A new protein factor that modulates assembly of microtubules in vitro. I. Purification and characterization.
A new protein factor that modulates microtubule assembly in a Ca2+ or Mg2+ concentration-dependent manner was extracted from porcine brain and purified by ammonium sulfate fractionation, acetone fractionation, chromatography on an affinity column fixed with microtubule proteins, and high speed liquid chromatography. The isolated protein was nearly homogeneous on SDS-polyacrylamide gel, appearing as a 94,000-dalton polypeptide. The protein gave a single symmetric peak with a relatively large Stokes radius on Sepharose 4B gel filtration. Moreover, it sedimented as a single homogeneous component with a sedimentation constant of nearly 5 S on sucrose density gradient centrifugation. These analyses indicated the homogeneity of the isolated protein and the asymmetry of its conformation. The purified protein factor slightly inhibited microtubule assembly under standard assembly condition. Increase in the concentration of either free Ca2+ or free Mg2+ markedly enhanced its inhibitory effect. Neither Ni2+ nor Mn2+ potentiated the inhibitory effect of the protein. The inhibition was reversible in a fashion dependent on the concentration of Ca2+ or Mg2+ and appeared to be stoichiometric rather than catalytic. The inhibitory activity was totally destroyed by trypsin digestion, but was very heat stable and was not lost on treatment with N-ethylmaleimide. This new protein factor may play an important role in regulation of microtubule assembly and/or function.
D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D013552:Swine
D009419:Nerve Tissue Proteins; D008274:Magnesium; D002118:Calcium