Efficient separation of Thermus aquaticus EF-Tu functional complexes

A new method for fast separation of the main functional complexes of the el ongation factor Tu from Thermus aquaticus has been developed. Binary comple xes EF-Tu * GDP and EF-Tu * GDPNP as well as the ternary complex EF-Tu * GD PNP * Leu similar to tRNA were separated from each other by means of HPLC o n a hydrophobic sorbent TSK-Gel Phenyl 5PW in a reverse gradient of ammoniu m sulfate. This technique is suitable for monitoring EF-Tu activity, charac terisation of the ratio between different EF-Tu forms in cell extracts, and isolation of individual EF-Tu complexes for structural and functional inve stigations. In order to illustrate the potentials of the method, we used HP LC on a TSK-Gel Phenyl 5PW matrix to determine the ratio between affinities of GDP and GDPNP for EF-Tu. We found that K-a(GDP) is about 27 times highe r than K-a(GDPNP) at 37 degreesC, the value being close to the one reported for Thermus thermophilus EF-Tu. (C) zool Academic Press.