Innocuous labeling of the subfragment-2 region of skeletal muscle heavy meromyosin with a fluorescent polyacrylamide nanobead and visualization of individual heavy meromyosin molecules.
We have studied transglutaminase-catalyzed incorporation of monodansylcadaverine and monobiotincadaverine into rabbit skeletal muscle heavy meromyosin (HMM). The incorporation of dansylcadaverine reached saturation at 4 mol per 1 mol of HMM. An electrophoretogram of the chymotryptic digest of the dansyl-labeled HMM revealed that the labeling took place primarily in the S-2 region of HMM. Atomic force microscopic images and electron micrographs of the complexes of the biotinylated HMM and UltraAvidin-coated fluorescent polyacrylamide nanoparticles revealed that the biotinylated site on S-2 was very close to the C-terminus (near the S-2/light meromyosin junction). In keeping with this result, together with HMM's key sites being localized on the S-1 region, the enzymatic conjugation of biotincadaverine had no influence upon the actin-activated ATPase activity of HMM or upon the ability of HMM to actuate sliding of actin filaments in in vitro motility assay. Attachment of an UltraAvidin-coated fluorescent nanobead to the biotinylated HMM also did not alter the motile activity of HMM. Thus, we can optically pinpoint individual HMM molecules in a sample, which will facilitate handling and manipulation of single HMM molecules and observation of their functional behavior.
Kunioka
Y
Y
Department of Physics, Faculty of Science, Kanazawa University, Ishikawa.
Ando
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T
D000180:Acrylic Resins; D000199:Actins / Q000737:chemistry; D000588:Amines; D000818:Animals; D001360:Avidin; D001710:Biotin / Q000031:analogs & derivatives; D002103:Cadaverine / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D018482:Muscle, Skeletal / Q000737:chemistry / Q000201:enzymology; D015879:Myosin Subfragments / Q000737:chemistry*; D009218:Myosins / Q000378:metabolism; D011817:Rabbits; D011503:Transglutaminases; D000099166:Polyacrylamides
D000180:Acrylic Resins; D000199:Actins; D000588:Amines; D001360:Avidin; D001710:Biotin; D002103:Cadaverine; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D009218:Myosins; D011503:Transglutaminases; D000099166:Polyacrylamides; C076022:5-(biotinamido)pentylamine; C008542:monodansylcadaverine