Cross-linking of contractile proteins from skeletal muscle by treatment with microbial transglutaminase.

The action on muscle proteins of microbial transglutaminase (MTGase), which catalyzes the formation of a "zero-length" covalent cross-link between glutamine and lysine residues in peptides, was studied in order to define a basis for future application of MTGase cross-linking to the study of muscle protein interaction. We examined the cross-linking of skeletal muscle myosin, myosin subfragments, actin, and myofibrils by treatment with MTGase and the possible side-effects of the cross-linking on the enzymic activity of myosin, and found that the rod portions of myosin in myosin filaments were quickly cross-linked to each other by the action of MTGase, but myosin subfragment 1 was not cross-linked to actin. The MgATPase activities at 0.5 M KCl of myosin, heavy meromyosin, subfragment 1, and subfragment 1-actin were not significantly affected by the MTGase reaction. A very small fraction of the head portion of heavy meromyosin was cross-linked to actin in their rigor complexes by MTGase, and the ATPase activity at 0.5 M KCl of the cross-linked heavy meromyosin-actin complexes was slightly enhanced.     

The sites on calmodulin cross-linked to myosin light chain kinase and troponin I with water-soluble carbodiimide

To elucidate the interaction of calmodulin with calmodulin binding proteins, we studied the location of the interaction sites on calmodulin by using a chemical cross-linking reagent. Calmodulin prepared from wheat germ was cross-linked to myosin light chain kinase and troponin-I with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked products were cleaved partially with cyanogen bromide and cross-linked sites were determined by peptide mapping analysis using SDS-urea polyacrylamide gel electrophoresis. Peptides which contain the cross-linked site were displaced from their position because of the attached fragments of myosin light chain kinase or troponin I. The peptide of calmodulin from the N-terminal to Met-73 in the cross-linked product with myosin light chain kinase had the same mobility as that of uncross-linked calmodulin on the map though the amount of the peptide was decreased in the cross-linked product. The peptide from the N-terminal to Met-110 in the cross-linked product was displaced from its position. Similar change in the mobility of the calmodulin peptides was also observed in the cross-linked products with troponin I. It was concluded, therefore, that at least one cross-linked site for myosin light chain kinase and one for troponin I were located between Met-73 and Met-110 of the wheat germ calmodulin.     

Chemical cross-linking of myosin. Disposition of the globular heads.

The interaction of a series of bifunctional reagents with skeletal muscle myosin has been studied. In the di-imido ester series dimethylmalonimidate failed to generate any cross-linked species, whereas the adipic and higher analogues gave dimers of myosin heavy chains. Analysis of free amino groups after reaction with these reagents and with the reducible species dimethyldithiobis(propionimidate) showed that no more than two to three cross-links per molecule were introduced. By contrast, the bifunctional reducible acylating agent, dithiobis(succinimidylpropionate), reacted with annihilation of about 10% of the amino groups under mild conditions that precluded the formation of intermolecularly linked species. Digestion of the intramolecularly cross-linked myosin with papain, followed by analysis of the fragments by gel electrophoresis, revealed extensive cross-linking between the globular heads of the myosin molecules. The subfragment 1 dimers regenerated subfragment 1 on reduction, as shown by the electrophoretic mobility and amino acid analysis. The extent of cross-linking, and therefore presumably the average relative orientation or freedom of the two heads, was unaffected by the addition of ADP and calcium ions. The internally cross-linked myosin retains practically its full calcium-activated adenosine triphosphatase activity, but in contrast to native myosin is soluble even at very low ionic strength. Circular dichroism measurements show that the alpha helical conformation is undisturbed in cross-linked myosin, but the sedimentation coefficient is considerably higher than that of the native protein, possibly due to freezing of the heads in a "closed" configuration. The light chaiins are not cross-linked to the heavy chains, except under extreme conditions that leads to intermolecular cross-linking and inactivation. The presence of calcium ions protects dithiobisnitrobenzoate light chains against degradation by papain.     

An enzymatically active cross-linked complex of calmodulin and rabbit skeletal muscle myosin light chain kinase

The interaction between bovine testes calmodulin and rabbit fast skeletal muscle myosin light chain kinase was investigated with the zero-length cross-linking reagent N,N'-dicyclohexylcarbodiimide. A cross-linked product of 110 kDa was produced only in the presence of Ca2+. The reaction mixture was separated on diethylaminoethyl cellulose, and a fraction containing the cross-linked complex of calmodulin and myosin light chain kinase was found to have an elevated kinase activity in the absence of Ca2+, which constituted approximately 50% of the maximally stimulated kinase activity of control, and additional kinase activity in the presence of Ca2+, which constituted the remaining 50% of control activity. Calmodulin added exogenously to the cross-linked complex had no effect on the measured Ca2+ dependence or the maximal extent of kinase activity, which is consistent with the cross-linking of calmodulin in close proximity to a regulatory region of myosin light chain kinase. Moreover, the results are consistent with a mechanism whereby the association of calmodulin is sufficient to stimulate kinase activity and the binding of Ca2+ to bound calmodulin increases catalytic efficiency.     

Actin cross-linking and inhibition of the actomyosin motor.

Intrastrand cross-linking of actin filaments by ANP, N-(4-azido-2-nitrophenyl) putrescine, between Gln-41 in subdomain 2 and Cys-374 at the C-terminus, was shown to inhibit force generation with myosin in the in vitro motility assays [Kim et al. (1998) Biochemistry 37, 17801-17809]. To clarify the immobilization of which of these two sites inhibits the actomyosin motor, the properties of actins with partially overlapping cross-linked sites were examined. pPDM (N,N'-p-phenylenedimaleimide) and ABP [N-(4-azidobenzoyl) putrescine] were used to obtain actin filaments cross-linked ( approximately 50%) between Cys-374 and Lys-191 (interstrand) and Gln-41 and Lys-113 (intrastrand), respectively. ANP, ABP, and pPDM cross-linked filaments showed similar inhibition of their sliding speeds and force generation with myosin ( approximately 25%) in the in vitro motility assays. In analogy to ANP cross-linking of actin, pPDM and ABP cross-linkings did not change the strong S1 binding to actin and the V(max) and K(m) parameters of actomyosin ATPase. The similar effects of these three cross-linkings reveal the tight coupling between structural elements of the subdomain 2/subdomain 1 interface and show the importance of its dynamic flexibility to force generation with myosin. The possibility that actin cross-linkings inhibit rate-limiting steps in motion and force generation during myosin cross-bridge cycle was tested in stopped-flow experiments. Measurements of the rates of mantADP release from actoS1 and ATP-induced dissociation of actoS1 did not reveal any differences between un-cross-linked and ANP cross-linked actin in these complexes. These findings are discussed in terms of the uncoupling between force generation and other aspects of actomyosin interactions due to a constrained dynamic flexibility of the subdomain 2/subdomain 1 interface in cross-linked actin filaments.     

The purification and characterization of a globular subfragment of Acanthamoeba myosin II that is fully active when cross-linked to F-actin

Acanthamoeba myosin II contains two heavy chains of Mr 185,000 and two pairs of light chains of Mr 17,500 and 17,000. We now report the purification of a globular proteolytic 103-kDa subfragment of myosin II which contained a 68-kDa NH2-terminal segment of the heavy chain and one pair of intact light chains. The myosin II head fragment expressed full Ca2+-ATPase activity but its actin-activated Mg2+-ATPase activity had a Vmax of only 0.07 s-1 compared to 1.9 s-1 (per head) for filaments of native unphosphorylated myosin II. The head fragment had a similar KATPase to that of filaments (5 versus 4 microM) and about 75% of the head fraction could bind to F-actin in the presence of ATP with a Kbinding of 5.6 microM. The Kbinding of the head fragment may be similar to that of individual heads in the native myosin II filaments although the experimentally determined apparent Kbinding for filaments is much lower, 0.3 microM. The head fragment was covalently cross-linked to F-actin in the absence of nucleotide using the zero length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked actin-myosin head complex hydrolyzed MgATP at a rate equivalent to Vmax for the active dephosphorylated native myosin II. These data indicate that the isolated head fragment had intact catalytic and actin-binding domains but that it bound to F-actin in the presence of ATP in a relatively inactive conformation. When covalently cross-linked to F-actin the head fragment was apparently locked into a catalytically fully active conformation.     

Covalent cross-linking of single fibers from rabbit psoas increases oscillatory power.

Single fibers from chemically skinned rabbit psoas muscle were treated with 1-ethyl-3-[3-dimethyl-amino)proyl]-carbodiimide (EDC) at 20 degrees C after rigor was induced. A 22-min treatment resulted in 18% covalent cross-linking between myosin heads and the thin filament as determined by stiffness measurements. This treatment also results in covalent cross-linking among rod portions of myosin molecules in the backbone of the thick filament. The fibers thus prepared are stable and do not dissolve in solutions at ionic strengths as high as 1,000 mM. The preparation was subjected to sinusoidal analysis, and the resulting complex modulus data were analyzed in terms of three exponential processes, (A), (B), and (C). Oscillatory work (process B) was much greater in the cross-linked fibers than in untreated ones in activating solutions of physiological ionic strength (200 mM); this difference was attributed to the decline of process (A) with EDC treatment. Consequently, the Nyquist plot of the EDC-treated preparation exhibited an insect-type response. We conclude that, under these conditions, both cross-linked and non-cross-linked myosin heads contribute to the production of oscillatory power. The cross-linked preparations also exhibited oscillatory work in high ionic strength (500-1,000 mM) solutions, indicating that cross-linked myosin heads are capable of utilizing ATP to produce work. We conclude that process (A) does not relate to an elementary step in a cross-bridge cycle, but it may relate to dynamics outside the cross-bridge such as filament sliding or sarcomere rearrangement.     

States of thin filament regulatory proteins as revealed by combined cross-linking/X-ray diffraction techniques

The regulatory protein system in the skeletal muscle thin filaments is known to exhibit three discrete states, called "off" or "blocked" (no Ca2+), "on" or "closed" (with Ca2+ alone) and "potentiated" or "open" (with strongly bound myosin head) states. Biochemical studies have shown that only weak interactions with myosin are allowed in the second state. Characterization of each state is often difficult, because the equilibria among these states are readily shifted by experimental conditions. To overcome this problem, we chemically cross-linked the skeletal muscle thin filament in the three states with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), in overstretched muscle fibers. The state of the regulatory proteins was monitored by measuring the intensity of the second actin layer-line (2nd LL) reflection in X-ray diffraction patterns. Structurally, the thin filaments cross-linked in the three states exhibited three corresponding discrete levels of 2nd LL intensities, which were not Ca2+-sensitive any more. Functionally, the thin filament cross-linked in the "off-blocked" state inhibited strong interaction with myosin head (subgfragment-1 or S1). The thin filament cross-linked in the "potentiated-open" state allowed strong interaction and full ATPase activity of S1 as described previously. The thin filament cross-linked in the "on-closed" state allowed strong interactions with S1 and actin-activated ATPase without enhancing the 2nd LL to the level of "potentiated-open" state, contrary to the expectations from the biochemical studies. The results demonstrate the potential of EDC as a tool for studying the states of calcium regulation, and the apparent uncoupling between the 2nd LL intensity and the function provides a new insight into the mechanism of thin filament regulation.     

Force generation and work production by covalently cross-linked actin-myosin cross-bridges in rabbit muscle fibers.

To separate a fraction of the myosin cross-bridges that are attached to the thin filaments and that participate in the mechanical responses, muscle fibers were cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and then immersed in high-salt relaxing solution (HSRS) of 0.6 M ionic strength for detaching the unlinked myosin heads. The mechanical properties and force-generating ability of the cross-linked cross-bridges were tested with step length changes (L-steps) and temperature jumps (T-jumps) from 6-10 degrees C to 30-40 degrees C. After partial cross-linking, when instantaneous stiffness in HSRS was 25-40% of that in rigor, the mechanical behavior of the fibers was similar to that during active contraction. The kinetics of the T-jump-induced tension transients as well as the rate of the fast phase of tension recovery after length steps were close to those in unlinked fibers during activation. Under feedback force control, the T-jump initiated fiber shortening by up to 4 nm/half-sarcomere. Work produced by a cross-linked myosin head after the T-jump was up to 30 x 10(-21) J. When the extent of cross-linking was increased and fiber stiffness in HSRS approached that in rigor, the fibers lost their viscoelastic properties and ability to generate force with a rise in temperature.     

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.     

Interaction of the N-terminus of chicken skeletal essential light chain 1 with F-actin

Skeletal myosin has two isoforms of the essential light chain (ELC), called LC1 and LC3, which differ only in their N-terminal amino acid sequence. The LC1 has 41 additional residues containing seven pairs of Ala-Pro, which form an elongated structure, and two pairs of lysines located near the N-terminus. When myosin subfragment-1 (S1) binds to actin, these lysines may interact with the C-terminus of actin and be responsible for the isoform specific properties of myosin. Here we employ cross-linking to identify the LC1 residues that are in contact with actin. S1 was reconstituted with various LC1 mutants and reacted with the zero-length cross-linker 1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide (EDC). Cross-linking occurred only when actin was in molar excess over S1. Wild-type LC1 could be cross-linked through the terminal alpha-NH2 group, as well as via the two pairs of lysines. In a mutant ELC, where the lysines were deleted but two arginines were introduced near the N-terminus, the light chain could still be cross-linked via the terminal alpha-NH2 group. When the charge was reduced in the N-terminal region while retaining the Ala-Pro rich region, the mutant could not be cross-linked. These results suggest that as long as the N-terminus contains charged residues and an Ala-Pro rich extension, the binding between LC1 and actin can occur.     

Proximity of regulatory light chains in scallop myosin

The distance between the regulatory light chains of the two heads of the scallop myosin molecule was estimated with the aid of two photolabile cross-linkers, benzophenone maleimide and p-azidophenacylbromide. These cross-linkers selectively alkylate thiol groups and have a maximum length of about 9 A. One of the two regulatory light chains of scallop myosin was removed by treatment of myofibrils at 10 degrees C with EDTA and replaced with a foreign regulatory light chain carrying a cross-linker. Cross-linking between the scallop and foreign regulatory light chains was effected by photolysis. This was demonstrated by incubating nitrocellulose transfers of sodium dodecyl sulfate/polyacrylamide gels of the photolyzed hybrid myofibrils with specific antibodies against the different light chains, followed by fluorescein isothiocyanate-125I-labeled secondary antibody. Scallop regulatory light chains cross-linked extensively (20 to 50%) with Mercenaria regulatory light chains (cysteine in position approximately 50) in solutions that induce rigor in skinned fibers (no ATP) and in relaxing solutions (ATP but no Ca2+). Neither the regulatory light chains of chicken skeletal myosin (cysteines 129 and 157) nor those of gizzard myosin (cysteine 108) were cross-linked to scallop regulatory light chains in either medium. These results indicate that the N-terminal portions of the myosin regulatory light chains can approach each other within 9 A or less, while the distance between the C-terminal halves exceeds 9 A, and support the view that the N termini of the regulatory light chains point toward the myosin rod. Since the relative distance between the regulatory light chains of the two myosin heads is not altered between rigor and rest, we suggest that motion of the essential light chains is mainly responsible for the observed difference in the relative positions of the regulatory and essential light chains between conditions of rigor and rest.     

Direct visualization by electron microscopy of the weakly bound intermediates in the actomyosin adenosine triphosphatase cycle.

We used a novel stopped-flow/rapid-freezing machine to prepare the transient intermediates in the actin-myosin adenosine triphosphatase (ATPase) cycle for direct observation by electron microscopy. We focused on the low affinity complexes of myosin-adenosine triphosphate (ATP) and myosin-adenosine diphosphate (ADP)-Pi with actin filaments since the transition from these states to the high affinity actin-myosin-ADP and actin-myosin states is postulated to generate the molecular motion that drives muscle contraction and other types of cellular movements. After rapid freezing and metal replication of mixtures of myosin subfragment-1, actin filaments, and ATP, the structure of the weakly bound intermediates is indistinguishable from nucleotide-free rigor complexes. In particular, the average angle of attachment of the myosin head to the actin filament is approximately 40 degrees in both cases. At all stages in the ATPase cycle, the configuration of most of the myosin heads bound to actin filaments is similar, and the part of the myosin head preserved in freeze-fracture replicas does not tilt by more than a few degrees during the transition from the low affinity to high affinity states. In contrast, myosin heads chemically cross-linked to actin filaments differ in their attachment angles from ordered at 40 degrees without ATP to nearly random in the presence of ATP when viewed by negative staining (Craig, R., L.E. Greene, and E. Eisenberg. 1985. Proc. Natl. Acad. Sci. USA. 82:3247-3251, and confirmed here), freezing in vitreous ice (Applegate, D., and P. Flicker. 1987. J. Biol. Chem. 262:6856-6863), and in replicas of rapidly frozen samples. This suggests that many of the cross-linked heads in these preparations are dissociated from but tethered to the actin filaments in the presence of ATP. These observations suggest that the molecular motion produced by myosin and actin takes place with the myosin head at a point some distance from the actin binding site or does not involve a large change in the shape of the myosin head.     

Gradients in the concentration and assembly of myosin II in living fibroblasts during locomotion and fiber transport.

Assembly and motor activity of myosin II affect shape, contractility, and locomotion of nonmuscle cells. We used fluorescent analogues and imaging techniques to elucidate the state of assembly and three-dimensional distribution of myosin II in living Swiss 3T3 fibroblasts. An analogue of myosin II that was covalently cross-linked in the 10S conformation and unable to assemble served as an indicator of the cytoplasmic volume accessible to 10S myosin II. Ratio-imaging of an analogue that can undergo 10S-->6S conversion versus the volume indicator revealed localized concentration of assembly-competent myosin II. In stationary serum-deprived cells and in cells locomoting at the edge of a wound, it was most concentrated in the peripheral cytoplasm, where fibers containing myosin II assemble, and least concentrated in the perinuclear cytoplasm, where they disassemble. Furthermore, fluorescence photobleaching recovery showed myosin II to be less mobile in the periphery than in perinuclear cytoplasm. These results indicate a gradient in the assembly of myosin II. Three-dimensional microscopy of living cells revealed that fibers containing myosin II were localized in the cortical cytoplasm, whereas myosin II was diffusely distributed in the deeper cytoplasm, suggesting that myosin II is assembled preferentially near the cell surface. Localized protein phosphorylation may play a role, because a kinase inhibitor, staurosporine, abolished the gradient of myosin II assembly.     

Regulation of the actin-activated ATPase activity of Acanthamoeba myosin I by cross-linking actin filaments

The actin-activated Mg2+-ATPase activity of phosphorylated Acanthamoeba myosin I was previously shown to be cooperatively dependent on the myosin concentration (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179). This observation was rationalized by assuming that myosin I contains a high-affinity and a low-affinity F-actin-binding site and that binding at the low-affinity site is responsible for the actin-activated ATPase activity. Therefore, enzymatic activity would correlate with the cross-linking of actin filaments by myosin I, and the cooperative increase in specific activity at high myosin:actin ratios would result from the fact that cross-linking by one myosin molecule would increase the effective F-actin concentration for neighboring myosin molecules. This model predicts that high specific activity should occur at myosin:actin ratios below that required for cooperative interactions if the actin filaments are cross-linked by catalytically inert cross-linking proteins. This prediction has been confirmed by cross-linking actin filaments with either of three gelation factors isolated from Acanthamoeba, one of which has not been previously described, or by enzymatically inactive unphosphorylated Acanthamoeba myosin I.     

Photocross-linking from dinitrophenylated SH1 in myosin head. II. Cross-linked site on 50-kDa fragment

We reported in the preceding paper [Muno, D., et al. (1987) J. Biochem. 101, 661-669] that the dinitrophenyl group exclusively introduced to SH1 on the 20-kDa fragment of myosin subfragment 1 was cross-linked to the 50-kDa fragment by irradiation, and that limited trypsinolysis of the cross-linked S1 generated an 83-kDa peptide, a cross-linking product between the 20- and 50-kDa fragments. This paper will deal with the location of the cross-linked residue on the 50-kDa fragment. When the 83-kDa fragment labeled at SH2 with a fluorogenic SH reagent was subjected to bromocyanolysis, a main fluorescent band, which implied a cross-linked peptide, appeared in the position with an apparent molecular mass of 18.5-kDa on SDS-PAGE. On the other hand, another cross-linked peptide was obtained from a complete tryptic digest of a 83-kDa fragment rich fraction. Amino acid sequence analysis of the two cross-linked peptides revealed that the DNP moiety attached at SH1 was cross-linked with a residue in the segment of the heavy chain spanning the 485-493 region from the N-terminus of the heavy chain.     

Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking

To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.     

Effect of pH on the cross-bridge arrangement in synthetic myosin filaments.

Synthetic thick filaments were cross-linked with dimethyl suberimidate at various pH values over the range pH 6.8---8.3. The rate of cross-linking myosin heads to the thick filament surface decreases significantly over a narrow pH range (7.4--8.0) despite the fact that the rate of the chemical reaction (amidination of lysine side chains) shows a positive pH dependence. The fall in rate cannot be ascribed to dissociation of the filament during the cross-linking reaction since the sedimentation boundary of the cross-linked filament (pH 8.3) remains unaltered in the presence of high salt (0.5 M). The decreased rate of cross-linking is also not caused by a shift in reactivity of a small number of highly reactive lysine groups, since the time course of cross-linking (pH 7.2) is unaffected by preincubation with a monofunctional imidate ester. Our results suggest that the heads of the myosin molecules move away from the thick filament surface at alkaline pH but are held close to the surface at neutral pH.     

Arrangement of myosin heads on Limulus thick filaments

The two myosin heads with a single surface subunit on thick filaments from chelicerate arthropod muscle may originate from the same, or from axially sequential molecules, as suggested by three-dimensional reconstructions. The resolution attained in the reconstructions, however, does not permit one to distinguish unequivocally between these two possible arrangements. We examined the effect of 0.6 M KCl on relaxed thick filaments separated from Limulus muscle and filaments in which nearest myosin heads were cross-linked by the bifunctional agent, 3,3'-dithio-bis[3'(2')-O-[6-propionylamino)hexanoyl]adenosine 5'-triphosphate (bis22ATP), in the presence of vanadate (Vi). In high salt, surface myosin dissolved from both native, relaxed filaments and those exposed to 1-2 mM dithiothreitol after cross-linking, but was retained on filaments with cross-linked heads. Since bis22ATP must form intermolecular bonds between myosin heads within each subunit to prevent myosin solubilization in high salt, we conclude that each of these heads originates from a different myosin molecule, as was previously predicted by the reconstructions.     

Fibrinoligase-catalyzed cross-linking of myosin from platelet and skeletal muscle.

Fibrinoligase (thrombin- and calcium-activated Factor XIII) from human plasma catalyzes the incorporation of dansylcadaverine and [¹⁴C]putrescine into myosin, prepared from either human platelets or rabbit skeletal muscle. At least 9 mol of amine is incorporated per mole of myosin of either type when the enzyme is used under saturating conditions. Both heavy and light chains of the platelet and muscle myosins incorporate dansylcadaverine and [ ¹⁴C]putrescine. However, in quantitative terms, the incorporation into the light chains of either type is much less than into the heavy chains. Profound fluorescent changes occurred when dansylcadaverine was bound to myosin. Highly cross-linked platelet and muscle myosin polymers form in the absence of added amines, indicating the presence of both acceptor and donor sites. ATPase activity was not altered by cross-linking of 50–60% of myosin. The nature of the cross-link in myosin was found to be a γ-glutamyl-?-lysine bond, with an average of 19 mol of dipeptide per mole of platelet myosin.