Role of nucleotides in tubulin polymerization: effect of guanylyl 5'-methylenediphosphonate.

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Abstract
Incubation of 48.000 .times. g rat brain supernatants for 30 min at 37.degree. with 1-2 mM guanylyl 5''-methylenediphosphonate [Gmp(CH2)pp] resulted in polymerization of 95-98% of the tubulin present. This was considerably more than the 50% polymerization that could be achieved with the natural nucleotide, GTP, under optimal conditions. Gmp(CH2)pp was also much more effective than GTP in inducing polymerization of purified tubulin. Assembly of microtubules with Gmp(CH2)pp occurred at tubulin concentrations 1/3 of those possible with GTP. With Gmp(CH2)pp, microtubule assembly did not require the high MW basic proteins needed with GTP. Polymerization of tubulin by Gmp(CH2)pp was neither prevented nor reversed by concentrations of Ca2+ (2 mM) that could either prevent microtubule assembly or disrupt already formed microtubules if the nucleotide used was GTP or guanylyl imidodiphosphate. When Ca2+ was added before or after microtubule assembly, EM of the Gmp(CH2)pp preparations revealed normal microtubules turning into twisted ribbons. Low temperature (4.degree. C) could prevent and disrupt the tubulin assembled with Gmp(CH2)pp although disruption proceeds much more slowly than when GTP was used.