Tubulin and Other Proteins from Squid Giant Axon

SDS-polyacrylamide gel electrophoresis of axoplasmic proteins and viscometric measurements of axoplasmic tubulin polymerization were performed using giant axons of the squid, Dory-teuthis bleekeri. The axoplasm contained major protein species having apparent chain weights of 200,000, 78,000, 59,000, 52,000, 49,000, and 43,000 daltons as well as high molecular weight components. The 200,000 dalton component and some of the 59,000 and 52,000 dalton components were found in the pellet after high speed centrifugation. Axoplasmic microtubule (MT) proteins were obtained by one cycle of temperature-dependent polymerization and depolymerization. SDS-polyacrylamide gel electrophoresis showed that the squid MT protein fraction consists mostly of tubulin and high molecular weight proteins but lacks the component corresponding to the major high molecular weight protein usually detected in porcine brain MT proteins. The a and β subunits of squid tubulin were shown to have the same mobilities as those of porcine brain tubulin. The viscometric measurements indicated that the optimal temperature for MT assembly was 25°C and optimal salt concentrations were 0.1 m for KC1 and KF, and 0.3 m for K glutamate. The ability of K halides to support assembly was in the order F−>Cl−>Br−>I−. I− was totally inhibitory for assembly. The assembled axoplasmic MT's were sensitive to SH-blocking reagent, Ca ions, colchicine, and cold, in the same way as porcine brain MT's. From axoplasm-extruded axons, a high molecular weight protein was extracted with 0.6 m KI or 10 mM CaCl2. This component was shown to be capable of promoting the polymerization of porcine brain tubulin dimers.