Molecular properties of elongation factor Tu fromStreptomyces aureofaciens andEscherichia coli

Abstract

Some molecular properties of the elongation factor Tu of protein synthesis purified in an aggregated state from gram-positive Streptomyces aureofaciens were studied and compared with those of Tu from gram-negative Escherichia coli. Electrofocussing under reducing conditions showed that the molecule of EF-Tu from S. aureofaciens has an isoelectric point shifted more to the acidic side compared with EF-Tu from E. coli. A comparison of amino acid composition revealed minor differences in the content of several amino acids in the two factors and showed that EF-Tu from S. aureofaciens contains four half-cystines per molecule. Under denaturing conditions only two mercapto groups reacted with 5,5'-dithiobis(2-nitrobenzoic acid). Limited tryptic digestion of aggregated EF-Tu from S. aureofaciens yields six fragments: the four main fragments are of a similar size as those of the E. coli factor. All fragments detected after trypsin digestion of S. aureofaciens EF-Tu were immunologically cross-reactive with antibodies against E. coli EF-Tu. However, even after 2 h of the reaction there still remains a small part of streptomycete factor uncleaved, which documents high resistance of aggregated EF-Tu towards trypsin.

Content, distribution and stability of protein-synthesis elongation factor Tu in subcellular fractions of vegetative cells and spores of Streptomyces aureofaciens

“…The difference in the effect of this protease on aggregated and non-aggregated EF-Tu supports our previous hypothesis [4,6] suggesting that the aggregation of EF-Tu in spores might function as a protective mechanism during differentiation.…”

“…The second band, representing a protein smaller by 4000-5000 Da, ranged in concentration from 10 to 50% of total EF-Tu depending on the batch. The size of the fragment was the same as that of the main fragment obtained during mild trypsinolysis of EF-Tu purified from vegetative cells [6]. We were able to identify two protein bands interacting with Anti-Tu in all the subcellular fractions from spores except for the membranes (not shown).…”

“…In our previous studies we compared some of the functional and structural properties of EF-Tu from Streptomyces aureofaciens vegetative mycelium and E. co# which revealed a high degree of functional and structural similarity between the two proteins [4][5][6]. Here we have identified the EF-Tu in spores of S. aureofaciens and we describe a membrane bound protease from spores which cleaves the EF-Tu in spore cell-free homogenates.…”

“…SDS polyacrylamide gel electrophoresis was performed according to Laemmli [11] with 10% acrylamide in the separating gel. EF-Tu was identified on the gels using an immunoblotting technique described previously [6]. Protein concentration was estimated by the method of Bradford [12] with bovine serum albumin as a standard.…”

The use of protein synthesis elongation factor EF‐Tu as internal calibration standard in two‐dimensional electrophoretic studies of differentiation in Streptomyces

“…We identified the protein on l-D and 2-D gels by Western blotting as described previously [7] and by comigration of the purified protein with the analyzed sample.…”