High affinity DNA-microtubule associated protein interaction.
We have isolated the MAP/tau proteins from twice-cycled chick brain microtubule preparations and demonstrated that they are responsible for the nitrocellulose DNA binding activity we and others have measured. Using the isolated MAP/tau proteins we then measured the apparent affinity constant K(app) for the homologous chick DNA interaction and found evidence for two equilibrium affinity classes-a K(app) = 6 x 10(7) M-1, responsible for the bulk of the DNA binding activity and a small (less than 10%) higher affinity K(app) = 10(8) - 10(9) M-1, likely due to sequence specific binding protein species. Using the same chick brain MAP-tau protein, a heterologous interaction with D. melanogaster DNA, was found to possess just the lower affinity class-K(app) = 2 x 10(7) M-1. Under stringent binding conditions we carried out equilibrium nitrocellulose filter binding experiments in a ternary reaction mixture at constant MAP/tau protein and 35S radiolabelled chick DNA concentration using increasing and excess concentrations of competitor DNAs of different sources. The order of competitor strengths found was-chick DNA greater than mouse DNA greater than D. melanogaster = E. coli. DNA. These data and specifically the homologous DNA: protein case being the strongest competitor corroborate our previous studies using total microtubule protein and provide new evidence for a conserved interaction of a small DNA sequence class with MAP/tau protein species. Moreover, these data allow us to conclude that the conserved DNA sequence: MAP/tau protein interactions do not critically depend upon any energetic feature co-involving tubulin for their properties since tubulin is absent from these preparations.
Marx
K A
KA
Department of Chemistry, University of Massachusetts, Lowell 01854.
D000818:Animals; D001667:Binding, Competitive; D001923:Brain Chemistry; D005591:Chemical Fractionation; D002645:Chickens; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D008869:Microtubule-Associated Proteins