Summary
Reaction of rabbit skeletal muscle F-actin with the lysine-directed photolabile cross-linker, N-5-azido-2-nitrobenzoyloxy succinimide was limited to Lysine-328 and Lysine-326, with Lysine-328 being labelled to a greater extent. Photolysis of the modified actin enhanced the actin-activated MgATPase activity of filamentous scallp myosin 3-4-fold more than unmodified actin, without affecting calcium sensitivity. Unphotolysed modified actin behaved as untreated actin, indicating that photolysis was essential for the effect. The actin-activated ATPase of filamentous rabbit myosin was similarly increased by photolysed N-5-azido-2-nitrobenzoyloxy succinimide-modified actin. After photolysis in either the monomeric (G-) or filamentous (F-) form, N-5-azido-2-nitrobenzoyloxy succinimide-modified actin moved as a monomeric (42 kDa) species on SDS gels, and depolymerized and polymerized readily, demonstrating that any cross-linking event produced by photolysis must be intramolecular. In contrast to the substantial increase in actin-activated ATPase activity observed when photolysed ANB-NOS-modified actin was added to filamentous myosin, the enhancement was not observed with the soluble HMM and S-1 fragments of myosin. Photolysed modified actin showed only poor movement on a rabbit HMM-coated surface in vitro motility assays. These results can be explained if the internally cross-linked G-actin subunits which comprise only a fraction of the actin population, either weaken the actin-actin contacts or have an increased affinity for myosin.
Similar content being viewed by others
Abbreviations
- ANB-NOS:
-
N-5-azido-2-nitrobenzoyloxy-succinimide
- G-actin:
-
monomeric actin
- F-actin:
-
filamentous actin
- S-1:
-
subfragment I
- HMM:
-
heavy meromyosin
- EGTA:
-
ethyleneglycol(bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)
- DTT:
-
dithiothreitol
- DMSO:
-
dimethyl sulfoxide
- NADodSO4 :
-
sodium dodecyl sulphate
- TEMED:
-
N,N,N′,N′-tetramethylethylenediamine
- TB:
-
tris(hydroxmethy)aminomethane boric acid
- Tris:
-
tris(hydroxymethy)aminomethane
- PTH:
-
phenylthiohydantoin
- TPCK:
-
N-tosyl-phenylalanine chloromethyl ketone
- HPLC:
-
highperformance liquid chromatography
- TFA:
-
trifluoroacetic acid
References
CHANTLER, P. D. & SZENT-GYÖRGYI, A. G. (1980) Regulatory Light Chains and Scallop Myosin: Full Dissociation, Reversibility and Co-operative Effects. J. Mol. Biol. 138, 473–92.
EGELMAN, E. H., FRANCIS, N. & DEROSIER, D. J. (1982) F-actin is a helix with a random variable twist. Nature 298, 131–5.
El-SALEH, S. C., THIERET, R., JOHNSON, P. & POTTER, J. D. (1984) Modification of Lys-237 on actin by 2,4-Pentanedione. J. Biol. Chem. 259, 11014–21.
HARRINGTON, W. (1971) A mechanochemical mechanism for muscle contraction. Proc. Natl. Acad. Sci. USA 68, 685–9.
HASELGROVE, J. C. (1972) X-ray evidence for a conformational change in the actin-containing filaments of vertebrate striated muscle. Cold Spr. Har. Symp. Quant. Biol. 37, 341–52.
HOLMES, K. C. & KABSCH, W. (1991) Muscle proteins: actin. Curr. Opin. Struc. Biol. 1, 270–80.
HUXLEY, H. E. (1969) The mechanism of muscle contraction. Science 164, 1356.
HUXLEY, H. E. (1972) Structural changes in the actin-and myosin-containing filaments during contraction. Cold Spr. Har. Symp. Quant. Biol. 37, 361–76.
HUXLEY, H. E. & BROWN, W. (1967) The low-angle X-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor. J. Mol. Biol. 30, 383–434.
HUXLEY, H. E. & HANSON, J. (1954) Changes in the cross-striations of muscle during contraction and stretch and their structural interpretation. Nature 173, 973–6.
HUXLEY, A. F. & NIEDERGERKE, R. (1954) Interference microscropy of living muscle fibres. Nature 173, 971–3.
KABSCH, W., MANNHERZ, H. G., SUCK, D., PAI, E. F. & HOLMES, K. C. (1990) Atomic structure of the actin: DNase 1 complex. Nature 347, 37–44.
JACKSON, A. P., WARRINER, K. E. & BAGSHAW, C. R. (1987) Measurement of single turnovers of scallop myosin ATPase in the filamentous state. Biochem. Soc. Trans. 15, 900–1.
LAEMMLI, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 80–1.
LEWIS, R. V., ROBERTS, M. F., DENNIS, E. A. & ALLISON, W. S. (1977) Photoactivated heterofunctional cross-linking reagents which demonstrate the aggregation state of phospholipase A2. Biochem. 16, 5650–4.
PROCHNIEWICZ, E. & YANAGIDA, T. (1990) Inhibition of sliding movement of F-actin by cross-linking emphasizes the role of actin structure in the mechanism of motility. J. Mol. Biol. 216, 761–72.
PROCHNIEWICZ-NAKAYAMA, E., YANAGIDA, T. & OOSAWA, F. (1983) Studies on conformation of F-actin in muscle fibres in the relaxed state, rigor and during contraction using fluorescent phalloidin. J. Cell Biol. 97, 1663–7.
PROCHNIEWICZ, E., KATAYAMA, E., YANAGIDA, T. & THOMAS, D. D. (1993) Cooperativity in F-actin: chemical modifications of actin monomers affect the functional interactions of myosin with unmodified monomers in the same actin filament. Biophys. J. 65, 113–23.
RAYMENT, I., HOLDEN, H. M., WHITTAKER, M., YOHN, C. B., LORENZ, M., HOLMES, K. C. & MILLIGAN, R. A. (1993) Structure of the actin-myosin complex and its implication for muscle contraction. Science 260, 58–65.
REES, M. K. & YOUNG, M. (1967) Studies on the isolation and molecular properties of homogeneous globular actin. J. Biol. Chem. 242, 4449–58.
SCHROCK, A. K. & SCHUSTER, G. B. (1984) Photochemistry of phenyl azide: chemical properties of the transient intermediates. J. Am. Chem. Soc. 106, 5228–34.
SCHRODER, R. R., MANSTEIN, D. J., JAHN, W., HOLDEN, H., RAYMENT, I., HOLMES, K. C. & SPUDICH, J. A. (1993) Three-dimensional atomic model of F-actin decorated with Dictyostelium myosin S1. Nature 364, 171–4.
SCHUTT, C. E. & LINDBERG, U. (1992) Actin as the generator of tension during muscle contraction. Proc. Natl. Acad. Sci. USA 89, 319–23.
SPUDICH, J. A. & WATT, S. (1971) The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J. Biol. Chem. 246, 4866–71.
STAFFORD, W. F.III, SZENTKIRALYI, E. M. & SZENT-GYÖRGYI, A. G. (1979) Regulatory properties of single headed fragments of scallop myosin. Biochemistry 24, 5273–80.
STOKES, D. L. & DERSOSIER, D. J. (1987) The variable twist of actin and its modulation by actin binding proteins. J. Cell Biol. 104, 1005–7.
SZENT-GYÖRGYI, A. (1951) In Chemistry of Muscle Contraction, 2nd ed., p. 162. New York: Academic Press.
SZILAGYI, L. & LU, R. C. (1982) Changes of lysine reactivities of actin in complex with myosin subfragment-1, tropomyosin and troponin. Biochim. Biophys. Acta. 709, 204–11.
TOYOSHIMA, Y. Y., KRON, S., McNALLY, E. M., NIEBLING, K. R., TOYOSHIMA, C. & SPUDICH, J. A. (1987) Myosin subfragment-1 is sufficient to move actin filaments in vitro. Nature 328, 536–9.
YAGI, N., & MATSUBARA, I. (1989) Structural changes in the thin filament during activation studied by X-ray diffraction of highly stretched skeletal muscle. J. Mol. Biol. 208, 359–63.
YANAGIDA, T., NAKASE, M., NISHIYAMA, K. & OOSAWA, F. (1994) Direct observation of motion of single F-actin filaments in the presence of myosin. Nature 307, 58–60.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kwon, H., Hardwicke, P.M.D., Collins, J.H. et al. Myosin filament ATPase is enhanced by intramolecularly cross-linked actin. J Muscle Res Cell Motil 15, 555–562 (1994). https://doi.org/10.1007/BF00121161
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00121161