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

Abstract

Incubation of 48,000 X g rat brain supernatants for 30 min at 37 degrees with 1-2 mM guanylyl 5'-methylenediphosphonate [Gmp(CH2)pp] results in polymerization of 95-98% of the tubulin present. This is considerably more than the 50% polymerization that can be achieved with the natural nucleotide, GTP, under optimal conditions. Gmp(CH2)pp is also much more effective than GTP in inducing polymerization of purified tubulin. Assembly of microtubules with Gmp(CH2)pp occurs at tubulin concentrations one-third of those possible with GTP. Furthermore, with Gmp(CH2)pp, microtubule assembly does not require the high molecular weight basic proteins needed with GTP. Polymerization of tubulin by Gmp(CH2)pp is neither prevented nor reversed by concentrations of calcium (2 mM) that can either prevent microtubule assembly or disrupt already formed microtubules if the nucleotide used is GTP or guanylyl imidodiphosphate. When Ca2+ is added before or after microtubule assembly, electron microscopy of the Gmp(CH2)pp preparations reveals normal microtubules turning into twisted ribbons. Low temperature (4 degrees) can both prevent and disrupt the tubulin assembled Gmp(CH2)pp although disruption proceeds much more slowly when GTP is used.

The free energy for hydrolysis of a microtubule-bound nucleotide triphosphate is near zero: all of the free energy for hydrolysis is stored in the microtubule lattice [published erratum appears in J Cell Biol 1995 Apr;129(2):549]

“…The most convincing evidence that GTP hydrolysis modulates microtubule dynamics has come from comparison of the behavior of microtubules containing nonhydrolyzable GTP analogues and GDP. The GTP analogue GMPCPP, which is susceptible to hydrolysis because the methylene bridge finks the alpha and beta phosphate residues, is unique in binding relatively (Zeeberg and Caplow, 1979). tightly to the tubulin E-site and in being more effective than GTP in promoting assembly (Sandoval et al, 1977;Hyman et al, 1992). It is of special interest that the rate of tubulin subunit dissociation from microtubules is dramatically increased when the gamma phosphate moiety of a bound NTP has been lost by hydrolysis.…”

“…4). The previous failure to form microtubnles in a reaction of purified tubulin with GMPCP (Sandoval, 1977) probably resuited from use of low protein concentrations. Video microscopy was used to measure the elongation rate with GMPCP.…”

Polymerization and Calcium Binding of the Tubulin-colchicine Complex in the GDP State

“…On the other hand, GTP can be substituted by non-hydrolyzable GTP analogues. [17][18][19] Further, it does not seem that the rate of GTP hydrolysis is in proportion to the rate of polymerization of the tubulin-colchicine complex and microtubule assembly from pure tubulin. 21,22,24) We found that GTP was hydrolyzed in the stage of tubulin oligomer formation before microtubule assembly.…”

“…16) At the non-exchangeable site, GTP is tightly bound to tubulin. Non-hydrolyzable GTP analogues [17][18][19] are available instead of GTP. In addition, it has been reported that GTP is required for the polymerization of the tubulin-colchicine complex as well as for microtubule formation.…”

Bundling of microtubules in transfected cells does not involve an autonomous dimerization site on the MAP2 molecule.

“…According to one interpretation, bundling of microtubules by MAP2 and tau involves their forming dimers in the cytoplasm and thus actively cross-linking the adjacent microtubules to which they are bound (Kanai et al, 1989;Lewis et al, 1989). The alternative view is based on the observation that microtubules in nonneuronal cells that do not express MAP2 or tau are also bundled by a variety of chemically dissimilar stabilising agents, including taxol, GTP analogues, and dimethyl sulfoxide (DMSO) (Sandoval et al, 1977;Schiff and Horwitz, 1980;Weisshaar et al, 1992). From this it has been argued that bundling by MAPs reflects their property of stabilizing microtubules, which are then bundled by other ubiquitous cellular proteins (Chapin et al, 1991) or because they have an intrinsic affinity for one another (Lee and Brandt, 1992) or because they are brought together by physical interactions within the cell (Matus, 1991;Weisshaar et al, 1992).…”