At pH 5.3 and 4.5 the half life of valyl-, threonyl-, leucyl- and seryl-tRNA from E. coli K 12 is significantly higher than at pH 6.8. While no changes were observed in the MAK elution patterns of valyl- and threonyl-tRNA, leucyl-tRNA was eluted in two peaks at pH 6.8 and 5.3 and in one broad peak at pH 4.5. Seryl-TRNA - two peaks at pH 6.8 - was separated in three peaks at pH 5.3 and 4.5. Rechromatography of these peaks at the other pH suggests the existence of at least four species of seryl-tRNA in E. coli K 12.
It is possible to determine the anomeric configuration of nucleosides by a simple, spectrophotometric assay, using the nucleoside phosphorylase activity of cell-free extracts from E. coli. β-nucleo-sides are split, a-anomers remain unchanged. For a single estimation 20-40 µg of nucleoside are required. 6-Azauracil- and 8-azaguanine-β-ᴅ-riboside and some nucleoside phosphates are resistant, a fact, which is of interest in view of the specificity of nucleoside phosphorylases.
The trifluoroacetylation of thymidine at room temperature was performed using trifluoroacetic acid phenylester in pyridine. A selective protection of the 5′-position was not possible: Even low molar quantities of the trifluoroacetylating agent gave rise to bis-trifluoroacetylation. The bis-trifluoroacetyl derivatives of thymidine and 5-bromo-deoxyuridine were purified by vacuum sublimation. The completely trifluoroacetylated deoxyribosides of uracil, 5-iodouracil and adenine underwent decomposition during sublimation.
Following treatment with the β-galactosidase inducer [methyl-3H] -thiogalactoside, an induceracceptor-complex was isolated from extracts of E. coli K 12 using DEAE cellulose chromatography. Enzymatic digestion with trypsin suggested that the inducer was bound to a protein component.
Specific radioactive peaks demonstrated acceptor activity in the inducible strains E. coli K 12 and ML 3, but different results were obtained using the non-inducible mutants ML 35, ML 308 and ML 309.
The potent inhibitor of TMG-induction, o-nitrophenylfucoside, reduced the radioactive acceptor peak and caused a similar inhibition of β-galactosidase synthesis, p-nitrophenylfucoside was ineffective.
Further evidence is presented for the in vitro formation of an inducer-acceptor-complex in cell free extracts of E. coli K 12.
Methylthio-β.ᴅ-galaktosid wird in E. coli K 12, sowie in den Mutanten ML 3 und ML 308 in vivo zu einem geringen Teil in einen Phosphorsäureester, wahrscheinlich das 6-Phosphat (TMG-P) umgewandelt. TMG-P wird von E. coli K 12 aufgenommen, wirkt jedoch nicht als Induktor des Lactose-Operons. Zellfreie Extrakte aus E. coli K 12 geben die gleiche Reaktion, wobei die in vitro-Reaktion durch anorganisches Phosphat und Phosphoenolpyruvat stimuliert wird.