title: Transglutaminase-induced cross-linking between subdomain 2 of G-actin and the 636-642 lysine-rich loop of myosin subfragment 1.
abstract: G-actin was covalently cross-linked with S1 in a bacterial transglutaminase-catalyzed reaction. The cross-linking sites were identified with the help of fluorescent probes and limited proteolysis as the Gln-41 on the DNase I binding loop of subdomain 2 in G-actin and a lysine-rich loop (residues 636-642) on the S1 heavy chain. The same lysine-rich loop was cross-linked to another region of G-actin in a former study (Combeau, C., D. Didry, and M-F. Carlier. 1992. J. Biol. Chem. 267:14038-14046). This indicates the existence of more than one G-actin-S1 complex. In contrast to G-actin, no cross-linking was induced between F-actin and S1 by the transglutaminase reaction. This shows that in F-actin the inner part of the DNase I binding loop, where Gln-41 is located, is not accessible for S1. The cross-linked G-actin-S1 polymerized upon addition of 2 mM MgCl2 as indicated by electron microscopy and sedimentation experiments. The filaments obtained from the polymerization of cross-linked actin and S1 were much shorter than the control actin filaments. The ATPase activity of the cross-linked S1 was not activated by actin, whereas the K+ (EDTA)-activated ATPase activity of S1 was unaffected by the cross-linking. The cross-linking between G-actin and S1 was not influenced by the exchange of the tightly bound calcium to magnesium; however, it was inhibited by the exchange of the actin-bound ATP to ADP. This finding supports the view that the structure of the DNase binding loop in ADP-G-actin is somewhere between the structures of ATP-G-actin and F-actin.
authors.affiliation: Department of Oral Biology, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.
authors.forename: L
authors.initials: L
authors.lastname: Eligula
authors.forename: L
authors.initials: L
authors.lastname: Chuang
authors.forename: M L
authors.initials: ML
authors.lastname: Phillips
authors.forename: M
authors.initials: M
authors.lastname: Motoki
authors.forename: K
authors.initials: K
authors.lastname: Seguro
authors.forename: A
authors.initials: A
authors.lastname: Muhlrad
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D008239:Lysine; D008854:Microscopy, Electron; D018482:Muscle, Skeletal / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011817:Rabbits; D012697:Serine Endopeptidases; D013211:Staphylococcus aureus / Q000201:enzymology; D013439:Sulfhydryl Reagents; D011503:Transglutaminases / Q000378:metabolism*; D014357:Trypsin
chemical_list: D000199:Actins; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D011503:Transglutaminases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D008239:Lysine