Separation of microtubule populations in rat brain homogenates by differential centrifugation

Subcellular fractions were separated from rat homogenates at 600 g for 5 min (P1), 15,000 g for 10 min (P2), 48,000 g for 60 min (P3), 100,000 g for 150 min (P4) and a final supernatant (S). They contained 5.0, 14.3, 14.7, 24.7 and 41.3% of the total tubulin in the homogenate measured by colchicine binding. The microtubules recovered in a pool of fractions P1 + P2 + P3 underwent a faster depolymerization than those of P4 when the sediments were suspended in buffered solutions, poor in the stabilizing agent glycerol. As was observed with the electron microscope, P4 was richer than the other fractions in microtubules associated with membranous structures (MTA) by filamentous lateral connections. Subcellular distribution and lability properties may be the result of the connection of microtubules with membranous structures; the attached membranes would decrease the sedimentation velocity of microtubules, and lateral bridges could increase microtubular stability. Differences in length were not a cause of microtubule separation since this parameter did not vary among the microtubule of the different fractions or between MTA and non-associated microtubules of the same fraction.