In vitro assembly and isolation of neurofilaments and microtubules from mammalian CNS.
Neurofilaments were assembled in vitro from the high speed supernatant of mammalian CNS homogenate in a disassembly buffer containing 4-morpholine-ethane sulfonic acid, MgCl2 and EGTA at 4 degrees C in the presence of 4 M glycerol. The assembled neurofilaments were depolymerized by dialysis against the disassembly buffer and repolymerized by the addition of glycerol to the clarified supernatant obtained afer disassembly. The filament assembly reaction was complete in less than 30 s as measured by turbidimetric changes at 415 nm and did not require any added nucleotide. No assembly of filaments was detected when using frozen tissue. The assembled filaments corresponded to the enrichment of neurofilament triplet, the 210,000, 160,000 and 70,000 dalton polypeptides on SDS-polyacrylamide gels and appeared morphologically and immunochemically identical to neurofilaments isolated by axonal flotation methods. These studies demonstrate in vitro assembly of neurofilaments under native conditions which raises the possibility that like microtubules, neurofilaments or a subpopulation of neurofilaments might be in a dynamic state of assembly--disassembly in situ.
D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D002417:Cattle; D003599:Cytoskeleton / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007381:Intermediate Filament Proteins / Q000302:isolation & purification / Q000378:metabolism; D007382:Intermediate Filaments / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008854:Microscopy, Electron; D008868:Microtubule Proteins / Q000302:isolation & purification / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D016900:Neurofilament Proteins; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013116:Spinal Cord / Q000378:metabolism* / Q000648:ultrastructure
D007381:Intermediate Filament Proteins; D008868:Microtubule Proteins; D016900:Neurofilament Proteins