Common antigenic determinants of the tubulin binding domains of the microtubule-associated proteins MAP-2 and tau.

The structural-functional aspects of the tubulin binding domain on the microtubule-associated protein MAP-2, and its relationship with the tubulin binding domain on tau, were studied using anti-idiotypic antibodies that react specifically with the epitope(s) on MAPs involved in their interaction with tubulin in addition to other tau and MAP-2 specific antibodies. Previous studies showed that MAP-2 and tau share common binding sites on tubulin defined by the peptide sequences alpha (430-441) and beta (422-434) of tubulin subunits. Furthermore, binding experiments revealed the existence of multiple sites for the interaction of the alpha- and beta-tubulin peptides with MAP-2 and tau. Most recent studies showed that the synthetic tau peptide Val187-Gly204 (VRSKIGSTENLKHQPGGG) from the repetitive sequence on tau defines a tubulin binding site on tau. Our present immunological studies using anti-idiotypic antibodies which interact with the synthetic tau peptide and antibodies against the Val187-Gly204 tau peptide indicate that MAP-2 and tau share common antigenic determinants at the level of their respective tubulin binding domains. These antigenic determinants appear to be present in the 35 kDa tubulin binding fragment of MAP-2 and in 18-20 kDa chymotryptic fragments containing the tubulin binding site(s) on MAP-2. These findings, along with structural information on these proteins, provide strong evidence in favor of the hypothesis that tubulin binding domains on MAP-2 and tau share similar structural features.     

MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain

BACKGROUND: MAP2 and tau are abundant microtubule-associated proteins (MAPs) in neurons. The development of neuronal dendrites and axons requires a dynamic interaction between microtubules and actin filaments. MAPs represent good candidates to mediate such interactions. Although MAP2c and tau have similar, well-characterized microtubule binding activities, their actin interaction is poorly understood. RESULTS: Here, we show by using a cosedimentation assay that MAP2c binds F-actin. Upon actin binding, MAP2c organizes F-actin into closely packed actin bundles. Moreover, we show by using a deletion approach that MAP2c's microtubule binding domain (MTBD) is both necessary and sufficient for both F-actin binding and bundling activities. Surprisingly, even though the MAP2 and tau MTBDs share high sequence homology and possess similar microtubule binding activities, tau is unable to bind or bundle F-actin. Furthermore, experiments with chimeric proteins demonstrate that the actin binding activity fully correlates with the ability to promote neurite initiation in neuroblastoma cells. CONCLUSIONS: These results provide the first demonstration that the MAP2c and tau MTBD domains exhibit distinct properties, diverging in actin binding and neurite initiation activities. These results implicate a novel actin function for MAP2c in neuronal morphogenesis and furthermore suggest that actin interactions could contribute to functional differences between MAP2 and tau in neurons.     

Capping and dynamic relation between domains 1 and 2 of gelsolin

Gelsolin is a protein that severs and caps actin filaments. The two activities are located in the N-terminal half of the gelsolin molecules. Severing and subsequent capping requires the binding of domains 2 and 3 (S2–3) to the side of the filaments to position the N-terminal domain 1 (S1) at the barbed end of actin (actin subdomains 1 and 3). The results provide a structural basis for the gelsolin capping mechanism. The effects of a synthetic peptide derived from the sequence of a binding site located in gelsolin S2 on actin properties have been studied. CD and IR spectra indicate that this peptide presented a secondary structure in solution which would be similar to that expected for the native full length gelsolin molecule. The binding of the synthetic peptide induces conformational changes in actin subdomain 1 and actin oligomerization. An increase in the polymerization rate was observed, which could be attributed to a nucleation kinetics effect. The combined effects of two gelsolin fragments, the synthetic peptide derived from an S2 sequence and the purified segment 1 (S1), were also investigated as a molecule model. The two fragments induced nucleation enhancement and inhibited actin depolymerization, two characteristic properties of capping. In conclusion, for the first time it is reported that the binding of a small synthetic fragment is sufficient to promote efficient capping by S1 at the barbed end of actin filaments. ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

NMR of a synthetic peptide spanning the triphosphate binding site of adenosine 5'-triphosphate in actin

The amino acid residues 114-118 in actin were found to be implicated strongly in the binding of nucleotide, and as would be expected for such an important binding site, they are located in a completely conserved region of the actin sequence. A 19-residue peptide with the actin sequence 106-124 was synthesized in order to span the putative triphosphate binding site. Proton NMR spectra of the actin peptide 114-118 in the presence and absence of ATP indicated that Arg-116 and Lys-118 are particularly involved in binding ATP. A strong binding of ATP to the peptide 106-124 also was measured. Tripolyphosphate bound to the peptide 106-124 somewhat more weakly than ATP. Binding involved residues 115-118 and 121-124, indicating the presence of a reverse turn between these segments. Proton resonances were assigned by using two-dimensional double quantum correlated spectroscopy, one-dimensional spin decoupling techniques, one-dimensional nuclear Overhauser enhancement difference spectroscopy, and pH titration. The alpha CH resonances of Ala-3 and Asn-6 are markedly shifted downfield with respect to values in small unstructured peptides due to their close proximity to the side chains of Pro-4 and Pro-7, respectively. Several other resonances display chemical shifts which are indicative of a structured environment. Assignment of the amide proton resonances in H2O and measurements of the coupling constant 3JHNCH and the chemical shifts of the amide protons reveal that much of the synthetic peptide, particularly the backbone, exhibits a highly structured environment and represents a good model for the triphosphate binding site in actin.     

A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau

The protein domain responsible for the interaction of tau with tubulin has been identified. Biophysical studies indicated that the synthetic peptide Val187-Gly204 (VRSKIG-STENLKHQPGGG) from the repetitive sequence on tau binds to two sites on the tubulin heterodimer and to one site on each of the microtubule-associated protein-interacting C-terminal tubulin peptides alpha(430-441) and beta(422-434). The binding data showed a relatively stronger interaction of Val187-Gly204 with beta(422-434) as compared to that with alpha(430-441). The interaction of this tau peptide with either alpha or beta tubulin peptides appears to be associated with conformational changes in both the tau and the tubulin peptides. The beta tubulin peptide also appears to induce a structural change of tau fragment Val218-Gly235. Interestingly, tau peptides Val187-Gly204 and Val218-Gly235 induced tubulin self-assembly in a cold-reversible fashion, and incorporated into the assembled polymers. The specificity of the interaction of the tau peptide was supported by the competition of tau protein for the interaction with the tubulin polymer. In addition, the tau peptide appears to contain the principal antigenic determinant(s) recognized by anti-idiotypic antibodies that react with the tubulin binding domains on microtubule-associated proteins. The present findings together with the demonstration of the presence of multiple sites for the binding of the alpha(430-441) and beta(422-434) tubulin fragments to tau, and the existence of repetitive sequences on tau, strongly support the hypothesis that the region of tau defined by the repetitive sequences is involved in its interaction with tubulin.     

Evidence that a 27-residue sequence is the actin-binding site of ABP-120

Proteolysis experiments of ABP-120 from Dictyostelium discoideum have previously demonstrated that removal of residues 89-115 from a tryptic peptide which retains actin binding activity, abolishes actin binding (Bresnick, A. R., Warren, V., and Condeelis, J. (1990) J. Biol. Chem. 265, 9236-9240). Antibodies made against a synthetic peptide of this 27-amino acid sequence (27-mer) specifically immunoprecipitate native ABP-120 from Dictyostelium high speed supernatants, demonstrating that the 27-mer sequence is on the surface of the molecule as expected for an active site. ABP-120 is inhibited in its binding to F-actin by Fab' fragments of the anti-27-mer IgG. Half-maximal inhibition occurs at an approximate molar ratio of 7 Fab' fragments/ABP-120 monomer. Viscoelastic measurements indicate that ABP-120 forms fewer cross-links with F-actin in the presence of the 27-mer synthetic peptide than in its absence. In F-actin cosedimentation assays, the binding of ABP-120 to actin is inhibited by the 27-mer synthetic peptide. Furthermore, the 27-mer synthetic peptide cosediments with F-actin, whereas a control hydrophobic peptide and a synthetic peptide of residues 69-88 of ABP-120 do not cosediment with F-actin. These observations suggest a direct involvement of the 27-mer sequence in the actin binding activity of ABP-120.     

Copper binding properties of a tau peptide associated with Alzheimer's disease studied by CD, NMR, and MALDI-TOF MS

We have previously reported the copper binding properties of R3 peptide (residues 318-335: VTSKCGSLGNIHHKPGGG, according to the longest tau protein) derived from the third repeat microtubule-binding domain of water-soluble tau protein. In this work, we have investigated copper binding properties of R2 peptide (residues 287-304: VQSKCGSKDNIKHVPGGG) derived from the second repeat region of tau protein. Similar to R3 peptide, R2 peptide also plays an important role in the formation of neurofibrillary tangles (NFTs) which is one of the two main biological characteristics of Alzheimer's disease (AD). Based on the copper binding properties of R2 peptide, the possible influences of the binding on the formation of NFTs were investigated. Results from circular dichroism (CD) spectra, nuclear magnetic resonance (NMR) spectroscopy, and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) suggest that the binding is pH-dependent and stoichiometry-determined. In addition, these results also reveal that R2 peptide adopts a monomeric alpha-helical structure in aqueous solutions at physiological pH after the addition of 1 mol equiv. of Cu2+. Since alpha-helix structure is responsible for the formation of paired helical filaments (PHFs) which aggregate into NFTs, it is hypothesized that Cu2+ induces R2 peptide to self-assemble into a PHFs-like structure. Hence, it is postulated that Cu2+ plays an important role in the aggregation of R2 peptide and tau protein and that copper binding to R2 peptide may be another possible involvement in AD.     

The specific NH2-terminal sequence Ac-EEED of alpha-smooth muscle actin plays a role in polymerization in vitro and in vivo

The blocking effect of the NH2-terminal decapeptide of alpha-smooth muscle (SM) actin AcEEED-STALVC on the binding of the specific monoclonal antibody anti-alpha SM-1 (Skalli, O., P. Ropraz, A. Trzeviak, G. Benzonana, D. Gillessen, and G. Gabbiani. 1986. J. Cell Biol. 103:2787-2796) was compared with that of synthetic peptides modified by changing the acetyl group or by substituting an amino acid in positions 1 to 5. Using immunofluorescence and immunoblotting techniques, anti-alpha SM-1 binding was abolished by the native peptide and by peptides with a substitution in position 5, indicating that AcEEED is the epitope for anti-alpha SM-1. Incubation of anti-alpha SM- 1 (or of its Fab fragment) with arterial SM actin increased polymerization in physiological salt conditions; the antibody binding did not hinder the incorporation of the actin antibody complex into the filaments. This action was not exerted on skeletal muscle actin. After microinjection of the alpha-SM actin NH2-terminal decapeptide or of the epitopic peptide into cultured aortic smooth muscle cells, double immunofluorescence for alpha-SM actin and total actin showed a selective disappearance of alpha-SM actin staining, detectable at approximately 30 min. When a control peptide (e.g. alpha-skeletal [SK] actin NH2-terminal peptide) was microinjected, this was not seen. This effect is compatible with the possibility that the epitopic peptide traps a protein involved in alpha-SM actin polymerization during the dynamic filament turnover in stress fibers. Whatever the mechanism, this is the first evidence that the NH2 terminus of an actin isoform plays a role in the regulation of polymerization in vitro and in vivo.     

Differential interaction of synthetic peptides from the carboxyl-terminal regulatory domain of tubulin with microtubule-associated proteins.

In previous studies we have demonstrated that a 4-kd tubulin fragment, including amino acid residues from Phe418 to Glu450 in alpha-subunit and Phe408-Ala445 of the beta-sequence, plays a major role in controlling tubulin interactions leading to microtubule assembly. The 4-kd carboxyl-terminal domain also constitutes an essential domain for the interaction of microtubule-associated proteins (MAPs). Removal of the 4-kd fragment facilitates tubulin self-association and renders the assembly MAP-independent. In order to define the substructure of the tubulin domain for MAP interaction, we have examined the binding of 3H-acetylated C-terminal peptides to MAP-2 and tau. Two synthetic peptides from the low-homology region within the 4-kd domain alpha (430-441) and beta (422-434) and the peptide, alpha (401-410) of the high-homology region adjacent to the 4-kd domain, were analyzed with respect to MAP interaction. The binding data showed a relatively strong interaction of MAP-2 with the beta (422-434) peptide and a weaker interaction of both MAPs components with alpha (430-441) tubulin peptide as analyzed by Airfuge ultracentrifugation and zone filtration chromatography. The homologous alpha (401-410) peptide did not bind to either MAP-2 or tau. Equilibrium dialysis experiments showed a co-operative binding of beta (422-434) peptide to multiple sites in tau. The alpha (430-441) peptide exhibited a stronger interaction for tau as compared with MAP-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microtubule-associated protein tau is phosphorylated by protein kinase C on its tubulin binding domain.

We have analyzed the in vitro phosphorylation of tau protein by Ca2+/calmodulin-dependent protein kinase, casein kinase II, and proline-directed serine/threonine protein kinase. These kinases phosphorylate tau protein in sites localized in different regions of the molecule, as determined by peptide mapping analyses. Focusing on the phosphorylation of tau by protein kinase C, it was calculated as an incorporation of 4 mol of phosphate/mol of tau. Limited proteolysis assays suggest that the phosphorylation sites could be located within the tubulin-binding domain. Direct phosphorylation of synthetic peptides corresponding to the cysteine-containing tubulin-binding region present in both fetal and adult tau isoforms demonstrates that serine 313 is modified by protein kinase C. Phosphorylation of the synthetic peptide by protein kinase C diminishes its binding to tubulin, as compared with the unphosphorylated peptide.     

A synthetic peptide of the N-terminus of actin interacts with myosin

Research reported from numerous laboratories suggested that the N-terminal region of actin contained one of the binding sites between actin and myosin. A synthetic peptide corresponding to residues 1-28 of skeletal actin was prepared by solid-phase peptide methodology. The formation of a complex between this peptide and myosin subfragment 1 (S1) was demonstrated by high-performance size-exclusion chromatography (pH 6.8). The actin peptide precipitated S1 at higher pH (7.4-8.2) but remained soluble when bound to heavy meromyosin (HMM) or S1 in the presence of F-actin. The actin peptide 1-28 bound to S1 and HMM and activated the ATPase activity in a manner similar to that of F-actin. These results demonstrate that the N-terminal region of actin, residues 1-28, contains a biologically important binding site for myosin.     

Estramustine-phosphate binds to a tubulin binding domain on microtubule-associated proteins MAP-2 and tau.

Estramustine-phosphate (EMP), a phosphorylated conjugate of estradiol and nor-nitrogen mustard binds to microtubule-associated proteins MAP-2 and tau. It was shown that this estramustine derivative inhibits the binding of the C-terminal tubulin peptide beta-(422-434) to both MAP-2 and tau. This tubulin segment constitutes a main binding domain for these microtubule-associated proteins. Interestingly, estramustine-phosphate interacted with the synthetic tau peptides V187-G204 and V218-G235, representing two major repeats within the conserved microtubule-binding domain on tau and also on MAP-2. This observation was corroborated by the inhibitory effects of estramustine-phosphate on the tau peptide-induced tubulin assembly into microtubules. On the other hand, the nonphosphorylated drug estramustine failed to block the MAP peptide-induced assembly, indicating that the negatively charged phosphate moiety of estramustine-phosphate is of importance for its inhibitory effect. These findings suggest that the molecular sites for the action of estramustine-phosphate are located within the microtubule binding domains on tau and MAP-2.     

Conformational and functional studies of three gelsolin subdomain-1 synthetic peptides and their implication in actin polymerization

Gelsolin, a calcium and inositol phospholipid-sensitive protein, regulates actin filament length. Its activity is complex (capping, severing, etc.) and is supported by several functional domains. The N-terminal domain alone (S1), in particular, is able to impede actin polymerization. Our investigations were attempted to precise this inhibitory process by using synthetic peptides as models mimicking gelsolin S1 activity. Three peptides issued from S1 and located in gelsolin—actin interfaces were synthesized. The peptides (15–28, 42–55, and 96–114 sequences) were tested for their conformational and actin binding properties. Although the three peptides interact well with actin, only peptide 42–55 affects actin polymerization. A detailed kinetic study shows that the latter peptide essentially inhibits the nucleation step during actin polymerization. In conclusion, the present work shows that the binding of a synthetic peptide to a small sequence located outside the actin—actin interface is essential in the actin polymerization process. © 1997 John Wiley & Sons, Inc. Biopoly 41: 647–655, 1997     

Characterization of f-actin tryptophan phosphorescence in the presence and absence of tryptophan-free myosin motor domain

The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140-293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (tau(1) and tau(2)) and increasing by approximately 20% the longest component (tau(3)). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (tau(4), twice as long as tau(3)) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique.     

Binding of copper (II) ion to an Alzheimer's tau peptide as revealed by MALDI-TOF MS, CD, and NMR

The tau protein plays an important role in some neurodegenerative diseases including Alzheimer's disease (AD). Neurofibrillary tangles (NFTs), a biological marker for AD, are aggregates of bundles of paired helical filaments (PHFs). In general, the alpha-sheet structure favors aberrant protein aggregates. However, some reports have shown that the alpha-helix structure is capable of triggering the formation of aberrant tau protein aggregates and PHFs have a high alpha-helix content. In addition, the third repeat fragment in the four-repeat microtubule-binding domain of the tau protein (residues 306-336: VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ, according to the longest tau protein) adopts a helical structure in trifluoroethanol (TFE) and may be a self-assembly model in the tau protein. In the human brain, there is a very small quantity of copper, which performs an important function. In our study, by means of matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy, the binding properties of copper (II) ion to the R3 peptide derived from the third repeat fragment (residues 318-335: VTSKCGSLGNIHHKPGGG) have been investigated. The results show that copper ions bind to the R3 peptide. CD spectra, ultraviolet (UV)-visible absorption spectra, and MALDI-TOF MS show pH dependence and stoichiometry of Cu2+ binding. Furthermore, CD spectra and NMR spectroscopy elucidate the copper binding sites located in the R3 peptide. Finally, CD spectra reveal that the R3 peptide adopts a mixture structure of random structures, alpha-helices, and beta-turns in aqueous solutions at physiological pH. At pH 7.5, the addition of 0.25 mol eq of Cu2+ induces the conformational change from the mixture mentioned above to a monomeric helical structure, and a beta-sheet structure forms in the presence of 1 mol eq of Cu2+. As alpha-helix and beta-sheet structures are responsible for the formation of PHFs, it is hypothesized that Cu2+ is an inducer of self-assembly of the R3 peptide and makes the R3 peptide form a structure like PHF. Hence, it is postulated that Cu2+ plays an important role in the aggregation of the R3 peptide and tau protein and that copper (II) binding may be another possible involvement in AD.     

The EGF receptor is an actin-binding protein

In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996.     

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.     

Bin1 directly remodels actin dynamics through its BAR domain

Endocytic processes are facilitated by both curvature‐generating BAR‐domain proteins and the coordinated polymerization of actin filaments. Under physiological conditions, the N‐BAR protein Bin1 has been shown to sense and curve membranes in a variety of cellular processes. Recent studies have identified Bin1 as a risk factor for Alzheimer's disease, although its possible pathological function in neurodegeneration is currently unknown. Here, we report that Bin1 not only shapes membranes, but is also directly involved in actin binding through its BAR domain. We observed a moderate actin bundling activity by human Bin1 and describe its ability to stabilize actin filaments against depolymerization. Moreover, Bin1 is also involved in stabilizing tau‐induced actin bundles, which are neuropathological hallmarks of Alzheimer's disease. We also provide evidence for this effect in vivo, where we observed that downregulation of Bin1 in a Drosophila model of tauopathy significantly reduces the appearance of tau‐induced actin inclusions. Together, these findings reveal the ability of Bin1 to modify actin dynamics and provide a possible mechanistic connection between Bin1 and tau‐induced pathobiological changes of the actin cytoskeleton.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

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.