Biomedical subjects

R Kassab

Publications and source records attributed to R Kassab.

At least 37 records · Page 2Linked to original sources

PubMed1994

The covalent maleimidobenzoyl-actin-myosin head complex. Cross-linking of the 50 kDa heavy chain region to actin subdomain-2.

We have identified the region of actin involved in the covalent coupling of maleimidobenzoyl-G-actin to the central 50 kDa segment of the myosin-S-1 heavy chain by analyzing the structure of the maleimidobenzoyl-G-actin-S-1 conjugate using selective proteolytic digestions, amino acid sequence determinations and novel cross-linking reactions between S-1 and different maleimidobenzoyl-G-actin derivatives. The cross-linking is shown to occur only on the stretch of residues 48-67 in actin subdomain-2 with Lys-50, residing on the outer part of the DNase-I-binding loop, as the most likely site of cross-linking. Because the maleimidobenzoyl-F-actin-S-1 complex undergoes the same coupling process, the data provide experimental evidence in favor of the recent model of the rigor F-actin-S-1 complex suggesting a close contact between structural elements of the lower domain of the 50 kDa fragment and the top of actin subdomain-2.

Actins↗

PubMed1994

Precise identification of the regulatory F-actin- and calmodulin-binding sequences in the 10-kDa carboxyl-terminal domain of caldesmon.

The precise location of the regulatory F-actin- and calmodulin-binding sites in the COOH-terminal sequence Trp659-Pro756 of gizzard caldesmon was investigated by subjecting the corresponding 10-kDa CNBr fragment, characterized earlier (Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238), to limited chymotryptic reactions conducted in the absence and presence of F-actin-tropomyosin. As a result, the F-actin-binding and actomyosin ATPase inhibitory activity was separated from the regulatory Ca(2+)-calmodulin-binding site. Seven chymotryptic peptides accounting for the entire primary structure of the CB10 fragment were isolated, and their complete amino acid sequences were established by combining NH2-terminal sequencing, mass spectrometry, and gel electrophoresis. Reversed-phase high performance liquid chromatography analyses of the binding of F-actin to these peptides revealed the 30-residue sequence Leu693-Trp722 as the unique crucial stretch for actin interaction and ATPase inhibition. This segment was also specifically protected by F-actin against proteolytic degradation. We further determined the functional properties of three synthetic peptides which successively cover the sequences Asn675-Lys695, Leu693-Trp722, and Arg711-Lys729. The first peptide segment specifically bound Ca(2+)-calmodulin as assessed by affinity chromatography and spectrofluorometry and should contain a potent novel calmodulin-binding subsite. The second immediately adjacent peptide inhibited the actomyosin ATPase in a tropomyosin-sensitive manner, as expected. In contrast, the third peptide displayed no detectable function. The results indicate that the overall sequence Asn675-Trp722 represents the essential regulatory unit of the COOH-terminal 10-kDa domain of caldesmon.

Actins↗

PubMed1994

Tropomyosin inhibits the glutaraldehyde-induced cross-link between the central 48-kDa fragment of myosin head and segment 48-67 in actin subdomain 2.

The glutaraldehyde-induced cross-linking of the F-actin-myosin head (S1) complex, previously described [Bertrand et al. (1988) Biochemistry 27, 5728-5736], was investigated in the presence of tropomyosin (Tm) alone or associated with troponin (Tn), at a Tm-Tn/actin/S1 molar ratio of 1:7:3. Among the two acto-S1 cross-linked products with apparent masses of 165 and 200 kDa generated in the absence of the regulatory proteins, only the 165-kDa adduct was formed in the presence of Tm. An identical result was obtained with and without Tn regardless of the presence of Ca2+ and/or Mg(2+)-ADP. The abolition of the 200-kDa cross-linked acto-S1 species was independent of the S1/actin ratio since even a 3-fold excess of S1 over actin, sufficient for fully turning on the thin filament, could not restore the 200-kDa covalent complex. In addition, the acto-S1 contacts cross-linked in either the 165- or 200-kDa product were not involved in the Ca(2+)-linked regulation of the acto-S1 ATPase activity, as the enzymatic activities of both types of complexes were regulated to the same extent by Ca2+/EGTA, in the presence of the regulatory proteins. Cross-linking experiments performed with [14C]glutaraldehyde showed that both covalent complexes were composed of 1 mol of actin bound to 1 mol of S1 heavy chain. The use of proteolytic actin or S1 derivatives together with the direct proteolysis of the acto-S1 covalent adducts revealed that Tm abolished the cross-link between the central 48-kDa fragment of the S1 heavy chain and Lys50 of actin subdomain 2 that is responsible for the formation of the 200-kDa entity, while it did not affect the cross-link between the S1 heavy chain segment of residues 636-642 and Arg28 of actin that generates the 165-kDa derivative. These results provide experimental clues for the interaction of S1 with actin subdomain 2 and show that this contact is implicated in the weak acto-S1 binding state. Furthermore they demonstrate the ability of Tm to affect the structure of actin subdomain 2 even in the presence of S1 bound in the rigor state.

Actins↗

PubMed1994

[Primary angioplasty at the stage of acute myocardial infarction].

The success of thrombolytic therapy for acute myocardial infarction is limited by failure of reperfusing 25% of occluded arteries, bleeding complications in 0.4 to 1% of patients and the possibility of recurrent ischemia. These problems can be overcome by the use of immediate angioplasty without previous thrombolytic therapy. Between February and December 1993, twelve patients (ten men and two women) admitted for acute myocardial infarction were treated by immediate angioplasty. Five patients had a contraindication to thrombolysis and seven had angioplasty as a deliberate choice. Successful recanalisation was obtained in 11 patients (92%) with one reocclusion at 24 hours. Two patients admitted in cardiogenic shock 12 hours after the onset of symptoms died lately with multiorgan failure. Only one case of clinical restenosis was observed and was redilated. In conclusion, immediate angioplasty without prior thrombolytic therapy is a rapid method of revascularisation with minimal risk and good outcome when it is used early after acute myocardial infarction.

Adult↗

PubMed1993

Photochemical cross-linking of the skeletal myosin head heavy chain to actin subdomain-1 at Arg95 and Arg28.

F-actin specifically substituted with the photocross-linker, p-azidophenylglyoxal, at Arg95 and Arg28 was isolated and characterized. Upon complexation with myosin subfragment-1 (S1) and photolysis at 365 nm, it was readily cross-linked to the S1 heavy chain with a yield of about 13-25%, generating four major actin-heavy-chain adducts with molecular masses in the range 165-240 kDa. The elevated Mg(2+)-ATPase of the covalent complexes displayed a turnover rate of 33 +/- 8 s-1 which is similar to the values reported earlier for other acto-S1 conjugates. The cross-linking between various proteolytic S1 and actin derivatives, combined with the fluorescent and immunochemical detection of the photocross-linked products, indicated that the arylnitrene group on Arg95 was inserted predominantly in the central 50-kDa region, whereas that attached to Arg28 mediated the selective cross-linking of the COOH-terminal 22-21-kDa fragments of the heavy chain, most probably by reacting at or near the connector segment between the 50-kDa and 20-kDa fragments. The rapid photoactivation and cross-linking to S1 of the substituted F-actin, which can be accomplished on a millisecond time scale, may serve to probe the structural dynamics of the interaction of the S1 heavy chain with subdomain-1 of actin during the ATPase cycle.

Actins↗

PubMed1992

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)

Actins↗

PubMed1992

Mapping of the functional domains in the amino-terminal region of calponin.

Three chymotryptic fragments accounting for almost the entire amino acid sequence of gizzard calponin (Takahashi, K., and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288) were isolated and characterized. They encompass the segments of residues 7-144 (NH2-terminal 13-kDa peptide), 7-182 (NH2-terminal 22-kDa peptide), and 183-292 (COOH-terminal 13-kDa peptide). They arise from the sequential hydrolysis of the peptide bonds at Tyr182-Gly183 and Tyr144-Ala145 which were protected by the binding of F-actin to calponin. Only the NH2-terminal 13- and 22-kDa fragments were retained by immobilized Ca(2+)-calmodulin, but only the larger 22 kDa entity cosedimented with F-actin and inhibited, in the absence of Ca(2+)-calmodulin, the skeletal actomyosin subfragment-1 ATPase activity as the intact calponin. Since the latter peptide differs from the NH2-terminal 13-kDa fragment by a COOH-terminal 38-residue extension, this difference segment appears to contain the actin-binding domain of calponin. Zero-length cross-linked complexes of F-actin and either calponin or its 22-kDa peptide were produced. The total CNBr digest of the F-actin-calponin conjugate was fractionated over immobilized calmodulin. The EGTA-eluted pair of cross-linked actin-calponin peptides was composed of the COOH-terminal actin segment of residues 326-355 joined to the NH2-terminal calponin region of residues 52-168 which seems to contain the major determinants for F-actin and Ca(2+)-calmodulin binding.

Amino Acid Sequence↗

PubMed1992

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)

Actins↗

PubMed1990

Construction, expression and unexpected regulatory properties of a tropomyosin mutant with a 31-residue deletion at the C-terminus (exon 9).

The cDNA coding for human skeletal muscle beta-tropomyosin was expressed in Escherichia coli to produce an unacetylated beta-tropomyosin. This cDNA was deleted from the sequence corresponding to the exon 9 and expressed in E. coli to produce an unacetylated beta-tropomyosin mutant lacking the C-terminal residues 254-284. The main structural and functional properties of the two isolated proteins, designated tropomyosin-1 and des-(254-284)-tropomyosin, respectively, were characterized in comparison with those of the genuine rabbit skeletal muscle alpha beta-tropomyosin. The folding and thermal stability of the three tropomyosins were indistinguishable. Tropomyosin-1, but not des-(254-284)-tropomyosin, was polymerized in the presence of troponin and did bind to actin in the presence of the troponin complex. Despite its weak binding to actin, des-(254-284)-tropomyosin displayed a regulatory function in the presence of troponin with a marked activation of the actomyosin subfragment-1 ATPase in the presence of Ca2+ and low concentrations of subfragment-1. The data were interpreted in the light of the allosteric models of regulation and suggest the involvement of the sequence coded by exon 9 in the stabilization by tropomyosin of the off state of the thin filament.

Actins↗

PubMed1990

Maleimidobenzoyl-G-actin: structural properties and interaction with skeletal myosin subfragment-1.

We have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt--and myosin subfragment 1 (S-1)-induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain (Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032). The far-ultraviolet CD spectrum and alpha-helix content of the MBS-actin were identical with those displayed by native G-actin. 45Ca2+ measurements showed the same content of tightly bound Ca2+ in MBS-actin as in G-actin and the EDTA treatment of the modified protein promoted the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl + 5 mM MgCl2 led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6 mg/mL, at 25 degrees C, pH 8.0. The MBS-F-actin formed activated the Mg2(+)-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was not to be at all affected by its specific covalent conjugation to S-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1990

Characterization of the carboxyl-terminal 10-kDa cyanogen bromide fragment of caldesmon as an actin-calmodulin-binding region.

A pair of 10-kDa peptides, designated CB-a and CB-b, was isolated by calmodulin-Sepharose chromatography from a total CNBr digest of turkey gizzard caldesmon. CB-a encompasses the COOH-terminal segment of residues 659-756, according to the sequence of adult chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R.G., and Lin, W.G. (1989) J. Biol. Chem. 264, 13873-13879), whereas CB-b comprises the same structure but was a few amino acids shorter at its COOH terminus. Both peptides cosedimented with F-actin, and their binding was increased by smooth muscle tropomyosin. The Kd values were 1.3 and 0.5 microM, in the absence and presence of tropomyosin, respectively, with a maximum binding capacity of 6.9 actins/mol of peptides. The CB-a/CB-b fragments inhibited, in a tropomyosin-sensitive and Ca2(+)-calmodulin-dependent manner, the skeletal actomyosin subfragment 1 ATPase activity to a level close but not identical to that observed for the parent caldesmon. Ca2(+)-calmodulin was selectively cross-linked to either caldesmon or the CNBr peptides with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide producing 1:1 covalent complexes that were retained neither by phenyl-Sepharose nor by immobilized calmodulin. Moreover, the cross-linked caldesmon bound weakly to F-actin and did not inhibit the actomyosin subfragment 1 ATPase in the absence of Ca2+. The results suggest that the CB-a/CB-b peptide region contains major regulatory determinants of caldesmon.

Actins↗

PubMed1990

Cross-linking of smooth muscle caldesmon to the NH2-terminal region of skeletal F-actin.

The cross-linking of the F-actin-caldesmon complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the presence of N-hydroxysuccinimide generated four major adducts which were identified on polyacrylamide gels. By cross-linking 3H-actin to 14C-caldesmon, these were found to represent 1:1 cross-linked complexes of actin and caldesmon displaying different electrophoretic mobilities. Tropomyosin did not noticeably affect the cross-linking process. The same four fluorescent species resulting from the cross-linking of caldesmon to F-actin labeled with N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide were subjected separately to partial cleavages with hydroxylamine or cyanogen bromide. These treatments yielded fluorescent 41- and 37-kDa fragments, respectively, from each cross-linked entity indicating unambiguously that caldesmon was cross-linked only to the NH2-terminal actin stretch of residues 1-12. This region is also known to serve for the carbodiimide-mediated cross-linking of the myosin subfragment-1 heavy chain (Sutoh, K. (1982) Biochemistry 21, 3654-3661). A covalent caldesmon-F-actin conjugate containing a protein molar ratio close to 1:19 was isolated following dissociation of uncross-linked caldesmon. It showed a low level of activation of the ATPase activity of skeletal myosin subfragment-1, and the binding of Ca2(+)-calmodulin to the derivative did not cause the reversal of the ATPase inhibition. In contrast, the reversible binding of caldesmon to F-actin cross-linked to myosin subfragment-1 did not inhibit the accelerated ATPase of the complex. The overall data point to the dual involvement of the actin's NH2 terminus in the inhibitory binding of caldesmon and in actomyosin interactions in the presence of ATP.

Actins↗

PubMed1989

Isolation, characterization and immunocytochemical localization of caldesmon-like protein from molluscan striated muscle.

A 140-kDa polypeptide present in the striated muscle of Pecten maximus and Sepia officinalis was purified to homogeneity and its main properties were investigated using biochemical and cytochemical approaches. The protein was found to be similar to chicken gizzard caldesmon. It is a heat-stable protein. It cross-reacts immunologically with anti-(gizzard caldesmon) antibody, binds to calmodulin-Sepharose in a Ca2+-dependent manner, cosediments with F-actin filaments and acts in the absence and presence of tropomyosin as a potent inhibitor of rabbit skeletal actomyosin Mg2+-ATPase. The immunocytochemistry of ultrathin sections revealed, at the light microscopy resolution level, that caldesmon-like protein is present in all types of muscles hitherto examined from invertebrates and vertebrates. However, according to the distribution and the intensity of the fluorescent reaction, we concluded that, under our experimental conditions, caldesmon is not homogeneously distributed and not located in the myofibrillar bands of striated muscles but rather in the sarcoplasmic elements, at the periphery of the fibres.

Animals↗

PubMed1989

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.

Actins↗

PubMed1989

Selective cleavage at lysine of the 50 kDa-20 kDa connector loop segment of skeletal myosin S-1 by endoproteinase Arg-C.

The reaction of endoproteinase Arg-C on the skeletal myosin head heavy chain was investigated through characterization of peptides and amino acid sequence analysis. The protease splits exclusively the 50 kDa-20 kDa junction at the lysine cluster spanning residues 639-641 and does not affect any other protease-sensitive region of the entire myosin heavy chain. The sensitivity of the cleavage to actin and nucleotide binding makes this protease a very specific conformational probe of S-1. The nicked S-1 derivative, containing an intact NH2-terminal 75 kDa fragment, may serve as a tool for gaining further insights into the domain structure and function of the myosin head.

Actins↗

PubMed1989

Coupling of nonpolymerizable monomeric actin to the F-actin binding region of the myosin head.

Polymerizations of skeletal G-actin induced by salt and myosin subfragment 1 (S-1) were suppressed by reaction of G-actin with m-maleimidobenzoyl-N-hydroxysuccinimide ester. The G-actin derivative, containing few intramolecular crosslinks and a free maleimide group, was covalently coupled in solution to the S-1 heavy chain. The resulting complex could no longer bind to F-actin. The SH-1 and SH-2 thiols of S-1 were not involved in the complexation and the covalent link was shown to be exclusively on the 50-kDa segment of the S-1 heavy chain. The specific conjugation of the two proteins followed formation of a reversibly associated pyrophosphate-sensitive binary complex which was characterized by different approaches. Potentially, these complexes may be useful in developing the crystallography of actin-bound S-1.

Actins↗

PubMed1988

Histidyls in lobster arginine kinase. 1H-NMR of native and ethoxyformylated enzyme, 1H- and 31P-NMR of its complexes with substrates and analogues.

The role of histidyls in lobster arginine kinase (EC 2.7.3.3) has been studied by 1H-NMR spectroscopy of the enzyme and its complexes with substrates or their analogues and 31P-NMR spectroscopy of complexes with ADP. Five histidyls were detected by 1H-NMR in native enzyme (His 1 to His 5). Three of them appeared possibly to be implicated in catalysis: His 3, whose pH/titration was affected by arginine binding, and His 1 and 4, shown from paramagnetic relaxation by Mn2+ to be close (less than or equal to 1.2 and less than or equal to 1.27 nm respectively) to the metal cofactor. His 4 was broadened beyond detection in the presence of any adenine nucleotide. In the enzyme reversibly inactivated by histidine ethoxyformylation, the modified histidyl was His 1. In the transition state analogue complex (in which NO3- mimics the transferred phosphoryl), Hill plots of histidyl pH/titration curves showed that His 1 and His 3 were both interacting with the same set of three titratable groups and hence spatially close. 31P-NMR demonstrated that ADP binding in this complex was unaffected by the chemical modification of His 1. It is concluded that His-ethoxyformyl-enzyme is inactive because ethoxyformyl-His 1 is unable to titrate. This is consistent with His 1 acting as the acid-base catalyst. However our results, which do not indicate any catalytic role of His 3, exclude any H-bonding of His 1 on either substrate. Involvement is needed of at least one other titratable residue for the proton evolved in the catalysis to exchange directly with the guanidino substrate.

Animals↗

PubMed1988

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)

Actins↗