Partial purification of neurofilament subunits from bovine brains and studies on neurofilament assembly.

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Abstract
The 200,000-dalton neurofilament subunit (P200) and the 160,000-dalton (P160) and 78,000-dalton (p78) neurofilament subunits were partially purified from bovine brain. Intact neurofilaments were prepared by high-speed and sucrose-zone centrifugation. The crude neurofilament was solubilized in 8 M urea solution containing pyridine, formic acid and 2-mercaptoethanol. The solubilized neurofilament was purified by carboxymethyl (CM) cellulose column and hydroxylapatitie column chromatography. The P200 was purified as separate from P160 and P78, but the P160 and P78 subunits were copurified on CM cellulose, hydroxylapatite, Bio-Cel A150m and Sephadex G-150 column chromatography. EM of these purified neurofilament subunits revealed the P200 subunit as a globular structure, and the P160 and P78 subunits as a rod-shaped structure extending up to 120 nm with a 8- to 12-nm width. In the presence of 200 mM KCl, 15 mM MgCl2 and 1 mM ATP, the purified subunits assembled into long filaments. Under the assembly condition, P160 and P78 subunits elongated up to 500 nm, but the longer filament formation required the presence of P200 subunits. The filaments formed in vitro were of 2 types: long straight filaments and intertwined knobby-type filaments. Apparently, P160 and P78 form the neurofilament backbone structure and P200 facilitates the assembly of the backbone units into longer filaments.