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Myosin filament ATPase is enhanced by intramolecularly cross-linked actin

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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.

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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

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Author notes

  1. Hyockman Kwon

    Present address: Program in Molecular Medicine, University of Massachusetts Medical Center, 0I605, Worcester, MA, USA

  2. Peter M. D. Hardwicke

    Present address: Medical Biochemistry, Southern Illinois University School of Medicine, 62901, Carbondale, Illinois, USA

Authors and Affiliations

  1. Department of Biology, Brandeis University, 02254, Waltham, MA

    Hyockman Kwon, Peter M. D. Hardwicke & Andrew G. Szent-Györgyi

  2. Department of Biological Chemistry, University of Maryland, 21201, Baltimore, MD, USA

    John H. Collins & Xinmei Zhao

  3. School of Medicine, University of Maryland, 21201, Baltimore, MD, USA

    John H. Collins & Xinmei Zhao

  4. Medical Biotechnology Center of Maryland Biotechnology Institute, University of Maryland, 21201, Baltimore, MD, USA

    John H. Collins & Xinmei Zhao

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  1. Hyockman Kwon
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  5. Andrew G. Szent-Györgyi
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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

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  • DOI: https://doi.org/10.1007/BF00121161

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