title: Covalent modification of G-actin by pyridoxal 5'-phosphate: polymerization properties and interaction with DNase I and myosin subfragment 1.
abstract: Pyridoxal 5'-phosphate (PLP), a lysine-specific reagent, has been used to modify G-actin. At pH 7.5, PLP reacted with 1.7-2 lysines on G-actin. Limited proteolytic digestion experiments indicated that, in agreement with previous works, essentially lysine-61 was modified in a 1:1 fashion by PLP, other lysines being much less reactive. A PLP-derivatized affinity label of ATP binding sites, AMPPLP, reacted with two additional lysines that do not appear to be located in the ATP site on G-actin. PLP-G-actin did not polymerize spontaneously up to 30 microM; however, it retained other essential native properties of G-actin. PLP-actin bound to the barbed ends of actin filaments with an equilibrium dissociation constant of 4 microM and prevented dilution-induced depolymerization like a capping protein. PLP-actin copolymerized with unmodified actin. The stability of F-actin copolymers decreased with the fraction of PLP-actin incorporated, consistent with a model within which the actin-PLP-actin interactions in the copolymer are 50-fold weaker, and PLP-actin-PLP-actin interactions are 200-fold weaker than regular actin-actin interactions. PLP-actin bound DNase I with an equilibrium association constant of 2 nM-1, i.e., 10-fold lower than that of unmodified actin. PLP modification did not affect the binding of G-actin to myosin subfragment 1. However, polymerization of PLP-actin by myosin subfragment 1 was not observed in low ionic strength buffers, whereas PLP-F-actin-S1 filaments, in which the stoichiometry PLP-actin:S1 is 1:1, were formed with an apparent critical concentration of 4.5 microM in the presence of 0.1 M KCl.
authors.affiliation: Laboratoire d'Enzymologie du CNRS, Gif-sur-Yvette, France.
authors.forename: C
authors.initials: C
authors.lastname: Combeau
authors.forename: M F
authors.initials: MF
authors.lastname: Carlier
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002021:Buffers; D002417:Cattle; D003850:Deoxyribonuclease I / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D015879:Myosin Subfragments / Q000378:metabolism*; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D011817:Rabbits; D013816:Thermodynamics
chemical_list: D000199:Actins; D002021:Buffers; D015879:Myosin Subfragments; D011732:Pyridoxal Phosphate; D003850:Deoxyribonuclease I