: a) an inactivated myosin fragment or the ATPase apyrase was used; b) MgATP was replaced by Mg-pyrophosphate; c) the ability of myosin to form “rigor” complex with actin has been abolished. It is concluded that the soluble myosin fragment is probably involved in the mechanochemical process associated with s.p..     

Interaction of alkali light chain 1 with the isolated 20-kilodalton fragment of myosin subfragment-1 heavy chain and F-actin

The binding of one of the alkali light chains of myosin, A1, with the isolated renatured 20-kDa fragment of myosin subfragment-1 heavy chain was demonstrated by means of difference UV absorption spectroscopy. The difference spectrum with either rabbit or chicken A1 showed two characteristic peaks at 279 and 287 nm indicating a perturbation of tyrosyl chromophores by the association with the 20-kDa fragment. The delta epsilon at 287 nm increased with an increase in the molar ratio of A1/20-kDa fragment and reached a maximum value at around equimolar ratio. The maximum delta epsilon value was approximately three times larger with rabbit A1 than with chicken A1. Based on the positions of Tyr residues in the amino acid sequences, the contact surface of A1 with myosin heavy chain was concluded to be spread over a large area of A1. The binding of 20-kDa fragment with F-actin was measured by following the increase in turbidity. The affinity appeared to increase several times in the presence of A1. A1 may possibly control the affinity of myosin for actin.     

Myosin phosphorylation triggers actin polymerization in vascular smooth muscle

A variety of contractile stimuli increases actin polymerization, which is essential for smooth muscle contraction. However, the mechanism(s) of actin polymerization associated with smooth muscle contraction is not fully understood. We tested the hypothesis that phosphorylated myosin triggers actin polymerization. The present study was conducted in isolated intact or beta-escin-permeabilized rat small mesenteric arteries. Reductions in the 20-kDa myosin regulatory light chain (MLC20) phosphorylation were achieved by inhibiting MLC kinase with ML-7. Increases in MLC20 phosphorylation were achieved by inhibiting myosin light chain phosphatase with microcystin. Isometric force, the degree of actin polymerization as indicated by the F-actin-to-G-actin ratio, and MLC20 phosphorylation were determined. Reductions in MLC20 phosphorylation were associated with a decreased force development and actin polymerization. Increased MLC20 phosphorylation was associated with an increased force generation and actin polymerization. We also found that a heptapeptide that mimics the actin-binding motif of myosin II enhanced microcystin-induced force generation and actin polymerization without affecting MLC20 phosphorylation in beta-escin-permeabilized vessels. Collectively, our data demonstrate that MLC20 phosphorylation is capable of triggering actin polymerization. We further suggest that the binding of myosin to actin triggers actin polymerization and enhances the force development in arterial smooth muscle.     

A mosaic multiple-binding model for the binding of caldesmon and myosin subfragment-1 to actin.

Binding of caldesmon to actin causes a decrease in the quantity of bound myosin and results in a reduction in the rate of actin-activated adenosine triphosphate hydrolysis. It is generally assumed that the binding of caldesmon and myosin to actin is a pure competitive interaction. However, recent binding studies of enzyme digested caldesmon subfragments directed at mapping the actin binding site of caldesmon have shown that a small 8-kD fragment around the COOH-terminal can compete directly with the myosin subfragment 1 (S-1) binding to actin; at least one other fragment that binds to actin does not inhibit the actin-activated adenosine triphosphate activity of myosin. That is, only a part of the caldesmon sequence may be responsible for directly blocking the binding of S-1 to actin. This prompts us to question the actual mode of binding of intact caldesmon and myosin S-1 to actin: whether the entire intact caldesmon molecule is competing with S-1 binding (pure competitive model) or just a small part of it (mosaic multiple-binding model). To answer this question, we measured the amount of myosin S-1 and caldesmon bound per actin monomer as a function of the total concentration of S-1 added to the system at constant concentrations of actin and caldesmon. A formalism for calculating the titration data based on the pure competitive model and a mosaic multiple-binding model was then developed. When compared with theoretical calculations, it is found that the binding of caldesmon and S-1 to actin cannot be pure competitive if no cooperativity exists between S-1 and caldesmon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between G-actin and myosin subfragment 1: immunochemical probing of the NH2-terminal segment on actin

The role of the N-terminal segment of actin in myosin-induced polymerization of G-actin was studied by using peptide antibodies directed against the first seven N-terminal residues of alpha-skeletal actin. Light scattering, fluorescence, and analytical ultracentrifugation experiments showed that the Fab fragments of these antibodies inhibited the polymerization of G-actin by myosin subfragment 1 (S-1) by inhibiting the binding of these proteins to each other. Fluorescence measurements using actin labeled with pyrenyliodoacetamide revealed that Fab inhibited the initial step in the binding of S-1 to G-actin. It is deduced from these results and from other literature data that the initial contact between G-actin and S-1 involves residues 1-7 on actin and residues 633-642 on the S-1 heavy chain. This interaction appears to be of major importance for the binding of S-1 and G-actin. The presence of additional myosin contact sites on G-actin was indicated by concentration-dependent recovery of S-1 binding to G-actin without displacement of Fab. The reduced Fab inhibition of S-1 binding to polymerizing and polymerized actin is consistent with the tightening of acto-S-1 binding at these sites or the creation of new sites upon formation of F-actin.     

Polymerization of G-actin by myosin subfragment 1

The polymerization of actin from rabbit skeletal muscle by myosin subfragment 1 (S-1) from the same source was studied in the depolymerizing G-actin buffer. The polymerization reactions were monitored in light-scattering experiments over a wide range of actin/S-1 molar rations. In contrast to the well resolved nucleation-elongation steps of actin assembly by KC1 and Mg2+, the association of actin in the presence of S-1 did not reveal any lag in the polymerization reaction. Light scattering titrations of actin with S-1 and vice versa showed saturation of the polymerization reaction at stoichiometric 1:1 ratios of actin to S-1. Ultracentrifugation experiments confirmed that only stoichiometric amounts of actin were incorporated into a 1:1 acto-S-1 polymer even at high actin/S-1 ratios. These polymers were indistinguishable from standard complexes of S-1 with F-actin as judged by electron microscopy, light scattering measurements, and fluorescence changes observed while using actin covalently labeled with N-(1-pyrenyl)iodoacetamide. F-actin obtained by polymerization of G-actin by S-1 could initiate rapid assembly of G-actin in the presence of 10 mM KC1 and 0.5 mM MgCl2 and showed normal activation of MgATPase hydrolysis by myosin.     

Specific cross-linking of the SH1 thiol of skeletal myosin subfragment 1 to F-actin and G-actin.

Recently, we reported that (maleimidobenzoyl)-G-actin (MBS-G-actin), which was resistant to the salt and myosin subfragment 1 (S-1) induced polymerizations, reacts reversibly and covalently in solution with the S-1 heavy chain at or near the strong F-actin binding region [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. Here, we have readily converted the MBS-G-actin into MBS-F-actin in the presence of phalloidin and salts. The binding of S-1 to the two actin derivatives carrying on their surface free reactive maleimidobenzoyl groups was investigated comparatively in cross-linking experiments performed under various conditions to probe further the molecular structure of the actin-heavy chain complex before and after the polymerization process. Like MBS-G-actin, the isolated MBS-F-actin, which did not undergo any intersubunit cross-linking, bound stoichiometrically to S-1, generating two kinds of actin-heavy chain covalent complexes migrating on electrophoretic gels at 180 and 140 kDa. The relative extent of their production was essentially dependent on pH for both G-and F-actins. At pH 8.0, the 180-kDa species was predominant, and at pH 7.0, the amount of the 140-kDa adduct increased at the expense of the 180-kDa entity. The cross-linking of MBS-F-actin to S-1 led to the superactivation of the MgATPase substantiating the ability of this derivative to stimulate the S-1 ATPase as the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism for the inhibition of acto-heavy meromyosin ATPase by the actin/calmodulin binding domain of caldesmon.

Caldesmon, an actin/calmodulin binding protein, inhibits acto-heavy meromyosin (HMM) ATPase, while it increases the binding of HMM to actin, presumably mediated through an interaction between the myosin subfragment 2 region of HMM and caldesmon, which is bound to actin. In order to study the mechanism for the inhibition of acto-HM ATPase, we utilized the chymotryptic fragment of caldesmon (38-kDa fragment), which possesses the actin/calmodulin binding region but lacks the myosin binding portion. The 38-kDa fragment inhibits the actin-activated HMM ATPase to the same extent as does the intact caldesmon molecule. In the absence of tropomyosin, the 38-kDa fragment decreased the KATPase and Kbinding without any effect on the Vmax. However, when the actin filament contained bound tropomyosin, the caldesmon fragment caused a 2-3-fold decrease in the Vmax, in addition to lowering the KATPase and the Kbinding. The 38-kDa fragment-induced inhibition is partially reversed by calmodulin at a 10:1 molar ratio to caldesmon fragment; the reversal was more remarkable in 100 mM ionic strength at 37 degrees C than in 20 or 50 mM at 25 degrees C. Results from these experiments demonstrate that the 38-kDa domain of caldesmon fragment of myosin head to actin; however, when the actin filament contains bound tropomyosin, caldesmon fragment affects not only the binding of HMM to/actin but also the catalytic step in the ATPase cycle. The interaction between the 38-kDa domain of caldesmon and tropomyosin-actin is likely to play a role in the regulation of actomyosin ATPase and contraction in smooth muscle.     

Antibody directed against the 142-148 sequence of the myosin heavy chain interferes with myosin-actin interaction.

It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.     

The binding of myosin subfragment 1 to actin can be measured by proteolytic rates method

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin subfragment 1 (S-1) were determined for free S-1, acto-S-1, and acto-S-1 in the presence of magnesium adenyl-5'-yl imidodiphosphate (Mg AMP-PNP) and MgATP under ionic strength conditions ranging from 30 to 124 mM. The percentage of S-1 bound to actin in the presence of Mg AMP-PNP and MgATP was calculated from these rates for each set of digestion experiments. Parallel experiments carried out in an Airfuge centrifuge on identical acto-S-1 solutions yielded independent information on the binding of S-1 to actin. The results of binding measurements by these two methods were in excellent agreement in all cases tested, covering the range from 15 to 95% binding of S-1 to actin. Tryptic digestions of synthetic mixtures of S-1 and p-phenylenedimaleimide S-1 in the presence of actin demonstrated that a two-component system of myosin heads with different affinities for actin can be resolved into its constituents by the proteolytic rates method. The results of this work justify applications of the proteolytic rates method to actomyosin binding studies in more complex systems.     

Dictyostelium myosin light chain kinase. Purification and characterization

A Dictyostelium myosin light chain kinase has been purified approximately 15,000-fold to near homogeneity. The purified kinase is a single polypeptide of approximately 34 kDa that phosphorylates only the 18-kDa Dictyostelium myosin regulatory light chain and itself among substrates tested. The enzyme was purified largely by ammonium sulfate fractionation and hydrophobic (butyl) interaction chromatography. Analysis using polyclonal antibodies raised against the purified 34-kDa protein confirms that this protein is responsible for myosin light chain kinase activity. Protein microsequence of the 34-kDa protein reveals conserved protein kinase sequences. The purified Dictyostelium myosin light chain kinase exhibits a Km for Dictyostelium myosin of 4 microM and a Vmax of 8 nmol/min/mg. Unlike other characterized myosin light chain kinases, this enzyme is not regulated by calcium/calmodulin. Western blot analysis demonstrates that the purified kinase is not a proteolytic fragment that has lost calcium/calmodulin regulation. The Dictyostelium myosin light chain kinase activity is not directly regulated by cyclic nucleotides. However, this kinase undergoes an intramolecular autophosphorylation that activates the enzyme.     

Conformational transitions in the myosin head induced by temperature, nucleotide and actin. Studies on subfragment-1 of myosins from rabbit and frog fast skeletal muscle with a limited proteolysis method

Tryptic digestion patterns reveal a close similarity of the substructure of frog subfragment-1 (S1) to that established for rabbit S1. The 97-kDa heavy chain of chymotryptic S1 of frog myosin is preferentially cleaved into three fragments with apparent molecular masses of 29 kDa, 49 kDa and 20 kDa. These fragments correspond to the 27-kDa, 50-kDa and 20-kDa fragments of rabbit S1, respectively; this is indicated by the sequence of their appearance during digestion, by the suppression by actin of the generation of the 49-kDa and 20-kDa peptides, and by a nucleotide-promoted cleavage of the 29-kDa peptide to a 24-kDa fragment and the 49-kDa peptide to a 44-kDa fragment, analogous to the nucleotide-promoted cleavage of the 27-kDa and 50-kDa fragments of rabbit S1 to the 22-kDa and 45-kDa peptides. The same changes in the digestion patterns as those produced by the presence of nucleotide (ATP or its beta,gamma-imido analog AdoP P[NH]P) at 25 degrees C were observed when the digestion was carried out at 0 degrees C in the absence of nucleotide. The low-temperature-induced changes were particularly well seen in the preparations from frog myosin. The presence of ATP or AdoP P[NH]P at 0 degrees C enhanced, whereas the complex formation with actin prevented, the low-temperature-induced changes. The results are consistent with there being two fundamental conformational states of the myosin head in an equilibrium that is dependent on the temperature, the nucleotide bound at the active site, and the presence or absence of actin.     

Functional characterization of skeletal F-actin labeled on the NH2-terminal segment of residues 1-28

Rabbit skeletal alpha-actin was covalently labeled in the filamentous state by the fluorescent nucleophile, N-(5-sulfo-1-naphthyl)ethylenediamine (EDANS) in the presence of the carboxyl group activator 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide (EDC). The coupling reaction was continued until the incorporation of nearly 1 mol EDANS/mol actin. After limited proteolytic digestion of the labeled protein and chromatographic identification of the EDANS-peptides, about 80% of the attached fluorophore was found on the actin segment of residues 1-28, most probably within the N-terminal acidic region of residues 1-7. A minor labeling site was located on the segment that consists of residues 40-113. No label was incorporated into the COOH-terminal moiety consisting of residues 113-375. The isolated EDANS-G-actin undergoes polymerization in the presence of salts but at a rate significantly greater than unlabeled actin. The EDANS-F-actin could be complexed to skeletal chymotryptic myosin subfragment 1 (S-1) and to tropomyosin. The complex formed between EDANS-F-actin and S-1 could not be further crosslinked by EDC but the two proteins were readily joined by glutaraldehyde as observed for native actin-S-1, suggesting that the EDANS-substituted carboxyl site is also involved in the EDC crosslinking of native actin to S-1. Moreover, the EDANS labeling of F-actin resulted in a 20-fold increase in the Km of the actin-activated Mg2+.ATPase of S-1. Thus, this labeling, while it did not much affect the rigor actin-S-1 interaction, changes the actin binding to the S-1-nucleotide complexes significantly. The selective introduction of a variety of spectral probes, like EDANS, or other classes of fluorophores, on the N-terminal region of actin, through the reported carbodiimide coupling reaction, would provide several different derivatives valuable for assessing the functional role of the negatively charged N-terminus of actin during its interaction with myosin and other actin-binding proteins.     

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.     

Interaction of 75-106 actin peptide with myosin subfragment-1 and its trypsin modified derivative

To explore the role of a hydrophobic domain of actin in the interaction with a myosin chain we have synthesized a peptide corresponding to residues 75-106 of native actin monomer and studied by fluorescence and ELISA the interaction (13+/-2.6x10(-6) M) with both S-1 and (27 kDa-50 kDa-20 kDa) S-1 trypsin derivative of myosin. The loop corresponding to 96-103 actin residues binds to the S-1 only in the absence of Mg-ATP and under similar conditions but not to the trypsin derivative S-1. Biotinylated C74-K95 and I85-K95 peptide fragments were purified after actin proteolysis with trypsin. The C74-K95 peptide interacted with both S-1 and the S-1 trypsin derivative with an apparent Kd(app) of 6+/-1.2x10(-6) M in the presence or absence of nucleotides. Although peptide fragment I85-K95 binds to S-1 with a Kd(app) of 12+/-2.4x10(-6) M, this fragment did not bind to the trypsin S-1 derivative. We concluded that the actin 85-95 sequence should be a potential binding site to S-1 depending of the conformational state of the intact 70 kDa segment of S-1.