Characterization of Tubulin Derived from Ginkgo Biloba and its Interaction with Rice Plant Kinesin

Abstract

Previously, we have expressed novel Rice plant specific kinesins and studied their biochemical characterization. The plant kinesins showed very unique properties. Especially ATPase activities of the kinesins were relatively much lower than that of conventional kinesin. Moreover, the kinesins did not show motile activity on the microtubules prepared from porcine brain. Although the structure of tubulin is well conserved, it is demonstrated that the plant tubulin has different characteristics from tubulin derived from animal. Therefore, plant kinesin may be more compatible with plant tubulin than animal tubulin.In this study, we prepared the plant tubulin from the pollen of Ginkgo biloba. Native tubulin was prepared from ground pollen by acetone treatment methods and purified with DEAE, Mono Q column and Gel filtration chromatography. We have also cloned cDNA of the α/β tubulin from the Ginkgo biloba leaves by RT-PCR. The recombinant α/β tubulins were expressed by E. coli. bacterial expression system. The recombinant tubulins were purified by Co2+ chelating column.Polymerization of the purified plant tubulin to microtubule was monitored by measuring the increase of absorption at 350 nm. Negative staining electron microscopic analysis also revealed the microtubule configuration. ATPase activity of the Rice plant kinesin, K16 was activated by plant tubulin more significantly than that of animal tubulin. These results suggested that plant kinesin is more compatible with plant kinesins than kinesin derived from animal.