GDP Inhibits the Activity of the Centrosome to Nucleate Microtubules.

Abstract

ABSTRACT. Microtubule-nucleating ability of the centrosome in the isolated mitotic apparatus was investigated under the condition that the centrosomal fragment was preincubated with guanine nucleotides or its derivatives. GTPand non-hydrolyzable GTP analogues such as GTP-fS or GMP-PNPconsistently supported high activity of the centrosome to nucleate microtubules. On the other hand, GDPsubstantially suppressed the microtubule-nucleating activity of the centrosomes. Evidence has been presented that this is not simply due to the inhibitory effect of GDPon microtubule assembly. This result is consistent with the idea that GTPbinding proteins such as 51 kDa protein and f-tubulin localized to the pericentriolar materials in the centrosome are competent for the nucleation of microtubules when it is saturated with GTP.

Nucleation of Astral-shaped Microtubules from Latex Beads Conjugated with MTOG Proteins

“…This may be why Figure 5 shows a weaker inhibition by GTP-γS than GDP. In a previous study (Nakazawa et al, 1994), the number of asters was not clearly distinguishable among assembly systems containing GTP, GTP-γS, or GMP-PNP. Therefore, the inhibition of the MtSt formation by GTP-γS in the present study would suggest that the inhibition was due not to the Mt nucleation but to the Mt growth.…”

“…The effect of GDP on the aster forming activity of MTOGs was preliminarily examined by Ohta et al (1989), who found that the aster formation was inhibited when p51 was saturated with GDP, and recently that p51 has a Kd of 1.1 µM for GTP and 0.15 µM for GDP (Ohta et al, 1999). That Mt assembly is inhibited by GDP-saturated p51 was later confirmed by Nakazawa et al (1994).…”

“…Extraction of Mt proteins from porcine brains and purification of tubulin dimers has been described previously (Maekawa et al, 1992;Nakazawa et al, 1994). Purified tubulin divers (ca.…”

“…The MTOG fraction was prepared from the unfertilized eggs of Clypeaster japonicus as described before (Toriyama et al, 1988), except that solubilization of the fraction was carried out in the presence of 1 mM GTP. We omitted the use of 50% glycerol because GTP was found to stabilize the aster-forming activity of the p51enriched fraction prepared by phosphocellulose column chromatography (Nakazawa et al, 1994). The MTOG fraction (hereafter referred to as p51) was stored at -80°C after being divided into 0.05 ml portions.…”

“…6) p51 was shown to be a GTP-binding protein (Ohta et al, 1990). The GTP-saturated form of p51 promoted Mt assembly but the GDP-saturated form of p51 was quite inhibitory for Mt assembly (Ohta et al, 1989;Nakazawa et al, 1994). These results suggested that p51 is a component of MTOGs and responsible for the formation of the mitotic apparatus.…”