title: A cross-linked profilin-actin heterodimer interferes with elongation at the fast-growing end of F-actin.
abstract: Profilin and beta/gamma-actin from calf thymus were covalently linked using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in combination with N-hydroxysuccinimide, yielding a single product with an apparent molecular mass of 60 kDa. Sequence analysis and x-ray crystallographic investigations showed that the cross-linked residues were glutamic acid 82 of profilin and lysine 113 of actin. The cross-linked complex was shown to bind with high affinity to deoxyribonuclease I and poly(l-proline). It also bound and exchanged ATP with kinetics close to that of unmodified profilin-actin and inhibited the intrinsic ATPase activity of actin. This inhibition occurred even in conditions where actin normally forms filaments. By these criteria the cross-linked profilin-actin complex retains the characteristics of unmodified profilin-actin. However, the cross-linked complex did not form filaments nor copolymerized with unmodified actin, but did interfere with elongation of actin filaments in a concentration-dependent manner. These results support a polymerization mechanism where the profilin-actin heterodimer binds to the (+)-end of actin filaments, followed by dissociation of profilin, and ATP hydrolysis and P(i) release from the actin subunit as it assumes its stable conformation in the helical filament.
authors.affiliation: Department of Cell Biology, the Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden.
authors.forename: Tomas
authors.initials: T
authors.lastname: Nyman
authors.forename: Rebecca
authors.initials: R
authors.lastname: Page
authors.forename: Clarence E
authors.initials: CE
authors.lastname: Schutt
authors.forename: Roger
authors.initials: R
authors.lastname: Karlsson
authors.forename: Uno
authors.initials: U
authors.lastname: Lindberg
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003285:Contractile Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D003460:Crystallization; D019281:Dimerization; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008958:Models, Molecular; D051304:Profilins; D020033:Protein Isoforms / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology; D013950:Thymus Gland / Q000378:metabolism
chemical_list: D000199:Actins; D003285:Contractile Proteins; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; D051304:Profilins; D020033:Protein Isoforms; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases