Studies on in vitro polymerization of tubulin from renal medullary extracts

Previous in vivo studies showed that microtubules are involved in the cellular action of vasopressin. In order to analyze the role of renal medullary microtubules, a system was developed which would allow the study of the assembly of tubulin in renal medulla extracts into microtubules in vitro. The assembly of tubulin into microtubules occurred in renal medullary cytosol (100 000 × g supernatant) under specific conditions which include pre-concentration of cytosol by ultrafiltration, the presence of ethylene glycol bis(2-aminoethyl)ether tetraacetic acid (EGTA) and 4 M glycerol, and warming at 37 °C. Formation of microtubules, which sedimented at 100 000 × g, was proved by (a) an increase in the apparent [³H]colchicine-binding activity of depolymerized pellets, (b) appearance of typical microbubules as shown by electron microscopy, and (c) by the increase in the quantity of microtubular protein analyzed by polyacrylamide gel electrophoresis. Vinblastine at a concentration of 10^-6 M completely blocked formation of microtubules. A slight increase of ionized calcium in the polymerization mixture also prevented microtubule assembly; this inhibitory effect of ionized calcium was present at concentrations as low as 10^-4 M. Blockade of microtubule assembly by the increase in concentration of ionized calcium or by vinblastine may be the basis of known inhibitory effect of these two agents upon the hydroosmotic effect of vasopressin in vivo.