Protein synthesis in yeast. II. Purification and properties of the elongation factor 1 from Saccharomyces cerevisiae
Dasmahapatra, B.; Skogerson, L.; Chakraburtty, K.
Journal of Biological Chemistry 256(19): 10005-10011
Elongation factor (EF) 1, a protein analogous in function to bacterial Tu, was isolated from the yeast S. cerevisiae and was purified over 270-fold from a high speed supernatant fraction. The homogeneity of the protein was shown by gel filtration and sedimentation equilibrium analysis of the native protein and by gel electrophoresis of the protein under denaturing conditions. The MW of EF1 was .apprx. 49,000 by gel filtration and by sodium dodecyl sulfate electrophoresis and 43,000 by sedimentation equilibrium analysis. Amino acid analysis showed high contents of lysine, arginine and proline, and lack of cysteine. Antibody raised against EF1 showed characteristic precipitation band on an Ouchterlony double diffusion plate. Polyphenylalanine synthesis and the binding reactions were inhibited by the immune serum. There was no detectable cross-reaction of anti-EF1 with the other 2 yeast elongation factors. Yeast EF1 demonstrated binding reactions with guanine nucleotides and with the aminoacyl-tRNA characteristics of the factor 1 activity. Stable binary and ternary complexes were isolated by gel filtration. Divalent cation and spermidine stimulated Phe-tRNA binding and polyphenylalanine synthesis. K+ and NH4+ ions were required for polymerization reactions but not for the binding. Yeast EF1 was highly labile in aqueous buffer but was stabilized by protease inhibitors and by 25% glycerol. EF1 from many eukaryotic sources exists as heterogeneous populations of multimeric forms ranging in MW of up to 1,000,000. EF1.beta., a protein analogous in function to the bacterial Ts, and another uncharacterized protein EF1.gamma., were isolated from some of the multimeric proteins. Yeast EF1 was shown only as a monomeric protein. Attempts to isolate high MW forms of EF1 by gel filtration were not successful. No EF1.beta. activity was detected in the yeast extract.