Rabbit skeletal myosin heads in solution, as observed by ultracentrifugation and freeze-fracture electron microscopy: dimerization and maximum chord

Bachouchi, Nacima; Gulik, Annette; Garrigos, Manuel; Morel, Jean E.

doi:10.1021/bi00343a040

Cited by 17 publications

(13 citation statements)

References 26 publications

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“…the isolated myosin heads can form dimers (2,10,11,17)]. We verified that the myosin was extremely pure: the index of polydispersity of our samples was typically close to zero [0.05 ( 0.05 (( SD; n ) 10)], which confirms that the head-head dimer was pure, at least for c > 0.72 mg/mL.…”

Section: Resultssupporting

confidence: 68%

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Native Myosin from Adult Rabbit Skeletal Muscle: Isoenzymes and States of Aggregation

et al. 1998

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The globular heads of skeletal muscle myosin have been shown to exist as isoenzymes S1 (A1) and S1 (A2), and there are also isoforms of the heavy chains. Using capillary electrophoresis, we found two dominant isoenzymes of the whole native myosin molecule, in agreement with what has previously been found by various techniques for native and nondenatured myosin from adult rabbits. Findings about possible states of aggregation of myosin and its heads are contradictory. By analytical ultracentrifugation, we confirmed the existence of a tail-tail dimer. By laser light scattering, we found a head-head dimer in the presence of MgATP. Capillary electrophoresis coupled with analytical ultracentrifugation and laser light scattering led us to refine these results. We found tail-tail dimers in a conventional buffer. We found tail-tail and head-head dimers in the presence of 0.5 mM MgATP and pure head-head dimers in the presence of 6 mM MgATP. All the dimers were homodimers. Naming the dominant isoenzymes of myosin a and b, we observed tail-tail dimers with isoenzyme a (TaTa) and with isoenzyme b (TbTb) and also head-head dimers with isoenzyme a (HaHa) and with isoenzyme b (HbHb).

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Section: Resultssupporting

confidence: 68%

“…Studies of the possible states of aggregation of myosin and its heads (LC2-free S1 1 or Mg‚S1, which contains LC2) report contradictory findings (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Among the different data we must cite Winkelman et al (14), who found that the crystallographic unit in S1 crystals is a S1 dimer.…”

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confidence: 98%

Native Myosin from Adult Rabbit Skeletal Muscle: Isoenzymes and States of Aggregation

et al. 1998

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“…It remains to be determined why the sedimentation velocity of SF, is affected significantly by changes in buffer conditions. How-2 While this report was in preparation, new evidence for dimer", formed from papain SF, was published (Bachouchi et al, 1985). Electron micrographs of SF, prepared by a freeze-fracture technique were presented, showing structures identified as dimer^.…”

Section: Discussionmentioning

confidence: 98%

Crosslinking of myosin subfragment 1 and heavy meromyosin by use of vanadate and a bis(adenosine 5'-triphosphate) analog

Munson¹,

Smerdon²,

Yount³

1986

Biochemistry

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The synthesis of a divalent ATP analogue [3,3'-dithiobis[3'(2')-O-[6-(propionylamino)hexanoyl]adenosine 5'-triphosphate] (bis22ATP)] is described in which two molecules of ATP are linked via esterification of their 3'(2')-hydroxyls to the linear dicarboxylic acid 3,3'-dithiobis[N-(5-carboxypentyl)-propionamide] [[HO2C(CH2)5NHC(O)(CH2)2S-]2]. This linkage introduces 22 atoms (a maximum of approximately 2.8 nm) between the ribose oxygens of two ATP molecules. Myosin subfragment 1 (SF1) or heavy meromyosin (HMM) readily cleave bis22ATP to bis22ADP. Upon subsequent addition of excess vanadate ion, both enzymes are rapidly inactivated by formation of a stable vanadate-bis22ADP complex at the active site. By adjustment of the reaction conditions, dimers of SF1 or HMM, both cross-linked with bis22ADP-vanadate, could be prepared. Dimers of SF1 could be separated from monomers by sucrose gradient centrifugation but not by gel filtration. These observations imply that the average Stokes radius of the dimer approximates that of the monomer, a result predicted only for monomers linked approximately side by side. Conversely, dimers of HMM were separated from HMM monomers by gel filtration, reflecting an increase in their Stokes radii. This increase, however, prevented resolution of HMM dimers from monomers by sucrose gradient centrifugation. These results and the molecular dimensions of bis22ATP suggest that the 3'-(2')-hydroxyl of ATP is no more than 1.3 nm from the surface of myosin and suggest further in the simplest interpretation that the active site is most likely located near the middle of the heads of myosin. Analytical sedimentation velocity experiments were performed in order to compare the sedimentation coefficient (s0(20),w) of the SF1 dimer formed by cross-linking to values predicted from ellipsoidal models of the dimer. The observed s0(20),w of the dimer was much closer to the range predicted for a side-to-side arrangement of SF1 monomers than the range predicted for two monomers linked end to end, a result consistent with the active site location suggested above. During the course of these experiments, unmodified SF1 was used as a control, and its sedimentation behavior was reexamined. We have corroborated the finding that the s0(20),w displayed by SF1 can be affected to a limited extent by the particular experimental parameters employed during centrifugation [Morel, J. E., & Garrigos, M. (1982) Biochemistry 21, 2679-2686].(ABSTRACT TRUNCATED AT 400 WORDS)

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“…Morel and Garrigos (1,2) found that crude LC2-free S1 (called below S1) can form dimers in solution. Bachouchi et al (3) directly visualized, by electron microscopy, myosin head dimers of LC2-free S1 1 and Mg.S1 (containing LC2). The existence of the dimeric state of S1 was not accepted for 12-13 years.…”

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confidence: 99%

“…Despite these results, the existence of dimers has been challenged by Munson et al (7) and Stafford III and Margossian (8). These authors probably overlooked the fact that the crystallographic unit in S1 crystals is a dimer (9), or considered that direct observation of the dimer by the techniques described by Bachouchi et al (3) and Winkelmann et al (9) may induce artifactual distortions, not occurring with hydrodynamic techniques. Furthermore, they could argue that the S1 routinely prepared in our laboratory was not sufficiently purified.…”

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confidence: 99%

Dimerization of Native Myosin LC2(RLC)-Free Subfragment 1 from Adult Rabbit Skeletal Muscle

Taouil

D'hahan

et al. 1998

Biochemistry

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We reinvestigated whether the native myosin LC2-free-subfragment 1 (S1) dimer exists by using viscometry, capillary electrophoresis, and laser light scattering. We found that the intrinsic viscosity of the monomer is [eta]m = 6.7 cm3/g and its translation diffusion coefficient is (c = 0) = 4.43 x 10(-)7 cm2/s. For the dimer, [eta]d = 19.8 cm3/g and (c = 0) = 2.54 x 10(-)7 cm2/s. Using the Svedberg equation and introducing the values of the sedimentation coefficients (5.05 S for the monomer and 6.05 S for the dimer), we find the following molecular weights: Mr,m = 108 000 Da and Mr,d = 213 000 Da, which agree well with previous determinations. Capillary electrophoresis successfully separated S1(A1) and S1(A2), in a monomer buffer, and S1(A1) and S1(A2) and a heterodimer S1(A1)-S1(A2), in a dimer buffer. An interesting feature of the monomer-dimer equilibrium is the presence of temperature transitions, whose positions and widths depend upon the buffer conditions. At low temperatures, a pure dimer was observed, whereas at high temperatures only the monomer was present. The dimerization site on both myosin and S1 is extremely labile.

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Rabbit skeletal myosin heads in solution, as observed by ultracentrifugation and freeze-fracture electron microscopy: dimerization and maximum chord

Cited by 17 publications

References 26 publications

Native Myosin from Adult Rabbit Skeletal Muscle: Isoenzymes and States of Aggregation

Native Myosin from Adult Rabbit Skeletal Muscle: Isoenzymes and States of Aggregation

Crosslinking of myosin subfragment 1 and heavy meromyosin by use of vanadate and a bis(adenosine 5'-triphosphate) analog

Dimerization of Native Myosin LC2(RLC)-Free Subfragment 1 from Adult Rabbit Skeletal Muscle

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