Purification of Euglena EF-1 L: A cytoplasmic factor that also functions on procaryotic and organellar ribosomes

Abstract

The low-molecular-weight form of the cytoplasmic protein synthesis elongation factor-1 (EF-1 L) from Euglena gracilis has been purified extensively from whole-cell extracts. A four-step purification procedure has been developed which results in a 45-fold enrichment in EF-1 L with 10% recovery of the total EF-1 activity present in the post-ribosomal supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the EF-1 L is greater than 90% pure. The purified factor is composed of a single subunit of molecular weight 56,000 as determined by gel filtration and polyacrylamide gel electrophoresis under denaturing conditions. Unlike EF-1s purified to date from other organisms, Euglena EF-1 L catalyzes polymerization on Escherichia coli and Euglena chloroplast ribosomes, as well as on wheat germ ribosomes. The activity of this factor on 70 S ribosomes is about 5% that observed on eucaryotic 80 S ribosomes. This level of catalytic activity is sufficient to obscure the activity of chloroplast EF-Tu and mitochondrial EF-Tu in whole-cell extracts of Euglena. The activity of EF-1 L as measured on either wheat germ or E. coli ribosomes is unstable in the absence of glycerol, is inhibited only slightly by 20 m m, N-ethylmaleimide, is not stimulated by E. coli EF-Ts, and is not inhibited by the antibiotic kirromycin. The relative affinity of EF-1 L for guanine nucleotides was also measured and it was observed that its affinity for GTP is approximately six- to eightfold greater than that for GDP.