Studies on Polypeptide Elongation Factors from Escherichia coli: II. PURIFICATION OF FACTORS Tu-GUANOSINE DIPHOSPHATE, Ts, AND Tu-Ts, AND CRYSTALLIZATION OF Tu-GUANOSINE DIPHOSPHATE AND Tu-Ts

Abstract

Abstract The polypeptide elongation factors Tu-GDP, Ts and Tu-Ts have been purified from Escherichia coli to homogeneous state as judged by criteria of ultracentrifugation and disc gel electrophoresis. The DEAE-Sephadex column chromatography of the ammonium sulfate fraction (37 to 64% saturation) of E. coli 150,000 x g supernatant yielded two peaks of T activity, i.e. Peaks I and II which are identified as Tu-Ts and Tu-GDP, respectively. Tu-GDP is purified from Peak II through Sephadex G-100 column chromatography and ammonium sulfate fractionation, and crystallized as long hexagonals with pyramidal bases. The crystalline Tu-GDP has a specific activity of 21,000 to 23,000 pmoles of [3H]GDP bound per mg of protein, or approximately 1 mole of GDP per 44,000 to 48,000 g of protein. Ts is purified from Peak I (Tu-Ts), after resolution to Tu-GDP and Ts with GDP, through chromatography on DEAE-Sephadex column in the presence of 10-5 m GDP. After ammonium sulfate fractionation, the homogeneous preparation of Ts is obtained. The purified Ts catalyzes the exchange of 320,000 to 340,000 pmoles of [3H]GDP with Tu-GDP per min at 0° per mg of protein. Tu-Ts complex is purified from Peak I through Sephadex G-100 and second DEAE-Sephadex column chromatography and crystallized as needles. An alternate procedure for the purification of Ts and Tu-GDP which involves the resolution of Tu-Ts complex into Ts and Tu-GDP at earlier step of purification (first ammonium sulfate fraction) has also been described.